Biophysica, Volume 2, Issue 4 (December 2022) – 20 articles

Over the last few decades, in vitro single-molecule manipulation techniques have enabled the use of force and displacement as controlled variables in biochemistry. Measuring the effect of mechanical force on the real-time kinetics of a biological process gives us access to the rates, equilibrium constants and free-energy landscapes of the mechanical steps of the reaction; this information is not accessible by ensemble assays. Optical tweezers are the current method of choice in single-molecule manipulation due to their versatility, high force and spatial and temporal resolutions. The aim of this review is to describe the contributions of our lab in the single-molecule manipulation field. We present here several optical tweezers assays refined in our laboratory to probe the dynamics and mechano-chemical properties of biological molecular motors and synthetic molecular devices at the single-molecule level. Full article

Directed cell migration is an essential building block of life, present when an embryo develops, a dendritic cell migrates toward a lymphatic vessel, or a fibrotic organ fails to restore its normal parenchyma. Directed cell migration is often guided by spatial gradients in a physicochemical property of the cell microenvironment, such as a gradient in chemical factors dissolved in the medium or a gradient in the mechanical properties of the substrate. Single cells and tissues sense these gradients, establish a back-to-front polarity, and coordinate the migration machinery accordingly. Central to these steps we find physical forces. In some cases, these forces are integrated into the gradient sensing mechanism. Other times, they transmit information through cells and tissues to coordinate a collective response. At any time, they participate in the cellular migratory system. In this review, we explore the role of physical forces in gradient sensing, polarization, and coordinating movement from single cells to multicellular collectives. We use the framework proposed by the molecular clutch model and explore to what extent asymmetries in the different elements of the clutch can lead to directional migration. Full article

Rapid, accurate, and label-free detection of biomolecules and chemical substances remains a challenge in healthcare. Optical biosensors have been considered as biomedical diagnostic tools required in numerous areas including the detection of viruses, food monitoring, diagnosing pollutants in the environment, global personalized medicine, and molecular diagnostics. In particular, the broadly emerging and promising technique of surface plasmon resonance has established to provide real-time and label-free detection when used in biosensing applications in a highly sensitive, specific, and cost-effective manner with small footprint platform. In this study we propose a novel plasmonic biosensor based on biaxially crumpled graphene structures, wherein plasmon resonances in graphene are utilized to detect variations in the refractive index of the sample medium. Shifts in the resonance wavelength of the plasmon modes for a given change in the RI of the surrounding analyte are calculated by investigating the optical response of crumpled graphene structures on different substrates using theoretical computations based on the finite element method combined with the semiclassical Drude model. The results reveal a high sensitivity of 4990 nm/RIU, corresponding to a large figure-of-merit of 20 for biaxially crumpled graphene structures on polystyrene substrates. We demonstrate that biaxially crumpled graphene exhibits superior sensing performance compared with a uniaxial structure. According to the results, crumpled graphene structures on a titanium oxide substrate can improve the sensor sensitivity by avoiding the damping effects of polydimethylsiloxane substrates. The enhanced sensitivity and broadband mechanical tunability of the biaxially crumpled graphene render it a promising platform for biosensing applications. Full article

Based on the coarse-grained model, we used molecular dynamics methods to calculate and simulate a semiflexible long ring–semiflexible short ring blended polymer system confined in a hard sphere. We systematically studied the distribution and motion characteristics of the long ring chain. The results show that when the short ring is short enough (L_short < 20), the long ring (L_long = 50) is separated from the blend system and then distributed against the inner wall. As the length of the short ring increases (L_short ≥ 20), the long ring can no longer be separated from the blending system. Moreover, we found that the long ring demonstrates a random direction of adherent walking behavior on the inner surface of the hard sphere. The velocity of the long ring decreases with the increase in the short ring length L_short. Specifically for L_short ≥ 20, the system does not undergo phase separation and the speed of the long ring decreases sharply along with the long ring distributed inside the confined bulk. This is related to the inner wall layer moving faster than the inside bulk of the restricted system. Our simulation results can help us to understand the distribution of macromolecules in biological systems in confined systems, including the restricted chromosome partitioning distribution and packing structure of circular DNA molecules. Full article

