In this paper I will address Dr. Sonne’s questions about will, agency, choice, consciousness, relevant brain regions, impacts of disorders and their therapeutics, and I will do this by referring to my theory, Dual-brain Psychology, which posits that within most of us there exist two mental agencies with different experiences, wills, choices, and behaviors. Each of these agencies is associated as a trait with one brain hemisphere (either left or right) and its composite regions. One of these agencies is more adversely affected by past traumas and is more immature and more symptomatic while the other is more mature and healthier. The theory has extensive experimental support through 17 peer-reviewed publications with clinical and non-clinical research. I will discuss how this theory relates to the questions that Dr. Sonne presented and will discuss also my published theory on the physical nature of subjective experience and its relation to the brain and how that theory interacts with DBP, and how the 2 theories relate to subjective experience, will, behavior, psychopathology and its treatment.

We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, reported previously for gel phase structures also extend to the physiological more relevant fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementary performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. This suggests that hydrocarbon chain interdigitation plays only a minor role in transbilayer coupling. For both cases of adaption to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.

Visual imagery, i.e., the mental experience of an object occurring in the absence of external visual stimulus, might encompass diverse content according to different observers. Further, subjectively experienced time is encoded in the later entorhinal cortex. Starting from these two observations, and considering Einstein’s account of spacetime, we show how, in terms of special relativity, the content of visual imagery is not stationary and fixed, rather depends on the standpoint of the observer. We elucidate how the subjective definition of time (perceived by our mind as static) might give rise to modifications in the length of the imagined object which are experimentally assessable and quantifiable. In particular, based on recent neuroscientific literature, we show how changes in our mental time windows are able to squeeze the visual content of mental imagery.

Principal component analysis (PCA) is used to reduce the dimensionalities of high dimensional datasets in a variety of research areas. For example, biological macromolecules, such as proteins, exhibit many degrees of freedom, allowing them to adopt intricate structures and exhibit complex functions by undergoing large conformational changes. Therefore, molecular simulations of and experiments on proteins generate a large number of structure variations in high dimensional space. PCA and many PCA-related methods have been developed to extract key features from such structural data, and these approaches have been widely applied for over 30 years to elucidate macromolecular dynamics. This review mainly focuses on the methodological aspects of PCA and related methods, and their applications for investigating protein dynamics.

Origin, mechanics and properties of the Solar System are analyzed in the framework of Complete Relativity. The analysis confirms the postulates and hypotheses of the theory with a high degree of confidence. During the analysis, some new hypotheses have emerged. These are discussed and confirmed with various degrees of confidence. To increase confidence or refute some hypotheses, experimental verification is necessary. Main hypotheses and conclusions are: - Solar System is a scaled Carbon isotope with a nucleus in a condensed (bosonic) state and components in various vertically excited states, - Earth is a living being of extremely introverted intelligence, life is common everywhere, albeit extroverted complex forms are present on planetary surfaces only during planetary neurogenesis, - anthropogenic climate change is only a part (trigger from one perspective) of bigger global changes on Earth and in the Solar System during planetary neurogenesis, - major extinction events are relative extinctions, a regular part of transformation and transfer of life in the process of planetary neurogenesis.

Under the oxidative stress condition, the small RNA (sRNA) Oxys that acts as essential post-transcriptional regulators of gene expression is produced and plays a regulatory function with the assistance of the RNA chaperone Hfq protein. Interestingly, experimental studies found that the N48A mutation of Hfq protein could enhance the binding affinity with OxyS while resulting in defection of gene regulation. But, how the Hfq protein interacts with sRNA Oxys and the origin of the stronger affinity of N48A mutation are both unclear. In this paper, molecular dynamics (MD) simulations were performed on the complex structure of Hfq and OxyS to explore their binding mechanism. The molecular mechanics generalized Born surface area (MM/GBSA) and interaction entropy (IE) method were combined to calculate the binding free energy between Hfq and OxyS sRNA, and the computational result is in excellent correlation with the experimental result. Per-residue decomposition of the binding free energy revealed that the enhanced binding ability of the N48A mutation mainly comes from the increased van der Waals interactions (vdW). This research explores the binding mechanism between Oxys and chaperone protein Hfq, and revealed the origin of the strong binding affinity of N48A mutation. The results provided important insights on the mechanism of gene expression regulation affected by protein mutations.

The goal of this short communication is to analyze a published discussion that states that long-term residing at a thermoneutral indoor temperature condition hinders human thermal acclimation capacities. According to current research, human thermal acclimation and acclimatization capacities can be easily gained through repeated heat and cold exposures mixed with physical activity over a period of days (often 3–21 days). Furthermore, heat and cold adaptations are not permanent, and heat acclimation would progressively fade away if frequent heat exposures (associated with physical work/exercise) were discontinued. People who have been heat acclimatized for a long period and live in tropical places may progressively lose their physiological and perceptual benefits when they shift to temperate zones. On the other hand, the decay of cold acclimation and cold acclimatization has not been well examined, demanding future research on this area. To summarize, there is no evidence to support the claim that extended exposure to thermoneutral conditions impairs human acclimatization abilities.

As a Complex System, our body acts as a whole system connected to the environmental incitements. It is ordered, coherent, tries to maintain the least possible entropy, saving the greatest amount of energy. We can observe its active systemic response to environmental information both when it is healthy and ill. To explain the dynamics of the systemic regulative network a theoretical model is proposed, with a comprehensive approach that allows seeing the entire regulative syste m as a continuous unicuum. The paper analyzes two points of view: 1) the connections between the quantum level and the classical one, through some principles of the QFT and through the Coherence Domains. The system is modeled as a field described by the wa ve function, with synchronous and consistent events, driven in a global computing by the quantum potential Q. The quantum potential implies the non locality, and it needs only ultra weak waves to occur, so it explains how the rapid and global activation of the organism in response to punctiform information work. The initial hypothesis is that some consistent quantum phenomena are amplified through the systemic regulative network until they become macroscopic observable. This is possible because of Coherence Domains. 2) The reactions of the different systemic networks to perturbations/punctiform information, with the first attempt to model and measure information in biology, going beyond the Shannon and Turing theories. Hopfield Networks and an informational point of view are used to address the crucial informational and organizational role of proteins and nucleic acids. With this new frame we could develop innovative therapeutic strategies, and also evolve new experimental way to make our clinical observation more precise.

In this manuscript we use an exactly solvable stochastic binary model for regulation of gene expression to analyse the dynamics of response to a treatment aiming to modulate the number of transcripts of RKIP gene. We demonstrate the usefulness of our method simulating three treatment scenarios aiming to reestablish RKIP gene expression dynamics towards pre-cancerous state: i. to increase the promoter’s ON state duration; ii. to increase the mRNAs’ synthesis rate; iii. to increase both rates. We show that the pre-treatment kinetic rates of ON and OFF promoter switching speeds and mRNA synthesis and degradation will affect the heterogeneity and time for treatment response. Hence, we present a strategy for reducing drug dosage by simultaneously targeting multiple kinetic rates. That enables a reduction of treatment response time and heterogeneity which in principle diminishes the chances of emergence of resistance to treatment. This approach may be useful for inferring kinetic constants related to expression of antimetastatic genes or oncogenes and on the design of multi-drug therapeutic strategies targeting master regulatory genes.

Human ether-a-go-go-related gene (hERG) potassium channel is well-known contributor to drug-induced cardiotoxicity and therefore an extremely important target when performing safety assessments of drug candidates. Ligand-based approaches in connection with quantitative structure active relationships (QSAR) analyses have been developed to predict hERG toxicity. Availability of the recent published cryogenic electron microscopy (cryo-EM) structure for the hERG channel opened the prospect for using structure-based simulation and docking approaches for hERG drug liability predictions. In recent time, the idea of combining structure- and ligand-based approaches for modeling hERG drug liability has gained momentum offering improvements in predictability when compared to ligand-based QSAR practices alone. The present article demonstrates uniting the structure-based SILCS (site-identification by ligand competitive saturation) approach in conjunction with physicochemical properties to develop predictive models for hERG blockade. This combination leads to improved model predictability based on Pearson’s R and percent correct (represents rank-ordering of ligands) metric for different validation sets of hERG blockers involving diverse chemical scaffold and wide range of pIC50 values. The inclusion of the SILCS structure-based approach allows determination of the hERG region to which compounds bind and the contribution of different chemical moieties in the compounds to blockade, thereby facilitating the rational ligand design to minimize hERG liability.

The nucleus has been studied for well over 100 years, and chromatin has been the intense focus of experiments for decades. In this review, we focus on an understudied aspect of chromatin biology, namely the chromatin fiber polymer's mechanical properties. In recent years, innovative work deploying interdisciplinary approaches including computational modeling, in vitro manipulations of purified and native chromatin have resulted in deep mechanistic insights into how the mechanics of chromatin might contribute to its function. The picture that emerges is one of a nucleus that is shaped as much by external forces pressing down upon it, as internal forces pushing outwards from the chromatin. These properties may have evolved to afford the cell a dynamic and reversible force-induced communication highway which allows rapid coordination between external cues and internal genomic function.

Understanding cell migration is a key step to unravel many physiological phenomena and predict several pathologies, like cancer metastasis. In particular, mechanical confinement has been proved to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in-vitro migration studies under mechanical confinement in microfluidic devices.

