Biophysica

The investigation of structure–function relationships in enzyme polysaccharide complexes provides a theoretical foundation for modulating enzyme properties and expanding their industrial applications. In this study, the interaction of cysteine proteases—bromelain, ficin, and papain—with a grafted chitosan–poly(N-vinylpyrrolidone) copolymers, Cs-g-PVP, was examined, and its effect on the catalytic and stability properties of the enzymes was assessed. Molecular docking and Fourier-transform infrared spectroscopy were used to analyze the topology of the resulting complexes and identify macromolecular fragments involved in binding. Based on the obtained results, it was hypothesized that complex formation would lead to a slight reduction in the catalytic activity of cysteine proteases. In vitro studies of the complexes confirmed this hypothesis, showing that the enzymes retained more than 63% of their proteolytic activity while their half-inactivation time during storage increased by up to ~12-fold. The investigated Cs-g-PVP copolymers demonstrated high efficiency as supports for the studied enzymes, capable of retaining up to 100% of the added enzymes. Full article

We present a corrosion-resistant quadrupole ion trap mass spectrometer (QIT-MS) for the direct detection of biosignature gasses in chemically reactive planetary atmospheres, such as Venusian clouds. The system employs a Paul trap with hyperbolic titanium alloy electrodes and alumina spacers for chemical durability and precise ion confinement. An yttria-coated iridium filament serves as the thermionic emitter within a modular electron gun capable of axial and radial ionization. Analytes are introduced through fused silica capillaries and crescent inlets into a miniature pressure cell. The testbed integrates high-voltage RF electronics, pressure-regulated sample delivery, and FPGA-based control for real-time tuning. Continuous operation in 98% sulfuric acid vapor for over three months demonstrated no degradation in emitter or sensor performance. Mass spectra revealed H₂SO₄ fragmentation and thermally induced decomposition up to 425 K. Spectral variations with filament current and electron energy highlight thermal and electron-induced dissociation dynamics. Operational modes include high-resolution scans and selective ion ejection (e.g., CO₂⁺, N₂⁺) to enhance the detection of PH₃⁺, H₂S⁺, and daughter ions. The compact QIT-MS platform is validated for future missions targeting corrosive atmospheres, enabling in situ astrobiological investigations through the detection of biosignature gasses such as phosphine and hydrogen sulfide. Full article

The tetrameric protein transthyretin (TTR) transports the hormone thyroxine in plasma and cerebrospinal fluid. Certain point mutations of TTR, including the Val122Ile mutation investigated here, destabilize the tetramer leading to its dissociation, misfolding, aggregation, and the eventual buildup of amyloid fibrils in the myocardium. Cioffi et al. reported the design and synthesis of a novel TTR kinetic stabilizing ligand, referred to here as TKS14, that inhibited TTR dissociation and amyloid fibril formation. In this study, molecular dynamics simulations were used to investigate the binding of TKS14 and eight TSK14 derivatives to the Val122Ile TTR mutant. For each complex, the ligand’s solvent accessible surface area (SASA), ligand–receptor hydrogen-bonding interactions, and the free energy of ligand-binding to TTR were investigated. The goal of this study was to identify the TSK14 functional groups that contributed to TTR stabilization. TKS14 was found to form a stable, two-point interaction with TTR by hydrogen bonding to Ser-117 residues in the inner receptor binding pocket and interacting through hydrogen bonds and electrostatically with Lys-15 residues near the receptor’s surface. The free energy of TKS14-TTR binding was −18.0 kcal mol⁻¹ and the ligand’s average SASA value decreased by over 80% upon binding to the receptor. The thermodynamic favorability of TTR binding decreased when TKS14 derivatives contained either methyl ester, amide, tetrazole, or N-methyl functional groups that disrupted the above two-point interaction. One derivative in which a tetrazole ring was added to TKS14 was found to form hydrogen bonds with Thr-106, Thr-119, Ser-117, and Lys-15 residues. This derivative had a free energy of TTR binding of −21.4 kcal mol⁻¹. Overall, the molecular dynamics simulations showed that the functional groups within the TKS14 structural template can be tuned to optimize the thermodynamic favorability of ligand binding. Full article

