Programme

L01

Two-Dimensional Electronic Spectroscopy Studies of Energy and Electron Transfer in Photosystems I and II

Parveen Akhtar¹, Thanh Nhut Do², Huang Long Nguyen², Howe-Siang Tan², Petar H. Lambrev¹

¹Institute of Plant Biology, Biological Research Centre, Szeged, Hungary
²Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore

Photosynthesis in plants, algae and cyanobacteria entails the excitation of pigment molecules in the light-harvesting antenna complexes and the transfer of the excitation energy to the reaction centres of photosystems (PS) I and II. With recent breakthroughs in structural biology, especially cryoelectron microscopy, not only the structures of individual pigment-protein complexes, but also of larger photosynthetic macromolecular assemblies have been revealed with near-atomic resolution. With this knowledge and using advanced ultrafast optical spectroscopy - in the femtosecond to nanosecond time range - we can map the energy and electron transfer in photosynthetic systems. This helps understanding how the protein environment enables efficient and tuneable light harvesting, which in turn could pave the way to higher productivity or novel solar energy technologies. In our recent studies we combined two-dimensional electronic spectroscopy (2DES) and structure-based theoretical modelling to reveal the light-harvesting dynamics in PS I and PS II. Taking advantage of the high temporal and spectral resolution of 2DES, we could separate the kinetics of energy transfer in the antenna from charge separation in the reaction centre of cyanobacterial PS I at 77 K and determine the primary charge separation time to be 0.6-0.8 ps. We also show that the kinetics of energy transfer in isolated light-harvesting antenna complexes are markedly different than in the PS II supercomplex, highlighting that protein-protein interactions and the native protein environment is key to efficient energy transfer in the photosynthetic membranes.

L02

UV-VIS Polarization Spectroscopy at Diamond B23 synchrotron beamline

Tamás Jávorfi, Rohanah Hussain, Tiberiu-Marius Gianga, Giuliano Siligardi

Diamond Light Source, Diamond House, OX11 0DE Didcot, United Kingdom

The B23 synchrotron radiation circular dichroism (SRCD) beamline at Diamond Light Source has been operational for over a decade now. The small spot size and the highly collimated light beam from the synchrotron source enabled us to interrogate small sample volumes in capillary tubes or to investigate dilute samples in long-pathlength cuvettes. A sample chamber, incorporating a motorized X-Y translation stage to accommodate 96-well plates, was developed for high-throughput screening. This arrangement was also used to investigate samples where precise positioning was required. This led to an increasing number of project proposals aiming for investigating solid samples, such as films or liquid crystals, with spatial inhomogeneity. Apart from collecting spectral information originating from different parts of the sample, mapping of spatial inhomogeneity, manifested in the absorption or CD intensity, was also required. Imaging capabilities were achieved by moving the stage in stepwise manner, scanning through an area of interest, and plotting the CD intensity, at fixed wavelength, as a function of sample position. In many cases, though, the higher degree of molecular order in solid samples, as opposed to liquids, the measured CD spectra were distorted by other polarization effects, such as linear dichroism (LD) and/or linear and circular birefringence (LB, CB). The only way to overcome this problem was the introduction of a Mueller Matrix Polarimeter (MMP) capable of measuring, at the same time, all 16 elements of the Mueller matrix, which fully describes the interaction of the sample with polarized light. Data can also be collected as a function of temperature (­170 ˚C to +300 ˚C) or magnetic field (1.3 T) parallel or perpendicular to the direction of the propagation of light. In the following presentation we are going to show some examples to demonstrate the capabilities of this latest addition to Diamond B23 beamline.

L03

Conformational flexibility in a photoactivated adenylate cyclase studied by small-angle X-ray scattering

Pécsi Ildikó¹, Bódis Emőke¹, Kengyel András¹, Pounot Kévin², Pernot Petra³, Tully Mark³, Schirò Giorgio², Weik Martin², Kapetanaki Sofia Maria², Lukács András¹

¹Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary
²Institute of Structural Biology, Grenoble, France
³European Synchrotron Radiation Facility, Grenoble, 38043 France

The photoactivated adenylate cyclase from the photosynthetic cyanobacterium Oscillatoria acuminata OaPAC is a homodimeric enzyme comprising of a N-terminal domain that senses blue light using flavin (BLUF)¹ and a C-terminal class III adenylate cyclase (AC) domain that catalyses the formation of cAMP from ATP (adenosine triphosphate)^2,3. cAMP is a universal regulator of metabolism and gene expression in all life forms⁴. Modulating the cellular concentration of cAMP has emerged in the focus of modern optogenetic applications and therapeutic approaches. Recent crystallographic studies have indicated that the activation mechanism involves only small movements. In this study, we apply small-angle X-ray scattering (SAXS)⁵ in combination with other biophysical techniques to investigate the substrate induced-conformational changes of OaPAC in solution. The implications of our work to the function of the enzyme are discussed.

References

1. Fujisawa, T. and Masuda, S. (2018) Light-induced chromophore and protein responses and mechanical signal transduction of BLUF proteins Biophys. Rev. 10, 327-337.
2. Ohki, M. et al. (2016) Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium Proc. Natl. Acad. Sci. 113, 6659-6664.
3. Ohki, M. et al. (2017) Molecular mechanism of photoactivation of a light-regulated adenylate cyclase Proc. Natl. Acad. Sci. 114, 8562-8567.
4. Zaccolo, M., Zerio, A., and Lobo, M.J. (2021) Subcellular Organization of the cAMP Signaling Pathway Pharmacol. Rev. 73, 278–309.
5. Da Vela Stefano and Svergun, D. (2020) Methods, development and applications of small-angle X-ray scattering to characterize biological macromolecules in solution Curr. Res. Struct. Biol. 2, 164-170.

L04

Structural and functional units associated with non-bilayer lipid phases of plant thylakoid membranes

Ondřej Dlouhý¹, Václav Karlický^1,2, Uroš Javornik³, Irena Kurasová¹, Ottó Zsiros⁴, Primož Šket³, Divya Kanna⁴, Kristýna Večeřová², Kinga Böde⁴, Otmar Urban², Edward S. Gasanoff^5,6, Janez Plavec^3,7,8, Vladimír Špunda^1,2, Bettina Ughy⁴, Győző Garab^1,4

¹University of Ostrava, Ostrava, Czech Republic
²Global Change Research Institute of the CAS, Brno, Czech Republic
³National Institute of Chemistry, Ljubljana, Slovenia
⁴Biological Research Centre, Szeged, Hungary
⁵Lomonosov Moscow State University, Moscow, Russia
⁶Chaoyang KaiWen Academy, Beijing, China
⁷EN-FIST Center of Excellence, Ljubljana, Slovenia
⁸University of Ljubljana, Ljubljana, Slovenia

The coexistence of bilayer (lamellar) and non-bilayer (non-lamellar) lipid phases in the two main energy-converting biological membranes – in isolated fully functional plant thylakoid membranes (TMs) and mammalian inner mitochondrial membranes (IMMs) – is now well established [1]. However, our understanding about the structural entities associated with different lipid phases is still rudimentary.

Here we investigated the effects of different lipases and proteinases on the polymorphic phase behavior of TMs, using ³¹P-NMR spectroscopy, and on structural and functional parameters of the photosynthetic machinery, via using biophysical and biochemical tools. We found that Phospholipase-A1 gradually destroyed all lipid phases (the lamellar phase, the two isotropic phases and the inverted hexagonal phase); the diminishment of the lamellar phase permeabilized the membranes; other effects, mainly on Photosystem II, lagged behind the loss of the original lipid phases. Wheat-germ lipase selectively eliminated the isotropic phases but did not disturb the structure and function of TMs – indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion and junctions, in accordance with the known fusogenic roles of non-bilayer lipids. Trypsin and Proteinase K selectively suppressed the H_II phase – suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides.

We conclude that the non-bilayer phases of TMs are found in subdomains separated from but interconnected with the bilayer. These findings – and similar data on IMMs – are interpreted within the frameworks of the Dynamic Exchange Model of the energy-converting membranes [1].

References

1. G Garab, LS Yaguzhinsky, O Dlouhý, SV Nesterov, V Špunda, ES Gasanoff (2022) Structural and functional roles of non-bilayer lipid phases of chloroplast thylakoid membranes and mitochondrial inner membranes. Prog Lipid Res 86: 101163

L06

Multiscale simulations of biomolecular systems

Matej Praprotnik^1,2

¹National Institute of Chemistry, Theory Department, Hajdrihova 19, Ljubljana, Slovenia
²University of Ljubljana, Faculty of Mathematics and Physics, Jadranska 19, Ljubljana, Slovenia

Open molecular systems exchange mass, momentum, and energy with their surroundings. In this talk, I will present simulation methodology that enables equilibrium molecular dynamics simulations in the grand-canonical ensemble as well as nonequilibrium fluid flow simulations. The flow is introduced as an external boundary condition while the equations of motion for the bulk remain unaltered. I will showcase the versatility of our multiscale approach on a few applications to biomolecular systems.

L07

Molecular Modeling Powered by Machine Learning

Stephan Thaler¹, Maximilian Stupp¹, Julija Zavadlav^1,2

¹Professorship of Multiscale Modeling of Fluid Materials, TUM School of Engineering and Design, Technical University of Munich, Munich, Germany
²Munich Data Science Institute & Munich Institute for Integrated Materials, Energy and Process Engineering, Technical University of Munich, Munich, Germany

Molecular modeling has become a cornerstone of many disciplines, including soft matter physics. However, the quality of predictions critically depends on the employed model that defines particle interactions. A class of models with tremendous success in recent years are neural network (NN) potentials due to their flexibility and capacity to learn many-body interactions. However, their accuracy critically depends on the training data and objective. Traditionally, these models are trained bottom-up via force matching. Top-down approaches that learn NN potentials directly from experimental data have received less attention, typically facing numerical and computational challenges when backpropagating through molecular dynamics (MD) simulations. We recently developed the Differentiable Trajectory Reweighting (DiffTRe) method, bypassing differentiation through the MD simulation. Leveraging thermodynamic perturbation theory, we avoid exploding gradients and achieve around two orders of magnitude speed-up in gradient computation for top-down learning. The effectiveness of DiffTRe is showcased on atomistic and coarse-grained models using diverse experimental observables, including thermodynamic, structural, and mechanical properties. Our approach opens the way to high-fidelity molecular models, particularly when bottom-up data is unavailable or insufficiently accurate.

L08

Estimation of model and variable complexity and quality

Bono Lučić¹, Viktor Bojović^2,3, Antonija Kraljević^4,5, Jadranko Batista⁶

¹Ruđer Bošković Institute, NMR Centre, Zagreb, Croatia
²Faculty of Electrical Engineering and Computing, University of Zagreb, Zagreb, Croatia
³Ruđer Bošković Institute, Centre for Informatics and Computing, Zagreb, Croatia
⁴University of Mostar, Faculty of Mechanical Engineering, Computing and Electrical Engineering, Mostar, Bosnia and Herzegovina
⁵University of Split, Postgraduate university study in biophysics, Split, Croatia
⁶University of Mostar, Faculty of Science and Education, Mostar, Bosnia and Herzegovina

When developing structure-property molecular models, it is desirable to pay attention to their simplicity and to include only informative variables (descriptors) in the models, i.e. those that contain useful and interpretable structural information. Moreover, almost every dataset used for model development contains some information that is redundant. Therefore, the protocol we use to validate the quality of the model is extremely important. One of the parts of the model validation procedures is the estimation and evaluation of the random accuracy resulting from the complexity (i.e. monotonicity) of the input data and the model quality, the background of which is mainly represented by the number of parameters optimised in the model. The complexity of the dichotomous variables representing the molecular descriptors and the property of the molecules to be modeled, as well as the values predicted by the model, will be assessed by estimating the number of possible permutations (permutation entropy) of values of a variable. Formulas were derived for new statistical measures that can be used to assess the quality and complexity of classification models and variables. In addition, formulae were derived for calculating the minimum and maximum possible accuracy/agreement of the model and for the average random accuracy/agreement. If we consider the case where predicted and experimental variables have identical distributions, we can obtain expressions for measuring the monotonicity of a variable. Recent results from this area of research will be presented and illustrated with examples of models developed to predict the structure of membrane proteins, the toxicity of organic compounds and the folding rates of proteins.

Acknowledgements

This research was supported by the Croatian Government and the EU through grants KK.01.1.1.01 (BioProCro) and KK.01.1.1.01.0009 (DATACROSS), and Croatian Science Foundation (grant DOK-2018).

L14

Single-molecule mechanics of proteins

Gabriel Žoldák

Center for Interdisciplinary biosciences, Technology and Innovation Park,University PJ Safarik in Kosice, Trieda SNP 1, 040 11, Slovakia

One of the ultimate goals in protein biophysics is obtaining three-dimensional structural dynamics with nanosecond time-resolution and large enough dynamic range to sample rare events that only occasionally appear over long time intervals. On the simulation side, all-atom molecular dynamics simulations are already providing full structural dynamics information on ns timescales, and recently even ms timescales have been reported [1]. However, no experimental method can currently provide a similarly detailed picture. Single-molecule force spectroscopy, while still far from this goal, provides the possibility to measure structural dynamics along a single reaction coordinate with microsecond resolution over a timescale of hours, and thus can uncover fine details of the underlying principles of protein nanomachines.

Protein nanomachines can be thought of as being built like man-made machines: static parts form a basic scaffold for movable parts whose motion is fuelled by an energy source, e.g., thermal motion, ligand binding, concentration gradients or chemical reactions. Using single-molecule force spectroscopy, we are now able to identify the basic parts of these “machines” i.e., functional mechanical elements of proteins. This knowledge will enhance our understanding of the design principles of these highly complex nano-scale objects, eventually enabling us to design our own man-made nanomachines.

For enzymes and ligand-binding proteins, the motion is fuelled by binding energy. For example, the closure of adenylate kinase upon ligand binding is due to favorable pairwise interactions and binding to a pre-optimized solvated binding pocket[2]. Upon ligand binding to the open crevice of the active site, flexible lid parts cover the binding pocket. This is energetically favorable partly due to the direct interaction with ligand and partly as a result of the change of the local structure around the binding site changes to favor interaction with the lids.

While minimization of binding free energy drives many processes in cells, it is not sufficient to direct unidirectional mechanical work or reaction cycles. Therefore, highly complex mechanical machines use the energy released during exergonic chemical reactions for mechanical functions (e.g., ATP hydrolysis) that need directionality (e.g., physical motion) or controlled timings. Single-molecule force spectroscopy experiments can characterize these essential function-related motions in great detail. Several examples how mechanical studies can contribute to our understanding of proteins will be discussed.

Acknowledgements

This work was supported by the research grants from the Slovak Grant Agency VEGA No. 1/0024/22, by the Slovak Research and Development Agency under contract no. APVV-18-0285, EU H2020 TWINNING program GA. No. 952333 project CasProt, BioPickmol, ITMS2014+: 313011AUW6 supported by the Operational Programme Integrated Infrastructure, funded by the ERDF.