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer’s and Parkinson’s disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques. Full article

In the 1960s, Biophysics was an unheard of scientific field in Spain, and even outside Spain, the distinction between Biophysics and Molecular Biology was not clear at the time. This paper describes briefly the developments that led to the foundation of the Spanish National Committee for Biophysics (1981) and of the Spanish Biophysical Society (1987), the incorporation of Spain into IUPAB and EBSA, and the normalized presence of Biophysics as a compulsory subject in undergraduate curricula in Spain. Full article

Technological advancement has produced a variety of instruments and methods to generate electron beams that have greatly assisted in the extensive theoretical and experimental efforts devoted to investigating the effect of secondary electrons with energies approximately less than 100 eV, which are referred as low-energy electrons (LEEs). In the past two decades, LEE studies have focused on biomolecular systems, which mainly consist of DNA and proteins and their constituents as primary cellular targets of ionizing radiation. These studies have revealed that compared to other reactive species produced by high-energy radiation, LEEs have distinctive pathways and considerable efficiency in inducing lethal DNA lesions. The present work aims to briefly discuss the current state of LEE production technology and to motivate further studies and improvements of LEE generation techniques in relation to biological electron-driven processes associated with such medical applications as radiation therapy and cancer treatment. Full article

An overview of the biophysics activity at the Department of Physics and Chemistry Emilio Segrè of the University of Palermo is given. For forty years, the focus of the research has been on the protein structure–dynamics–function paradigm, with the aim of understanding the molecular basis of the relevant mechanisms and the key role of solvent. At least three research lines are identified; the main results obtained in collaboration with other groups in Italy and abroad are presented. This review is dedicated to the memory of Professors Massimo Ugo Palma, Maria Beatrice Palma Vittorelli, and Lorenzo Cordone, which were the founders of the Palermo School of Biophysics. We all have been, directly or indirectly, their pupils; we miss their enthusiasm for scientific research, their deep physical insights, their suggestions, their strict but always constructive criticisms, and, most of all, their friendship. This paper is dedicated also to the memory of Prof. Hans Frauenfelder, whose pioneering works on nonexponential rebinding kinetics, protein substates, and energy landscape have inspired a large part of our work in the field of protein dynamics. Full article

Tauopathies, including Alzheimer’s disease (AD), are a group of neurodegenerative disorders characterized by pathological aggregation of microtubule binding protein tau. The presence of tau neurofibrillary tangles, which are insoluble β-sheet fibrils, in the brain has been the histopathological hallmark of these diseases as their level correlates with the degree of cognitive impairment. However, recent studies suggest that tau oligomers, which are soluble proteins that are formed prior to insoluble fibrils, are the principal toxic species impairing neurons and inducing neurodegeneration. Targeting toxic tau oligomers is challenging, as they are mostly unstructured and adopting multiple conformations. The heterogeneity of tau oligomers is further illustrated by the different oligomeric species formed by various methods. The current models and technologies to study tau oligomerization represent important resources and avenues to push the forefront of elucidating the true toxic tau species. In this review, we will summarize the distinct tau oligomers generated using different strategies and discuss their conformational characteristics, neurotoxicity, relevance to pathological phenotypes, as well as their applications in drug discovery. This information will provide insights to understanding heterogeneous tau oligomers and their role as molecular targets for AD and related tauopathies. Full article

The RBL-2H3 mast cell immunological synapse dynamics is often simulated with reaction–diffusion and Fokker–Planck equations. The equations focus on how the cell synapse captures receptors following an immune response, where the receptor capture at the immunological site appears to be a delayed process. This article investigates the physical nature and mathematics behind such time-dependent delays. Using signal processing methods, convolution and cross-correlation-type delay capture simulations give a χ-squared range of 22 to 60, in good agreement with experimental results. The cell polarization event is offered as a possible explanation for these capture delays, where polarizing rates measure how fast the cell polarization event occurs. In the case of RBL-2H3 mast cells, polarization appears to be associated with cytoskeletal rearrangement; thus, both cytoskeletal and diffusional components are considered. From these simulations, a maximum polarizing rate ranging from 0.0057 s⁻² to 0.031 s⁻² is obtained. These results indicate that RBL-2H3 mast cells possess both temporal and spatial memory, and cell polarization is possibly linked to a Turing-type pattern formation. Full article