Unlike electron microscopy, which can achieve very high resolutions, but to date can only be used to study static structures, time-resolved X-ray diffraction from contracting muscles can, in principle, be used to follow the molecular movements involved in force generation on a millisecond timescale albeit at moderate resolution. However, previous X-ray diffraction studies of resting muscles have come up with structures for the head arrangements in resting myosin filaments that are different from the apparently ubiquitous interacting heads motif (IHM) found by single particle analysis of electron micrographs of isolated myosin filaments from a variety of muscle types. This head organization is supposed to represent the super-relaxed state of the myosin filaments where ATP usage is minimized. Here we have tested whether the interacting heads motif structures will satisfactorily explain the observed low-angle X-ray diffraction patterns from resting vertebrate (bony fish) and invertebrate (insect flight) muscles. We find that the interacting heads motif does not, in fact, explain what is observed. Previous X-ray models fit the observations much better. We conclude that the X-ray diffraction evidence has been well interpreted in the past and that there is more than one ordered myosin head state in resting muscle. There is, therefore, no reason to question some of the previous X-ray diffraction results on myosin filaments; time-resolved X-ray diffraction should be a reliable way to follow crossbridge action in active muscle and may be one of the few ways to follow molecular changes in myosin heads on a millisecond timescale as force is actually produced.

Background. The treatment of coronary stenosis relies on invasive high risk surgical assessment to generate the fractional flow reserve diagnostics index, a ratio of distal to proximal pressures in respect of coronary atherosclerotic plaque causing stenosis. Non-invasive methods are therefore a need of the times. This study proposes an extensible mathematical description of the coronary vasculature that permits rapid estimation of the coronary fractional flow reserve. Methods. By adapting an existing closed loop model of human coronary blood flow, the effects of large vessel stenosis and microvascular disease on fractional flow reserve were quantified. Several simula-tions generated flow and pressure information which was used to compute fractional flow re-serve under a spectrum of conditions including focal stenosis, diffuse stenosis, and microvascular disease. Sensitivity analysis stratified the influence of model parameters on the index. The model was simulated as coupled non-linear ordinary differential equations and numerically solved us-ing an implicit higher order method. Results. Large vessel stenosis affected fractional flow re-serve. The model predicts that the presence, rather than severity, of microvascular disease affect coronary flow deleteriously. Sensitivity analysis revealed that heart rate may not affect the index. Conclusions. The model provides a computationally inexpensive instrument for future in silico coronary blood flow investigations as well as clinical-imaging decision making. A combination of focal and diffuse stenosis appears to be essential in reducing the index. In addition to pressure measurements in the large epicardial vessels, diagnosis of microvascular disease is essential. The independence of the index with respect to heart rate suggests that computationally inexpensive steady state simulations may provide sufficient information to reliably compute the index.

In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: "In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43”. In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1β), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in the last years, it was observed a huge increase in the number of MPs solved by cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB). However, sample preparation remains a significant challenge in the field. Here, we evaluated in the MPs solved by cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å the most critical parameters for sample preparation: i) the surfactant used for protein extraction from the membrane, ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, iii) the vitrification method employed, and iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs, but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.

Group IV phospholipase A₂α (cPLA₂α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA₂α to the Golgi involves the N-terminal C2 domain whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA₂α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigate how membrane curvature influences the homeostatic role of cPLA₂α in vesicular trafficking. The cPLA₂α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA₂α membrane penetration. We show that cPLA₂α undergoes C2 domain-dependent oligomerization on membranes in vitro and in A549 cells. We found that the association of the cPLA₂α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA₂α C2 domain generates cholesterol enriched Golgi-derived vesicles independently of cPLA₂α catalytic activity. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.

Intermolecular interactions are the fabrics underlying almost all processes in living organisms, where two cornerstone concepts, intermolecular binding affinity (K d ) and binding energy (ΔG), have long been established to physically describe the strengths of biomolecular interactions, e.g., drug-target K d and ΔG to describe the strength of drug-target interaction. The past two-three years saw a big step forward in the use of artificial intelligence (AI) in structural biology (e.g., AlphaFold for protein structure prediction) and drug discovery & design. In light of the roles of K d and ΔG in drug discovery & design, the speed of this AI progress raises a question of what’s next for its practical application in the pharmaceutical industry, in addition to a system-wide account of biomolecular structures and motions. Last August, the concept of a general intermolecular binding affinity calculator (GIBAC) was for the first time coined and proposed in an MDPI-published preprint. Here, this article puts forward an updated conceptual and practical framework of GIBAC, including its inception, definition, construction, practical applications, technical challenges and limitations, and future directions. Moreover, this article argues that the time is now ripe for the construction of such an accurate, precise and efficient GIBAC to be on the agenda of the entire drug discovery & design community, to ensure its applicability & reliability, and to enhance its value in drug R&D in future.

There is a paucity of data on the progression of track cycling sprinters, and the evolution of training and performance over a training cycle. Following prior research showing relationships between sprint cycling power and endurance cycling power, we compared these relations over a 3-month period. Our hypothesis is large improvements in power would come from training either for sprint power or sprint capacity, and this would be reflected in the data. A total of publicly available 25 datasets. These data were plotted against the line of best fit for 30-s and 2-min power to assess if training towards the line led to better 30-s power. We observe the best performances came from riders who start below the 30-s vs 2-min power line of best fit, with greater capacity and ability to ride fast over a sprint series, and progressing towards focusing on specific power in the final block leading into a competition. These results support the hypothesis of combined capacity and power training based on rider-specific relationship to the line of best fit between these measures versus a strictly maximal power training focus.

The translocator protein (TSPO) is a transmembrane protein present in the three domains of life. Its functional quaternary structure consists of one or more subunits. In mouse, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.

All living organisms require some metal ions for their energy production as well as metabolic and biosynthetic processes. Within cells, metal ions are involved in the formation of adducts interact with metabolites and macromolecules (proteins and nucleic acids). The proteins that require binding to one or more metal ions to be able to carry out their physiological function are called metalloproteins. About one third of all protein structures in the Protein Data Bank involve metalloproteins. Over the past few years there has been a tremendous progress in the number of computational tools and techniques making use of 3D structural information to support the investigation of metalloproteins. This trend has been boosted also by the successful applications of neural networks and deep learning approaches in molecular and structural biology at large. In this review, we discuss recent advances in the development and availability of resources dealing with metalloproteins from a structure-based perspective. We start by addressing tools for the prediction of metal-binding sites (MBSs) using structural information on apo-proteins. Then, we provide an overview of methods for and lessons learned from the structural comparison of MBSs in a fold-independent manner. We then move to describing databases of metalloprotein/MBS structures. Finally, we summarize recent DL applications enhancing the functional interpretation of metalloprotein structures.

Identifying the correct dosage and time are key factors to successful implementation of anthelmintic. Comparing differential evolution of infection between anthelmintic treated animals against untreated ones, we present a mathematical model that first calibrates data collected and analyzed over an extended period of 10 years (2011-2019), and then predicts the dynamical evolution of gastrointestinal parasites in livestock, focusing specifically on Stronglye \& Coccidia oocysts, the two prime negative contributors to cattle health, measured using the standard Eggs-Per-Gram (EPG) index. The model incorporates information about all three critical regimes of infection - low infection regime ($< 50$ EPG), medium infection regime ($50-100$ EPG) and high infection regime ($>100$ EPG), including fatally large doses of infection ($> 500$), and probabilistically estimates the variation in animal weight due to infection propagation. A key success of our model is its ability to accurately predict the appropriate anthelmintic treatment times for cattle from a numerical solution of the model presented. The generic model can be applied to other agroclimatic conditions and can serve as a major diagnostic tool for anthelmintic strategizing.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Its treatment includes antiarrhythmic drugs (AADs) to modulate the function of cardiac ion channels. However, AADs have been limited by proarrhythmic effects, non-cardiovascular toxicities as well as often modest antiarrhythmic efficacy. Theoretical models showed that combined blockade of Nav1.5 (and its current INa) and Kv1.5 (and its current, IKur) ion channels yield a synergistic anti-arrhythmic effect without effect on ventricles. We focused on Kv1.5 and Nav1.5 to search for structural similarities in their binding site (BS) for flecainide (a common blocker and widely prescribed AAD), as a first step for prospective rational multi-target directed ligand (MTDL) design strategies. We presented a computational workflow for flecainide BS comparison in a flecainide-Kv1.5 docking model and a solved structure of flecainide-Nav1.5 complex. The workflow includes docking, molecular dynamics, BS characterization and pattern matching. We identified a common structural pattern in flecainide BS for these channels. The latter belongs to the inner cavity and consist of a hydrophobic patch and a polar region, involving residues from S6 helix and P-loop. Since the rational MTDL design for AF is still incipient, our findings could advance multi-target atrial-selective strategies for AF treatment.

Ebolavirus has a membrane envelope decorated by trimers of a glycoprotein (GP), which is responsible for host cell attachment and membrane fusion. Therefore, GP is a primary target for antiviral drugs development. Here, this article reports the first, to my knowledge, set of structural analysis of all Ebolavirus GP structures as of March 10, 2020, and also a brief update of the structurally identified electrostatic features of the Ebolavirus GP structures in both apo (unliganded) state and also in bound states with a series of small compounds, including a variety of approved drugs. With this comprehensive set of structural analysis, this article puts forward a hypothesis of two Achilles' heels of Ebolavirus GP structure, where the formation of two interfacial salt bridges, instead of destabilizing the prefusion conformation of Ebolavirus GP, constitutes a positive contribution towards the structural rigidification of the prefusion conformation of the Ebolavirus GP structure, thereby acting against GP-mediated Ebolavirus cell entry and/or preventing fusion between the viral and endosome membranes.

In previous work, a quantum mathematical formalism associated an element of experience with a single sensory neuron, as a local reduction of a global mental state. In contrast to the binary objective states of neuronal polarisation/depolarisation, neuronal experience was modeled as a continuous variable, the instantaneous value of which could only be estimated statistically from an ensemble of evoked responses to stereotyped stimulus presentation. In the present work, the quantum operations formalism of energy dissipation through amplitude damping is adopted to explain how smooth evolution of conscious experience might arise from discrete spikes and discontinuous synaptic transmission between neurons.