References

1. K. Lindorff-Larsen, S. Piana, RO. Dror, DE. Shaw DE (2011) Science, 334, 517-520.
2. B. Pelz, G. Žoldák, F. Zeller, M. Zacharias, M. Rief (2016) Nature Comm. 7, 10848, 1-9

L15

Cancer-derived small extracellular vesicles as modulators of target cells mechanics

Beatrice Senigagliesi^1,2, Pietro Parisse^1,3, Loredana Casalis¹

¹Elettra Sincrotrone Trieste, Trieste, Italy
²Università degli studi di Trieste, Trieste, Italy
³CNR-IOM, Trieste, Italy

Small extracellular vesicles (sEVs) are nano-sized vesicles which transfer bioactive molecules to recipient cells and play a crucial role in intercellular communication in health and disease. However, their specific role in disease spreading is still unclear. Here we focused on the role of sEVs in metastatic cancer, in particular in the modulation of the mechanical properties of target cells. We isolated and thoroughly characterized sEVs derived from triple-negative breast cancer (TNBC) cell lines, and measured the morphological/biomechanical effects induces by physiological amounts of sEVs on other breast cell lines.

Our results suggest that TNBC-derived small EVs are able to directly modify recipient cells by inducing a decrease in cell stiffness, rearrangements in cytoskeleton, focal adhesions, cellular morphology and chromatin condensation, and an increase in Yap downstream gene expression. Testing the biomechanical response of cells after EV addition might represent a new functional assay in metastatic cancer framework that can be exploited for future application both in diagnosis and therapy.

L16

Biophysics approach in anticancer therapies: Studying anticancer drug interactions with extracted and model cell membranes by Langmuir films and computer simulations

María Pedrosa^1,2, Pablo Graván-Jiménez^2,3, Jesús Peña-Martín^2,3, Julia Maldonado-Valderrama^1,2, Matej Kanduč⁴, Arturo Moncho-Jordá^1,5, María José Gálvez-Ruiz^1,2

¹Biocolloids and Fluid Physics Group, Applied Physics Department, University of Granada, Granada, Spain. mpedrosab@ugr.es
²Excellence Research Unit “Modeling Nature”, University of Granada, Granada, Spain
³Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain
⁴Slovenian Biophysical Society, Department for Theoretical Physics, Jožef Stefan Institute, Ljubljana, Slovenia
⁵Instituto “Carlos I” de Física Teórica y Computacional, Universidad de Granada, Granada, Spain

The rational design of novel anticancer drugs or their nanocarriers requires the complex knowledge of the action mechanism of the anticancer drugs, for which it is relevant to analyze the interactions between drugs and cell membranes. Langmuir monolayers have been widely used to mimic half of a cell membrane to study these interactions under controlled conditions, typically using lipidic models matching membrane cell compositions. However, replicating the complex and variant composition of cell membranes is an arduous task, so the formation of Langmuir films with membranes extracted from real cells needs to be explored.

As a first approach, in this work a healthy and a tumor cell membrane were modeled by mixing a saturated lipid dipalmitoylphosphatidylcholine and high cholesterol proportion, and a lower proportion of cholesterol and an unsaturated lipid sphingomyelin, respectively. The effect of the anticancer compound curcumin was evaluated in both models by analyzing the compression isotherms, and by BAM and AFM imaging. The results show that curcumin disrupts the cancerous model provoking repulsive forces, and causing destabilization and fluidizing, whereas it improves cohesion in the healthy one.

These findings were completed through all-atoms Molecular Dynamics computer modeling. The healthy and tumor monolayers were simulated on a water-vacuum interface and curcumin molecules were placed at different locations of the monolayers to elucidate the most energetically favorable position of the Cur.

As an improvement to better match the real systems, it was possible to create stable Langmuir films of membranes extracted from human breast adenocarcinoma cells (line MCF-7). Doxorubicin anticancer drug was then introduced into the subphase while recording the changes in surface pressure to observe its effect on the membrane films. This novel result confirms it is possible to evaluate interactions in real cell membranes by using Langmuir monolayers.

Acknowledgments

Project RTI2018-101309-B-C21 funded by MCIN/AEI/10.13039/501100011033/FEDER. MPB thanks the FPU19/02045 fellowship funded by MCIN/AEI/10.13039/501100011033 and FSE. This work has been done in the framework of the doctoral of AAG in the Doctoral Programme in Physics and Space Sciences (B09/56/1) of the University of Granada. JMV acknowledges support from project PID2020-116615RA-I00 funded by MCIN/ AEI /10.13039/501100011033. This work was also partially supported by the Biocolloid and Fluid Physics Group (ref. PAI-FQM115) of the University of Granada (Spain).

References

1. Hąc-Wydro K, Dynarowicz-Łątka P. Colloids Surf., B. 2010, 76: 366–369.
2. Nobre T.M, Pavinatto F.J, Caseli L, Barros-Timmons A, Dynarowicz-Łątka P, Oliveira O.N. Thin Solid Films. 2015, 593: 158–188
3. MateronEM, Nascimento GF, Shimizu FM, Câmara AS, Sandrino B, Faria RC, et al. Colloids Surf., B. 2020. 196: 111357.
4. 4. Peetla C, Bhave R, Vijayaraghavalu S, Stine A, Kooijman E, Labhasetwar. Mol Pharm. 2010, 7(6):2334-48.
5. Javanainen, M., Lamberg, A., Cwiklik, L., Vattulainen, I., & Ollila, O. S. Langmuir, 2018, 34: 2565–2572.

L17

Imaging the infection cycle of T7 at the single virion level

Bálint Kiss^1,2, Luca Annamária Kiss¹, Zsombor Dávid Lohinai¹, Dorottya Mudra¹, Hedvig Tordai¹, Levente Herényi¹, Gabriella Csík¹, Miklós Kellermayer^1,2

¹Department of Biophysics and Radiation Biology, Semmelweis University
²ELKH-SE Biophysical Virology Research Group

T7 phages are E. coli-infecting viruses that find and invade their target with high specificity and efficiency. The exact molecular mechanisms of the T7 infection cycle are yet unclear. As the infection involves mechanical events, single-particle methods are to be employed to alleviate the problems of ensemble averaging. Here we used TIRF microscopy to uncover the spatial dynamics of the target recognition and binding by individual T7 phage particles. In the initial phase, T7 virions bound reversibly to the bacterial membrane via two-dimensional diffusive exploration. Stable bacteriophage anchoring was achieved by tail-fiber complex to receptor binding which could be observed in detail by atomic force microscopy (AFM) under aqueous buffer conditions. The six anchored fibers of a given T7 phage displayed isotropic spatial orientation. Viral infection led to the onset of an irreversible structural program in the host which occurred in three distinct steps. First, bacterial cell surface roughness, as monitored by AFM, increased progressively. Second, membrane blebs formed on the minute time scale (average ~5 min) as observed by phase-contrast microscopy. Finally, the host cell was lysed in a violent and explosive process that was followed by the quick release and dispersion of the phage progeny. DNA ejection from T7 could be evoked in vitro by photothermal excitation, which revealed that genome release is mechanically controlled to prevent premature delivery of host-lysis genes. The single-particle approach employed here thus provided an unprecedented insight into the details of the complete viral cycle.

L09

A dynamic preferred direction model for the self-organization dynamics of bacterial microfluidic pumping

Daniel Svenšek

Faculty of Mathematics and Physics, National Institute of Chemistry, Ljubljana, Slovenia

Since the pioneering work by Kim and Breuer [1] it is known that some flagellated bacteria like Serratia marcescens, when deposited and affixed onto a surface to form a bacterial carpet, self-organize in a collective motion of the flagella that is capable of pumping fluid through microfluidic channels.

We set up a continuum model comprising two macroscopic variables, that is capable of describing this self-organization mechanism as well as quantifying it to the extent that an agreement with the experimentally observed channel width dependence of the pumping is reached as a particular challenge [2].

The activity is introduced through a collective angular velocity of the helical flagella rotation, which is an example of a dynamic macroscopic preferred direction [3].

Our model supports and quantifies the view that the self-coordination is due to a positive feedback loop between the bacterial flagella and the local flow generated by their rotation. Moreover, our results indicate that this biological active system is operating close to the self-organization threshold, where magnitude fluctuations of the self-organized order are substantial.

This finding is rather unexpected and may hint to a general premise that biological systems are typically rather noisy --- and are therefore also quite robust against noise.

References

1. M. J. Kim and K. S. Breuer, Small 4, 111-118 (2008).
2. D. Svensek, H. Pleiner, and H. R. Brand, Soft Matter 15, 2032-2042 (2019).
3. D. Svensek, H. Pleiner, and H. R. Brand, Phys. Rev. Lett. 111, 228101 (2013).

L10

From isles of Königsberg to islets of Langerhans: Calcium oscillations in networks of beta cells

Marko Gosak^1,2, Jurij Dolenšek^1,2, Patrick E. MacDonald³, Andraž Stožer¹

¹Institute of Physiology, Faculty of Medicine, University of Maribor, Maribor, Slovenia
²Department of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia
³Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada

Islets of Langerhans are multicellular endocrine organs that regulate whole-body energy homeostasis. Through secretion of insulin, they control postprandial storage and interprandial usage of energy-rich nutrients. Intercellular electrical coupling through gap junctions is the basis for coordinated responses. Increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. Several novel techniques have been proposed to assess intercellular cooperation, including the fruitful combination of multicellular imaging and network science introduced by our group. I will first provide a short introduction to the basic principles of network theory and define the measures quantifying the functional connectivity. Then I will sketch the methodological approaches to construct functional beta cell networks and concentrate on recent findings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. The combination of these imaging techniques and network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus. Finally, it is applicable to other fields where functional multicellular calcium imaging or recording of other biological signals can be performed on a large number of cells working in concert.

L11

Boundary scattering and the spatial distribution of confined active matter

András Búzás¹, Pál Ormos¹, Roberto Di Leonardo^2,3, Gaszton Vizsnyiczai^1,4

¹Institute of Biophysics, Biological Research Centre Szeged, Eötvös Loránd Research Network, Szeged, Hungary
²Dipartimento di Fisica, Sapienza Università di Roma, Rome, Italy
³NANOTEC-CNR, Soft and Living Matter Laboratory, Institute of Nanotechnology, Rome, Italy
⁴Department of Biotechnology, University of Szeged, Szeged, Hungary

The field of active matter aims to study nonequilibrium systems composed of agents, so called active particles, that continuously consume internal or external energy to propel themselves. Such agents can be found from the microscopic to the macroscopic scales: biomolecular motors, synthetic microswimmers, moving or swimming cells, drones, birds, humans. Active matter systems provide test subjects for studying out-of-equilibrium statistical physics and can also be harnessed for practical applications such as self-pumping fluids or microscale transport [1-3]. Unique behaviours (e.g. collective motion) can emerge from the particles’ interactions with each other and their environment. It was shown that the motion of active particles confined in microfluidic channels can be directed by shaping the geometry of the confinement [4-5].

Here we present a study on how active particles spread within two-dimensional confinements, and what are the general conditions for achieving directed motion and accumulation of particles. While active particles, like swimming bacteria, typically display a wall aligning behaviour in confinement [4-5], our aim is to provide a more general understanding, and therefore we examine a range of different boundary “scattering” angle distributions with numerical simulations. Using phototactic microalgae cells (Euglena gracilis) trapped in two-dimensional light patterns we also present a confined active matter system whose particles display a non-aligning boundary scattering, and compare our observations with simulated results. Furthermore, we introduce a novel analytical method for the calculation of the spatial probability density distribution of confined active particles.

Supported by the ÚNKP-21-SZTE-539 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

References

1. Nature Active Matter Collection, https://www.nature.com/collections/hvczfmjfzl
2. Gompper, Gerhard, et al. The 2020 motile active matter roadmap. Journal of Physics: Condensed Matter 32.19 (2020): 193001.
3. Varghese, M., Baskaran, A., Hagan, M. F., & Baskaran, A. (2020). Confinement-induced self-pumping in 3D active fluids. Physical Review Letters, 125(26), 268003.
4. Galajda, P., Keymer, J., Chaikin, P., & Austin, R. (2007). A wall of funnels concentrates swimming bacteria. Journal of bacteriology, 189(23), 8704-8707.
5. Katuri, J., Caballero, D., Voituriez, R., Samitier, J., & Sanchez, S. (2018). Directed flow of micromotors through alignment interactions with micropatterned ratchets. ACS nano, 12(7), 7282-7291.

L12

Elucidation of DMSO effects on catalytic activity of halohydrin dehalogenase HheC by molecular dynamics

Višnja Stepanić¹, Zlatko Brkljača^1,3, Nevena Milčić², Ivo Crnolatac¹, Zvjezdana Findrik Blažević² and Maja Majerić Elenkov¹

¹Ruđer Bošković Institute, Zagreb, Croatia
²University of Zagreb, Faculty of Chemical Engineering and Technology, Zagreb, Croatia
³Present Address: Selvita Ltd., 10000 Zagreb, Croatia

Homotetrameric halohydrin dehalogenase from Agrobacterium radiobacter AD1, HheC is extensively used for the industrial green synthesis of enantiopure building blocks. It naturally catalyses reversible dehalogenation of vicinal haloalcohols, but it is utilized with a whole range of unnatural nucleophiles in epoxide ring-opening reactions. In order to increase solubility of lipophilic epoxides and conversion efficiency, addition of various solvent is explored.

The results of study of effects of widely explored solvent DMSO (dimethyl sulfoxide) on catalytic activity of HheC will be presented. Besides determination of kinetic parameters, differential scanning calorimetry (DSC) and dynamic light scattering (DLS), molecular dynamics (MD) is used to elucidate mechanisms of DMSO action on HheC. We carried out MD simulations (GROMACS ) on natural tetrameric and hypothetical monomeric HheC in water as well as in 20% and 50% (v/v) DMSO/aqueous environment. The tetramer HheC exhibits remarkable conformational tolerance towards DMSO up to 30% and it instantly aggregates at 50% DMSO, but its catalytic activity exponentially decreases with DMSO addition. 5% DMSO inhibits the HheC activity by half. The MD demonstrates that while subunit conformations slightly changes with DMSO addition, distinct sheering of the main structural motifs between subunits occurs, with changes proceeding from more localized (20%) to more extended and collective (50%). However, no dissociation (up to 300 ns) was observed in accordance with DSC and DLS results, but buried surface area increases and the catalytic site becomes more constrained. DMSO is found to replace H₂O molecules in catalytic site forming alternately H-bonds with the catalytic amino acid residues S132 and Y145, and to form small clusters around the protein.

