SARS-CoV-2 virion physicochemical characteristics pertinent to abiotic substrate attachment.

The structure, size, and main physicochemical characteristics of the SARS-CoV-2 virion with the spike transmembrane protein corona were discussed. Using these data, diffusion coefficients of the virion in aqueous media and in air were calculated. The structure and dimensions of the spike protein derived from molecular dynamic modeling and thorough cryo-electron microscopy measurements were also analyzed. The charge distribution over the molecule was calculated and shown to be largely heterogeneous. Although the stalk part is negatively charged, the top part of the spike molecule, especially the receptor binding domain, remains positively charged for a broad range of pH. It is underlined that such a charge distribution promotes the spike corona stability and enhances the virion attachment to receptors and surfaces, mostly negatively charged. The review is completed by the analysis of experimental data pertinent to the spike protein adsorption at abiotic surfaces comprising nanoparticle carrier particles. It is argued that these theoretical and experimental data can be used for developing quantitative models of virus attachment to surfaces, facilitating adequate analysis of future experimental results.

An evaluation of the construction of the device along with the software for digital archiving, sending the data, and supporting the diagnosis of cervical cancer.

Cervical cancer is still an important cause of mortality among women in a number of countries. There are effective methods of prevention and early diagnosis, but they require well-trained medical professionals including cytologists. Within this project, we built a prototype of a new device together with implemented software using U-NET and CNN architectures of neural networks (ANN), to convert the currently used optical microscopes into fully independent scanning and evaluating systems for cytological samples. To evaluate the specificity and sensitivity of the system, 2058 (2000 normal and 58 abnormal samples) consecutive liquid-based cytology (LBC) samples were analysed. The observed sensitivity and specificity to distinguish normal and abnormal samples was 100%. We observed slight incompatibility in the evaluation of the type of abnormality. The use of ANN is promising for increasing the effectiveness of cervical screening. The low cost of neural network usage further increases the potential areas of application of the presented method. Further refinement of neural networks on a larger sample size is required to evaluate the software.

Adsorption/Desorption Transition of Recombinant Human Neurotrophin 4: Physicochemical Characterization.

Bulk physicochemical properties of neurotrophin 4 (NT-4) in electrolyte solutions and its adsorption/desorption on/from mica surfaces have been studied using dynamic light scattering (DLS), microelectrophoresis, a solution depletion technique (enzyme-linked immunosorbent assay, ELISA), and AFM imaging. Our study presents a determination of the diffusion coefficient, hydrodynamic diameters, electrophoretic mobility, and isoelectric point of the NT-4 under various ionic strength and pH conditions. The size of the NT-4 homodimer for an ionic strength of 0.015 M was substantially independent of pH and equal to 5.1 nm. It has been found that the number of electrokinetic charges per NT-4 molecule was equal to zero for all studied ionic strengths at pH 8.1, which was identified as the isoelectric point (iep). The protein adsorption/desorption on/from mica surfaces was examined as a function of ionic strength and pH. The kinetics of neurotrophin adsorption/desorption were evaluated at pH 3.5, 7.4, and 11 by direct AFM imaging and the ELISA technique. A monotonic increase in the maximum coverage of adsorbed NT-4 molecules with ionic strength (up to 5.5 mg/m2) was observed at pH 3.5. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA). Our measurements revealed a significant role of ionic strength, pH, and electrolyte composition in the lateral electrostatic interactions among differently charged NT-4 molecules. The transition between adsorption/desorption processes is found for the region of high pH and low surface concentration of adsorbed neurotrophin molecules at constant ionic strength. Additionally, results presented in this work show that the adsorption behavior of neurotrophin molecules may be governed by intrasolvent electrostatic interactions yielding an aggregation process. Understanding polyvalent neurotrophin interactions may have an impact on the reversibility/irreversibility of adsorption, and hence they might be useful for obtaining well-ordered protein layers, targeting the future development of drug delivery systems for treating neurodegenerative diseases.

Surface fine structure influence on saturated random packings.