Nonionizing millimeter-waves (MMW) are reported to interact with cells in a variety of ways. Possible mechanisms of the inhibited cell division effect were investigated using 85-105 GHz MMW irradiation within the ICNIRP (International Commission on Non-Ionizing Radiation Protection) non-thermal 20 mW/cm² safety standards. ~1.0 mW/cm² exposure over 5-6 hours treatment on 50 cells/μl samples of Saccharomyces cerevisiae model organism, resulted in 62% growth rate reduction compared to control (sham). The effect was specific for 85-105 GHz range and energy dose and cell density dependent. Irradiation of wild type and Δrad52 (DNA damage repair gene) deletion cells presented no differences of colony growth profiles indicating non-thermal MMW treatment does not cause genetic alterations. Dose versus response relations studied using a standard horn antenna (~1.0 mW/cm²) and compared to that of a compact waveguide (17.17 mW/cm²) for increased power delivery resulted in complete termination of cell division via non-thermal processes supported by temperature rise measurements. Combinations of MMW mediated Structure Resonant Energy Transfer (SRET), membrane modulations eliciting signaling effects, and energetic resonance with biomolecules were indicated to be responsible for the observations reported. Our results provide novel mechanistic insights enabling innovative applications of nonionizing radiation procedures for eliciting targeted biomedical outcomes.

Complex diseases such as cancer are caused by changes in the Gene Regulatory Networks. Systems that model the complex dynamics of these networks along with adapting to real gene expression data are closer to reality and can help understand the creation and treatment of cancer. In this paper, for the first time, modelling of gene regulatory networks is performed using delayed nonlinear variable order fractional systems in the state space by a new tool called GENAVOS. This tool uses gene expression time series data to identify and optimize system parameters. This software has several tools for analyzing system dynamics. The results show that the nonlinear variable order fractional systems have very good flexibility in adapting to real data. We found that regulatory networks in cancer cells actually have a larger delay parameter than in normal cells. It is also possible to create chaos, periodic and quasi-periodic oscillations by changing the delay, degradation and synthesis rates. Our findings indicate a profound effect of time-varying order on these networks, which may be related to a type of cellular memory due to epigenetic and environmental factors. We showed that by changing the delay parameter and the variable order function for a normal cell system, its behavior changes and becomes quite similar to the behavior of a cancer cell. This work also confirms the effective role of the miR-17-92 cluster in the cancer cell cycle. GENAVOS is available at https://github.com/hanif-y/GENAVOS with its user guide and MATLAB codes.

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting variety of hydrophobic ligands including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth and this can be attributed to its estrogen-binding ability. Despite AFP has long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP-ligand interaction remain obscure. In our study we constructed homology-based 3D model of human AFP (HAFP) with the purpose to perform docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol) and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on ligand docked scoring function, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity sites were located in (i) a tunnel formed within HAFP subdomains IB and IIA and (ii) opposite side of the molecule in a groove originating from cavity formed between domains I and III, while (iii) the third low-affinity site was found at the bottom of the cavity. 100 ns MD simulation allowed studying their geometries and showed that HAFP-estrogen interactions occur due to van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP-antiestrogen binding. MM/GBSA rescoring method estimated binding free energies (ΔG_bind) and showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP-ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues along two disulfide bonds, Cys224-Cys270 and Cys269-Cys277 have key roles in both HAFP-estrogen and HAFP-antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein-ligand interactions and anti-cancer therapy strategies based on ER-binding ligands.

From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.

Connexin 43 (Cx43) is expressed in kidneys and constitutes a feedforward mechanism leading to inflammation in other tissues where they form hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analyses of ethidium uptake and thiobarbituric acid reactive species revealed that TNF-α plus IL-1β increase Cx43 hemichannel activity and oxidative stress in MES-13 cells, a cell line derived from mesangial cells. The latter also was accompanied by a reduction in gap junctional communication, whereas western blotting analysis showed a progressive increase of phosphorylated MYPT (a substrate of RhoA/ROCK) and Cx43 upon TNF-α/IL-1β treatment. Additionally, inhibition of RhoA/ROCK strongly diminished the TNF-α/IL-1β-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

In the same manner that there are several statements of the second law of thermodynamics but all of them are equivalent, it is possible that most of the entropic methods applied to the nervous system and brain in particular share a similar outcome, or essence, that help understand some aspects of the structure of cognition. In this short review focused on certain aspects of the entropic metrics some results are examined that indicate the fundamental importance of the natural tendency towards a maximal energy distribution for brain activity to be optimal and thus cognition to emerge.

Although there is a common physiological notion that the origin of the membrane potential is attributed to transmembrane ion transport, it is theoretically possible to explain its generation by the mechanism of ion adsorption. It was previously suggested that the ion adsorption mechanism led even to the potential formulas which are even identical to either the famous Nernst equation or Goldman-Hodgkin-Katz equation. Our further analysis shown in this paper indicates that the potential formula based on the ion adsorption mechanism leads to one equation which is the function of material surface charge density and the material surface potential. Furthermore, we confirmed that the equation holds in all the different experimental systems we studied. Although we have not succeeded in elucidating why such an equation is established, the equation appears to be the key equation governing the characteristics of the membrane potential regardless of the systems in question.

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences enriched with thymine bases represent the signal inducing abortive transcription. On the other hand, certain repetitive sequence elements broadly embedded in promoter regions constitute the signal inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we also suggest that repetitive sequence elements of promoter DNA modulate the rigidity of its double-stranded form, which profoundly influences the reaction coordinates of the productive initiation via pausing.

Man has always been interested in animal electricity, which seems to be measured in every living cell. He has been fascinated by trying to elucidate the mechanisms by which this potential is created and maintained. Biology is the science that seeks to explain this mystery. Biology is based on basic sciences such as physics or chemistry. The latter, in turn, make systematic use of mathematics to measure, evaluate and predict certain phenomena and to develop "laws" and models that are as general as possible while respecting, as closely as possible, observations and facts. The Nernst equation was one of the pillars of electrochemistry. Biology also uses this same equation as one of the indispensable bases for the computation of membrane potential. Man has established a cellular model that highlights this equation in several forms. However, we are going to show by various means that this model is inadequate or even inapplicable.

Nonionizing millimeter-waves (MMW) interact with cells in a variety of ways. Here the inhibited cell division effect was investigated using 85-105 GHz MMW irradiation within the ICNIRP (International Commission on Non-Ionizing Radiation Protection) non-thermal 20 mW/cm2 safety standards. We irradiated using radiation with a power density of about 1.0 mW/cm2 over 5-6 hours on 50 cells/μl samples of Saccharomyces cerevisiae model organism. This resulted in 62% growth rate reduction compared to the control (sham). The effect was specific for 85-105 GHz range, and was energy and cell density dependent. Irradiation of wild type and Δrad52 (DNA damage repair gene) deleted cells presented no differences of colony growth profiles indicating non-thermal MMW treatment does not cause permanent genetic alterations. Dose versus response relations studied using a standard horn antenna (~1.0 mW/cm2) and compared to that of a compact waveguide (17.17 mW/cm2) for increased power delivery resulted in complete termination of cell division via non-thermal processes supported by temperature rise measurements. Combinations of MMW mediated Structure Resonant Energy Transfer (SRET), membrane modulations eliciting signaling effects, and energetic resonance with biomolecules are conjectured to be responsible for the observations reported. Our results suggest innovative applications of nonionizing radiation procedures for yeast related diseases and other targeted biomedical outcomes.

The extremely controversial conclusions about the function of human interferon-gamma (hIFNγ) C-terminus as well as the lack of a consistent model explaining its role in the receptor binding prompted us to scrutinize the interaction of hIFNγ with its extracellular receptor hIFNGR1 in different scenarios by means of molecular dynamics simulations. We find that the two molecules alone fail to form a stable complex but the presence of heparan-sulfate-like oligosaccharides largely facilitates the process by both demobilizing the highly flexible C-termini of the cytokine and assisting in the proper positioning of its globule between the receptor subunits. An antiproliferative-activity essay on cells depleted from surface sulfation confirms qualitatively the simulation-based multistage complex-formation model. Our results reveal the key role of HS and its proteoglycans in all processes involving hIFNγ signalling.

Three finger toxins (3FTX) are a group of peptides that affect multiple receptor types. One group of proteins affected by 3FTX are nicotinic acetylcholine receptors (nAChR). Structural information on how neurotoxins interact with nAChR is limited and are confined to a small group of neurotoxins. Therefore, in silico methods are valuable in understanding the interactions between 3FTX and different nAChR subtypes, but there are no established protocols to model 3FTX – nAChR interactions. We developed a homology modeling and protein docking protocol to address this issue and tested its success on three different systems. First, neurotoxin peptides co-crystallized with acetylcholine binding protein (AChBP) were re-docked to assess whether Rosetta protein – protein docking can reproduce the native poses. Second, experimental data on peptide binding to AChBP was used to test whether the docking protocol can qualitatively distinguish AChBP-binders from non-binders. Finally, we docked eight peptides with known α7 and muscle-type nAChR binding properties to test whether the protocol can explain the differential activities of the peptides at the two receptor subtypes. Overall, our docking protocol succeeded in predicting both qualitative and specific aspects of 3FTX binding to nAChR and shed light on some unknown aspects of 3FTX binding to different receptor subtypes.

The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, TALE and CRISPR-Cas9 revealed common rules, which can help the optimization of activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression, and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor systematically optimized designer transcription factors to the needs of specific applications.

Therapeutically effective treatments of cancer are limited. To calibrate the efficiency of the novel technique we recently discovered to modulate cancer cell viability using tuned electromagnetic fields; H1299 human lung cancer cells were irradiated in a sweeping regime of W-band (75-105 GHz) millimeter waves (MMW) at 0.2 mW/cm² (2 W/m²). Effects on cell morphology, cell death and senescence were examined and compared to that of non-tumorigenic MCF-10A human epithelial cells. MMW irradiation led to alterations of cell and nucleus morphology of H1299 cells, significantly increasing mortality and senescence over 14 days of observation. Extended irradiation of 10 minutes duration resulted in complete death of exposed H1299 cell population within two days, while healthy MCF-10A cells remained unaffected even after 16 minutes of irradiation under the same conditions. Irradiation effects were observed to be specific to MMW treated H1299 cells and absent in the control group of non-irradiated cells. MMW irradiation affected nuclear morphology of H1299 cells only and not of the immortalized MCF-10A cells. Irradiation with low intensity MMW shows an antitumor effect on H1299 lung cancer cells. This method provides a novel treatment modality enabling targeted specificity for various types of cancers.