L13

Investigating the competitive regulation of Las17 through Agent-Based modelling

Lewis Hancock, Kathryn Ayscough, Mike Williamson

The University of Sheffield, School of Biosciences, Sheffield, United Kingdom

Clathrin-mediated endocytosis (CME) is a mechanism used by eukaryotic cells to move membrane-associated elements into the cytosol. Proteins are recruited to form a patch that is later invaginated and pinched off to produce a vesicle [1]. A key step in the progression of new patches involves the de novo nucleation of actin filaments. Las17, a largely natively unstructured actin-binding protein, is thought to regulate this process, although it is not known how [2].
The central region of Las17 has recently been shown to bind and nucleate actin via a series of largely uncharacterised polyproline binding sites which are also known to weakly bind the SH3 domains of several patch-associated proteins [3,4]. Human WASP family proteins (homologues of Las17) have been linked to several neurodegenerative diseases [5,6]. These domains can be thought of as a ‘cloud’ of SH3s which changes in composition as endocytosis progresses and more domains are recruited. Some proteins even possess multiple SH3 domains [7]. A pure experimental characterisation of these interactions would prove challenging due to the high number of binding sites and proteins involved.
Agent-based modelling can provide a valuable insight into (i) how the combined effect of weakly binding SH3 domains can deliver robust endocytic control via their joint effect on Las17 activity and (ii) why some of these proteins interact through a tandem of SH3 domains. Here, we built a parallel coded, agent-based computational model within the FLAMEGPU modelling environment [8] which, through its modular nature, allows for an in-depth investigation of this regulation. The program is being supplemented by key binding data such as affinities obtained using Microscale thermophoresis (MST) and Biolayer interferometry (BLI).

References

1. Goode, B. L., Eskin, J. A. & Wendland, B. Actin and endocytosis in budding yeast. Genetics 199, 315–58 (2015).
2. Urbanek, A. N., Smith, A. P., Allwood, E. G., Booth, W. I. & Ayscough, K. R. A novel actin-binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3. Curr Biol 23, 196–203 (2013).
3. Tyler, J. J., Allwood, E. G. & Ayscough, K. R. WASP family proteins, more than Arp2/3 activators. Biochem Soc Trans 44, 1339–1345 (2016).
4. Feliciano, D., Tolsma, T. O., Farrell, K. B., Aradi, A. & di Pietro, S. M. A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis. Traffic 16, 379–97 (2015).
5. Kitamura, Y. et al. Possible involvement of Wiskott–Aldrich syndrome protein family in aberrant neuronal sprouting in Alzheimer’s disease. Neuroscience Letters 346, 149–152 (2003).
6. Kumar, S. et al. Compound heterozygous variants in Wiskott-Aldrich syndrome like (WASL) gene segregating in a family with early onset Parkinson’s disease. Parkinsonism & Related Disorders 84, 61–67 (2021).
7. Tong, A. H. Y. et al. A Combined Experimental and Computational Strategy to Define Protein Interaction Networks for Peptide Recognition Modules. Science (1979) 295, 321–324 (2002).
8. Paul Richmond, Dawn Walker, Simon Coakley & Daniela Romano. High performance cellular level agent-based simulation with FLAME for the GPU. Briefings in Bioinformatics 11, 334–347 (2010).

L18

Nanomaterial Quarantining by Lung Epithelial Cells and in vitro Prediction of Chronic Inflammation after Nanomaterial Inhalation

Tilen Koklič¹, Hana Kokot¹, Boštjan Kokot¹, Aleksandar Sebastijanović¹, Rok Podlipec¹, Iztok Urbančič¹, Tobias Stoeger², Janez Štrancar¹

¹Laboratory of biophysics, Jožef Stefan Institut, Ljubljana, Slovenia
²Institute of Lung Biology and Disease, Helmholtz Zentrum München, Germany

In the framework of the European project H2020 SmartNanoTox, in cooperation with leading European toxicologists, we showed that TiO₂ nanotubes cause the formation of composites of nanotubes, lipids, and fibrogenic proteins. We named the phenomenon nano-quarantine. The value of the work was enhanced by the fact that the process of nano-quarantine formation for several different nanoparticles was also observed in mice. This led to the discovery of one of the first in vivo validated in vitro/in silico tests to predict chronic inflammation without the use of animals. The co-authored article by 34 authors was chosen in 2020 as the cover of a chapter in the prestigious journal Advanced Materials, known for its distinct interdisciplinarity [1].

References

1. H. Kokot, B. Kokot, A. Sebastijanović, C. Voss, R. Podlipec, P. Zawilska, T. Berthing, C. Ballester-López, P. Høgh Danielsen, C. Contini, M. Ivanov, A. Krišelj, P. Čotar, Q. Zhou, J. Ponti, V. Zhernovkov, M. Schneemilch, Z. Doumandji, M. Pušnik, P. Umek, S. Pajk, O. Joubert, O. Schmid, I. Urbančič, M. Irmler, J. Beckers, V. Lobaskin, S. Halappanavar, N. Quirke, A. P. Lyubartsev, U. Vogel, Tilen Koklič*, T. Stoeger*, J. Štrancar*. Prediction of Chronic Inflammation for Inhaled Particles: The Impact of Material Cycling and Quarantining in the Lung Epithelium, Advanced Materials, 32, 2003913 (2020).

L19

Stimuli-responsive polymeric nanocarriers

Matej Kanduč

Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia

Thermoresponsive hydrogels have become integral building blocks of ‘smart’ functional materials in modern applications. For many developments, such as drug delivery or nano catalytic carrier systems, selective transport of molecules (ligands or reactants) inside the polymeric matrix are key processes that must be controlled and tuned for the desired material function. I will discuss our recent computer simulation studies of atomistic and coarse-grained polymer models that helped uncover the molecular principles of permeability and selectivity in hydrogel permeation. We found that dense hydrogels are incredibly selective, owing to a delicate balance between the partitioning and diffusivity of the molecules. These properties are sensitively tuned by the penetrant size, shape, and chemistry, leading to vast cancellation effects, which nontrivially contribute to the permeability.

L20

Viscosity measurements using flexible microstructures

Jana Kubackova¹, Cyril Slabý², Denis Horvath³, Andrej Hovan², Gergely T. Iványi^4,5, Gaszton Vizsnyiczai⁴, Lóránd Kelemen⁴, Alena Strejčková⁶, Zoltán Tomori¹, Gregor Bánó²

¹Department of Biophysics, Institute of Experimental Physics SAS, Košice, Slovakia
²Department of Biophysics, Faculty of Science, P. J. Šafárik University, Košice, Slovakia
³Center for Interdisciplinary Biosciences, TIP, P. J. Šafárik University, Košice, Slovakia
⁴Biological Research Centre, Institute of Biophysics, ELKH, Szeged, Hungary
⁵Faculty of Science and Informatics, University of Szeged, Szeged, Hungary
⁶Dep. of Chem., Biochem. and Biophys., Univ. of Veterinary Med. and Pharm., Košice, Slovakia

Micro-rheological measurements of small-volume liquid samples are of high importance. It is our goal to develop new concepts for significant down-scaling of the viscosity measurements. Flexible microstructures are prepared by Two-Photon Polymerization Direct Laser Writing (TPP-DLW), a 3-dimensional microfabrication method of CAD-designed objects. In TPP-DLW a pulsed (femtosecond) laser beam is tightly focused into the liquid photoresist material to induce polymerization locally. The laser focus is scanned along a pre-defined trajectory inside the microstructure volume. Spatial resolution on the order of 100 nm can be reached. The mechanical properties of the polymerized microstructures are chosen by selecting the photoresist material and setting the polymerization parameters. Highly flexible microstructures composed of a microsphere attached to a nanowire cantilever were prepared in this work from Ormocomp, a biocompatible hybrid organic-inorganic photoresist.

The shape of low-stiffness flexible microstructures immersed into liquid media is deformed depending on the flow conditions of the surrounding liquid. The dynamics of the deformation depends, besides others, on the viscosity of the liquid. This effect is used to construct micron-sized viscometers that are capable of viscosity measurements in sub-microliter volumes. An optical tweezer is used to trap the microsphere attached to the cantilever and displace it from the relaxed position. The fluid viscosity is derived from the overdamped recovery motion of the microstructure after switching the trapping laser off. The data analysis relies on the microstructure mechanical model which takes the nanowire viscoelastic properties into account.

Acknowledgements

This work was funded by the Slovak Research and Development Agency (grants APVV-18-0285 and APVV-21-0333), the Slovak Ministry of Education (grant VEGA 2/0094/21), the EU H2020 TWINNING program GA. No. 952333 project CasProt, the Operational Program Integrated Infrastructure, funded by the ERDF (Project: OPENMED, ITMS2014+: 313011V455), the joint project of Slovak and Hungarian Academies of Sciences (NKM-53/2021) and the National Research Development and Innovation Fund (FK138520). G.V. acknowledges funding from the Eötvös Lóránd Research Network under the grant agreement No. SA-75/2021.

L21

MATRIX STED - Many eyes see clearer: enhancing resolution, signal quality and brightness

Martin Meschkat^1*, Mary Grace Velasco³, Gero Schloetel¹, Jasmin Rehmann², Frederic Weidling¹, Björn Thiel¹, Christian A. Wurm¹, Matthias Reuss¹, Jan Vavra¹

¹Abberior Instruments, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
²Abberior, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
³Abberior Instruments America, Max Planck Way 1, Jupiter, Florida 33458, USA
^*Correspondence: martin.meschkat@abberior-instruments.com

STED microscopy is a well-established method to improve the resolution of fluorescence microscopes. Nevertheless, improving resolution implies a reduction of the volume of the effective point-spread-function and thus images may appear dimmer, because the signal from fewer dye molecules is integrated at any scan position Since STED mostly acts in or close to the focal plane, the signal seemingly decreases there, while background signal throughout the rest of the sample is mostly unchanged. In other words, the signal-to-background ratio often decreases when doing STED on samples that are thicker than a few microns. This holds true especially for areas of the sample which are densely packed, i.e. areas of high protein and label concentration. The result is that often the interesting parts of the sample are drowned in fluorescent background.
Here, we present a method to sense and subtract background using an array-based detector in connection with deformable mirror based adaptive optics. We show that such a detector can be harnessed to discriminate in-focus signal from out-of-focus background using only a single 2D (xy) image from the sample. This significantly increases optical sectioning and signal-to-background ratio and enables high-resolution 2D and 3D STED microscopy on thick samples and densely labeled structures. To demonstrate the advantages offered by MATRIX STED and adaptive optics, we present super-resolution STED images of several biologically relevant samples such as cilia in cultured mammalian cells or the visualization of crowded and overlapping structures like brain slice tissue. The presented results highlight the superior signal-to-background ratio over conventional STED microscopy with the potential to gain more insights about the structure and biology of studied specimen.

L22

Structural and Conformational Dynamics of a Disordered Protein Motif

Mónika Ágnes Tóth¹, Péter Gaszler^{1, 2}, Andrea Teréz Vig¹, Veronika Takács-Kollár¹, Rauan Sakenov¹, Réka Pintér¹, Beáta Bugyi^{1, 2, #}

¹University of Pécs, Medical School, Department of Biophysics, Pécs Szigeti str 12. H-7624
²Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee, Pécs, Hungary
^# correspondence: beata.bugyi@aok.pte.hu

SALS (sarcomere length short) is a Drosophila-specific sarcomere regulatory protein. [1] It contributes to establishing sarcomere length and organization. The absence of SALS is lethal in the embryonic age. This may be due to the shortening of sarcomeric actin filament length or the disruption of their order. SALS, according to our bioinformatics analysis, is an intrinsically disordered protein (IDP). IDPs are biologically active proteins that, however, do not have a well-defined three-dimensional structure. They possess specific physicochemical properties different from those characteristics of ordered proteins (e.g., hydrophilic/charged: hydrophobic amino acid ratio, thermal stability, electrophoretic mobility). Only two discernable motifs were identified in SALS consisting of a few ten amino acids, called Wiscott-Aldrich syndrome homology 2 (WH2) domains. WH2 domains are intrinsically disordered protein regions (IDR) of low structural complexity. Considering their role, they possess actin-binding properties. Depending on the number and sequence of domains, proteins containing WH2 can exhibit multifunctional properties. Based on our functional analysis of the SALS WH2 domains (SALS-WH2), both WH2 domains interact with actin and influence actin homeostasis by shifting the monomer:filament ratio towards monomeric actin. [2] The structural and conformational dynamical properties of the SALS WH2 are not yet known. Therefore, we further aimed to characterize these properties using in silico and experimental approaches. Our bioinformatic analysis suggests that the SALS WH2 domains have IDR elements. Our prediction-based results were experimentally verified by fluorescence spectroscopy and thermal analysis.

Acknowledgments

New National Excellence Program of the Ministry for Innovation and Technology ÚNKP-21-3-II-PTE-997 (PG), University of Pécs, Medical School, KA-2021-30 (AV). We thank József Mihály (Institute of Genetics, Biological Research Centre) for the SALS plasmid.

References

1. Bai J, Hartwig JH, Perrimon N. SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth. Dev Cell.2007 Dec;13(6):828-42.
2. Tóth MÁ, Majoros AK, Vig AT, Migh E, Nyitrai M, Mihály J, Bugyi B. Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein. J Biol Chem. 2016 Jan 8;291(2):667-80.

L23

Quorum sensing response of single bacterial cells studied by a microfluidic mother machine

Ágnes Ábrahám^1,2, Krisztina Nagy¹, Eszter Csákvári^1#, László Dér¹, Imre Pap^1,2, Rebeka Lukács¹, Vanda Varga-Zsíros^1##, Péter Galajda¹

¹Institute of Biophysics, Biological Research Centre, Szeged, Hungary
²Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Szeged, Hungary
Current affiliations:
^#Division for Biotechnology, Bay Zoltán Nonprofit Ltd. for Applied Research, Szeged, Hungary 
^##Institute of Biochemistry, Biological Research Centre, Szeged, Hungary

Social interactions are common and essential in natural microbial ecosystems. Among these, quorum sensing is one of the most important forms of bacterial communication. Quorum sensing is used to regulate (and synchronize) gene expression of a population according to cell density. It involves the production and detection of small excreted signal molecules (autoinducers), and controls multiple functions, e.g. bioluminescence, metabolic pathways, motility, biofilm formation, sporulation and virulence.

We applied a microfluidic “mother machine” device to trap single cells of Pseudomonas aeruginosa bacteria and expose them to waves of autoinducer signal molecules. We studied the quorum sensing response on single cell and population level by means of a GFP-based fluorescence reporter system. We described the kinetics of the response and explored cell to cell variations. Furthermore we tracked cell size, division and cell relatedness and explored their importance in quorum sensing. We applied a quantitative model based on the molecular mechanisms behind quorum sensing to explain the experimental data.

