Biophysica

Silver(I) ions and organometallic complexes thereof are well-established antimicrobial agents. They have been employed in medical applications for centuries. It is also known that some bacteria can resist silver(I) treatments through an efflux mechanism. However, the exact mechanism of action remains unclear. All-atom force-field simulations can provide valuable structural and thermodynamic insights into the molecular processes of the underlying mechanism. Lennard-Jones parameters of silver(I) have been available for quite some time; their applicability to properly describing the binding properties (affinity, binding distance) between silver(I) and peptide-based binding motifs is, however, still an open question. Here, we demonstrate that the standard 12-6 Lennard-Jones parameters (previously developed to describe the hydration free energy with the TIP3P water model) significantly underestimate the interaction strength between silver(I) and both methionine and histidine. These are two key amino-acid residues in silver(I)-binding motifs of proteins involved in the efflux process. Using free-energy calculations, we calibrated non-bonded fix (NBFIX) parameters for the CHARMM36m force field to reproduce the experimental binding constant between amino acid sidechain fragments and silver(I) ions. We then successfully validated the new parameters on a set of small silver-binding peptides with experimentally known binding constants. In addition, we monitored how silver(I) ions increased the α-helical content of the LP1 oligopeptide, in agreement with previously reported Circular Dichroism (CD) experiments. Future improvements are outlined. The implementation of these new parameters is straightforward in all simulation packages that can use the CHARMM36m force field. It sets the stage for the modeling community to study more complex silver(I)-binding processes such as the interaction with silver(I)-binding-transporter proteins. Full article

Purpose: The purpose of this study is to analyze the relationship between nuclear charge (Z), atomic mass (A), LET (linear energy transfer for maximal relative biological effectiveness (RBE)) for accelerated ions based on the hypothesis that for each ion, LET_U is related to their nuclear radius. Methods: Published LET_U data for proton, helium, carbon, neon, silicon, argon, and iron ions and their Z and A numbers are fitted by a power law function (PLF) and compared with PLF based on atomic cross-sections and nuclear dimensions for spherical or spheroidal atomic nuclei. The PLF allows for isoeffective RBE estimations for different ions at any value of LET based on the LET_U estimations. For any two ions, A and B, and a specified bioeffect obtained at LET_A, the equivalent isoeffective LET_B, is estimated using ${LET}_B={LET}_A.\left({\displaystyle \frac{{LET}_{U\left[B\right]}}{{LET}_{U\left[A\right]}}}\right)$. Results: The data-fitting program provided the following results: ${LET}_U=78.1.A^{0.26}$, and ${LET}_U=86.6.Z^{0.29}$, where 78.1 and 86.6 keV.μm⁻¹ are the proton LET_U values (i.e., without proton cellular range limit considerations). Goodness-of-fit tests are similar for each model, but the proton estimations differ. These exponents are lower than 0.66 and 0.33 (those for nuclear cross-sections and spherical nuclear radii, respectively), but suggest prolate nuclear shapes in most of the ions studied. Worked examples of estimating isoeffective LET values for two different ions are provided. Conclusions: The fitted power law relationships between LET_U and Z or A are broadly equivalent and compatible with prolate nuclear shapes. These models may offer a more rational basis for future ion-beam radiobiology research. Full article

Cardiovascular diseases have become the leading cause of death in developed countries. Among these, some are related to disruptions in the electrical synchronization of cardiac tissue leading to arrhythmias such as atrial flutter, ventricular tachycardia, or ventricular fibrillation. Their origin is diverse and involves several spatial and temporal scales, ranging from nanoscale ion channel dysfunctions to tissue-level fibrosis and ischemia. Mathematical models play a crucial role in elucidating the mechanisms underlying cardiac arrhythmias by simulating the electrical and physiological properties of cardiac tissue across different spatial scales. These models investigate the effects of genetic mutations, pathological conditions, and anti-arrhythmic interventions on heart dynamics. Despite their varying levels of complexity, they have proven to be important in understanding the triggers of arrhythmia, optimizing defibrillation protocols, and exploring the nonlinear dynamics of cardiac electrophysiology. In this work, we present diverse modeling approaches to the electrophysiology of cardiac cells and share examples from our own research where these approaches have significantly contributed to understanding cardiac arrhythmias. Although computational modeling of the electrical properties of cardiac tissue faces challenges in integrating data across multiple spatial and temporal scales, it remains an indispensable tool for advancing knowledge in cardiac biophysics and improving therapeutic strategies. Full article