Random packings of disks on a mesh are studied numerically using random sequential adsorption algorithm. The mesh is built of straight horizontal and vertical one-dimensional lines of a given distance between them. The packing fraction and structure as well as the kinetics of packing growth dependence on mesh size are analyzed to provide information, whether surface inhomogeneity will affect the properties of random packings. It has been shown that the number of disks in a packing slightly decreases with growing distance between mesh lines while the kinetics may change significantly even for very dense meshes. As packings obtained in random sequential adsorption resemble monolayers produced by irreversible adsorption processes, results of this study show that by measuring properties of a random packing it may be possible to determine fine structure of an underlying surface.

Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability.

BACKGROUND: Bacteriophage survives in at least two extremes of ionic environments: bacterial host (high ionic-cytosol) and that of soil (low ionic-environmental water). The impact of ionic composition in the micro- and macro-environments has not so far been addressed in phage biology. RESULTS: Here, we discovered a novel mechanism of aggregation/disaggregation transitions by phage virions. When normal sodium levels in phage media (150 mM) were lowered to 10 mM, advanced imaging by scanning electron microscopy, atomic force microscopy and dynamic light scattering all revealed formation of viral packages, each containing 20-100 virions. When ionic strength was returned from low to high, the aggregated state of phage reversed to a dispersed state, and the change in ionic strength did not substantially affect infectivity of the phage. By providing the direct evidence, that lowering of the sodium ion below the threshold of 20 mM causes rapid aggregation of phage while returning Na+ concentration to the values above this threshold causes dispersion of phage, we identified a biophysical mechanism of phage aggregation. CONCLUSIONS: Our results implicate operation of group behavior in phage and suggest a new kind of quorum sensing among its virions that is mediated by ions. Loss of ionic strength may act as a trigger in an evolutionary mechanism to improve the survival of bacteriophage by stimulating aggregation of phage when outside a bacterial host. Reversal of phage aggregation is also a promising breakthrough in biotechnological applications, since we demonstrated here the ability to retain viable virion aggregates on standard micro-filters.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Tip-enhanced Raman spectroscopy reveals the structural rearrangements of tau protein aggregates at the growth phase.

Tau protein aggregates inside neurons in the course of Alzheimer's disease (AD). Because of the enormous number of people suffering from AD, this disease has become one of the world's major health and social problems. The presence of tau lesions clearly correlates with cognitive impairments in AD patients, thus, tau is the target of potential treatments for AD, next to amyloid-ß. The exact mechanism of tau aggregation has not been understood in detail so far; especially little is known about the structural rearrangements of tau aggregates at the growth phase. The research into tau conformation at each step of the aggregation pathway will contribute to the design of effective therapeutic approaches. To follow the secondary structure of individual tau aggregates at the growth phase, we applied tip-enhanced Raman spectroscopy (TERS). The nanospectroscopic approach enabled us to follow the structure of individual aggregates occurring in the subsequent phases of tau aggregation. We applied multivariate data analysis to extract the spectral differences for tau aggregates at different aggregation phases. Moreover, atomic force microscopy (AFM) allowed the tracking of the morphological alterations for species occurring with the progression of tau aggregation.

Mechanisms of fibrinogen adsorption at solid substrates at lower pH.

Adsorption of fibrinogen was theoretically studied using the three-dimensional random sequential adsorption (RSA) model. Fibrinogen molecule shape was approximated by the bead model considering the presence of flexible side arms. Various cases were considered inter alia, the side-on adsorption mechanisms and the simultaneous side-on/end-on adsorption mechanism. The latter mechanisms is pertinent to fibrinogen adsorption at lower pH (below isoelectric point of 5.8) where the entire molecule is positively charged. Extensive calculations enabled one to determine the jamming surface concentration (coverage) of molecules adsorbed under the side-on and end-on orientations as well as the total coverage. For the simultaneous side-on/end-on model the maximum surface concentration was 7.29 × 10(3) µm(-2) corresponding to the protein coverage of 4.12 mg m(-2) (without considering hydration). Additionally, the surface blocking functions for different adsorption regimes were determined and analytically approximated for the entire range of coverage by the interpolating polynomials. Using these blocking functions, fibrinogen adsorption kinetics for diffusion controlled transport conditions was evaluated. Comparison of these theoretical results with experimental data was made. It was demonstrated that the simultaneous side-on/end-on model properly reflects the maximum coverage of fibrinogen adsorbed on latex particles determined via the electrokinetic (electrophoretic mobility) and AFM measurements. Also, streaming potential measurements of fibrinogen adsorption kinetics on mica were successfully interpreted in terms of this model. The theoretical results derived in this work have implications for basic science providing information on mechanisms of anisotropic protein adsorption.