Optical properties of the cornea are responsible for correct vision, ultrastructure allows optical transparency and biomechanical properties governs the shape, elasticity or stiffness of the cor-nea affecting ocular integrity and intraocular pressure. Therefore, optical aberrations, corneal transparency, structure and biomechanics play a fundamental role in the optical quality of hu-man vision, ocular health and refractive surgery outcomes. However, the convergence of those properties is not yet reported at macroscopic scale within the hierarchical structure of the cornea. This work explores the relationships between biomechanics, structure and optical properties (corneal aberrations and optical density) at macrostructural level of the cornea through dual Placido-Scheimpflug imaging and air-puff tonometry systems in a healthy young adult popula-tion. Results showed convergence between optical transparency, corneal macrostructure and biomechanics.

We research the interaction between six representative carbon-based nanoparticles (CBNs) and 20 standard amino acids through all-atom molecular dynamics simulations. The six carbon-based nanoparticles are fullerene(C60), CNT55L3, CNT1010L3, CNT1515L3, CNT2020L3, and two-dimensional-graphene(Graphene33). Their curvatures decrease sequentially, and all of CNT are single-walled carbon nanotubes. We have observed that as the curvature of CBNs decreases, the adsorption effect of 20 amino acids with them has an increasing trend. In addition, we also used multi-dimensional clustering to analyze the adsorption effects of 20 amino acids on six carbon-based nanoparticles. We observed that the π-π interaction still plays an extremely important role in the adsorption of amino acids on carbon-based nanoparticles. Individual long-chain amino acids and “Benzene-like” Pro also have a strong adsorption effect with carbon-based nanoparticles.

It is widely, but not universally, believed that the lipids of the plasma membrane are not uniformly distributed, but that "rafts'' of sphingolipids and cholesterol float in a "sea'' of unsaturated lipids. The physical origin of such heterogeneities is often attributed to a phase coexistence between the two different domains. We argue that this explanation is untenable for several reasons. Further we note that the results of recent experiments are inconsistent with this picture. However they are quite consistent with an alternate explanation, namely that the plasma membrane is an emulsion of the two kinds of regions. To show this, we briefly review a simplified version of this theory and its phase diagram. We also explicate the dependence of the predicted domain size on four physical parameters. Among them are the spontaneous curvature of the membrane and its bending modulus and surface tension. Taking values of the latter two from experiment, we obtain domain sizes for several different cell types that vary from 58 to 88 nm.

We identified a single nucleotide variation (SNV) (c.1264A&gt;G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein, never mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions, in the presence of auxiliary subunit KCNE1, results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect, the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A-B linker of Kv7.1, which was not shown before to be crucial for channel functioning.

Artificial membrane systems can serve as models to investigate molecular mechanisms of different cellular processes, including transport, pore formation, and viral fusion. However, the current simulacrums such as SUVs, GUVs, and the supported lipid bilayers suffer from issues, namely high curvature, heterogeneity, and surface artefacts, respectively. Freestanding membranes provide a facile solution to these issues, but current systems developed by various groups use silicon or aluminium oxide wafers for fabrication that involves access to a dedicated nanolithography facility and high cost while conferring poor membrane stability. Here, we report the development, characterization and applications of an easy-to-fabricate suspended lipid bilayer (SULB) membrane platform leveraging commercial track-etched porous filters (PCTE) with defined microwell size. Our SULB system offers a platform to study the lipid composition-dependent structural and functional properties of membranes with exceptional stability in a high throughput fashion. With dye entrapped in PCTE microwells by SULB, we show that sphingomyelin significantly augments the activity of pore-forming toxin, Cytolysin A (ClyA) and the pore formation induces lipid exchange between the bilayer leaflets. Further, we demonstrate high efficiency and rapid kinetics of membrane fusion by dengue virus in our SULB platform. Our suspended bilayer membrane mimetic offers a novel platform to investigate a large class of biomembrane interactions and processes.

Chemistry has been viewed as one of the most fruitful near-term applications to science of quantum computing. Recent work in transitioning classical algorithms to a quantum computer has led to great strides in improving quantum algorithms and illustrating their quantum advantage. Much less effort has been placed on how one finishes these calculations by using the results from the quantum computer (on the active region of the molecule) and embeds them back into the remainder of the molecule in order to determine the properties of the entire molecule. Such strategies are critical if one wants to expand the focus to biochemical molecules that contain active regions that cannot be properly explained with classical algorithms on classical computers. While we do not solve this problem here, we provide an overview of where the field is going to enable such problems to be tackled in the future.

In celIs, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule “tubulin code,” which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit – despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament.

This work is the first part of a series of papers on a comprehensive analysis of the processes of prebiotic self-organization and protophotosynthesis on the surface of semiconductor minerals and in systems of natural dispersed semiconductors. Comprehensive analysis within the framework of the "semiconductor world" concept allows integrating a variety of models on a single physical basis - from ZnS world and FeS world (based on inorganic semiconductors) to the PAH world and aromatic world (including organic semiconductors). Thus, we do not put forward a new alternative hypothesis of chemical evolution - a new "chemical world", but only integrate the evolutionary and geochemical criteria of different "chemical worlds" into a single "physical world", which gives one the opportunity to reconstruct and predict the directions of chemical evolution according to the uniform principles of physical chemistry. In the first part of this series we consider photoinduced self-organization and "photo-controlled" evolution of the early protobiological systems performing solar energy conversion on the surface of dispersed semiconductor minerals capable of (photo)catalyzing and photosensitizing prebiological processes. The latter include a transition from the elementary cycles of photocatalytic reactions on the surface of semiconductors to protophotometabolic cycles and protophotosynthesis, from photophysical processes on the surface of mineral semiconductors to photoinduced membrane potentials, from photocontrolled sorption on the surface of such minerals to the formation of photosensitive protomembranes and selection of photosynthetic structures. The evolutionary approach to the analysis of protobiological mechanisms and protobioenergetics within the framework of the "semiconductor world" concept provides a new approach to the study of the last common point of divergence of protobiological systems, where the emergence principles of different energy supply schemes (like the energy source-specific photoautotrophy and substrate-specific chemoautotrophy) merge at the physicochemical level. The proposed integrating scheme is consistent with most biological, geological, and physicochemical concepts, which ensures its complete cross-checking and internal consistency.

Fluorescence microscopy provides an unparalleled tool for imaging biological samples. However, producing high-quality volumetric images quickly and without excessive complexity remains a challenge. Here, we demonstrate a simple multi-camera structured illumination microscope (SIM) capable of simultaneously imaging multiple focal planes, allowing for the capture of 3D fluorescent images without any axial movement of the sample. This simple setup allows for the acquisition of many different 3D imaging modes, including 3D time lapses, high-axial-resolution 3D images, and large 3D mosaics.

A recent paper demonstrates the importance of the linker region joining the two SNARE motifs of the neuronal t-SNARE SNAP25 for maintaining rates of secretion with roles for distinct segments in speeding fusion pore expansion (Shaaban et al., 2019, Elife. 8). Remarkably, lipid perturbing agents rescue a palmitoylation-deficient phenotype that includes slow fusion pore expansion, suggesting that protein-protein interactions have a role not only in bringing together the granule or vesicle membrane with the plasma membrane but also in orchestrating protein-lipid interactions leading to the fusion reaction. Furthermore, biochemical investigations demonstrate the importance of the C-terminal domain of the linker in the formation of the plasma membrane t-SNARE acceptor complex for synaptobrevin2 (Jiang, et al., 2019, FASEB J. 33:7985-7994;Shaaban et al., 2019, Elife. 8). This insight, together with biophysical and optical studies from other laboratories (Wang, et al., 2008, Molecular Biology of the Cell. 19:3944-3955; Zhao, et al., 2013, Proc Natl Acad Sci U S A. 110:14249-14254) suggests that the plasma membrane SNARE acceptor complex between SNAP25 and syntaxin and the resulting trans SNARE complex with the v-SNARE synaptobrevin form just milliseconds before fusion.

Lack of sleep is a factor that disrupts the receptors' reception of information from the environ-ment and contributes to the emergence of problems with maintaining balance. The main pur-pose of this study was to find out whether Computer Dynamic Posturography could be a useful tool in distinguishing between people who sleep well from people with insomnia. The study participants were 76 male students who were divided into groups based on the results obtained from the Pittsburgh Sleep Quality Questionnaire. In each group, postural stability had been tested using three main tests: Strategy Organization Test SOT, Motor Control Test MCT and Ad-aptation Test ADT. The results of the analysis show that the obtained results differ in the exam-ined groups under the SOT test. Among people with insomnia, higher values of the tested pa-rameters were noted, than with people who sleep well, which translates into a worse ability to maintain balance. The greatest impact is observed when using eyesight and a vestibular system to maintain a stable posture. It was confirmed that Computer Dynamic Posturography is used to differentiate between people who sleep well from people with insomnia in the group of men. Lack of sleep significantly disturbs postural stability.