L25

Ruthenium dendrimers – a potential drug carriers for cancer therapy

Zuzana Garaiová¹, Sylwia Michlewska², Veronika Šubjaková¹,  Maksim Ionov², Iveta Waczuliková¹, Francisco Javier de la Mata ^3,4,5, Maria Bryszewska², Joseph Wang⁶ ,Tibor Hianik¹

¹Comenius University, Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics,Bratislava, Slovakia
²University of Lodz, Department of General Biophysics and Laboratory of Microscopic Imaging & Specialized Biological Techniques, Faculty of Biology and Environmental Protection, Lodz, Poland
³University of Alcalá, Department of Organic and Inorganic Chemistry, and Research Institute in Chemistry “Andrés M. del Río” (IQAR), Madrid, Spain
⁴Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),Spain
⁵Institute “Ramón y Cajal” for Health Research (IRYCIS), Spain
⁶University of California San Diego, Department of Nanoengineering, La Jolla, California, 92093, United States

Dendrimers represent a group of synthetic polymer nanoparticles that gain an interest as potential drug carriers. These radially branched molecules reminding tree-like structures possess terminal functional groups suitable for drug conjugation as well as internal cavities which can harbor guest molecules [1]. Dendrimers that contain metal atoms such as ruthenium have been synthetized and investigated for complexation with conventional anticancer drugs [2], anticancer small interfering RNA [3] followed by the examination of their interactions with various cell lines. It has been shown that ruthenium functionalities can enhance the cytotoxicity to cancer cells.

This contribution is focused on biophysical characterization of a new class of fluorescently labeled metallodendrimers based on ruthenium possessing anticancer activity (FITC-CRD₁₃). These dendrimers have been combined with graphene oxide modified gold nanowires and investigated for ultrasound propelled delivery towards breast cancer cells using fluorescence microscopy [4]. In addition, encapsulation of FITC-CRD₁₃ into liposomal vesicles will be also discussed.

In summary the dendritic nanoparticles and the presence of ruthenium in their structure is promising tool for a design of new drug delivery systems with improved antitumor potential.

Acknowledgments

This work has been financially supported by Science Grant Agency VEGA, project No. 1/0756/20; by Agency for Promotion Research and Development, project No. SK-PL-21-0073 and SK-BY-RD-19-0019; by KEGA, project No. 041UK-4/2020 and by NAWA International Academic Partnership Programme EUROPARTNER.

References

1. Aurelia Chis, A., et al, Molecules 2020, 25(17):3982
2. Michlewska, S. et al., Dalton Trans., 2021, 50: 9500-9511
3. Michlewska, S. et al., Journal of Inorganic Biochemistry 2018, 181: 18-27
4. Garaiova, Z., et al. Clinical Oncology and research 2019, 2(4): 2-5

L26

LSM 980 with Airyscan 2 - Shaping high-end imaging for your research

Christine Strasser

Carl ZEISS AG., CH

To analyze life with as little disturbance as possible, you must use low labeling density for your biological models. This requires excellent imaging performance combined with low phototoxicity and high speed. LSM 980, your platform for confocal 4D imaging, is optimized for simultaneous spectral detection of multiple weak labels with the highest light efficiency.

L27

Biophysical virology of SARS-COV-2 and its variants

Miklós S. Z. Kellermayer¹, Bálint Kiss¹, Dominik Sziklai¹, Dorottya Mudra¹, Levente Herényi¹, Bernadett Pályi², Zoltán Kis^2,3

¹Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó str. 37-47., Budapest, H-1094 Hungary
²National Biosafety Laboratory, National Public Health Center, Albert Flórián Rd 2-6., Budapest, H-1097 Hungary
³Department of Medical Microbiology, Semmelweis University, Nagyvárad Sq. 4., H-1089 Hungary
Email: kellermayer.miklos@med.semmelweis-univ.hu

The development of advanced experimental methodologies, such as optical tweezers, scanning-probe and super-resolved optical microscopies, has led to the evolution of single-molecule biophysics, a field of science that allows direct access to the mechanistic detail of biomolecular structure and function. The extension of single-molecule methods to the investigation of viruses permits unprecedented insights into their properties and behavior. Here we investigated the nanoscale biophysical properties of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. This enveloped ssRNA virus displays a corona-shaped layer of spikes which play fundamental role in the infection process. By imaging and mechanically manipulating individual, native SARS-CoV-2 virions with atomic force microscopy, we show that their surface displays a dynamic brush owing to the flexibility and rapid motion of the spikes. The virions are highly compliant and able to recover from drastic mechanical perturbations. Their global structure is remarkably temperature resistant, but the virion surface becomes progressively denuded of spikes upon thermal exposure. The dynamics and the mechanics of SARS-CoV-2 are likely to affect its stability and interactions. Variants of the virus possess increased infectivity, but the exact mechanisms behind this phenomenon are not fully understood. We imaged and mechanically manipulated individual, wild-type, alpha- and delta-variant SARS-CoV-2 virions. The variants appear to be significantly smaller in their radii than the wild type virus particles. Considering that the surface and volume of the spherical virions scale with the second and third power of the radius, respectively, our results reveal an increased specific surface (surface/volume ratio) in the variant virus particles. Thus, while the infectivity of SARS-CoV-2 relies on the dynamics and the mechanics of the virus, it may be influenced by the specific surface as well.

L28

Immunometabolism of Multi-System Inflammatory Syndrome in Children (MIS-C) Related to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Mojca Pavlin^1,2, Repas Jernej¹, Janžič Larisa³, Maša Bizjak⁴,Avčin Tadej^4,5, Kopitar Andreja Nataša³

¹University of Ljubljana, Faculty of Medicine, Institute of Biophysics, Ljubljana, Slovenia, mojca.pavlin@mf.uni-lj.si
²University of Ljubljana, Faculty of Electrical Engineering, Group for Nano and Biotechnological applications, Ljubljana, Slovenia
³University of Ljubljana, Faculty of Medicine, Laboratory for Cellular Immunology, Institute of Microbiology and Immunology, Ljubljana, Slovenia
⁴University Medical Center Ljubljana, Children’s Hospital, Department for Allergology, Rheumatology and Clinical Immunology, Ljubljana, Slovenia
⁵University of Ljubljana, Faculty of Medicine, Pediatrics, Ljubljana, Slovenia

Metabolic pathways of the immune cells has been recently recognized to be one of the crucial parameters involved in immune cells functionality. Measurements of cellular bioenergetics in live cells can aid to understanding of the biochemical alterations in immune cells as a response to specific pathological condition. Several studies showed alterations in the mitochondrial respiration and glycolysis of immune cells in the acute condition of multisystem inflammatory syndrome in children (MIS-C). MIS-C is known to occur only in a small fraction of children after SARS-CoV-2 infection, however the mechanisms of this syndrome are still not fully understood. We have performed detailed bioenergetics analysis of peripheral blood mononuclear cells (PBMC) obtained from MIS-C patients in the post-acute phase to observe potential long-term immunometabolic alterations in immune cells. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in PBMCs, abCD3/CD28 activated PBMC and in separated monocytes and lymphocytes. Results were compared to the group of healthy children. In parallel detail immunephenotyping by flow cytometry was performed.

While both immunephenotyping and immunometabolic analyses showed that in most parameters the immune cells in post-acute MIS-C return to the normal state, there were some significant alterations, in terms of more metabolically active abCD3/CD28 stimulated T lymphocytes. Among other alterations, the percentages of activated T cells were significantly increased, followed by an increased proportion of double negative T cells (DNTs) in post-acute MIS-C. The antigen-presenting cells; monocytes, dendritic cells (DC) play an important role in MIS-C pathology. We have observed significantly reduced levels of plasmacytoid DC and classic monocytes .This demonstrates that some alterations in the immune system still exists in the post-acute MIS-C phase two to three months after acute MIS-C condition.

L29

Viral RNA as a randomly branched polymer

Domen Vaupotič,¹ Angelo Rosa,² Luca Tubiana,³ Anže Božič¹

¹Department of Theoretical Physics, Jožef Stefan Institute, Ljubljana, Slovenia
²Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy
³Department of Physics, University of Trento, Trento, Italy

RNA viruses pack functionality into small genomes, and often use different properties of RNA to carry out distinct parts of their lifecycle. Recent experimental and theoretical insights have shown that viral RNAs are tasked with a variety of functions on which optimal viral assembly depends, and many of them are tied to the secondary and tertiary structure of RNA. For instance, It has recently been observed that the genomes of positive-sense, single-stranded RNA (+ssRNA) icosahedral (spherical) viruses tend to be overall more physically compact than random RNA sequences of comparable length and composition. Despite this, even a small percent of synonymous mutations suffices to destroy this compactness. These results strongly support the idea that viral genomes code, at least in part, for their physical properties. We address the question of which properties of the sequence and structure of the genomes of +ssRNA viruses make them unusually compact and what is the relationship between them. To do so, we draw on the concepts from the theory of branched polymers, which allows us to obtain polymer scaling exponents from individual RNA secondary structures by mapping them onto graphs.  We compare the results obtained for the genomes of +ssRNA viruses to random RNA sequences of different nucleotide and dinucleotide compositions. Our study aims to elucidate the branching properties of random and viral RNAs compared to other branched polymers and the properties of their sequence which give rise to them. Apart from understanding the principles by which RNAs can encode for their physical characteristics such as size, these properties can then be selectively targeted to disrupt the packaging efficiency during self-assembly of virions and thus interfere with the virus’ life-cycle.

L30

Adsorption of SARS-CoV-2 spike protein onto the surface of materials

Mehdi Sahihi, Jordi Faraudo

Institut de Ciencia de Materials de Barcelona (ICMAB-CSIC), Campus de la UAB, E-08193 Bellaterra, Barcelona, Spain

The interaction of virion with inanimate surfaces could be the main source of indirect infection. Here, we used MD simulation method to investigate the interaction of the SARS-CoV-2 spike protein with surface of polymers, metals and carbon-based materials. Indeed, the main motivations for this work are: i) to clarify the molecular and atomic details of the interaction between SARS-CoV-2 virus and different materials; ii) to reveal the difference between up and down conformations of the spike protein for interaction with surfaces and iii) to classify the available results for interaction of SARS-CoV-2 spike protein with different materials in a reasonable manner.

The results show that spike protein adsorbed onto the surfaces of investigated materials with following mechanism: the protein adjusted its spatial conformation in a couple of time steps, then started to interact with the surfaces rapidly, and finally achieved the equilibrium state with readjusted conformation. However, the final conformations and interaction energy of the protein on the surface of materials are completely different. We can imagine four different types of materials based on their interaction with SARS-CoV-2 spike protein: i) materials that have no special effect on infective viral particles e.g., polymeric materials (RMSD and No. contacts are below 10 Å and 100, respectively); ii) materials that may inactivate SARS-CoV-2 virus but are less likely to accumulate infective viral particles e.g., metals and carbon-based materials (RMSD above 10 Å and No. contacts below 100); iii) materials with ability to capture and accumulate the infective viral particles but cannot to inactivate them and are able to inhibit transmission e.g., skin models (RMSD below 10 Å and No. contacts above 100) and iv) materials that not only have high affinity for capturing the SARS-CoV-2 virus but also, may denature the spike protein and inactivate the virus (RMSD and No. contacts above 10 Å and 100, respectively).

L31

A possible role of transferrin in severe COVID-19-associated diseases

Elek Telek^1,†, Zoltán Ujfalusi^1,†, Gábor Kemenesi^2,3,4, Brigitta Zana^2,3,4, Ferenc Jakab^2,3,4, Gabriella Hild⁵, András Lukács¹, Gábor Hild ¹

¹Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary
²Szentágothai Research Centre, Virological Research Group, University of Pécs, Pécs, Hungary
³Faculty of Sciences, Institute of Biology, University of Pécs, Pécs, Hungary
⁴National Laboratory of Virology, University of Pécs, Pécs, Hungary
⁵Languages for Biomedical Purposes and Communication, Medical School, University of Pécs, Pécs, Hungary
⁶Department of Medical Imaging, Clinical Centre, University of Pécs, Pécs, Hungary
^† The authors contributed equally to this work.

We studied the effect of SARS-CoV-2 on human whole blood by differential scanning calorimetry. The analysis of the thermal transition curves showed that the melting temperature of the transferrin-related peak decreased in the presence of SARS-CoV-2. The ratio of the under-curve area of the two main peaks was greatly affected, while the total enthalpy of the heat denaturation remained nearly unchanged in the presence of the virus. These results indicate that SARS-CoV-2, through binding to transferrin, may influence its Fe³⁺ uptake by inducing thermodynamic changes. Therefore, transferrin may remain in an iron-free apo-conformational state, which depends on the SARS-CoV-2 concentration.

P01

Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

Ágnes Ábrahám^1,2, Krisztina Nagy¹, László Dér¹, Imre Pap^1,2, Eszter Csákvári^1,3, Lóránd Kelemen¹ and Péter Galajda¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²University of Szeged, Doctoral School of Multidisciplinary Medical Science, Szeged, Hungary
³Bay Zoltán Nonprofit Ltd. for Applied Research, Szeged, Hungary

Bacterial populations are heterogeneous, which can help them to survive in a changing environment. To explore how phenotypic differences appear in genetically identical cells instead of population-based studies we need single-cell approaches.

Using microfluidic techniques, we are able to develop platforms, where we can change the environment in a controlled manner and monitor cell-to-cell differences.

In this work we use two devices. One of them is the Mother Machine, which consists of a main channel and an array of side channels. Through the main channel we constantly pump nutrient rich medium and in the side channels we can trap cells and follow their relatedness until the flow washes out the outer cells from the narrow channels. One interesting property of this system is that we can define mother cells, which are the cells deepest in the dead-end growth channels. The aging old pole makes them special compared to other cells and we can follow them throughout the whole experiment.

In our work one application of this device is to study quorum sensing on a single cell level. For this purpose we use Pseudomonas aeruginosa mutant, which cannot produce but can detect QS signal molecules and react to them. This strain contains a reporter plasmid, so the fluorescence level of cells gives us information about their quorum state. Through medium flow we add signal molecules in a cyclic manner and observe single cells and the phenotypic heterogeneity in their quorum sensing.

In our lab we develop a new device, the so-called Baby Machine, where we combine microfluidics with optical tweezers. The main part of this system is an array of single cell traps. In this device after the division of a trapped cell one daughter cell remains in the trap while the other drops out and falls into the next empty trap. After several divisions all the traps are filled with the progeny of a single cell. With this device we could collect and study hundreds of cell generations.

P03

Calcium-dependent internal dynamics of calmodulin

Klaudia Onica^1,2, Gusztáv Schay¹, Franci Merzel³, Károly Liliom¹, Erika Balog¹

¹Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
²Pázmány Péter Catholic University, FITB, Budapest, Hungary
³National Institute of Chemistry, Laboratory for Molecular Modelling, Ljubljana, Slovenia

Calmodulin (CaM) plays a crucial role in calcium signalling-driven intracellular processes, controlling the function of more than a hundred different enzymes and proteins, such as phosphodiesterase, myosin light chain kinase, and CaM-dependent kinases and takes part in processes such as smooth muscle contraction, metabolism, and memory formation. How CaM differentiates among the possible target molecules still remains unclear.