Random packing of spheres in Menger sponge.

Random packing of spheres inside fractal collectors of dimension 2 < d < 3 is studied numerically using Random Sequential Adsorption (RSA) algorithm. The paper focuses mainly on the measurement of random packing saturation limit. Additionally, scaling properties of density autocorrelations in the obtained packing are analyzed. The RSA kinetics coefficients are also measured. Obtained results allow to test phenomenological relation between random packing saturation density and collector dimension. Additionally, performed simulations together with previously obtained results confirm that, in general, the known dimensional relations are obeyed by systems having non-integer dimension, at least for d < 3.

The role of the cortex in indentation experiments of animal cells.

We present a model useful for interpretation of indentation experiments on animal cells. We use finite element modeling for a thorough representation of the complex structure of an animal cell. In our model, the crucial constituent is the cell cortex-a rigid layer of cytoplasmic proteins present on the inner side of the cell membrane. It plays a vital role in the mechanical interactions between cells. The cell cortex is modeled by a three-dimensional solid to reflect its bending stiffness. This approach allows us to interpret the results of the indentation measurements and extract the mechanical properties of the individual elements of the cell structure. During the simulations, we scan a broad range of parameters such as cortex thickness and Young's modulus, cytoplasm Young's modulus, and indenter radius, which define cell properties and experimental conditions. Finally, we propose a simple closed-form formula that approximates the simulated results with satisfactory accuracy. Our formula is as easy to use as Hertz's function to extract cell properties from the measurement, yet it considers the cell's inner structure, including cell cortex, cytoplasm, and nucleus.

Nanoscale insights into the local structural rearrangements of amyloid-ß induced by bexarotene.

A better understanding of the abnormal protein aggregation and the effect of anti-aggregation agents on the fibrillation pathways and the secondary structure of aggregates can determine strategies for the early treatment of dementia. Herein, we present a combination of experimental and theoretical studies providing new insights into the influence of the anti-aggregation drug bexarotene on the secondary structure of individual amyloid-ß aggregates and its primary aggregation. The molecular rearrangements and the spatial distribution of ß-sheets within individual aggregates were monitored at the nanoscale with infrared nanospectroscopy. We observed that bexarotene limits the parallel ß-sheets formation, known to be highly abundant in fibrils at later phases of the amyloid-ß aggregation composed of in-register cross-ß structure. Moreover, we applied molecular dynamics to provide molecular-level insights into the investigated system. Both theoretical and experimental results revealed that bexarotene slows down the protein aggregation process via steric effects, largely prohibiting the antiparallel to parallel ß-sheet rearrangement. We also found that bexarotene interacts not only via the single hydrogen bond formation with the peptide backbone but also with the amino acid side residue via a hydrophobic effect. The studied model of the drug-amyloid-ß interaction contributes to a better understanding of the inhibition mechanism of the amyloid-ß aggregation by the small molecule drugs. However, our nanoscale findings need to meet in vivo research requiring different analytical approaches.

Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.

Random sequential adsorption on fractals.

Irreversible adsorption of spheres on flat collectors having dimension d < 2 is studied. Molecules are adsorbed on Sierpinski's triangle and carpet-like fractals (1 < d < 2), and on general Cantor set (d < 1). Adsorption process is modeled numerically using random sequential adsorption (RSA) algorithm. The paper concentrates on measurement of fundamental properties of coverages, i.e., maximal random coverage ratio and density autocorrelation function, as well as RSA kinetics. Obtained results allow to improve phenomenological relation between maximal random coverage ratio and collector dimension. Moreover, simulations show that, in general, most of known dimensional properties of adsorbed monolayers are valid for non-integer dimensions.

Random sequential adsorption: An efficient tool for investigating the deposition of macromolecules and colloidal particles.