Since 2020, the attention of the scientific community has been focused on the overwhelming COVID-19 pandemic, the infectious disease caused by the coronavirus that has affected populations worldwide. The alarming number of deaths and the severity of the symptoms have driven studies aimed at combating this disease. One of the key components in the development of this disease is the protein M^Pro, responsible for the replication and transcription of the virus, making it an excellent biological target in research efforts seeking an effective treatment for the disease. Furthermore, studies have shown that vanadium complexes, such as bis(N′,N′-dimethylbiguanide)oxovanadium (IV), VO(metf)₂∙H₂O, exhibit highly promising effects for the treatment of COVID-19. This molecule contains a ligand known as metformin, which also holds a prominent place as a potential agent in the treatment of this disease due to its antiviral properties. Therefore, an investigation into the interactions between these two systems (M^Pro+Vanadium Complex and M^Pro+Metformin) is pertinent given the significance of these two molecules. Thus, computational studies such as molecular docking and classical molecular dynamics are considered advantageous, assisting in this comparative study, as well as providing a deeper understanding of the interactions that occur within each of them. Full article

Nature equipped red blood cells (RBCs) with diverse mechanical properties, which makes it possible to examine blood with different RBC properties (size, shape, aggregability, deformability). We investigated whether the shelf life of cow blood (stiff RBCs, low aggregability) is longer compared with pig blood (deformability/aggregability comparable to human) due to a delay in RBC clustering and decomposition. Blood was drawn from conscious pigs and cows in their familiar environment to reduce stress and stored 30 days at +7 °C. RBCs remained intact in cow samples whereas pig samples became hemolytic after day 20. White blood cells and platelets decreased with similar percentages in both species. Hematocrit (HCT) decreased due to RBC shrinking in bovine samples and due to RBC decay in porcine samples. Blood viscosity increased in both species although HCT decreased. In porcine samples, shear thinning decreased progressively, indicating a gradual loss of sample cohesion with storage. Yield stress and storage modulus decreased with hemolysis. In HCT-native cow samples, shear thinning, yield stress, and storage modulus showed high intraindividual variability, but the mean values did not change over the time course. In HCT-adjusted (38%) cow samples, solidification occurred after day 7, followed by a reduction in cohesion and shear thinning until the end of storage. Full article

The thermodynamic study of protein folding shows the generation of a narrow range of ΔG° values, as a net result of large changes in the ΔH° and TΔS° values of the folding process. The obvious consequence of this narrow range of values is that a linear enthalpy–entropy relationship, showing apparent enthalpy–entropy compensation (EEC), is clearly observed to be associated with the study of protein folding. Herein, we show the ΔH°, TΔS°, and ΔG° values for a set of 583 data from protein folding processes, at various temperatures, as calculated by using the Gibbs–Helmholtz equations. This set of thermodynamic data was calculated from the melting temperature (Tm), the melting enthalpy (ΔHm), and the change in heat capacity (ΔCp°) values, all of them associated with the heat-induced protein unfolding processes and included in the ProTherm Data Base. The average values of enthalpy (ΔH°av), entropy (TΔS°av), and free energy (ΔG°av) for the folding process were calculated within the range of temperature from 0 °C to the average value of Tm. The values and temperature dependency of TΔS°av within this temperature range are practically equal to those corresponding to ΔH°av, while ΔG°av remains small and displaying a curve with a minimum at about 10 °C and a value of ΔG° = −30.9 kJ/mol at the particular temperature of 25 °C. The large negative value of TΔS°av, together with the also large and negative value of ΔCp°av, suggests large conformational changes and important EEC, thus causing the small average value of ΔG° for protein folding, which is enough to guarantee both protein stability and molecular flexibility to allow for adaptation to the chemical potentials of the environment. Our analysis suggests that EEC may be the quantum-mechanical evolutive mechanism to make functional proteins adaptative to environmental temperature and metabolite concentrations. The analysis of protein folding data, compared with those of protein–ligand interaction, allows us to suggest strategies to overcome EEC in the design of new drugs. Full article