From the structural point of view CaM is a two-domain enzyme containing four calcium-binding sites, the two domains being connected by a linker region.

Using molecular dynamics (MD) here we present how calcium saturation alters the internal dynamics of calmodulin, something with the potential to influence its target-molecule recognition.

P05

Different phosphorylation states of native titin filaments visualized by atomic force microscopy

Andrea Balogh-Molnár, Dominik Sziklai, Zsolt Mártonfalvi

Department of Biophysics and Radiation biology, Semmelweis University, Budapest, Hungary

During muscle stretch, elastic or passive force develops which is mainly determined by the giant protein titin that forms the third filament system of muscle sarcomeres. The magnitude of this force is mainly dependent on the elasticity of the titin filaments, which is primarily determined by the structure of the polypeptide chain. However, it is suggested that post-translational modifications of titin, such as phosphokinase activity, regulate the sarcomeric passive force development. Mechanical studies on single myofibrils revealed that different protein kinases alter the passive tension of muscle, surprisingly, in opposite ways. This suggests that phosphorylation of sarcomeric proteins by various kinases is an essential regulatory mechanism of passive force. However, the extrapolation of these findings to titin’s phosphorylation has not been studied so far at the single-molecule level. To reveal titin’s contribution to the alterations of passive force due to phosphorylation, single-molecule experiments must be carried out on individual titin molecules, where the effect of each relevant phosphorylation state can be tested individually. In our work, we isolated individual titin molecules from rabbit skeletal muscle. The isolated native titin molecules showed high levels of phosphorylation, when stained with phosphoprotein gel stain. The isolated molecules were treated with lambda protein phosphatase to alter the in situ phosphorylation state of the polymer. To investigate the effect of the various phosphorylation states on titins structure, we visualized surface-bound titin molecules by atomic force microscope and found that the C-terminal region of dephosphorylated titins collapses into a compact, coiled structure.

P07

Energy Transfer from Phycobilisomes to Photosystem I in a Model System

Avratanu Biswas, Zita Szegletes, Petar H. Lambrev,Parveen Akhtar

Institute of Plant Biology, Biological Research Centre, Szeged, Hungary

Cyanobacteria are ubiquitously found oxygenic photosynthetic organisms using phycobilins as primary light-harvesting pigments. The phycobilins are covalently bound to the phycobiliproteins phycocyanin (PC) and allophycocyanin (APC), which together with colorless linker proteins are assembled into phycobilisomes (PBS) - giant water-soluble light-harvesting complexes that effectively absorb light in the green-orange wavelength region. The PBS are connected to the thylakoid membranes containing the two photosystems, PSI and PSII, enabling efficient excitation energy transfer (EET), thereby extending the absorption capacity of chlorophyll in the photosystems. The EET routes and dynamics from the PBS to PSI and PSII are still a matter of debate, especially regarding the connectivity of PBS and PSI. In this work, we aimed to test whether PBS can directly transfer energy to PSI in an in vitro model system. We present an experimental evidence for the functional connectivity of isolated PBPs and PBS to surface-immobilized PSI. Steady-state and time-resolved fluorescence spectroscopy showed efficient quenching of the fluorescence of isolated PC and intact PBS by PSI. We could estimate that up to 80% of the antenna excitations are transferred to chlorophyll suggesting that the externally added antenna can effectively supplement the absorption cross-section of PSI. On one hand, these results experimentally verify the notion that PBS function as an antenna feeding excitations directly to both photosystems in cyanobacteria. On the other hand, they demonstrate the possibility of using PBPs for extending the absorption cross-section of chlorophyll in biohybrid solar applications.

P11

Membrane organization and mobility of chimeric antigen receptors bear functional consequences for CAR T cell activation

Marianna Csaplár¹, Árpád Szöőr¹, György Vereb^1,2

¹Faculty of Medicine, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary
²MTA-DE Cell Biology and Signalling Research Group, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Chimeric antigen receptor (CAR) modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disappointing, indicating the necessity of their molecular level optimization. While incorporation of CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved long-term efficacy of T cell products, their influence on early tumor engagement has not yet been elucidated.

We studied the antigen independent self-association and membrane diffusion kinetics of 1^st (.z) 2^nd (CD28.z, 41BB.z) and 3^rd (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using semi-superresolution AiryScan microscopy and Fluorescence Correlation Spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CAR dimers formed large, high-density submicron clusters, 41BB containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. 1^st, 2^nd and 3^rd generation CARs exhibited gradually increasing lateral diffusion as separation of their CD3z domain from the plane of the membrane increased. Assessed by confocal microscopy, both highly mobile small clusters of CD28.41BB.z and preassembled large clusters of .z CARs showed more effective immediate CD3z phosphorylation and pLck recruitment to the immunological synapse than CD28.z or 41BB.z with interim mobility. However, Electric Cell-substrate Impedance Sensing revealed that the CD28.41BB.z CAR performs worst in eliminating adherent tumor cells while the .z CAR is superior to all others. Thus, molecular structure, membrane organization and mobility appear as important design parameters predicting efficient immune synapse formation and target elimination. However, expansion capacity of CAR T cells, which necessitates costimulation, also needs to be considered.

P13

In silico methods from structural analysis to virtual screening: SULT1 Isoenzymes substrate binding and selectivity

Dániel Tóth^1,2, Bálint Dudas^2,3, David Perahia³, Maria A. Miteva², Erika Balog¹

¹Department of Biophysics and Radiation Biology, Semmelweis University, Hungary
²Inserm U1268 MCTR, CiTCoM UMR 8038 CNRS - Université Paris Cité, France
³Laboratoire de biologie et pharmacologie appliquee, Ecole Normale Superieure Paris-Saclay, France

In this Franco-Hungarian partnership, we utilise multiple in silico methods to better understand the selectivity of Sulfotransferase enzymes (SULTs), a family of cytosolic globular proteins in the chain of metabolism. By catalysing a sulfate transfer from their co-factor, 3′-Phosphoadenosine 5′-Phosphosulfate (PAPS) they eliminate a large variety of small molecules like drugs, hormones and neurotransmitters. Even though the tertiary structure across the family is very similar, the substrates they recognize vary considerably in size and composition. Moreover, these enzymes can be found in the body from the neuroglia to the hepatic cells with different purpose and target molecules. This selectivity, can be very well modelled with the methods used by our laboratories.

In the Biophysics Institute at Semmelweis University we employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited Normal Modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. This allowed exploring an extended conformational space of PAPS-bound protein, which has not been achieved up to now. In the Faculty of Pharmacy, Université Paris Cité, we used these structures clustered into ensembles and combined them with categorised ligands, performing Virtual Screening. Based on these results, we broadened our research to use the same approach for the SULT1A3 with different substrate pools, which contained specific and non-specific substrates and inhibitors for each of the enzymes.

These results show the selectivity is likely to be governed by certain amino acid sidechains in 1A3 by opening the binding pocket to an unfavourable conformation for the most common ligands of 1A1, thus acting as efficient selectors. These results can be used in the future to develop an algorithm for machine learning, that can differentiate and even recognize new substrates, thus helping in the development of ADME-Tox profiling of novel drug candidates.

P15

Optimized exosome isolation protocol based on ultrafiltration and size exclusion chromatography

D. Džubinská¹, J. Frýdlová², P. Přikryl², M. Zvarík¹, I. Waczulíková¹

¹Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics of the Comenius University, Mlynská dolina F1, 842 48 Bratislava, Slovakia
²Institute of Pathological Physiology, First Faculty of Medicine, Charles University, U Nemocnice 5, 128 53 Prague 2, Czech Republic

Exosomes, a type of extracellular vesicles, are nano-sized membranous vesicles secreted by every type of cell and play an important role in the intracellular communication [1]. They were shown to carry a cell-specific cargo of proteins, lipids, and genetic materials, and represent potential use of that cargo as non-invasive diagnostic and prognostic biomarkers [2]. All exosome research is limited by the use of disparate isolation methods, in addition, every isolation method provides different exosomes yield that may be contaminated with protein aggregates or different type of vesicles [1].

In our work we focused on optimization of the protocol to isolate extracellular vesicles from urine. We have developed a modified protocol for exosomes isolation, based on coupling of size exclusion chromatography and ultrafiltration. Presence of isolated exosomes and their purity was verified by Western blot analysis of common exosomes markers. The size exclusion chromatography and ultrafiltration method was compared with the most used isolation technique – ultracentrifugation. Appropriate selection of the isolation method, considering their advantages and disadvantages, is very important for further analysis and use of extracellular vesicles. Exosomes isolation and characterization is crucial for discovery of cancer biomarkers and for developing an efficient diagnostic tool in the future.

Key words: exosomes, size exclusion chromatography, ultrafiltration, ultracentrifugation

Acknowledgements

This work was supported by projects KEGA, project 041UK-4/2020, by the Cooperation Program, research area of Medical Diagnostics and Basic Medical Sciences of the Ministry of Education, Youth and Sport of the Czech Republic, UK/117/2022, NAWA, project EUROPARTNER PPI/APM/2018/1/00007/U/001, APVV, grants SK-BY-RD-19-0019 and SK-PL-21-0073.

References

1. Sidhom, K., Obi, P. O., & Saleem, A. (2020). A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?. International journal of molecular sciences, 21(18), 6466. https://doi.org/10.3390/ijms21186466
2. Zhang, Y., Liu, Y., Liu, H. et al. Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci 9, 19 (2019). https://doi.org/10.1186/s13578-019-0282-2

P17

Label-free optical biosensor method for detailed analysis of bacteria repellent and adhesive surfaces

Eniko Farkas¹, Robert Tarr^1,2, Tamás Gerecsei^1,3, Andras Saftics¹, Kinga Dóra Kovács^1,3, Balazs Stercz⁴, Judit Domokos⁴, Beatrix Peter¹, Sandor Kurunczi¹, Inna Szekacs¹, Attila Bonyár², Anita Bányai⁵, Péter Fürjes⁵, Szilvia Ruszkai-Szaniszló⁶, Máté Varga⁶, Barnabás Szabó⁶, Eszter Ostorházi⁴, Dóra Szabó⁴, Robert Horvath¹

¹Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Budapest, Hungary
²Department of Electronics Technology, Faculty of Electrical Engineering and Informatics, Budapest University of Technology and Economics, Budapest, Hungary
³Department of Biological Physics, Eötvös Loránd University, Budapest, Hungary
⁴Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
⁵Microsystems Lab, Institute of Technical Physics and Materials Science, Centre for Energy Research, Budapest, Hungary
⁶77 Elektronika Ltd., Budapest, Hungary

In the field of biosensors and design of biomedical devices it is getting more important to develop and characterize bacterial repellent surfaces and bacterial adhesive coatings [1, 3]. However, the conventional approaches are lacking of in-depth analysis and comparison of various solutions. In response to this problem, surface analysis by applying label-free optical waveguide lightmode spectroscopy (OWLS) instrument is well suitable. This biosensor is able to detect rapidly and efficiently the optical properties of the surface with 100–150 nm depth sensitivity [2-3].

In the present work, the OWLS method is presented with in-depth characterization of bacteria repellent and bacterial adhesive surfaces. We investigated five common blocking agents to block E. coli adhesion; bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures). As a result, the PAcrAM-P provided the best blocking capability with the bacteria concentration up to 10⁷ cell/mL. Thereafter, this blocking agent was employed to E. coli specific antibodies, which were chosen by enzyme-linked immunosorbent assay (ELISA) and then applied in the OWLS analysis as well. Furthermore, we tested various immobilization methods to bind these specific antibodies. We created Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries and tried simple physisorption too. The parameters of the used agents were determined by considering the kinetic data of adhesion, the surface mass density and the protein orientation revealed by the OWLS analysis. Using this method and analysis, we found the best solution to specific bacteria binding with Pacram blocked polycolonal antibody, immobilized with protein A. As a conclusion, we found that the surface sensitivity of the best performing antibody and blocking agent is reached 70 cells/mm². [3]

Acknowledgments

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (ERC_HU, VEKOP 2.2.1-16, ELKH topic-fund, Élvonal KKP_19 and KH grants, PD 131543 and TKP2022-EGA-04 –INBIOM TKP Programs financed from the NRDI Fund). This work was also supported by 77 Elektronika Ltd. by their supplying of antibodies and reagents.

References

1. Péter, B., Farkas, E., et. al. Biosensors 2022, 12, 188.
2. Saftics, A., et. al. Adv. Colloid Interface Sci.2021, 294, 102431–102433.
3. Farkas, E., et. al. Biosensors 2022, 12, 56.

P19

BRAF modulates cell-cell adhesion through PECAM-dependent mechanotransduction

Éva Gráczer, Katalin Pászty, Csilla Lehoczky, Andrea Varga

Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary

Endothelial cells are continuously exposed to external forces. In addition, during inflammation or metastasis a leukocyte or a cancer cell can exert tension on the endothelial monolayer. Change in tension is sensed by cell adhesion molecules, such as PECAM-1, and is translated to specific cellular responses, such as actin reorganization, in a process called mechanotransduction. We have shown that endothelial cell-specific ablation of BRAF, a kinase frequently activated in cancer, inhibits metastatic spread. BRAF modulates cytoskeletal reorganization, which impinges on cell-cell adhesion to allow cancer cell migration through the endothelial monolayer. The question arises whether BRAF, through regulation of the actin cytoskeleton, can influence PECAM-dependent mechanotransduction. To test this hypothesis, we used PECAM-1-antibody-coated magnetic beads together with a permanent magnet to apply a continuous force (about 40 pN) on the endothelial monolayer and followed reorganization of the actin cytoskeleton as well as remodelling of cell-cell junctions. PECAM-1-mediated adhesion alone led to an increased peripheral actin ring formation, which was followed by stress fiber formation upon application of force. These fibers were spanning through the whole cell and were marked at their ends with vinculin, a cellular marker of remodelling adherens junctions. Endothelial BRAF depletion did not affect the adhesion step, but accelerated force-dependent stress fiber formation. The BRAF inhibitor PLX8394, which was shown to reduce BRAF-RAF1 heterodimer formation, could phenocopy the effect of BRAF ablation. The role of BRAF in PECAM-dependent mechanotransduction was independent on its kinase activity, since the MEK-specific inhibitor U0126 did not accelerate force-dependent stress fiber formation. These results suggest that inhibition of BRAF-RAF1 heterodimerization protects endothelial cell-cell junctions from opening, through PECAM-mediated force-dependent junctional remodelling.