Random Sequential Adsorption (RSA) is one of the most efficient theoretical models used to investigate adsorption of macromolecules and particles, with a long-standing tradition in the field of colloid and interface science. In the first part of this paper, we demonstrate how the RSA model can be applied to interpret the experimental data and extract information about the density of the adsorption monolayer, the kinetics of its growth, and microstructural properties such as pair-correlation function and monolayer roughness. We briefly summarized the most important generalizations of the RSA model for monolayers and reviewed its extensions considering, e.g., various particle shapes, the introduction of electrostatic interaction, or adsorption on non-uniform substrates. We thoroughly scrutinized the extended RSA model developed for bilayer and multilayer formation. We collected the mean saturated packing fractions of various two- and three-dimensional objects and provided the most accurate result for two-dimensional disk packing. In the second part of this paper, we summarize various numerical algorithms and techniques that allow one to effectively implement RSA algorithms. We describe efficient methods for detecting intersections of various shapes and techniques enabling generation of strictly saturated RSA packings built of a wide range of different shapes. We hinted at how an inherently sequential RSA scheme can be parallelized. Finally, we critically discuss the limitations of the model and possible directions for future studies.

Plasmonic hot spots reveal local conformational transitions induced by DNA double-strand breaks.

DNA double-strand breaks (DSBs) are typical DNA lesions that can lead to cell death, translocations, and cancer-driving mutations. The repair process of DSBs is crucial to the maintenance of genomic integrity in all forms of life. However, the limitations of sensitivity and special resolution of analytical techniques make it difficult to investigate the local effects of chemotherapeutic drugs on DNA molecular structure. In this work, we exposed DNA to the anticancer antibiotic bleomycin (BLM), a damaging factor known to induce DSBs. We applied a multimodal approach combining (i) atomic force microscopy (AFM) for direct visualization of DSBs, (ii) surface-enhanced Raman spectroscopy (SERS) to monitor local conformational transitions induced by DSBs, and (iii) multivariate statistical analysis to correlate the AFM and SERS results. On the basis of SERS results, we identified that bands at 1050 cm-1 and 730 cm-1 associated with backbone and nucleobase vibrations shifted and changed their intensities, indicating conformational modifications and strand ruptures. Based on averaged SERS spectra, the PLS regressions for the number of DSBs caused by corresponding molar concentrations of bleomycin were calculated. The strong correlation (R2 = 0.92 for LV = 2) between the predicted and observed number of DSBs indicates, that the model can not only predict the number of DSBs from the spectra but also detect the spectroscopic markers of DNA damage and the associated conformational changes.

Mechanisms of fibrinogen adsorption at solid substrates.

Adsorption of fibrinogen, modeled as a linear chain of touching beads of various sizes, was theoretically studied using the random sequential adsorption (RSA) model. The adsorption process was assumed to consist of two steps: (i) formation of an irreversibly bound fibrinogen monolayer under the side-on orientation, which is independent of the bulk protein concentration and (ii) formation of the reversibly bound, end-on monolayer, whose coverage was dependent on the bulk concentration. Calculation based on the RSA model showed that the maximum surface concentration of the end-on (reversible) monolayer equals N(⊥∞) = 6.13 × 10(3) µm(-2) which is much larger than the previously found value for the side-on (irreversible) monolayer, equal to N(∞) = 2.27 × 10(3) µm(-2). Hence, the maximum surface concentration of fibrinogen in both orientations is determined to be 8.40 × 10(3) µm(-2) corresponding to the protein coverage of 5.70 mg m(-2) assuming 20% hydration. Additionally, the surface blocking function (ASF) was determined for the end-on fibrinogen adsorption, approximated for the entire range of coverage by the interpolating polynomial. For the coverage approaching the jamming limit, the surface blocking function (ASF) was shown to vanish proportionally to (θ(⊥∞) - θ(⊥))(2). These calculation allowed one to theoretically predict adsorption isotherms for the end-on regime of fibrinogen and adsorption kinetics under various transport conditions (diffusion and convection). Using these theoretical results, a quantitative interpretation of experimental data obtained by TIRF and ellipsometry was successfully performed. The equilibrium adsorption constant for the end-on adsorption regime was found to be 8.04 × 10(-3) m. On the basis of this value, the depth of the adsorption energy minimum, equal to -17.4 kT, was predicted, which corresponds to ΔG = -41.8 kJ mol(-1). This is in accordance with adsorption energy derived as the sum of the van der Waals and electrostatic interactions. Besides having significance for predicting fibrinogen adsorption, theoretical results derived in this work also have implications for basic science providing information on mechanisms of anisotropic protein molecule adsorption on heterogeneous surfaces.