Temperature influences the life of many organisms in various ways. A great number of them live under conditions, where their ability to adapt to changes in temperature can be vital and largely determine their fitness. Understanding the mechanisms and principles underlying this ability to adapt can be of great advantage, for example, to improve growth conditions for crops and increase their yield. In times of imminent, increasing climate change, this becomes even more important, in order to find strategies and help crops cope with these fundamental changes. There is intense research in the field of acclimation, that comprises fluctuations of various environmental conditions, but most acclimation research focuses on regulatory effects and the observation of gene expression changes within the examined organism. As thermodynamic effects are a direct consequence of temperature changes, these should necessarily be considered in this field of research, but are often neglected. Also, compensated effects might be missed, even though they are equally important for the organism, since they do not cause observable changes, but rather counteract them. In this work, using a systems biology approach, we demonstrate that even simple network motifs can exhibit temperature dependent functional features, resulting from the interplay of network structure and the distribution of activation energies over the involved reactions. The demonstrated functional features are (i) the reversal of fluxes within a linear pathway, (ii) a thermo-selective branched pathway with different flux modes and (iii) the increased flux towards carbohydrates in a minimal calvin cycle that was designed to demonstrate temperature compensation within reaction networks. By this, we expand the scope of thermodynamic modelling of biochemical processes by addressing further possibilities and effects, following established mathematical descriptions of biophysical properties.

For more than 70 years, biologists and biophysicists have been trying to unravel the mystery that exists regarding the saltatory conduction of so-called myelinated neurons. Albert Einstein used the train metaphor to explain the theory of relativity. It is possible to use a similar metaphor to better understand this transient functioning of the neuron: the action potential. We will, once again, use a train to demonstrate unequivocally that the action potential does not jump from node of Ranvier to node of Ranvier (noR) as we thought it would. It is possible to describe that the neuron uses an elegant method to increase the speed of transmission of the neural message. It is also important to conclude that this increase in speed, contrary to the common idea, has a certain energy cost that is proportional to speed and in accordance with thermodynamics.

In this perspective, we give an overview of several less explored structural and optical characterisation techniques useful for biomaterials. New insights into the structure of natural fibres such as spider silk can be gained with minimal sample preparation. Electromagnetic radiation (EMR) over a broad range of wavelengths (from X-ray to THz) provides information of the structure of the material at cor- respondingly different length scales (nm-to-mm). When the sample features, such as the alignment of certain fibres, cannot be characterised optically, polarisation analysis of the optical images can provide further information on feature alignment. The 3D complexity of biological samples necessitates that there be feature measure- ments and characterisation over a large range of length scales. We discuss the issue of characterising complex shapes by analysis of the link between the color and struc- ture of spider scales and silk. For example, it is shown that the green-blue color of a spider scale is dominated by the chitin slab’s Fabry-P ́erot type reflectivity rather than the surface nanostructure. The use of a chromaticity plot simplifies complex spectra and enables quantification of the apparent colors. All the experimental data presented herein are used to support the discussion on the structure-color link in the characterisation of materials.

In computational neuroscience, spiking neurons are often analyzed as computing devices that register bits of information, with each action potential carrying at most one bit of Shannon entropy. Here, I question this interpretation by using Landauer's principle to estimate an upper limit for the quantity of thermodynamic information that can be dissipated by a single action potential in a typical mammalian neuron. I show that an action potential in a typical mammalian cortical pyramidal cell can carry up to approximately 3.4e11 natural units of thermodynamic information, or about 4.9e11 bits of Shannon entropy. This result suggests that an action potential can process much more information than a single bit of Shannon entropy.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (Attenuated Total Reflection – Fourier Transform Infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (Nuclear Magnetic Resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

Pore Forming Toxins (PFTs), formed mainly by virulence factors of bacteria, belongs to Pore Forming Protein (PFP) family. Secreted as soluble monomers, they bind specific targets in membranes where their oligomerization and insertion place. Lysenin, a member of the PFTs, forms and oligomer after sphingomyelin binding, the so-called prepore, which become inserted forming a pore after a conformational change triggered by a pH decrease. In crowded conditions, oligomers tends to stay in prepore form because the prepore-to-pore transition is sterically blocked. In this study, we investigate the effect of calcium ions in those crowded conditions, finding that calcium act as a trigger for lysenin insertion. We localize the residues responsible for calcium sensitivity in a small α-helix. Our results are not only one of the few complete structural descriptions of prepore-to-pore transitions but the very first that involves a calcium triggering mechanism. The presence of glutamic or aspartic acids in the insertion domains could be an indication that calcium may be a general trigger for PFTs and more generally PFP.

Liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, critical in the controlling of many biological processes through forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to demix in solution forming the protein condensed droplets. The molecular interactions regulating its LLPS were investigated at the protein fusion equilibrium stage, where the droplets stopped growing. We found the molecules in the droplet were still liquid-like but with enhanced intermolecular helix-helix interaction in the LCD. The protein would start to aggregate after about 200 minutes of lag time and aggregate slower than at the condition when the protein does not phase separate or the molecules have a reduced intermolecular helical interaction. A structural transition intermediate towards protein aggregation was also discovered involving a decrease of the intermolecular helix-helix interaction and a reduction in the helicity. Therefore, LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

(1) Background: The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood.; (2) Methods: Here we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch-clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. Besides this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties.; (3) Results: Both, Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single digit range, can be detected by transcriptome analysis of reticulocytes. Proteomics analysis of RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch-clamp method. While for the Gárdos channel the activity is higher in reticulocytes compared to mature RBCs, for Piezo1 the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch-clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs.; (4) Conclusions: We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Spatial biology is a rapidly growing research field which focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for identification of multiple targets in situ. Nonetheless, the number of genes that can be reliably visualized is limited by the diffraction of light. Here, we investigate the effect of structured illumination (SIM), a super-resolution microscopy approach, over the performance of single gene transcript detection in spatial transcriptomics experiments. We performed direct mRNA-targeted hybridization in situ sequencing for multiple genes in mouse coronal brain tissue sections. We evaluated spot detection performance in widefield and confocal images versus those with SIM in combination with 20X, 25X and 60X objectives. In general, SIM increases the detection efficiency of gene transcripts spots compared to widefield and confocal modes. For each case, the specific fold increase in localizations is dependent on gene transcripts density and the numerical aperture of the objective used, which showed to play an important role especially for densely clustered spots. Taken together, our results suggest that SIM has the capacity to improve spot detection and overall data quality in spatial transcriptomics.

Cancer stem cells remain a challenge to isolate and characterize because of their plastic phenotype. Using a microfluidic lab-on-a-chip based on ultra-high frequency dielectophoresis, we measured the electromagnetic signature of colorectal cancer cells and demonstrated that cancer stem cells show a distinct and lower electromagnetic signature than differentiated cells. The release of extracellular vesicles from tumor cells can drive tumor progression and metastasis development. As extracellular vesicles from cancer stem cells carry more aggressive content, we treated colorectal cancer cells with these vesicles to test whether the lab-on-a-chip can detect a change in phenotype. The electromagnetic signature of treated cells is modified in comparison to untreated cells and sometimes even when no biological change is observed. The lab-on-a-chip provides rapid and relevant result without prior labeling compared to conventional biological approaches. It could be useful in the clinic for early detection of cancer stem cells in the tumor mass and for monitoring the aggressive potential of extracellular vesicles in the bloodstream in order to adapt therapeutic management and prevent relapse.

The stage of a super-early scale-invariant Universe is considered on the basis of the Poincaré–Weyl gauge theory of gravity in a Cartan–Weyl space-time. An approximate solution has been found that demonstrates an inflationary behavior of the scale factor and, at the same time, a sharp exponential decrease in the effective cosmological constant from a huge value at the beginning of the Big Bang to an extremely small (but not zero) value in the modern era, which solves the well-known “cosmological constant problem”.

Hyper-radiosensitivity (HRS) is the increased sensitivity to low doses of ionizing radiation observed in most cell lines. We previously demonstrated that HRS is permanently abolished in cells irradiated at a low dose rate (LDR), in a mechanism dependent on transforming growth factor β3 (TGF-β3). In this study, we aimed to elucidate the activation and receptor binding of TGF-β3 in this mechanism. T-47D cells were pre-treated with inhibitors of potential receptors and activators of TGF-β3, along with addition of small extracellular vesicles (sEVs) from LDR primed cells, before their radiosensitivity was assessed by the clonogenic assay. The protein content of sEVs from LDR primed cells was analyzed with mass spectrometry. Our results show that sEVs contain TGF-β3 regardless of priming status, but only sEVs from LDR primed cells remove HRS in reporter cells. Inhibition of the matrix metalloproteinase (MMP) family prevents removal of HRS, suggesting an MMP-dependent activation of TGF-β3 in the LDR primed cells. We demonstrate a functional interaction between TGF-β3 and activin receptor like kinase 1 (ALK1), by showing that TGF-β3 removes HRS through ALK1 binding, independent of ALK5 and TGF-βRII. These results are an important contribution to a more comprehensive understanding of the mechanism behind TGF-β3 mediated removal of HRS.

Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) enables determination of room-temperature structures without causing radiation damage. Using an optical pump-probe or mix-and-injection, SFX enables the intermediate state visualization of a molecular reaction. In SFX experiments, serial and stable microcrystal delivery to the X-ray interaction point is vital for reasonable data collection and efficient beam time. The Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) facility established SFX instruments at a nanocrystallography and coherent imaging (NCI) experimental station. Various sample delivery methods, including injection, fixed-target scanning, and hybrid methods, have been developed and applied to collect XFEL diffraction data. Herein, we review the currently available sample delivery methods for SFX at the NCI experimental station at the PAL-XFEL. This article will help PAL-XFEL users access the SFX system for their experiments.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes producing adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial FOF1-ATP synthases those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light to the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized FOF1-ATP synthases from spinach chloroplasts at different salinity. The structural data were obtained by SAXS and showed dimerization in response to changes in ionic strength. The best model describing SAXS data was two ATP-synthases connected through F1/F1’ parts, presumably via their δ-subunits, forming dimers with the “I” shape. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the FOF1 monomers comprising such dimers are embedded in different thylakoid membranes. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast FOF1-ATP synthase for preventing of ATP hydrolysis in dark, when salinity in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast FOF1 and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

Functional magnetic resonance imaging (fMRI) is an indirect measure of neural activity with the hemodynamic response function (HRF) coupling it with unmeasured neural activity. The HRF, modulated by several non-neural factors, is variable across brain regions, individuals and populations. Yet, a majority of resting-state fMRI connectivity studies continue to assume a non-variable HRF. In this article, with supportive prior evidence, we argue that HRF variability cannot be ignored as it substantially confounds within-subject connectivity estimates and between-subjects connectivity group differences. We also discuss its clinical relevance with connectivity impairments confounded by HRF aberrations in several disorders. We present limited data on HRF differences between women and men, which resulted in a 15.4% median error in functional connectivity estimates in a group-level comparison. We also discuss the implications of HRF variability for fMRI studies in the spinal cord. There is a need for more dialogue within the community on the HRF confound, and we hope that our article is a catalyst in the process.