P21

In situ structure and nanomechanics of pulmonary fibrosis collagen fibrils

Dóra Haluszka¹, Tamás Nagy², Eszter Regős³, Judit Pápay³, Veronika Müller², Miklós Kellermayer¹

¹Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
²Semmelweis University, Department of Pulmonology Budapest, Hungary
³Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest, Hungary

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease, dominated by the accumulation of fibroblasts and extracellular matrix proteins, including collagen, leading to irreversible loss of lung function. IPF ethology is unknown, however various factors such as toxins, smoking or radiation exposure might be associated with its development. Understanding structural changes of the fibrotic lungs needs characterization of collagen with innovative techniques. Multiphoton microscopy, by way of its second harmonic generation (SHG), allows label-free imaging of collagen fibrils, and atomic force microscopy (AFM) can reveal their high-resolution topographical features and mechanical properties.

Although normal type I collagen has been relatively well characterized, its topographical and mechanical properties in IPF are little known. In our work we performed a detailed analysis of collagen obtained from paraffin embedded histology samples of normal and IPF lung tissue to reveal their structural and mechanical properties.

Sections exhibited high SHG and autofluorescence (AF) signal intensity, which indicated the accumulation of collagen and elastin fibres. By using Fast Fourier Transformation (FFT), collagen fibril orientation index (COI) was calculated, which indicated the random arrangement of collagen bundles in the fibrotic tissue. In AFM experiments the detailed topographical structure of fibrotic collagens was identified, then force maps were recorded with nanoindentation method. Collagen fibrils of IPF sections displayed a broad distribution of Young’s moduli, pointing at an increased stiffness compared with controls.

In summary, IPF leads to significant changes in the properties of the accumulated collagen fibrils which is manifested in a shift in their optical behaviour and nanomechanical properties. Conceivably, the random fibril arrangement and increased stiffness play important role in the emergence of clinical symptomatology.

P23

Structure and nanomechanics of electrospun nanofibers

Imre Hegedüs, Rita Pázmány, Voniatis Constantinos, Domokos Máthé, Miklós Kellermayer, Angéla Jedlovszky-Hajdú

Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

Nanofibers are nanoscale fibrous structures composed of synthetic or natural polymers that carry the prospect of wide-spread use in biomaterial development. The use of electrospinning has opened novel means of efficient nanofiber preparation with tunable qualities. Nanofibers are prepared by ejecting a viscous polymer solution, from a syringe into a high-voltage electric field emerging between a needle (attached to the syringe), towards a grounded target. As the polymer solution flies towards the target, as a whipping jet, the solvent evaporates, then a solidified meshwork is formed on the target surface.

Here the nanoscale structural and mechanical properties of individual nanofibers, such as height (vertical diameter), surface roughness, and tangential Young modulus (Y_t), which is perpendicular to the axis, were investigated with atomic force microscopy (AFM). We compared the properties of four different polymeric systems: polyvinyl alcohol (PVA), polycaprolactone (PCL), polysuccinimide (PSI), and polycaprolactone/polysuccinimide hybrid (PSI/PCL).

The height (i.e., diameter) of the fibers varied between 400-800 nm. The surface of PVA and PCL fibers was less rough (root mean square or rms about 300 nm, ISO scale N4) than that of PCL or PSI/PCL fibers (rms 350-500 nm, ISO N5). Based on Y_t values, PVA (0.5-1.5 GPa) and PSI (0.5-3.5 GPa) were more rigid than PCL (0.1-0.5 GPa). Y_t of the hybrid nanofibers varied between 0.1-3.5 GPa, which suggests that it composed of its components in random spatial distribution. The physical parameters (diameter, surface roughness, elasticity) of nanofibers depend strongly on the quality of its material. The parameters also have site-dependent distribution on the submicron scale depending on the local composition, e.g. Y_t values of PSI/PCL. The single-fiber analysis employed in this work provides a unique glimpse into the physical properties of nanofibers, allowing us to fine-tune the macroscopic qualities of the prepared polymer mesh.

Acknowledgements

This research was supported by NKFIH FK 137749, TKP2021-EGA-23 and EFOP-3.6.3-VEKOP-16-2017-00009. This work was also funded by grants from the Hungarian National Research, Development and Innovation Office (National Heart Program NVKP-16-1-2016-0017; Thematic Excellence Programme of Semmelweis University in the BIOImaging Excellence thematic priority). HCEMM, a Teaming grant associated to the European Molecular Biology Laboratories, has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 739593.

P25

Time-resolved singlet oxygen phosphorescence detection in live cell suspensions

Andrej Hovan¹, Viktória Pevná¹, Veronika Huntošová², Pavol Miskovsky², Gregor Bánó¹

¹P. J. Šafárik University, Department of Biophysics, Košice, Slovak Republic
²P. J. Šafárik University, Center for Interdisciplinary Biosciences, Košice, Slovak Republic

Singlet oxygen and its cytotoxic effect play a key role in photodynamic therapy of cancer. The production of singlet oxygen is done by energy transfer between photo-activated drug molecules (photosensitizers) and molecular oxygen. After absorption of the light, the photosensitizer is excited to the singlet state S1, which populates the triplet state T1 through inter-system crossing. The triplet state photosensitizer reacts with an oxygen molecule, which is transferred to the highly reactive singlet oxygen state. The range of cytotoxic activity of singlet oxygen is determined by its lifetime and diffusion rate. Previous research in this area has not clarified the value of singlet oxygen lifetime in cells. There is currently no consensus in the scientific community on the proper method of measuring and evaluating the lifetime of singlet oxygen in cells. The results of various scientific groups range from several tens or hundreds of nanoseconds [1] up to few micro-seconds [2]. The main objective of our work was to determine the lifetime of singlet oxygen in cell suspensions and to contribute to this key issue of photodynamic therapy.

The decay of singlet oxygen phosphorescence intensity was measured following nanosecond pulsed excitation of Hypericin, the natural photosensitizer, embedded into live cells of the SKBR3 and U-87 MG cell lines. The excitation wavelength was set to the Hypericin absorption maximum near 600 nm. The lifetime of Hypericin triplet state plays an important role in understanding the kinetics of singlet oxygen phosphorescence. Due to this fact, the Hypericin triplet state was monitored by transient absorption measurements using a 532 nm laser.

The singlet oxygen phosphorescence kinetics were analysed to determine the singlet oxygen lifetime. Our preliminary results indicate that the effective lifetime of singlet in the studied cells is in between the minimal and maximal values presented by other authors [1,2].

Acknowledgements

This work was supported by Slovak Research and Development Agency through the project APVV-20-0340 and by the grant agency of the Ministry of Education, Science, Research, and Sport of the Slovak Republic (grants no. VEGA 1/0557/20 and no. VEGA 1/0074/22), OPENMED (Open Scientific Community for Modern Interdisciplinary Research in Medicine) ITMS2014+: 313011V455 from the Operational Program Integrated Infrastructure funded by the ERDF, and EU H2020 TWINNING program GA. No. 952333 project CasProt.

References

1. M. Niedre, M. S. Patterson, and B. C. Wilson, Photochemistry and Photobiology, 75 (2002), 382-391.
2. M. K. Kuimova, G. Yahioglu, and P. R. Ogilby, Journal of the American Chemical Society, 131 (2009), 332-340.

P27

Comparative analyses of the gelsolin homology domains of Gelsolin and Flightless-I

Tamás Huber^1,3, Péter Gaszler^1,3, Péter Bukovics¹, Réka Pintér¹, Rauan Sakenov¹, Andrea Teréz Vig¹, Mónika Ágnes Tóth¹, Veronika Takács-Kollár¹, Venukumar Vemula², Marko Ušaj², Alf Månsson², Beáta Bugyi^1,3

¹University of Pécs, Medical School, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary
²Linnaeus University, Department of Chemistry and Biomedical Sciences, SE-39182, Kalmar, Sweden
³Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee

Flightless-I is a unique member of the gelsolin (GSN) superfamily alloying six gelsolin homology (GH) domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton. However, its biochemical activities in actin dynamics regulation are still largely elusive. To better understand its biological functioning, we performed a comparative analysis of GSN and Flightless-I by in vitro fluorescence spectroscopy and single filament TIRF microscopy approaches. We found that Flightless-I inhibits actin assembly by high-affinity (∼ nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼ µM) monomer binding and does not sever actin filaments in vitro. Flightless-I was found to interact with actin and affect actin dynamics in a calcium-independent fashion. Notably, our functional analyses indicate that GSN and Flightless-I respond to calcium differently implying different conformational characteristics of the GH domains in the two proteins. Bioinformatics analyses predict that the sequence elements responsible for calcium activation of GSN are not conserved in the GH domains of Flightless-I. Consistently, the use of intrinsic and extrinsic fluorescent probes revealed that unlike that of GSN the conformational behavior of the GH domains Flightless-I was not significantly affected by calcium-binding. Altogether, our work reveals different calcium-response and predicts distinct modes of activation of GSN and Flightless-I.

New National Excellence Program of the Ministry for Innovation and Technology ÚNKP-21-3-II-PTE-997 (PG), University of Pécs, Medical School, KA-2021-30 (AV). We thank József Mihály (Institute of Genetics, Biological Research Centre) for the Flightless-I plasmids and Robert C. Robinson (Okoyama University) for the GSN plasmid.

P29

Structural in-depth analysis of iron complexes of plant gall polyphenols by optical spectroscopic techniques and DFT calculations

Alba Espina¹, S. Sanchez-Cortes^2,3, M. V. Cañamares², Z. Jurašeková¹

¹P. J. Šafárik University, Faculty of Science, Department of Biophysics, Košice, Slovakia
²CSIC, Institute of the Structure of Matter, Madrid, Spain
³P. J. Šafárik University, Technology and Innovation Park, Center for Interdisciplinary Biosciences, Košice, Slovakia

Phenolic compounds are the most abundant secondary metabolites in plants demonstrating many beneficiary properties and activities. Special attention deserves the phenolic compounds existing in plant galls. In particular, oak galls contain a large amount of tannic acid and gallic acid. Generally, one of the main chemical properties of polyphenols is the high affinity to link metals leading to the formation of metal complexes. Therefore, these compounds were the bases for the preparation of iron gall inks (IGIs). Although Raman and the SERS spectroscopy were employed in the analysis of the chemical structure of many phenols, less attention was devoted to an eventual structural characterization of IGIs by using the information provided by the Raman technique. The main reasons are the intrinsic complexity of the studied materials and the lack of appropriated and valid assignments of the vibrational bands.

In this work, a structural analysis of polyphenol complexes with iron at several conditions is reported. The investigated polyphenols were tannic acid (TA), gallic acid (GA), pyrogallol (PY), and syringic acid (SA) being components and molecular models of the gallnuts usually employed in the past in the fabrication of IGIs. PY and SA were employed as models to study the interaction of iron with similar structures to the GA one, and, more precisely, to evaluate the importance of the presence of both the carboxylic and the –OH groups in the benzene ring. This work was done by using Raman, FTIR, UV-Vis absorption, and fluorescence spectroscopy under different conditions: pH, aging, and stoichiometry. Besides, DFT calculations were performed for the first time on the gallic acid complex with iron to elucidate the structure of the IGIs, as well as to aid in the normal mode assignment of the IGIs Raman bands.

Acknowledgments

This work was supported by the project OPENMED from the EU structural funds, and by the project CasProt financed from the Horizon 2020 EU program.

P33

Nanoinjection of fluorescent nanoparticles to single live cells by robotic fluidic force microscopy

Tamás Gerecsei^1,*, Tamás Visnovitz^2,3,*, Kinga Dóra Kovács¹, Beatrix Peter¹, Sándor Kurunczi¹, Anna Koncz², Krisztina Németh², Dorina Lenzinger², Krisztina V. Vukman², Péter Lőrincz⁴, Inna Székács¹, Edit I. Buzás^2,5,6,**, Robert Horvath^1,**

¹Nanobiosensorics Laboratory, Centre of Energy Research, ELKH, Budapest, Hungary
²Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
³Department of Plant Physiology and Molecular Plant Biology, ELTE Eötvös Loránd University, Budapest, Hungary
⁴Department of Anatomy, Cell and Developmental Biology, ELTE Eötvös Loránd University, Budapest, Hungary
⁵HCEMM-SU Extracellular Vesicle Research Group, Budapest, Hungary
⁶ELKH-SE Translational Extracellular Vesicle Research Group, Budapest, Hungary
*,** equal contributions
**corresponding authors

Direct injection of fluorescent nanoparticles into the cytoplasm of living cells can provide new insights into the intracellular fate of various different fluorescently labelled biologically active particles. Here we used fluorescent nanoparticles to prove the feasibility of nanoinjection into single live HeLa cells by using robotic fluidic force microscopy (FluidFM). This injection platform offers the advantage of high cell selectivity and efficiency. We confirmed the successful injection of both GFP encoding plasmids and GFP tagged fluorescent nanoparticles to the cells by confocal microscopy. We were able track the nanoparticles in the living cells for 20 hours. The injected nanoparticles were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, the FluidFM platform is suitable for targeting single living cells by fluorescently labelled biologically active particles and may lead to information about the intracellular cargo delivery at a single-cell level.

P37

Measuring the effect of the PHSRN synergy peptide on living cells with label-free optical biosensors

Beatrix Magyaródi¹, Kinga Dóra Kovács^1,2, Attila Bonyár³, Ildikó Szabó^4,5, Szilvia Bősze^4,5, Inna Székács¹, Róbert Horváth¹

¹Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary
²ELTE Eötvös Loránd University, Department of Biological Physics, Budapest, Hungary
³Department of Electronics Technology, Faculty of Electrical Engineering and Informatics, Budapest University of Technology and Economics, Budapest, Hungary
⁴MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary
⁵National Public Health Center, Albert Flórián út 2-6, 1097 Budapest, Hungary

The PHSRN is also known as the synergy peptide, its sequence consists of 5 amino acids (prolyl-histidyl-seryl-arginyl-asparagine) and can be found on the extracellular protein fibronectin. This peptide promotes cell adhesion in the presence of RGD; however, alone it is not enough to induce cellular adhesion. In contrast, pure RGD displaying surfaces trigger cellular adhesion. [1, 2] The PHSRN peptide synergistically enhances cell adhesion when RGD is present in low surface concentrations, so cells adhere weakly to the surface. At high RGD concentrations, the presence of the synergy peptide negatively affects cell adhesion. PHSRN allows cells to adhere to surfaces with low RGD density. [1] Cell division is significantly higher with the use of PHSRN-G6-RGDS on the surface, with nearly twice the cell density, than with only RGDS or with PHRSN-RGDS. The distance between RGDS and PHSRN and their orientation are also important factors for these two motifs in enhancing cell division. [2]

Until now, the effects of PHSRN on cell adhesion has only been studied under a microscope and with the peptides attached to a surface. The primary goal of our work is to study the effect of PHSRN in solutions at various concentrations affecting HeLa cancer cell adhesion on RGD displaying surfaces. During our experiments, we used a label-free optical biosensor, which is excellent for monitoring cell adhesion kinetics. [3] The main question is whether the PHSRN peptide has the same synergistic effect in solution as it has when attached to the surface.

Acknowledgments

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (VEKOP, ELKH topic-fund, Élvonal KKP_19 and KH grants, TKP2022-EGA-04 program financed from the NRDI Fund). IS and SB thanks for the support of the EFOP-1.8.0-VEKOP-17-2017-00001.