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques of 3D microscopy and super-resolution microscopy (SRM) with an optical resolution down to the sub-nanometer range have recently been realized. However, most of these achievements have been made with fixed specimens, i.e., direct information about the dynamics of the biosystem studied was not possible. This stimulated the development of live cell microscopy imaging approaches, including Low Illumination Fluorescence Microscopy, Light Sheet (Fluorescence) Microscopy (LSFM), or Structured Illumination Microscopy (SIM). Here, we discuss perspectives, methods, and relevant light doses of advanced fluorescence microscopy imaging to keep the cells alive at low levels of phototoxicity. Full article

Microplastics (MPs) are widespread environmental pollutants that have drawn significant attention due to their possible health risks to humans and animals, as well as their extensive presence in ecosystems. Recent growing evidence highlights a remarkable relationship between MPs and extracellular vesicles (EVs), nanoscale particles involved in intercellular communication. The purpose of this review was to investigate how the relationships between MPs and EVs can affect cellular functions and how this interaction could impact environmental conditions leading to broader ecological risks. The interaction patterns and bioactivity of both MPs and EVs are strongly influenced by biophysical characteristics such as hydrophobicity, surface charge, and particle size, which have received particular attention from the scientific community. Recent studies indicate that MPs affect EV distribution and their capacity to function appropriately in biological systems. Additionally, MPs can modify the molecular cargo of EVs, which may result in alterations of cell signaling pathways. Understanding the interactions between MPs and EVs could provide important opportunities to comprehend their potential effects on human health and environmental systems, especially when it comes to cancer development, endocrine, metabolic, and inflammatory disorders, and ecological disruptions. This review emphasizes the necessity of multidisciplinary research to clarify the molecular and biophysical mechanisms regulating the interaction between MPs and EVs. Full article

Targeted Auger emitters are being considered as a cancer treatment owing to the high linear energy transfer of Auger electrons. When targeted to cancers, this allows for a highly efficient treatment with a low risk of damage to surrounding healthy tissue. The purpose of this study was to determine the most DNA-damaging Auger emitters from a range of radionuclides, some of which are clinically utilised. A Monte Carlo method-based software (Geant4-DNA version 10.7) was used to determine the energy deposition and number of DNA double-strand breaks from Auger (and internal conversion) electrons imposed on a tetranucleosome. The Auger emitters, ¹¹⁹Sb and ¹⁰³Pd, have similar or slightly greater damaging properties compared to ¹²³I, ¹¹¹In, and ⁸⁹Zr. ^193mPt demonstrated the greatest therapeutic potency. Whilst ¹²⁵I was highly damaging, its relatively long half-life (60 days) makes it less desirable for clinical use. Geant4-DNA modelling identified the radionuclide ^193mPt as being highly favourable for use in radiotherapy. Full article