Fungal diseases represent a significant threat to global agriculture, leading to substantial crop losses and endangering food security worldwide. Conventional chemical fungicides, while effective, are increasingly criticized for their detrimental environmental impacts, including soil degradation, water contamination, and the disruption of non-target organisms. Additionally, the overuse of these fungicides has accelerated the emergence of resistant fungal strains, further challenging disease management strategies. In response to these issues, bio-nanofungicides and nano-biofungicides have emerged as a cutting-edge solution, combining biocompatibility, environmental safety, and enhanced efficacy. These advanced formulations integrate bio-based agents, such as microbial metabolites or plant extracts, with nanotechnology to improve their stability, controlled release, and targeted delivery. Chitosan, silica, and silver nanoparticles were extensively studied for their ability to encapsulate bioactive compounds or because of their outstanding antifungal activity, while minimizing environmental residues. Recent studies demonstrated the potential of nano-based fungicides to address critical gaps in sustainable agriculture, with promising applications in integrated pest management systems. Here, we summarize the last advances in the development of bio-nanofungicides and nano-biofungicides and analyze the main differences between them. In addition, challenges such as large-scale production, regulatory approval, and comprehensive risk assessments are discussed. Full article

Cell-based therapy is a promising direction for the treatment of various diseases. However, it is associated with several problems, primarily related to reproducibility and standardization. In this context, the development of new methods for the production of cell-based preparations is of particular relevance. Recently, a novel technology named ‘crossing’ has been developed. It comprises the multi-stage vibrational processing of two closely spaced test tubes containing the initial substance and a neutral carrier (water or lactose). As a result, the neutral carrier acquires some properties of the initial substance, and artificial products, vibrational iterations, are obtained. Some vibrational iterations are also capable of exerting a modifying effect on the initial substance (or its target in the body), changing its physico-chemical/biological properties. Earlier, we demonstrated the possibility of obtaining vibrational iterations from biological molecules (antibodies). In this study, we evaluated the biological effects of vibrational iterations obtained by the crossing technology using cells grown in culture. This work shows that vibrational iterations obtained from CHO-S cell culture affect the ability of CHO-S cells to utilize glucose in the presence of insulin. The data demonstrate the prospect of developing fundamentally new biological drugs based on vibrational iterations, including for the treatment of diabetes mellitus. Full article

Neurodegenerative disorders include Alzheimer’s and Parkinson’s, both of which lead to progressive loss of neurons resulting in the severe loss of cognitive and motor functions. These diseases are among the heavy burdens on global healthcare systems largely because there is no cure, and current treatments apply almost entirely to controlling symptoms rather than disease progression. Recent advances in 3D printing and bioprinting technologies now open the way to overcome these challenges and form patient-specific models and therapeutical tools closely simulating the complex environment of the human brain. It then further illustrates how this technological integration with the aid of 3D printing, coupled with microfabrication and biosensing technologies, transforms drug-screening platforms as well as develops customization in medicine. For example, one can form highly intricate and multi-materially composed structures to better facilitate one’s study or test into some new therapeutic possibilities using methodologies of stereolithography and selective laser sintering. Moreover, 3D printing allows the creation of organ-on-a-chip models that simulate brain-like conditions, which may help identify specific biomarkers and evaluate new options of therapy. On the other hand, bioprinting methods based on neural cells combined with scaffolds mimicking native tissue dramatically transform regenerative medicine. New pathways in neural tissue development and implantable devices are now being brought forth, which can be tailored to the needs of individual patients. These advances bring not only greater precision in terms of the therapy that can be delivered but also 3D printing of implantable microelectrodes able to determine real-time biomarkers responsible for neurodegenerative diseases. Thus, this review highlights the robust impact that might be brought forth on the diagnosis and treatment of these neurodegenerative diseases via 3D printing technologies toward more effective management and personal solutions for healthcare. Full article

The description of the organization of microorganisms in terms of emergent “social” interactions has long been a fascinating and challenging subject, in both biology and sociology. In these organisms, the role of the individual is far less dominant than that of the community, which operates as a sort of superorganism. The coordination is achieved through a communication mechanism known as quorum sensing. Quorum sensing coordinates and regulates various biological aspects of a microbial community, such as the expression of pathogenicity factors, biofilm formation, and the production of secondary metabolites, among others. These processes rely on the coordinated behavior of the entire bacterial population, enabling them to adapt and thrive withing a specific ecological niche under its unique biological, physical and chemical conditions. Finally, quorum sensing also allows the community to control the development of potentially harmful individuals, thus preserving the cooperativeness of the community. This study uses an agent-based quorum sensing model to explore the relationship between metabolic functions and social behavior in bacteria. In particular, we identify two metabolic parameters whose variations provide a broad panorama of possible social characteristics. Furthermore, the proposed QS model allows us to reproduce, at least qualitatively, some experimental results regarding the competition between some strains with different social characteristics. Finally, we examine how an ideal polyculture responds to variations in the metabolic characteristics of its components. Specifically, we identify a particularly stable condition in which the components cooperate to maximize the overall health of the colony. We refer to this state as resonance for life. Full article