Various alternative compounds have been investigated to prevent icing, one of which includes poly(vinyl) alcohol (PVA), which has shown promising anti-freeze effects. However, determining the optimal structures and formulations of PVA for anti-icing applications has remained a challenge. Building upon our previous work, which used molecular dynamics simulations to assess the effects of hydroxyl group separation distance on ice nucleation, in this work, PVA was modified based upon the structures of antifreeze glycoproteins (AFGPs) found in Antarctic fish, and examined as a potential antifreeze compound. Four different PVA samples with different degrees of hydrolysis were fabricated and subsequently examined for their effects on ice crystallization. The results showed that the modified PVA samples with degrees of hydrolysis of 76% and 66% had an effect on ice crystallization, delaying ice crystallization by an average of approximately 20 min, and even preventing ice crystallization altogether in a small portion of the sample. Meanwhile, other samples with degrees of hydrolysis of 100% and 34% either showed no effect on ice crystallization, shortened the ice crystallization time, and appeared to promote ice nucleation. Full article

This study shows that the luminescent diagnostic of oral fluid allows the determination of the severity of inflammatory markers after implantation. The noninvasive diagnostic method, which is used, allows the rapid detection of the stages of development of the inflammatory process after intraosseous implantation and prevents the development of complications in the postoperative period. Full article

Bioprinting is an emerging tissue engineering method used to generate cell-laden scaffolds with high spatial resolution. Bioprinting parameters, such as pressure, nozzle size, and speed, highly influence the quality of the bioprinted construct. Moreover, cell suspension density and other critical biological parameters directly impact the biological function. Therefore, an approximation model that can be used to find the optimal bioprinting parameter settings for bioprinted constructs is highly desirable. Here, we propose a type-2 fuzzy model to handle the uncertainty and imprecision in the approximation model. Specifically, we focus on the biological parameters, such as the culture period, that can be used to maximize the output value (mineralization volume 21.8 mm³ with the same culture period of 21 days). We have also implemented a type-1 fuzzy model and compared the results with the proposed type-2 fuzzy model using two levels of uncertainty. We hypothesize that the type-2 fuzzy model may be preferred in biological systems due to the inherent vagueness and imprecision of the input data. Our numerical results confirm this hypothesis. More specifically, the type-2 fuzzy model with a high uncertainty boundary (30%) is superior to type-1 and type-2 fuzzy systems with low uncertainty boundaries in the overall output approximation error for bone bioprinting inputs. Full article

Viral populations are large and highly heterogeneous. Despite the evolutionary relevance of such heterogeneity, statistical approaches to quantifying the extent to which viruses maintain a high genotypic and/or phenotypic diversity have been rarely pursued. Here, we address this issue by analyzing a nucleotide-to-protein sequence map through deep sequencing of populations of the Q$\beta$ phage adapted to high temperatures. Tens of thousands of different sequences corresponding to two fragments of the gene coding for the viral replicase were recovered. A diversity analysis of two independent populations consistently revealed that about $40\%$ of the mutations identified caused changes in protein amino acids, leading to an almost complete exploration of the protein neighborhood of (non-silent) mutants at a distance of one. The functional form of the empirical distribution of phenotype abundance agreed with analytical calculations that assumed random mutations in the nucleotide sequence. Our results concur with the idea that viral populations maintain a high diversity as an efficient adaptive mechanism and support the hypothesis of universality for a lognormal distribution of phenotype abundances in biologically meaningful genotype–phenotype maps, highlighting the relevance of entropic effects in molecular evolution. Full article