The soma, dendrites and axon of neurons may display calcium-dependent release of transmitters and peptides. Such release is named extrasynaptic for occurring in the absence of synaptic structures. This review describes cooperative actions of three calcium sources on somatic exocytosis. Emphasis is given to the release of serotonin by the classical serotonergic leech Retzius neuron, which has allowed detailed studies of each step between excitation and exoctytosis. Trains of action potentials induce transmembrane calcium entry through L-type channels. If the frequency of action potentials is above 5 Hz, summation of calcium transients upon individual action potentials increases the intracellular calcium concentration to activate calcium–induced calcium release. The amplified calcium wave activates motochondrial ATP synthesis that fuels the transport of vesicles to the plasma membrane. Serotonin that is released activates autoreceptors coupled to phospholipase C. Production of IP3 produces release of calcium that sustains the large-scale exocytosis. The swiss-clock workings of the release machinery for somatic exocytosis has a striking disadvantage. The essential calcium-releasing endoplasmic reticulum that lays between resting vesicles and the plasma membrane becomes an obstacle for the vesicle transport. Such architecture reduces drastically the thermodynamic efficiency of the vesicle transport and elevates its energy cost..

In its prefusion state, the SARS-CoV-2 Spike protein (similarly to other class I viral fusion proteins) is metastable, which is considered to be an important feature for optimizing or regulating their functions. After the binding process of its S1 subunit (S1) with the ACE2, the Spike protein (S) suffers a dramatic conformational change where S1 splits from the S2 subunit, which then penetrates the membrane of the host cell, promoting the fusion of the viral and cell membranes. This results in the infection of the host cell. In a previous work, we showed -using large scale molecular dynamics simulations- that the application of external electric fields (EF) induces drastic changes and damage in the receptor-binding domain (RBD) of the wild type Spike protein, as well of the Alpha, Beta and Gamma variants, leaving a structure which cannot be recognized any more by ACE2. In this work, we first extend the study to the Delta and Omicron variants and confirm the high sensitivity and extreme vulnerability of S to moderate EF (as weak as 104 V m-1), but, more importantly, we also show that, in contrast, the postfusion state of the spike protein does not suffer structural damage even if electric field intensities four orders of magnitude higher are applied. These results provide a solid scientific basis to confirm the prefusion state metastability roots of the SARS-CoV-2 Spike protein susceptibility to be damaged by EF. After the virus docks to the ACE2 receptor, the stable and robust postfusion conformation develops, which exhibits a similar resistance to EF (damage threshold higher than 108 V m-1) like most globular proteins.

This study investigates the metabolic parallels between stimulated pancreatic beta cells and cancer cells, emphasizing glucose and glutamine metabolism. Addressing the significant public health challenges of Type 2 Diabetes Mellitus (T2DM) and cancer, we aim to deepen understanding of the mechanisms driving insulin secretion and cellular proliferation. Our analysis of anaplerotic cycles and NADPH's role in biosynthesis elucidates their vital functions in both processes. Additionally, we find that both cell types share an antioxidative response mediated by the Nrf2 signaling pathway, glutathione synthesis, and UCP2 upregulation. Notably, UCP2 facilitates the transfer of C4 metabolites, enhancing reductive TCA cycle metabolism. We also uncover that hypoxic responses are transient in beta cells post-stimulation but persistent in cancer cells. By synthesizing these insights, our research suggests novel therapeutic targets for T2DM, highlighting the shared metabolic strategies of stimulated beta cells and cancer cells. This comparative analysis not only illuminates the metabolic complexity of these conditions but also emphasizes the crucial role of metabolic pathways in cell function and survival, offering fresh perspectives for tackling T2DM and cancer challenges.

G-protein coupled receptors (GPCRs), one of the largest superfamilies of cell-surface receptors, are heptahelical integral membrane proteins that play critical roles in virtually every organ system. G-protein-coupled receptors operate in membranes rich in cholesterol, with an imbalance in cholesterol level within the vicinity of GPCR transmembrane domains affecting the structure and/or function of many GPCRs, a phenomenon that has been linked to several diseases. These effects of cholesterol could result in indirect changes by altering the mechanical properties of the lipid environment or direct changes by binding to specific sites on the protein. There are a number of studies and reviews on how cholesterol modulates class A GPCRs, however, this area of study is yet to be explored for class C GPCRs, which are characterized by a large extracellular region and often form constitutive dimers. This review highlights specific sites of interaction, functions, and structural dynamics involved in the cholesterol recognition of the class C GPCRs. We summarize recent data from some typical family members to explain the effects of membrane cholesterol on the structural features and functions of Class C GPCRs and speculate on their corresponding therapeutic potential.

SARS-CoV-2 belongs to the group of RNA viruses with a pronounced tendency to mutate. Omicron BA.2.75 is a subvariant believed to be able to suppress the currently dominant BA.5. Omicron BA.2.75 is characterized by a greater infectivity compared to earlier Omicron variants. However, Gibbs energy of biosynthesis of virus particles is slightly less negative compared to those of other variants. Thus, the multiplication rate of Omicron BA.2.75 is lower than that of other variants. This leads to slower accumulation of newly formed virions and less damage to host cells, indicating evolution of SARS-CoV-2 towards decreasing pathogenicity.

Physical properties like shape, volume and size affect the dynamics of biological systems. Along these lines, we focus on the topological properties of biological fluids and their biochemical and physiological outcomes. We take as a paradigmatic example the salivary fluid and describe how its topological features may affect the physiopathology of the oral cavity. Topological approaches assess the general properties of saliva, ignoring small-scale physical details such as density, flow rate, stiffness, viscosity. Specifically, the mucin aggregates scattered in the salivary fluid can be tackled in terms of topological holes, i.e., vortical clusters that modify the direction, flow, impulse, local rate-of-change and velocity of saliva. While the current methodological approaches are inclined to remove the effects of impurities assessing systems as homogeneous structures, we argue that the occurrence of mucins breaks up the salivary fluid’s homogeneity, leading to unexpected biophysical modifications. We suggest that every collected salivary sample is not reliable for accurate clinical and experimental investigation, since it displays highly local as well as variable chemical, physical and biological features, not reflecting the current physiological state of the oral cavity. Therefore, the assessment of a single salivary sample is not fully reproducible and cannot provide information about the biophysical, enzymatic and microbiological content of the whole saliva. In sum, the very topological features of the saliva - such as volume, shape, antipodal cells, vortex area, whirling fluid mass, segmentation, discretization, triangulation, node numbering - produce unnoticed biological consequences and network connectivity features with intriguing operational implications.

A novel physiologically based algorithm (PBA) for the computation of fractional flow reserve (FFR) in coronary artery trees (CATs) using computational fluid dynamics (CFD) is proposed and developed. The PBA is based on the extension of Murray's law and additional inlet conditions prescribed iteratively, and is implemented in OpenFOAM for testing and validation. 3D models of CATs are created using CT scans and computational meshes, and the results are compared to in-vasive coronary angiographic (ICA) data to validate the accuracy and effectiveness of the PBA. The discrepancy between calculated and experimental FFR is within 2.33-5.26% in steady-state and transient simulations, respectively, when convergence is reached. The PBA is a reliable and physiologically sound technique compared to the current lumped parameter model (LPM), which is based on empirical scaling correlations and requires nonlinear iterative computing for conver-gence. The accuracy of the PBA method is further confirmed using the FDA nozzle, which demonstrates good alignment with CFD-validated values.

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron regulatory hormone is a 25 amino acid peptide with 4 intramolecular disulfide bonds, circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin, extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 1010.1 M-1 for Zn(II) ions, the Cd(II) ions bind with affinities of 1015.2 M-1 and 1014.1 M-1. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. These studied empowered us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for better thermostability.

A human brain can communicate with another one’s brain using quantum entanglement. Similar particles can entangle without interaction. Sensory communication between two individuals from far distances is still not known. Former studies primarily have been performed regarding brain neurons' quantum behavior. The test was performed on two individuals far from each other. These individuals were put under the same music on the basis of brain neuroplasticity property. By activating the brain reward system, aligned thinking was time scheduled in these individuals’ brains. On the basis of entanglement, compressed information was sent and received between these two individuals.

Ice-binding proteins are crucial for adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside through the nucleation process. Unlike most other works, our paper is focused on ice nucleation rather than the behavior of the already existing ice. The nucleation kinetics is studied both theoretically, with special attention paid to ice nucleation on ice-binding surfaces, and experimentally. For experimental studies, we use the ice-binding protein mIBP83, a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein. We show that mIBP83 does not affect the ice nucleation temperature in the buffer in test tubes, but hinders the impact of potent ice nucleators of various chemical natures, namely CuO powder and ice-nucleating bacteria Pseudomonas syringae. Additional experiments on human cells show that mIBP83 is concentrated in some regions, but only in cooled cells. Thus, the antifreeze protein not only binds to ice, but also blocks various sites that act or can act as ice nucleators. Such ice-preventing binding may be the crucial biological task of antifreeze proteins.

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here we report that among eighteen representatives of the multifunctional S100 protein family only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (Kd) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent Kd values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1-0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatic analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect efficacy of anti-TNF treatment in various diseases.