Cellular heterogeneity, an inherent feature of biological systems, plays a critical role in processes such as development, immune response, and disease progression. Human mesenchymal stem cells (hMSCs) exemplify this heterogeneity due to their multi-lineage differentiation potential. However, their inherent variability complicates clinical use, and there is no universally accepted method for detecting and quantifying cell population heterogeneity. Dielectrophoresis (DEP) has emerged as a powerful electrokinetic technique for characterizing and manipulating cells based on their dielectric properties, offering label-free analysis capabilities. Quantitative information from the DEP spectrum, such as transient slope, measure cells’ transition between negative and positive DEP behaviors. In this study, we employed DEP to estimate transient slope of various cell populations, including relatively homogeneous HEK-293 cells, heterogeneous hMSCs, and cancer cells (PC3 and DU145). Our analysis encompassed hMSCs derived from bone marrow, adipose, and umbilical cord tissue, to capture tissue-specific heterogeneity. Transient slope was assessed using two methods, involving linear trendline fitting to different low-frequency regions of the DEP spectrum. We found that transient slope serves as a reliable indicator of cell population heterogeneity, with more heterogeneous populations exhibiting lower transient slopes and higher standard deviations. Validation using cell morphology, size, and stemness further supported the utility of transient slope as a heterogeneity metric. This label-free approach holds promise for advancing cell sorting, biomanufacturing, and personalized medicine. Full article

Cyclodextrins (CDs) are natural cyclic oligosaccharides with the ability to form inclusion complexes with various organic substances. In this paper, we investigate the potential of CD complex formation to enhance the antibacterial activity and antioxidant properties of poorly soluble bioactive agents, such as chalcones, chromenes, stilbenoids and xanthylium derivatives, serving as potential adjuvants, in comparison with standard antiseptics. The interaction of these bioactive agents with the hydrophobic pocket of methyl-β-cyclodextrin (MCD) was confirmed using spectroscopic methods such as UV-vis, FTIR, ¹H and ¹³C NMR, mass-spectrometry. CD-based delivery system allows for combining multiple active agents, improving solubility, antibacterial efficacy by enhancing penetration into target bacterial cells (E. coli selectivity demonstrated via confocal microscopy). Novel compounds of chalcones and stilbenoids derivatives additionally enhance efficacy by inhibiting bacterial efflux pumps, increasing membrane permeability, and inhibiting bacterial enzymes, and showed a synergy when used in combination with metronidazole. The intricate relationship between the structural characteristics and functional properties of chalcones and stilbenoids in terms of their antibacterial and antioxidative capabilities is revealed. The substituents within aromatic rings significantly influence this activity, where position of electron-donating methoxy groups playing a crucial role. Among chalcones, stilbenoids, ana xanthyliums, the compounds caring a benzodioxol ring, analogous to natural bioactive compounds like apiol, dillapiol, and myristicin, emerge as prominent antibacterial activity. To explore the possibility to create theranostic formulations, we used fluorescent markers to visualize target cells, antiseptics to provide antibacterial activity, and bioactive agents as chalcones acting as adjuvants. Additionally, new antioxidant compounds were found such as Xanthylium derivative (R351) and chromene derivative: 1-methyl-3-(2-amino-3-cyano-7-methoxychromene-4-yl)-pyridinium methanesulfate: the pronounced antioxidant properties of these substances are observed comparable to quercetin in the efficiency. Rhodamine 6G, gentian violet, and Congo Red exhibit good antioxidant properties, although their activity is an order of magnitude lower than that of quercetin. However, they have remarkable potential due to their multifaceted nature, including the ability to visualize target cells. The most effective theranostic formulation is the combination of the antibiotic (metronidazole) + dye/fluorophore (methylene blue/rhodamine 6G) for visualization of target cells + adjuvant (chalcones or xanthylium derivatives) for antiinflammation effect. This synergistic combination, results in a promising theranostic formulation for treating bacterial infections, with enhanced efficiency, selectivity and minimizing side effects. Full article