Physics of free climbing.

The theory of stochastic processes provides theoretical tools which can be efficiently used to explore the properties of noise-induced escape kinetics. Since noise-facilitated escape over the potential barrier resembles free climbing, one can use the first-passage time theory in an analysis of rock climbing. We perform the analysis of the mean first-passage time in order to answer the question regarding the optimal, i.e., resulting in the fastest climbing, rope length. It is demonstrated that there is a discrete set of favorable rope lengths assuring the shortest climbing times, as they correspond to local minima of mean first-passage time. Within the set of favorable rope lengths there is the optimal rope giving rise to the shortest climbing time. In particular, more experienced climbers can decrease their climbing time by using longer ropes.

Methods for Studying Endometrial Pathology and the Potential of Atomic Force Microscopy in the Research of Endometrium.

The endometrium lines the uterine cavity, enables implantation of the embryo, and provides an environment for its development and growth. Numerous methods, including microscopic and immunoenzymatic techniques, have been used to study the properties of the cells and tissue of the endometrium to understand changes during, e.g., the menstrual cycle or implantation. Taking into account the existing state of knowledge on the endometrium and the research carried out using other tissues, it can be concluded that the mechanical properties of the tissue and its cells are crucial for their proper functioning. This review intends to emphasize the potential of atomic force microscopy (AFM) in the research of endometrium properties. AFM enables imaging of tissues or single cells, roughness analysis, and determination of the mechanical properties (Young's modulus) of single cells or tissues, or their adhesion. AFM has been previously shown to be useful to derive force maps. Combining the information regarding cell mechanics with the alternations of cell morphology or gene/protein expression provides deeper insight into the uterine pathology. The determination of the elastic modulus of cells in pathological states, such as cancer, has been proved to be useful in diagnostics.

Increasing AFM colloidal probe accuracy by optical tweezers.

A precise determination of the cantilever spring constant is the critical point of all colloidal probe experiments. Existing methods are based on approximations considering only cantilever geometry and do not take into account properties of any object or substance attached to the cantilever. Neglecting the influence of the colloidal sphere on the cantilever characteristics introduces significant uncertainty in a spring constant determination and affects all further considerations. In this work we propose a new method of spring constant calibration for 'colloidal probe' type cantilevers based on the direct measurement of force constant. The Optical Tweezers based calibration method will help to increase the accuracy and repeatability of the AFM colloidal probe experiments.

Circulating tumour-derived microvesicles in plasma of gastric cancer patients.

Cell membrane microfragments called microvesicles (MV) originating from different cells are circulating in the blood of healthy subjects and their elevated numbers are found in different diseases, including cancer. This study was designed to characterise MV present in plasma of gastric cancer patients. Since majority of MV in blood are platelets-derived (PMV), plasma samples deprived of PMV were used. In comparison to control, the number of MV in patients was significantly elevated in all stages, higher in more advanced disease. Patients' MV showed an increased membrane expression of CCR6 and HER-2/neu. The proportion of MV carrying some leucocyte determinants was low and similar in patients and control. Transmission electron microscopy showed their substantial heterogeneity in size and shape. The size determined by dynamic light scattering analysis confirmed this heterogeneity. The MV size distribution in patients was broader within the range of 10-800 nm, while in control MV showed 3-mode distribution within the range of 10-400 nm. Atomic force microscopy confirmed MV size heterogeneity with implication that larger objects represented aggregates of smaller microparticles. Patients' MV exhibited increased absolute values of zeta potential, indicating a higher surface charge. Tumour markers HER-2/neu, MAGE-1, c-MET and EMMPRIN were detected both in control and patients' samples with stronger expression in the latter. Significantly higher expression of MAGE-1 and HER-2/neu mRNA was observed in individual patients. All together, it suggests that at least some MV in plasma of gastric cancer patients are tumour-derived. However, their role in cancer requires further studies.