Mitochondria are highly dynamic organelles that constantly undergo fusion and fission events to maintain their shape, distribution, and cellular function. Mitofusin 1 and 2 proteins are two dynamin-like GTPases involved in the fusion of outer mitochondrial membranes (OMM). Mitofusins are anchored to the OMM through their transmembrane domain and possess two heptad repeat domains (HR1 and HR2) in addition to their N-terminal GTPase domain. The HR1 domain was found to induce fusion via its amphipathic helix, which interacts with the lipid bilayer structure. The lipid composition of mitochondrial membranes can also impact mitochondrial fusion. However, the precise mode of action of lipids in mitochondrial fusion is not fully understood. In this study, we have examined the role of the mitochondrial lipids phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidic acid (PA) in membrane fusion induced by the HR1 domain, both in the presence and absence of divalent cations (Ca2+ or Mg2+). Our results show that PE, as well as PA in the presence of Ca2+, effectively stimulate HR1-mediated fusion, while CL has a slight inhibitory effect. By considering the biophysical properties of these lipids in the absence or presence of divalent cations, we infer that the interplay between divalent cations and specific cone-shaped lipids creates regions with packing defects in the membrane, which provides a favorable environment for the amphipathic helix of HR1 to bind to the membrane and initiate fusion.

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE) where its fluorescence properties are used to assess the retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE, and lipofuscin-laden-cells to visible light, and determine whether an abundant component of lipofuscin, docosahexaenoate (DHA) can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible leads to a decrease of its long-wavelength fluorescence at about 610 nm with concomitant growth of the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure to light of lipofuscin-laden cells leads to loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes of fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra together with quantitation of intensity of long-wavelength fluorescence can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, thereby useful for screening the retina for increased oxidative damage and early AMD-related changes.

Transition between the unfolded and native states of the ordered globular proteins is accompanied by accumulation of several intermediates, such as pre-molten globule, wet molten globule, and dry molten globule. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under the conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin from the synaptic cleft, which is an essential process plays an important role in depression. In addition to substrate serotonin, hSERT is also the target of drugs of abuse like cocaine and clinically used antidepressants such as escitalopram and paroxetine. To date, few studies attempt to investigate the unbinding mechanism underlying the orthosteric and allosteric modulation of hSERT. The high-resolution X-ray structure of hSERT resolved recently enables us to theoretically study the unbinding of the above four ligands against the S1 or S2 site of hSERT, by means of molecular docking, molecular dynamics (MD) and potential of mean force (PMF) simulations. We proved that for either the S1 or S2 site, the other three ligands (cocaine, escitalopram and paroxetine) are much more favorable than the original substrate serotonin, whether in kinetics along the unbinding pathways or in thermodynamics at the equilibrium states. Furthermore, the S1 site is much more favorable than the S2 site, for each ligand. Interestingly, inspection revealed that there are ~ 3Å lengths between the allosteric site of serotonin and cocaine, and an unseen un-binding pathway for escitalopram at the S1 site except for verification of the broadest trail.

Proteomic analysis revealed the preservation of many proteins in the “Heslington brain” (which is at least 2,600-year-old brain tissue uncovered within the skull excavated in 2008 from a pit in Heslington, Yorkshire, England). Five of these proteins (“main proteins”), heavy, medium, and light neurofilament proteins (NFH, NFM, and NFL), glial fibrillary acidic protein (GFAP), and myelin basic (MBP) protein are engaged in the formation non-amyloid protein aggregates, such as intermediate filaments and myelin sheath. We used a wide spectrum of bioinformatics tools to evaluate the prevalence of functional disorder in several related sets of proteins, such as “main proteins” and their 44 interactors, as well as all other protein identified in the Heslington brain. These analyses revealed that all five “main proteins”, half of their interactors and almost one third of the Heslington brain proteins are expected to be mostly disordered. Furthermore, most of the remaining proteins are expected to contain sizable disordered regions. This is in contrary the expected substantial (if not complete) elimination of the disordered proteins from the Heslington brain. Therefore, it seems that the intrinsic disorder of NFH, NFM, NFL, GFAP, and MBP, their interactors and many other proteins might play a crucial role in preserving the Heslington brain by forming tightly folded brain protein aggregates, in which different parts are glued together via the disorder-to-order transitions.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation on electrocardiograms (ECGs), and a high risk of life-threatening ventricular arrhyth-mia and sudden cardiac death. In BrS patients, except for SCN5A, mutations in other responsible genes are poorly elucidated. We identified a new missense mutation, c.1189C>T (p.R397C), in the KCNH2 gene in asymptomatic male proband with diagnosed BrS. We performed patch-clamp analysis on I(Kr) reconstituted with this KCNH2 mutation in Chinese hamster ovary cells and compared the phenotype with the wild type. We found that the R397C mutation increased the I(Kr) density. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

The dentate gyrus (DG) is part of the hippocampal formation and is essential for important cognitive processes such as navigation and memory. The oscillatory activity of the DG network is believed to play a critical role in cognition. DG circuits generate three main rhythms: theta, beta, and gamma, which participate in the specific information processing performed by DG neurons. In the temporal lobe epilepsy (TLE), cognitive abilities are impaired, which may be due to drastic alterations in the DG structure and network activity during epileptogenesis. The theta rhythm and theta coherence are especially vulnerable in dentate circuits; disturbances in DG theta oscillations and their coherence may be responsible for general cognitive impairments observed during epileptogenesis. Some researchers suggested that the vulnerability of DG mossy cells is a key factor in the genesis of TLE, but others did not support this hypothesis. The aim of the review is not only to present the current state of the art in this field of research but to help pave the way for future investigations by highlighting the gaps in our knowledge to completely appreciate the role of DG rhythms in brain functions. Disturbances in oscillatory activity of the DG during TLE development are described in detail that may be a diagnostic marker in the treatment of this disease.

Stabilization of a G-quadruplex (G4) in the promotor of the c-MYC proto-oncogene leads to inhibition of gene expression, and it thus represents a potentially attractive new strategy for cancer treatment. However, most G4 stabilizers show little selectivity among the many G4s present in the cellular complement of DNA and RNA. Intriguingly, a crescent-shaped cell-penetrating thiazole peptide, TH3, preferentially stabilizes the c-MYC G4 over other promotor G4s, but the mechanisms leading to this selective binding remain obscure. To investigate these mechanisms at the atomic level, we performed an in silico comparative investigation of the binding of TH3 and its analogue TH1 to the G4s from the promotors of c-MYC, c-KIT1, c-KIT2, and BCL2. Molecular docking and molecular dynamics simulations, combined with in-depth analyses of non-covalent interactions and bulk and per-nucleotide binding free energies, revealed that both TH3 and TH1 can induce the formation of a sandwich-like framework through stacking with both the top and bottom G-tetrads of the c-MYC G4 and the adjacent terminal capping nucleotides. This framework produces enhanced binding affinities for the c-MYC G4 relative to other promotor G4s, with TH3 exhibiting an outstanding binding priority. Van der Waals interactions were identified to be the key factor in complex formation in all cases. Collectively, our findings fully agree with available experimental data. Therefore, the identified mechanisms leading to specific binding of TH3 towards the c-MYC G4 provide valuable information to guide the development of new selective G4 stabilizers.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common one has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain DNA of living cells. To do so, it needs to interact with and permeate through cell membranes. The pH of biological media may vary quite significantly and this influences H33342 ionization state, and may change its rate of permeation through biomembranes. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 &lt; pH &lt; 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics simulations. While probe insertion occurs for all ionization states, with similar overall equilibrium location, orientation was found to depend on the probe charge, with the neutral form lying mostly parallel and the doubly charged displaying a significant tilt relative to the membrane plane. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy of interaction is negative and is larger at lower pH values. This suggests efficient hydrogen bonding between the probe and the lipid, namely for the protonated +2 form. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from water and octanol partition. This highlights the distinction between lipophilicity and hydrophobicity, and the importance of considering association with lipid bilayers when predicting the affinity for biomembranes.

The generation and maintenance of membrane potential is considered a fundamental element of the cellular machinery. They are based on elements of physics and chemistry. Mathematics is used to validate the equations and evaluate the results. However, this computation process can lead to a misunderstanding of the phenomena involved or to the masking of crucial events. It is even possible that the calculation no longer corresponds to the hypothesis considered. Membrane theory is no exception to these trivial considerations, which are essential for normal theory validation.

For the first time, the effect of a significant change in the resistance of the aquatic environment of a population of protozoan microorganisms under the influence of radiofrequency electromagnetic radiation from cell phones has been experimentally studied. The results of the experiment demonstrate the biological nature of the changes occurring in the “water population of protozoa” system, which indicates that the impact of radiation directly affects the cell. This conclusion is confirmed by the same order of magnitude time of information exchange between individual individuals of the population in the calculation and in the experiment. An extremely low concentration of protozoa in the experiment was established, equal to 1000 pcs./liter, at which the indicated effect is observed.An unambiguous dependence of the biological effect in the “water – amoeba population” system on the average power of electromagnetic radiation of cell phones, both in standby mode and in talk mode, has been established. It is concluded that the presented method of changing the physical properties of the aquatic environment of a population of protozoa under the influence of electromagnetic fields can be a model experiment in identifying the mechanisms of interaction of biological systems with electromagnetic radiation, determining the response time of biological systems to external electromagnetic influence, and establishing the degree of influence of radiation in the 4G, 5G ranges.

Emerging applications of optical technologies are driving the development of miniaturised light sources, which in turn require the fabrication of matching micro-optical elements with sub-1 mm cross sections and high optical quality. This is particularly challenging for spatially-constrained biomedical applications where reduced dimensionality is required, such as endoscopy, optogenetics, or optical implants. Planarisation of a lens by the Fresnel lens approach was adapted for a conical lens (axicon) and was made by direct femtosecond 780 nm/100 fs laser writing in SZ2080™polymer with photo-initiator. Optical characterisation of the positive and negative fraxicons is presented. Numerical modeling of fraxicon optical performance under illumination by incoherent and spatially extended light sources is compared with the ideal case of plane wave illumination. Considering the potential for rapid replication in soft polymers and resists, this approach holds great promise for the most demanding technological applications.