The effect of sodium alginate on the denaturation and aggregation behavior of bovine serum albumin and hen egg-white lysozyme was studied. Large amounts of polysaccharide increase the thermal stability of albumin due to the weak binding interactions. At the same time, sodium alginate can reduce the quantity of amyloid fibrils formed by albumin under denaturing conditions, which is a consequence of the stabilization of the native protein form by glycan binding. In the case of lysozyme, the polysaccharide has no influence on the thermal stability of the protein in 2 M guanidinium hydrochloride. However, the inhibition of fibril formation with an increase in the lag time was observed, which is explained by the binding of sodium alginate to lysozyme fibrils, but not to the protein monomer. The molecular nature of the binding interactions between alginate and the studied proteins was elucidated using molecular docking and known experimental structures of glycan–protein complexes. Full article

Cells compartmentalize biochemical processes using physical barriers in the form of membranes. Eukaryotes have a wide diversity of membrane-based compartments that can be used in this context, with the main ones being the extracellular membrane, which separates the inside from the outside of the cell, and the nuclear membrane, which separates the nucleus from the cytoplasm. The nuclear membrane not only isolates and protects the DNA and the transcription and replication processes from the other processes that are occurring in the cytoplasm but also has an active role in the regulation of cellular signaling. The TGF-$\beta$ pathway is one of the most important and conserved signaling cascades, and it achieves compartmentalization using a well-tuned balance between the import and export rates of the active and inactive forms of key proteins. Thus, compartmentalization serves as an additional regulatory mechanism, physically isolating transcription factors from their targets, influencing the dynamics and strength of signal transduction. This contribution focuses on this biophysical layer of regulation, using the TGF-$\beta$ pathway to illustrate the molecular mechanisms underlying this process, as well as the biological consequences of this compartmentalization. We also introduce a simplified mathematical formulation for studying the dynamics of this process using a generalized approach. Full article

The interactions and structural organization of water molecules play a crucial role in a wide range of physical, chemical, and biological processes. The ability of water to form hydrogen bonds (H-bonds) underpins its unique properties and enables it to respond dynamically to various environmental factors. These interactions at the molecular level may affect vital processes like protein folding, enzyme activity, and cellular organization. The presence of solutes and spatial constraints can alter the H-bonding network of water, and these effects are ubiquitous in the biological environment. In this study, we analyzed the fluorescence of 2-acetyl-6-(dimethylamino)naphthalene (ACDAN) fluorescence emission in water solutions containing kosmotropic and chaotropic salts and in agar hydrogels. Recently, this dye has proven invaluable in studying water network structure and dynamics, as its fluorescence signal changes based on the local dielectric environment, revealing variations in the dipolar relaxation of water. Our results show that ACDAN spectral response correlates with the degree of water ordering, providing important insights into solute–water interactions and water dynamics in free and confined environments. Full article

Tissue spheroids are self-organised 3D cellular aggregates that serve as a versatile platform in tissue engineering. While numerous high-throughput methods exist to characterise the cellular function of tissue spheroids, equivalent techniques for the mechanical characterisation are still lacking. In this review, we focus on tissue fusion— a simple, fast, and inexpensive method to characterise the rheology of tissue spheroids. We begin by discussing the implications of tissue rheology in development and disease, followed by a detailed explanation of how the phenomenon of arrested coalescence can be used to explore the rheology of tissue spheroids. Finally, we present different theoretical models that, when combined with experimental data, allow us to extract rheological information. Full article

Vertex models have become essential tools for understanding tissue morphogenesis by simulating the mechanical and geometric properties of cells in various biological systems. These models represent cells as polygons or polyhedra, capturing cellular interactions such as adhesion, tension, and force generation. This review explores the ongoing evolution of computational vertex models, highlighting their application to complex tissue dynamics, including organoid development, wound healing, and cancer metastasis. We examine different energy formulations used in vertex models, which account for mechanical forces such as surface tension, volume conservation, and intercellular adhesion. Additionally, this review discusses the challenges of expanding traditional 2D models to 3D structures, which require the inclusion of factors like mechanical polarisation and topological transitions. We also introduce recent advancements in modelling techniques that allow for more flexible and dynamic cell shapes, addressing limitations in earlier frameworks. Mechanochemical feedback and its role in tissue behaviour are explored, along with cutting-edge approaches like self-propelled Voronoi models. Finally, the review highlights the importance of parameter inference in these models, particularly through Bayesian methods, to improve accuracy and predictive power. By integrating these new insights, vertex models continue to provide powerful frameworks for exploring the complexities of tissue morphogenesis. Full article