References

1. Franziska C. Schenk, Heike Boehm, Joachim P. Spatz, and Seraphine V. Wegner, „Dual-Functionalized Nanostructured Biointerfaces by Click Chemistry”, Langmuir 2014, pp. 6987-6905
2. Mitsuhiro Ebara, Masayuki Yamato, Takao Aoyagi, Akihiko Kikuchi, Kiyotaka Sakai, and Teruo Okano „A Novel Approach to Observing Synergy Effects of PHSRN on Integrin–RGD Binding Using Intelligent Surfaces”, Advanced Material 2008, 20, pp. 3034–3038
3. Norbert Orgovan, Beatrix Peter, Szilvia Bősze, Jeremy J. Ramsden, Bálint Szabó & Robert Horvath „Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor”, Scientific Reports volume 4, Article number: 4034 (2014)

P39

Grating Coupled Interferometry (GCI) for kinetic interaction analysis of small molecules and their target proteins

Barbara Majoros¹, Beatrix Péter¹, Imre Boldizsár^2,3, Szilvia Bősze^4,5, Inna Szekacs¹, Sándor Kurunczi¹, Robert Horvath¹

¹Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute of Technical Physics and Materials Science, Budapest, Hungary
²Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Budapest, Hungary
³Department of Pharmacognosy, Semmelweis University, Budapest, Hungary
⁴ELKH-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, Budapest, Hungary
⁵National Public Health Center, Budapest, Hungary

The development of optical biosensors has accelerated rapidly in recent years, with the aim of creating devices with better sensitivities and overall performances. Characterizing the binding kinetics of small molecules (molecular weight less than 500 Da) to their targets is an important aim accelerated by the development of new drug candidates. During our work, we employed the grating-coupled interferometry (GCI) [1, 2, 3] based WAVEdelta device (Creoptix AG, CH) with a surface sensitivity of 0.01 pg/mm². Distinct advantages of the technology are i) its very high surface sensitivity, allowing the detection of small molecules [4] and even ions as demonstrated recently [5], ii) the 4-channel arrangement making parallel measurements possible and iii) the capability of kinetic measurements in a completely label-free manner. iv) The kinetic parameters can be obtained from a single concentration employing a small amount of substances using a novel repeated analyte pulses of increasing duration (RAPID) methodology. From kinetic analysis, one can determine binding strength and kinetic rate constants for the actual binding event. It is also important to emphasize that the reference channel can be employed to subtract any nonspecific binding signals, often present in surface binding events.

The kinetic interaction between small molecular substances and body proteins is very important in drug research. Drug substances travel through the bloodstream to reach the area where they can express their effects, often transported by human serum albumin (HSA). Therefore, it is important to understand the interactions between HSA and small molecule drugs. The strength of the protein-drug interactions can be characterized by the equilibrium dissociation constant, which is about providing information on the concentration ratio of bound and free drugs.

During our work we demonstrate the excellent capabilities of GCI in this area by presenting the real time binding experiments of some model substances.

Acknowledgments

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (VEKOP, ELKH topic-fund, Élvonal KKP_19 and KH grants, TKP2022-EGA-04 and PD 131543 program financed from the NRDI Fund and OTKA NKFIH K-135712).

References

1. P. Kozma, A. Hámori, K. Cottier, S. Kurunczi and R. Horváth, “Grating coupled interferometry for optical sensing”. Applied Physics B, vol 97, p. 5-8. doi: 10.1007/s00340-009-3719-1., 2009
2. P. Kozma, A. Hámori, S. Kurunczi, K. Cottier and R. Horváth. “Grating coupled optical waveguide interferometer for label-free biosensing”. Sensors and Actuators B: Chemical, 155 (2), p. 466-450., doi: 10.1016/j.snb.2010.12.045., 2011
3. D. Patko, K. Cottier, A. Hamori and R. Horvath, “Single beam grating coupled interferometry: high resolution miniaturized label-free sensor for plate based parallel screening”. Optics Express, Vol. 20, No. 21, p. 23162-23173., doi: 10.1364/OE.20.023162., 2012
4. B. Peter, A. Saftics, B. Kovacs, S. Kurunczi, and R. Horvath, “Oxidization increases the binding of EGCG to serum albumin revealed by kinetic data from label-free optical biosensor with reference channel”. Analyst, Issue 2, pp. 588–595, doi:10.1039/c9an01779h., 2020
5. H. Jankovics, B. Kovacs, A. Saftics et. al. “Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers”. Scientific Reports, 10. 22253. doi: 10.1038/s41598-020-79226-w., 2020

P41

Neuroinductive effects of graphene film on dental stem cells

Jelena Milašin¹, Boško Toljić¹, Jelena Simonović¹, Maja Milošević¹, Miloš Lazarević¹, Jasna Vujin², Mina Perić³, Radmila Panajotović²

¹Department of Human Genetics School of Dental Medicine, University of Belgrade, Belgrade 11000, Serbia
²Graphene Laboratory, Center for Solid State and New Materials, Institute of Physics, University of Belgrade, Belgrade 11000, Serbia
³Center for Laser Microscopy, Faculty of Biology, University of Belgrade, Belgrade 11000, Serbia

One of the most promising, easily accessible stem cell dental niche is the soft tissue of the top of immature tooth root (apical papilla). Over 90% of cells from apical papilla are stem cells (SCAP), showing a high potential for neural differentiation. Carbon-based nanomaterials, such as graphene film, could enhance SCAP transition into neural lineage as these materials possess advanced mechanical, electrical and neuroinductive properties. Our aim was to evaluate the influence of graphene film on neuro-differentiation process of SCAP. SCAP were isolated from third molars, and grown in complete medium (CM) under standard conditions. Their stemness was confirmed by the expression of specific markers detected by flow-cytometry and differentiation into different cell types. They were subdivided into three experimental groups: Group A was seeded on graphene film and grown in CM whereas cells seeded and grown on uncoated plastic dish were subjected to a neuroinduction medium (group B) or CM (group C). After 7 days cells were evaluated under light, confocal and atomic force microscopy (AFM). Additionally, the expression of neural markers was assessed by qPCR. SCAP stemness was demonstrated by flow-cytometry, which revealed high percentage of CD73, CD90 and CD 105 marker expression, and by differentiation into osteo-, adipo-, and chondro- cell lineages. Light microscopy and AFM showed that SCAP grown on graphene presented long, slender projections suggestive of neuron-like cells. Higher expression of the neural markers (Ngn2, NF-M, nestin, MAP2 tested by real-time PCR and confocal microscopy -MAP2) was found in group A compared to group B, while the lowest was found in group C. The predisposition of SCAPs to differentiate toward neural lineages, as well as the neuroinductive properties of graphene film, should warrant further studies of SCAP in conjunction with this nanomaterial, with the aim of finding a superior solution for autologous neuroregenerative therapy.

P43

Exploring the Particle Swarm Optimization: a novel simulation approach addressing larger biological systems

János Horváth^1,2, Ferenc Bogár³, András Dér¹, Zoltán Násztor¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²Doctoral School of Physics, University of Szeged, Szeged, Hungary
³MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary

The Particle Swarm Optimization (PSO) is a population-based method for the optimization of non-linear functions. Utilizing an Artificial Intelligence (AI) approach, the PSO is usually considered a part of the family of Evolutionary Algorithms, however it lacks genetic operators, such as recombination or mutation. During the algorithm a large set (swarm) of candidate solutions (particles) are moving in a pre-defined search space to find the best possible value for a fitness function (optimization). On the course of the run, the particles adjust their trajectory taking into account their own, known best position (personal best fitness function value) and also the global, known best position of the swarm.

The PSO method could be used in solving problems involving simulation systems containing large biomolecules. To this end, our research group implemented the PSO in a Python code (PredStruct program), in which we consider molecules: one is tagged as “Surface”, whereas the other one as “Protein”. However, there are no restrictions regarding these molecules, either of them could be chosen to be a lipid bilayer, a graphene sheet, a protein, a crystal-like surface etc. The goal of our program is to provide an estimated structure with respect to how the two molecules “stick” together. In our case the candidate solutions are a set of row vectors containing translations and rotations of the Protein with respect to the Surface. The fitness function can be chosen to be the binding free energy between the two molecules or the overall energy minimum of the system.

Utilizing the program’s large-scale docking-like aspect, structures as large as human serum albumin and a correspondingly sized graphene oxide sheet could be treated, or the photoactive yellow protein on various protein surfaces. The result provided by PredStruct is an „educated guess” for such large systems which could not be treated with classical MD (initial value problem, random walk) or docking (size) tools.

P45

Label-free discovery of natural compounds as target biomolecules in cellular adhesion and migration

Beatrix Péter¹, Inna Székács¹, Szilvia Bősze^2,3, Imre Boldizsár^4,5, Gábor M. Kovács^4,6, Robert Horvath¹

¹Nanobiosensorics Group, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary
²ELKH-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary
³National Public Health Center, Albert Flórián út 2-6, 1097 Budapest, Hungary
⁴Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, 1117 Budapest, Hungary
⁵Department of Pharmacognosy, Semmelweis University, Üllői út 26, 1085 Budapest, Hungary
⁶Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary

Various plant tissues can be applied for medical applications because of their accumulation of special bioactive metabolites with diverse molecular structures. Natural products with their broad chemical diversity and bioactivity spectrum are sought after by the pharmaceutical industry and they continue to provide new structures with promising effects and to offer templates for the development of scaffolds of novel drug candidates. Therapeutics search for natural substances that are beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites [1]. In general, the so-called classical labelling techniques are used to test their effect on cellular adhesion and migration. However, labels or dyes, which may disturb the samples. Furthermore, natural compounds usually have small molecular weight where labeling can be problematic or even impossible, especially if their binding pocket is small or embedded. Label-free biosensors are emerging tools to investigate the mode of action of small molecules as well. They eliminate all of the disadvantages of the classical techniques. In the field of natural compound research, novel methods, for example, grating-coupled interferometry (GCI), resonant waveguide grating (RWG) and holographic microscopy can be applied. In this work, we provide examples for revealing the effects of natural compounds on cellular adhesion and migration and the binding between transfer proteins and active substances is also presented by the mentioned label-free methods [2-4].

Acknowledgments

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (VEKOP, ELKH topic-fund, Élvonal KKP_19 and KH grants, TKP2022-EGA-04 and PD 131543 program financed from the NRDI Fund, and OTKA NKFIH K-135712). IB, GMK and SB thanks for the support of the EFOP-1.8.0-VEKOP-17-2017-00001.

References

1. B. Péter et al. Biomedicines, 2021, 9, 1781.
2. B. Péter et al. Analyst, 2020, 145, 588-595.
3. B. Péter et al. Sci. Rep., 2017, 7, 42220.
4. B. Péter et al. J. Biomed. Opt., 2015, 20, 067002.

P47

Label-free optical biosensor and FluidFM for monitoring the adhesion of cells with enzymatically digested glycocalyx

Imola Rajmon^1,2, Kinga Dóra Kovács^1,2, Nicolett Kanyó¹, Inna Székács¹, Bálint Szabó², Robert Horvath¹

¹Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary
²ELTE Eötvös Loránd University, Department of Biological Physics, Budapest, Hungary

The role of the glycocalyx (GLX) in cellular adhesion is not yet clear. Most cancer cells have an altered glycocalyx structure, presumably playing a role in cancer development and metastasis. In our work, we investigated the enzymatic digestion of specific glycocalyx components by chondroitinase ABC on cancer cells that adhered to RGD (arginine-glycine-aspartic acid) motif displaying surfaces. We used a surface-sensitive label-free resonant waveguide grating (RWG) based optical biosensor, and fluidic force microscopy (FluidFM) to characterize cell adhesion [1, 2]. Earlier studies found that there is a difference between the adhesion forces after intense and mild glycocalyx digestion [2]. We made experiments using the same enzyme concentration with HeLa cells under three different conditions. In the first one, we injected the enzyme into the buffer before the cells adhered to the surface, while in the second one we waited for 1.5 hours and the cells mostly adhered before we added the enzyme [2]. Control experiments were also conducted. We started with the recording of the high-resolution kinetic data by the biosensor and after 1.5 hours we moved the same cells into the FluidFM measuring unit, where we recorded the detachment force values between some chosen individual cells and their substrate [1]. We concluded that using this concentration of enzyme decreases the adhesion force, which is smaller if we digest the glycocalyx after cell spreading. We arrived at the conclusion that log-normal population distribution functions fit better than normal functions on our single adhesion force and energy data [1]. We also experienced that in this case, the measured adhesion forces are time-dependent, while in the other two experiments this effect is not detectable.

Acknowledgements

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (VEKOP, ELKH topic-fund, Élvonal KKP_19 and KH grants, TKP2022-EGA-04 program financed from the NRDI Fund).

References

1. Milan Sztilkovics, Tamas Gerecsei, Beatrix Peter, Andras Saftics, Sandor Kurunczi, Inna Szekacs, Balint Szabo, Robert Horvath. Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy. Sci Rep 10, 61 (2020). https://doi.org/10.1038/s41598-019-56898-7
2. Nicolett Kanyo, Kinga Dora Kovacs, Andras Saftics, Inna Szekacs, Beatrix Peter, Ana R. Santa-Maria, Fruzsina R. Walter, András Dér, Mária A. Deli, Robert Horvath. Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data. Sci Rep 10, 22422 (2020). https://doi.org/10.1038/s41598-020-80033-6

P49

Effect of SMIFH2 on the structural and functional dynamics of FH2 domain of DAAM

Rauan Sakenov¹, Péter Bukovics¹, Veronika Takács-Kollár¹ Péter Gaszler¹, Beáta Bugyi^1,2

¹University of Pécs, Medical School, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary
²Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee, Pécs, Hungary
Email: rauansakenov@gmail.com

Small molecular inhibitor of formin homology domain 2 (SMIFH2) was developed as a cytostatic anticancer agent, which inhibits the actin activity of FH2 domains of formin proteins, including human DAAM1 (Disheveled associated activator of morphogenesis) and mouse Dia1 (mDia1). To investigate the structural and functional aspects of SMIFH2 binding to the FH2 domain of DAAM (Drosophila), we conducted in-silico docking studies and steady-state fluorescence spectroscopy experiments.

The functional aspects of the interaction were assayed in bulk pyrenyl actin fluorescence-based polymerization assays and the half maximal inhibitory concentration of SMIFH2 was derived. Our in silico docking analysis revealed the highly site-specific binding of SMIFH2 to chain A of the FH2 domain for all selected formins. We found that SMIFH2 can form different interactions with some of the highly conserved tryptophans and tyrosine residues important in the oligomerization of the FH2 domain. The analysis of fluorescence emissions of tryptophan residues of the DAAM FH2 domain in the presence of SMIFH2 showed ~4 nm blueshift, which indicates that the binding of SMIFH2 to DAAM FH2 can result in conformational alterations influencing intrinsic tryptophan fluorescence. Finally, the results of Stern-Volmer analysis of acrylamide quenching of tryptophan fluorescence in DAAM FH2 at 10-30^oC revealed mixed static and dynamic mechanisms of quenching with the prevalence of dynamic mechanism.