This study focuses on biochemical pathways of complex biochemical formation, taking into account various thermodynamic parameters that change as the complexity and molecular weight of complex molecules increase. We conducted a study of the co-direction of changes in thermodynamic quantities such as $lg\left[K_d\right]$, $T\Delta S$, $\Delta (\Delta W)$, and $lg\left(\mathrm{cond}\right(W\left)\right)$ during the transition from a monomer to a dimer and then to a trimer and tetramer. In this work, we assume that the co-direction of changes in thermodynamic quantities as the final molecular formation being achieved signals a higher affinity of molecules among themselves than there is for a biochemical formation, which is characterized by the lack of coordination of the biochemical pathway directions of the final molecular compound. As the studied molecular complexes, we took [LGP2-8dsRNA-LGP2], [VP35]₂-dsRNA-[VP35]₂, and MARV NPcore proteins with peptides and the complex of MJ20 with antigens from the Bundibugyo strain of Ebola virus. Calculations of biochemical reaction paths were conducted. Full article

Within the field of physiology, it is widely recognized that the constant flow of mobile ions across the plasma membrane generates membrane potential in living cells. This understanding is a part of the membrane theory. Despite this, membrane theory does not account for the role of ion adsorption (or desorption) processes in generating membrane potential, even though ion adsorption is a key concept in basic thermodynamics. Presently, the study of physiology lacks integration with thermodynamic principles. The membrane theory posits that living cells can differentiate between Na⁺ and K⁺ by means of channels and pumps. Thus, Na⁺ and K⁺ differentially impact the membrane potential. On the other hand, the Hofmeister effect, an older and less prominent thermodynamic theory, proposes that Na⁺ and K⁺ have varying adsorption levels to biomolecules, potentially accounting for their distinct effects on membrane potential even without the involvement of channels and pumps. This concept, distinct from the traditional membrane theory and grounded in ion adsorption (desorption) alongside the Hofmeister effect, might elucidate the process of membrane potential formation. This ion adsorption (desorption) and Hofmeister effect-based idea relates to the previously overlooked Association-Induction Hypothesis (AIH). Our experimental measurements of membrane potentials using artificial cell models highlight that ion adsorption activity and the Hofmeister effect have a comparable impact on the generation of membrane potential as ion flow in the conventional physiological model, assisted by channels and pumps. Full article

ATP analogues are essential tools in enzymology and structural biology, but the structural and functional implications of their chemical modifications on nucleotide-binding proteins are often underappreciated. To address this, we evaluated a panel of ATP analogues, focusing on thiosubstituted and fluorinated molecules, using the AAA+ ATPase p97 as a benchmark system. Hydrolysis stability and impact on protein conformation, binding modes, and kinetics of enzymatic catalysis were assessed by protein-detected methyl NMR and ligand-detected ¹⁹F NMR in solution, as well as ³¹P solid-state NMR of nucleotides within protein sediments. ATPγS and AMP-PNP emerged as the most suitable analogues for preserving pre-hydrolysis states over extended periods, despite undergoing gradual hydrolysis. In contrast, both AMP-PCP and α/β-thiosubstituted analogues failed to induce native protein conformations in p97. Notably, we demonstrate a novel real-time NMR setup to explore the effect of nucleotide mixtures on cooperativity and the regulation of enzymes. Additionally, aromatic fluorine TROSY-based ¹⁹F NMR shows promise for direct ligand detection in solution, even in the context of large macromolecular complexes. These findings provide critical guidance for selecting ATP analogues in functional and structural studies of nucleotide-binding proteins. Full article

Electrostatic charge significantly influences microorganism–surface interactions, including viral adhesion and transmission. While bacterial surface charges are well characterized using electrophoretic mobility and X-ray photoelectron spectroscopy (XPS), similar studies for viruses are limited. This work bridges the gap by estimating the negative surface charge of the Phi6 bacteriophage using XPS data. A novel approach is applied, combining chemical functionalities derived from XPS with a system of equations to quantify surface polysaccharides, proteins, hydrocarbons, and negatively charged groups (RCOO⁻ and R₂PO₄⁻). The results indicate a predominance of proteins on the viral surface and a pH-dependent negative charge: phosphate groups dominate at low pH (1–3), while both groups contribute equally at pH 4–9. These findings provide a deeper understanding of virus–surface interactions and underscore the importance of pH in modulating viral surface charge. This method, which surpasses traditional electrophoretic mobility techniques, offers new perspectives for studying viral adhesion and developing improved antiviral materials and disinfection strategies. Full article

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