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence. Full article

We have investigated the nature of the interaction of small organic drug molecules with lipid membranes of various compositions. Using infrared spectroscopy and differential scanning calorimetry methods, we studied the role of the structure of the active molecule in interaction with the membrane using the example of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylcholine:cardiolipin (DPPC:CL) liposomes. We discovered the key role of the heterocycle in interaction with the polar part of the bilayer and the network of unsaturated bonds in interaction with the hydrophobic part. For rifampicin and levofloxacin, the main binding sites were phosphate and carbonyl groups of lipids, and in the case of anionic liposomes we found a slight penetration of rifampicin into the hydrophobic part of the bilayer. For rapamycin, experimental confirmation of the localization of the molecule in the region of fatty acid chains was obtained, and perturbation in the region of phosphate groups was demonstrated for the first time. The process of phase transition of liposomal forms of rifampicin and levofloxacin was studied. DPPC liposomes accelerate the phase transition when loaded with a drug. DPPC:CL liposomes are less susceptible to changes in the phase transition rate. Full article

Entropy of multivariate distributions may be estimated based on the distances of nearest neighbours from each sample from a statistical ensemble. This technique has been applied on biomolecular systems for estimating both conformational and translational/rotational entropy. The degrees of freedom which mostly define conformational entropy are torsion angles with their periodicity. In this work, tree structures and algorithms to quickly generate lists of nearest neighbours for periodic and non-periodic data are reviewed and applied to biomolecular conformations as described by torsion angles. The effect of dimensionality, number of samples, and number of neighbours on the computational time is assessed. The main conclusion is that using proper data structures and algorithms can greatly reduce the complexity of nearest neighbours lists generation, which is the bottleneck step in nearest neighbours entropy estimation. Full article

The field of supramolecular peptides self-assembly has undergone outstanding growth since the early 1990s after the serendipitously discovery by Shuguang Zhang of an ionic self-complementary peptide as a repeating segment in a yeast protein. From then on, the field expanded at an accelerating pace and these self-assembled materials have become an integral part of a broad plethora of designer supramolecular nanomaterials useful for different applications ranging from 3D tissue cell cultures, regenerative medicine, up to optoelectronics. However, the supramolecular peptide based-nanomaterials available thus far for regenerative medicine still lack the dynamic complexity found in the biological structures that mediate regeneration. Indeed, self-assembling peptide (SAPs) suffer from poor mechanical stability, losing mechanical properties at low strains. Just like the extracellular matrix (ECM) of living systems, the chemical structure of the SAP-biomaterials should concurrently contain non-covalent and covalent bonds, bringing, respectively, infinite and finite lifetimes of interactions to obtain a reversibly dynamic matrix. In this review, will be highlighted the major advantages and current limitations of SAP-based biomaterials, and it will be discussed the most widely used strategies for precisely tune their mechanical properties (stiffness, resilience, strain-failure, stress resistance), describing recent and promising approaches in tissue engineering, regenerative medicine, and beyond. Full article

In this work, we report a non-destructive and non-contacting ultrasound system with a novel air-coupled transducer to continuously monitor the drying process of prickly pear (nopal) pads in a lab environment. Compared with conventional imaging and spectroscopic methods or electrical-based approaches, ultrasound-based methods are non-invasive, cost-effective, and suitable for large volume evaluation. The time-dependent elastic modulus of the cactus can be obtained and monitored by using our proposed ultrasonic method. The evaluated elastic modulus behavior shows a good agreement with the destructive testing results in the existing literature. With further development, the proposed method can be used for in vivo plant health monitoring. Full article

The results of studies on the assessment of new biomaterials from juvenile teeth for further use in surgical dentistry for bone tissue repair are presented in this work. The comparative assessment of these materials and brefomatrices used in dentistry was carried out. It was shown that spectral properties of new biomaterials from juvenile dentin were similar to the spectral properties of brefomatrices from cortical tissue according to the developed discriminant model of the characteristic changes of Raman line intensities. The calculated accuracy of the discriminant model was 82.7 ± 3.2%. Full article