The variable heavy chain fragments derived from camelid antibodies, called VHHs or nanobodies, have recently shown promise as high-affinity reagents. They offer higher stability compared to conventional antibodies and fragments thereof. Furthermore, their smaller size (~15–20 kDa) allows better targeting of molecules localized inside the cell and in crowded environments, like tissues and protein aggregates. Despite these advantages, nanobody clones screened using phage display can suffer from poor soluble expression, which we hypothesized is due to the presence of hydrophobic hotspots on their surface. In this work, we propose a novel, computationally guided workflow for screening and production of nanobody binders for optimized expression. After an initial round of phage display screens against our target (K-Ras), we modeled the lead candidates to generate spatial aggregation propensity (SAP) maps to highlight the hydrophobic hotspots with single amino acid resolution, which were subsequently used to guide mutagenesis of the binders for soluble expression. We followed two approaches to perform point hydrophilic mutations: (i) performing point hydrophilic mutations in the hydrophobic hotspots; (ii) combining point mutation resulting from a round of random mutagenesis that show favorable SAP scores. Both approaches led to a remarkable increase in soluble expression, which allowed production and characterization of their binding to their target (K-Ras) on soluble ELISA and biolayer interferometry. We observed that the latter approach resulted in clones with stronger binding affinity compared to the former approach. Our results emphasize the need to perform a round of random mutagenesis to identify point mutations, which can then be used in an in silico guided pipeline to identify the right combination of mutations for high soluble expression. Full article

Chemokine ligands play a pivotal role in immune response by mediating cell migration and coordinating cellular processes through interactions with chemokine receptors. Understanding their sequence and structural integrity is crucial for elucidating their biological functions and potential therapeutic applications. In this study, we investigate the dimer interface between two distinct homodimer topologies: CXC and CC homodimers. Despite nearly identical monomeric structures, the rigid CXC interface is characterized by interactions between the N-loop/β-sheet regions, while the more flexible CC interface involves interactions through the unstructured N-terminal regions. Our structural and biophysical analyses indicate no significant differences in the free energy of folding (2–8 kcal/mol) and binding (10–14 kcal/mol) between the two homodimer topologies, showing that their free energy is primarily driven by sequence. We hypothesize that the biological signal is driven by the malleability of the dimer, depending on the binding interface. Understanding these structural dynamics opens new possibilities for designing chemokine-based therapeutics to modulate immune responses in diseases such as cancer, inflammation, and autoimmune disorders. Full article

We revisit the seminal model of glycolysis first proposed by Sel’kov more than fifty years ago. We investigate the onset of instabilities in biological systems described by the Sel’kov model in order to determine the conditions of the model parameters that lead to bifurcations. We analyze the glycolysis reaction under the circumstances when the diffusivity of both ATP and ADP reactants are taken into account. We estimate the critical value of the model’s single compact dimensionless parameter, which is responsible for the onset of reaction instability and the system’s symmetry breaking. It appears that it leads to spatial inhomogeneities of reactants, leading to the formation of standing waves instead of a homogeneous distribution of ATP molecules. The consequences of this model and its results are discussed in the context of the Warburg effect, which signifies a transition from oxidative phosphorylation to glycolysis that is correlated with the initiation and progression of cancer. Our analysis may lead to the selection of therapeutic interventions in order to prevent the symmetry-breaking phenomenon described in our work. Full article