Wolfenden and his coworkers observed the astronomical numbers for the catalytic proficiency of some enzymes. We connected that pinnacle of biological evolution to the universal thermodynamic evolution. We added or multiplied a random noise with chosen rate constants to explore the correlation between dissipation and enzyme efficiency for ten enzymes: beta-galactosidase, glucose isomerase, β-lactamases from three bacterial strains, ketosteroid isomerase, triosephosphate isomerase, and carbonic anhydrase I, II, and T200H. The turnover number kcat and catalytic efficiency kcat/KM are proportional to the overall entropy production – the main parameter from irreversible thermodynamics. For most enzymes with the Michaelis-Menten type cycle kinetics, the best increase in the forward kcat/KM follows after increasing the equilibrium constant of substrate-enzyme association. The Discussion section emphasizes the role of biological evolution in harvesting order (high enzyme efficiency) from disorder (high noise and dissipation). It also connects the applications of the maximum partial entropy production theorem in optimizing enzyme kinetics (D. Juretić "Bioenergetics - A Bridge across Life and Universe") with the present total entropy production role analysis. De novo enzyme design and various attempts to speed up the rate-limiting catalytic steps may profit from our theoretical insights.

The review presents the most developed by date hypotheses on the mechanisms of the influence of low-frequency magnetic fields (LFMF) on organisms. Biophysical models that explain the resonance-like responses of biological systems to LFMF with a specific frequency and amplitude are described. Two groups can be distinguished among these models: one considers ions-cofactors of proteins as the primary targets for the LFMF influence, and the other regards the magnetic moments of particles in biomolecules. Attention is paid to the dependence of resonance-like LFMF effects on the cell type. A radical-pair mechanism of the magnetic field's influence on biochemical processes is described with the example of cryptochrome. Conditions for this mechanism's applicability to explain the biological effects of LFMF are given. A model of the influence of LFMF on radical pairs in biochemical oscillators, which can explain the frequency-amplitude efficiency windows of LFMF, is proposed.

Dormancy is a physiological state that confers winter hardiness to and orchestrates phenological phase progression in temperate perennial plants. Weather fluctuations caused by climate change increasingly disturb dormancy onset and release in many plant species including tree crops leading to aberrant growth, flowering, and fruiting. Currently, research in this field is impeded by the lack of affordable non-invasive methods for on-line monitoring of dormancy. We report on an automatic framework for low-cost, long-term, and scalable dormancy studies in deciduous plants. The proposed method is based on continuous near-field sensing of the photosynthetic activity of shoots via pulse-amplitude modulated chlorophyll fluorescence sensors connected remotely to a data processing system. The resulting high-resolution time series of JIP-test parameters indicative of the responsiveness of the photosynthetic apparatus to environmental stimuli are subjected to frequency-domain analysis. The proposed approach allows to overcome the variance coming from diurnal changes of insolation and to derive estimations on the depth of dormancy. Our approach was validated over three seasons in an experimental apple (Malus × domestica Borkh.) orchard by collating the non-invasive estimations with the results of traditional methods (growing of the cuttings obtained from the tress at different phases of dormancy) and the output of commonly used chilling requirement models. We discuss the advantages of the proposed monitoring framework such as prompt detection of freeze damages along with its potential limitations.

Bovine lactoferrin (bLf) is a milk-derived protein that exhibits multiple biological activities and has been explored towards different therapeutic applications. Since this molecule is susceptible to degradation and some of its properties depend on its tertiary structure, the encapsulation of bLf in stimuli-responsive therapeutic formulations provides an added value to potentiate its biological activities when administered. Plasmonic magnetoliposomes emerge as promising nanosystems for dual hyperthermia (magneto-photothermia) and local therapy, since the combination of magnetic and gold nanoparticles (NPs) in a single nanosystem (multifunctional liposomes) enables the targeting and controlled release of encapsulated drugs. In this work, plasmonic magnetoliposomes (PMLs) containing manganese ferrite nanoparticles (28 nm size) and gold nanoparticles (~ 5 nm size) functionalized with 11-mercaptoundecanoic acid or octadecanethiol, were synthesized and loaded with bLf. The structural, magnetic and optical properties of the nanoparticles were measured by TEM, SQUID and UV/vis/NIR absorption spectroscopy. Specific Absorption Rate was determined to assess the capabilities for magnetic and photothermal hyperthermia. Finally, Saccharomyces cerevisiae was used as an eukaryotic cellular model to assess the biological activity and the mechanism of entry of bLf-loaded PMLs, through counting of colony forming units and fluorescence microscopy, respectively. The results demonstrate that PMLs are mainly internalized through an energy- and temperature-dependent endocytic process, though the contribution of a diffusion component cannot be discarded. Most notably, only bLf-loaded PMLs exhibit cytotoxicity with an efficiency similar to free bLf, attesting their promising potential for bLf delivery in the context of therapeutic interventions.

Recent advancements in the study of the protein complex photosystem II have clarified the sequence of events leading to the formation of oxygen during the S3->S4->S0 tran-sition, wherein the inorganic Mn4Ca(µ-O)6(OHx)4 cluster finishes photo-catalyzing the water splitting reaction (Greife et al, Nature 2023, 617, 623-628; Bhowmick et al, Nature 2023, 617, 629-636) . During this final step a tyrosine radical (TyrZ), stable for a couple of milliseconds, oxidizes a cluster bound oxygen while the hydrogen bonding patterns of nearby waters shift a proton away. A treatment of this redox reaction within the context of accepted transition state theories predicts rate constants that are significantly higher than experimentally recovered values (10^12s^-1 versus 10^3s^-1). In an effort to understand this disparity, temperature dependent experiments have revealed large entropic con-tributions to the rates with only a moderate energy of activation. We suggest that the entropic source may be related to the observed proton rearrangements, and further possible near isoenergetic variations in the nearby extended h-bonding network de-laying the realization of an ‘ideal’ transition state. In the following we explore this re-lation in the context of Eyring’s transition state theory and Marcus’ electron transfer theory, and evaluate their compatibility with the experimental evidence.

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant 'closed' conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an 'open' form which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high throughput (HTP) 96-well SAXS assay setup. This method enables us to determine the Kopen value, which is used to compare ligands’ effect on Plg conformation directly. Based on our analysis using Plg GII, we have found that the Kopen for ε-aminocaproic acid (EACA) is approximately three times greater than that of Tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin and receptor Plg‑RKT. Our findings suggest that, besides the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.

A picture is worth a thousand words. Many branches of Science have been historically benefited with plots and visual analyses (lately, image processing and deep learning) alongside with traditional number crunching. In Molecular Biophysics, one such problem is the structure validation problem in proteins which stands with a history of plot-tools being effectively serving the complex problem to its complete resolution. Spanning across six decades, validation of protein structures (from experimental to modeled) dates back to the legendary Ramachandran Plot (with its never ending growth and modern-day applications) to the relatively recent innovation of the Complementarity Plot (CP), establishing the dual nature of complementarity as the physical basis of both binding and folding of proteins. Lately, CP has been extended to serve as a trustworthy free energy predictor utilizing supervised learning in the form of a comprehensive web-server (EnCPdock: https://www.scinetmol.in/EnCPdock/) that can be directly used in the design of protein interfaces. The commentary recapitulate the history of structure validation with a special emphasis on plot tools, highlighting key features and important discoveries worth re-visiting.

Due to the progression of antimicrobial resistance, Photodynamic (aPDT) and Sonodynamic (SDT) Therapies arose as promising approaches for microbial control. Recently, the combination of both therapies, called Sonophotodynamic Therapy (SPDT), have shown greater effects than the single treatments. This study evaluated the effects of aPDT, SDT and SPDT mediated by curcumin on the metabolism, adhesion capacity, biofilm formation ability and structural alteration in the Staphylococcus aureus biofilm. Moreover, possible antimicrobial mechanisms involved, such as the production of reactive oxygen species (ROS) and the degradation spectrum of curcumin by light, ultrasound and light+ultrasound, were measured. SPDT was more effective to inactivate the biofilm than aPDT and SDT. All treatments reduced the adhesion ability of the bacteria and it is probably related to the metabolism cell reduction. Regarding to the biofilm formation ability, all treatments reduced the total biomass. One possible mechanism involved in the inactivation is the ROS production observed. Curcumin had a significantly decay in its absorbance with the application of light, and a slighter one caused by the ultrasound. Finally, all treatments impacted the biofilm components and structure, reducing the cells and matrix. In conclusion, SPDT was more effective in the inactivation, and all treatments had the same effect in the virulence factors evaluated.

Giant unilamellar vesicles (GUVs) are membrane model used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol dry lipid film is formed. Drying of the lipid film induce Cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals, where these crystals does not participate in formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose novel electroformation protocol that includes rapid solvent exchange, plasma cleaning and spin-coating methods to produce GUVs. We tested the protocol focusing on vesicles with high Chol content using different spin-coating durations and vesicle types deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles made out of phosphatidylcholine/Chol mixture was 30 s. Mulitlamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores were deposited on electrode for 1.5/1 Chol/phosphatidylcholine lipid mixture and the results were compared. Electroformation using all three deposited vesicle types gave a high GUVs yield, but deposition of LUVs obtained by extrusion of MLVs through 100 nm membrane pores gave the most reproducible results. Using deposition of these LUVs, we produced GUVs with high yield for 6 different Chol concentrations (from 0 % to 71.4%). Using protocol that includes dry lipid film GUVs yield was lower and Chol demixing artifact was induced, proving that the lipid film should never undergo drying when Chol content is high.