In conclusion, our comparative sequence alignment of docking results of SMIFH2 to chain A of the FH2 domain of mDia1, DAAM1 and DAAM (Drosophila) revealed its highly specific binding to the regions of the FH2 domain of all investigated formins, which are functional in intra- and interchain interactions. These in-silico data were corroboated by in vitro fluorometric measurements.

Keywords: SMIFH2, formin homology-2 domain, actin, proteins as drug targets, mechanism of action of SMIFH2, molecular modelling and computational structural biophysics

P51

Conformations of NADH in solutions: Ultrafast fluorescence kinetics measurements analysed by machine learning

Ferenc Sarlós, Rita Nagypál, Áron Sipos, Géza I. Groma

Institute of Biophysics, Biological Research Centre Szeged, Eötvös Loránd Research Network, Szeged, Hungary

The structure of many different enzyme-bound forms of the essential coenzyme nicotinamide adenine dinucleotide are well characterized by X-ray diffraction data. Due to the limitations of this technique in solution, the unbound forms of the molecule need to be characterized by alternative methods, like time-resolved fluorescence spectroscopy. In NADH the relative position of the nicotinamide and adenine group has primary importance on the fluorescence kinetics of the excited nicotinamide group. In aqueous solution the molecule exists in an equilibrium of closed and open conformations, while the presence of methanol favours the latter.

The fluorescence kinetics of NADH was measured in water and methanol environments using fluorescence upconversion and time-correlated single photon counting in a large 50 fs – 10 ns time window at different wavelengths for both environments.To avoid the uncertainties of exponential fitting, the experimental data were fitted by a quasi-continuous set of time constants, applying regularization terms for favouring sparse solutions, i.e., a minimum number of nonzero amplitudes. For fine tuning the level of sparsity we developed a machine-learning method based on cross-validation and Bayesian optimization. This approach was found a powerful method for fluorescence kinetics analysis, avoiding any arbitrary or random parameters.

According to the above analysis the fast (<100 ps) part of the kinetics can be characterized by an unusually complex, three-step vibrational relaxation process. The slow part is well modelled either by the conventional distinct exponential terms or by distributed kinetics, corresponding to the equilibrium of a very high number conformational states.

P53

Prediction of chronic inflammation for inhaled particles: the impact of material cycling and quarantining in the lung epithelium

Hana Majaron^1,2, Boštjan Kokot¹,³, Aleksandar Sebastijanović^1,2, Carola Voss⁴, Rok Podlipec^1,5, Patrycja Zawilska¹, Trine Berthing⁶, Carolina Ballester López⁴, Pernille Høgh Danielsen⁶, Claudia Contini⁷, Mikhail Ivanov⁸, Ana Krišelj¹, Petra Čotar^1,9, Qiaoxia Zhou^4,10, Jessica Ponti¹¹, Vadim Zhernovkov¹², Matthew Schneemilch⁷, Zahra Manel Doumandji¹⁴, Mojca Pušnik¹³, Polona Umek¹, Stane Pajk^1,13, Olivier Joubert¹⁴, Otmar Schmid⁴, Iztok Urbančič¹, Martin Irmler¹⁵, Johannes Beckers^15,16,17, Vladimir Lobaskin¹⁸, Sabina Halappanavar¹⁹, Nick Quirke⁷, Alexander P. Lyubartsev⁸, Ulla Voge⁶, Tilen Koklič¹, Tobias Stoeger⁴, Janez Štrancar¹

¹Department of Condensed Matter Physics, Jozef Stefan Institute, Ljubljana, Slovenia
²Jožef Stefan International Postgraduate School, 1000 Ljubljana, Slovenia
³Faculty of Natural sciences and Mathematics, University of Maribor, Maribor, Slovenia
⁴Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764 Neuherberg, Germany
⁵Ion Beam Center, Helmholz Zentrum Dresden Rossendorf, Dresden, Germany
⁶National Research Centre for the Working Environment, Copenhagen Ø, Denmark
⁷Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London, United Kingdom
⁸Department of Materials and Environmental Chemistry, Stockholm University, SE-10691 Stockholm, Sweden
⁹Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia
¹⁰Department of Forensic Pathology, Sichuan University, Chengdu, China
¹¹European Commission, Joint Research Centre (JRC), Ispra, Italy
¹²School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
¹³Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
¹⁴Institut Jean Lamour, CNRS-Université de Lorraine, Nancy, France
¹⁵Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany
¹⁶German Center for Diabetes Research (DZD), Neuherberg, Germany
¹⁷Chair of Experimental Genetics, Center of Life and Food Sciences, Weihenstephan, Technische Universität München, Freising, Germany
¹⁸School of Physics, University College Dublin, Belfield, Dublin 4, Ireland
¹⁹Health Canada, Ottawa, Canada

Nanomaterial-induced diseases cannot be reliably predicted because of the lack of clearly identified causal relationships, in particular between acute exposures and chronic symptoms. By applying advanced microscopies and omics to in vitro and in vivo systems, together with in silico molecular modelling, we have here determined that the chronic inflammatory response arises due to the counteracting of a newly discovered nanomaterial quarantining and nanomaterial cycling among different lung cell types after a single exposure to nanomaterial. Besides its profound implications for cost-efficient animal-free predictive toxicology, our work also paves the way to a better mechanistic understanding of nanomaterial-induced cancer, fibrosis, and other chronic diseases.

P55

Dynamically facilitated water transport in hSGLT

Marko Sever, Franci Merzel

Theory Department, National Institute of Chemistry, Ljubljana, Slovenia

The hSGLT protein is an important representative of the sodium solute symporters belonging to the family of secondary active transporters critical for to the homeostasis of sugar, sodium and water in the cell. The underlying transport mechanism of hSGLT is based on switching between inward- (IF) and outward-facing (OF) conformations, known as the alternating access model, which is crucial for the sugar transport but has also been postulated for water permeation.  However, the nature of water transport remains unclear.

Using a large number of equilibrium all-atom molecular dynamics simulations of hSGLT performed on different hSGLT systems corresponding to different conformational states including complexes with sugars, ions and inhibitors, we evaluate water permeability as a function of physico-chemical factors. By further analysing channel water, its energetics, local and collective mobility and its correlation with the collective motion of protein domains we demonstrate the facilitating role of protein domain motion resembling the rocking-bundle type of motion. Our results confirm high values of permeability in both (IF and OF) conformational states which is consistent with experimental results and supports the hypothesis of passive water transport in hSGLT.

P57

The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

Cyril Slabý¹, Janka Kubacková², Denis Horvath³, Andrej Hovan¹, Gergely T. Iványi^4,5, Gaszton Vizsnyiczai⁵, Lóránd Kelemen⁵, Gabriel Žoldák³, Zoltán Tomori², Gregor Bánó¹

¹ P. J. Šafárik University, Faculty of Science, Department of Biophysics, Košice, Slovakia
² Institute of Experimental Physics SAS, Department of Biophysics, Košice, Slovakia
³P. J. Šafárik University, Center for Interdisciplinary Biosciences, TIP, Košice, Slovakia
⁴University of Szeged, Faculty of Science and Informatics, Szeged, Hungary
⁵Biological Research Centre, Institute of Biophysics, ELKH, Szeged, Hungary

Nanowires fabricated of photopolymer materials are the building blocks of many microstructure applications. Such nanowires can be prepared by two-photon polymerization direct laser writing (TPP), one of the basic and precise microstructure fabrication techniques used in biomedical and microfluidic applications [1]. In our previous research, we used a simple viscoelastic mechanical model to describe the bending recovery motion of deflected nanowire cantilevers in Newtonian liquids [2]. The inverse problem was targeted recently [3]. It was our goal to determine the nanowire physical characteristics based on the experimental recovery motion data. Explicit formulas were derived to calculate the nanowire viscoelastic material properties.

A holographic optical tweezer setup was used to deflect 16 µm long photopolymer nanowire cantilevers made of the Ormocomp photoresist immersed in aqueous glucose solutions. The measurements were repeated in solutions of different concentrations. After the initial deflection, the laser tweezer was switched off and the structure started to recover to its original, relaxed shape. In agreement with the model predictions, the recovery data obtained by video-tracking could be well fitted with a double-exponential time-dependence.

The effective elastic modulus of the studied nanowires was determine to be two orders of magnitude lower than measured for the bulk material. Besides that, the intrinsic viscosity of the nanowire was obtained. Interestingly, this viscosity changes significantly with the glucose concentration, which indicates significant porosity of the nanowire material.

Acknowledgements

This work was funded by the Slovak Research and Development Agency (grants APVV-18-0285, APVV-21-0333) and the Slovak Ministry of Education (grants VEGA 2/0094/21 and 2/0101/22) and internal grant of PF UPJŠ (grant vvgs-pf-2021-1771). This publication is also the result of the implementation of the project OPENMED (Open Scientific Community for Modern Interdisciplinary Research in Medicine) ITMS2014+: 313011V455 from the Operational Program Integrated Infrastructure funded by the ERDF and by the EU H2020 TWINNING program GA. No. 952333 project CasProt.

References

1. Otuka, N. Tomazio, K. Paula, C. Mendonça. Polymers 13 (2021) 1994.
2. Kubacková et al., Applied Physics Letters 117 (2020) 013701.
3. Kubacková, C. Slabý, D. Horvath, A. Hovan, G. T. Iványi, G. Vizsnyiczai, L. Kelemen, G. Žoldák, Z. Tomori, G. Bánó. Nanomaterials 11 (2021) 2961.

P59

Behind the anisotropy imaging using RCM

Gábor Steinbach¹, Dávid Nagy², László Zimányi²

¹Cellular Imaging Laboratory, Biological Research Centre, ELKH, Szeged, Hungary
²Institute of Biophysics, Biological Research Centre, ELKH, Szeged, Hungary

Both the differential polarization attachment for LSMs and Re-scan Confocal Microscopy (RCM) facilitate imaging structures at the molecular level [1]. For smaller structures, increasing the available resolution can be fundamental. The re-scan confocal microscopy (RCM) provides 4 times better signal to noise ratio (compared to conventional PMTs) using an sCMOS camera, moreover, due to the second scanner, the image resolution increases by the factor of 1.4, while the axial resolution is identical with the LSMs. With additional polarization elements, the RCM enables the 2D and 3D microscopic mapping of the anisotropy of samples via measuring fluorescence-detected linear dichroism (FDLD). The usage of the liquid crystal modulator for the RCM and the high frequency modulation (photoelastic modulator- PEM) are synchronised with the imaging. The DP-LSMs and the pRCM (RCM equipped with polarization attachment) are suitable for obtaining unique structural information on the anisotropic molecular organization of biological samples and intelligent [2,3] materials in 2D and 3D.

References

1. Steinbach et al. Re-scan confocal microscope modified for anisotropy imaging – as a part of a differential polarization system. European Biophysics Journal 48: 457–463. 2019. doi: 10.1007/s00249-019-01365-4
2. Simonović Radosavljević J et al. Anisotropic molecular organization of plant cell walls revealed by differential polarization imaging techniques. Int. J. Mol. Sci. 22, 7661, 2021. doi: 10.3390/ijms22147661
3. Pleckaitis M et al. Structure and principles of self-assembly of giant “sea urchin” type sulfonatophenyl porphine aggregates. Nano Research, 2022. doi: 10.1007/s12274-021-4048-x

P61

Rheological behaviour of whole human blood upon interaction with amphiphilic phosphorous dendrons

Š. Šutý¹, K. Ládiová¹, M. Kopáni², P. Vitovič³, D. Shcharbin⁴, M. Ionov⁵, J. P. Majoral⁶, M. Bryszewska⁵, I. Waczulíková¹

¹Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia
²Institute of Medical Physics, Biophysics, Informatics and Telemedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia
³Department of Simulation and Virtual Medical Education, Faculty of Medicine, Comenius University, Bratislava, Slovakia
⁴Institute of Biophysics and Cell Engineering of NASB, Minsk, Belarus
⁵Faculty of Biology and Environmental protection, Department of General Biophysics, University of Lodz, Poland
⁶Laboratoire de Chimie de Coordination Du CNRS, Toulouse, France

One group of nanoparticles widely studied for their theranostic potential is represented by dendritic assemblies, more specifically dendrimers and dendrons - synthetic hyperbranched polymers. The toxicity of conventional anticancer drugs can be repressed using amphiphilic dendron-based polymers with a unique hyperbranched nanosized structure, able to accommodate substantial amount of drugs. However, events such as a premature release of the drug from nanodelivery systems still occasionally occur, which leads to adverse interactions with blood components. In our study we used a first generation dendron (D1) and a second generation dendron (D2) at concentrations of 2 and 10 µM which have been tested in cell studies. Blood compatibility of any nanosystem intended for medical use must be carefully evaluated before clinical application.  Since blood is considered a non-Newtonian, shear thinning, thixotropic and viscoelastic fluid, we focused on flow behaviour and viscosity changes upon interaction of blood with D1 and D2. We used a rotational modular compact rheometer MCR 102 in the range of shear rate of 0.1 to 1000 s^-1 to mimic circulation in various in vivo conditions. We observed an increase in viscosity at both D1 and D2 concentrations. This might have potentially serious consequences under in vivo conditions ranging from problems with perfusion in body tissues to thrombosis and embolism. Our findings can help optimize dendron-based nanotherapeutics. We conclude that amphiphilic phosphorous dendrons interact with whole human blood, and alter blood flow and apparent viscosity.

Acknowledgements

This work was supported by the grant agencies: KEGA 041UK-4/2020; NAWA International Academic Partnership Programme EUROPARTNER, PPI/APM/2018/1/00007/U/001; APVV SK-BY-RD-19-0019 and SK-PL-21-0073, and partly by the State Committee on Science and Technology of the Republic of Belarus (SCST RB) B20-SLKG-002 and Comenius University Grant UK/437/2021. The results of this study were partly published in Pharmaceutics 2022, 14, 1596 (DOI: 10.3390/pharmaceutics14081596)

P63

The calcium regulated actin remodelling in apoptotic cells

Alexandra Hencz^1,2, Edina Szabó-Meleg¹, Muhammad Yaqoob Dayo¹, Ardora Bilibani¹, Szilvia Barkó¹, Miklós Nyitrai¹, Dávid Szatmári¹

¹University of Pécs, Medical School, Department of Biophysics, Pécs, Hungary
²University of Pécs, Medical School, Institute of Physiology, Pécs, Hungary

Primer stress response of cells is Ca²⁺ influx, then among others the cytoskeletal system quick remodelling can change the cell motility, division and transport processes which finally can lead to the apoptosis. Our interest is focused on that how the gelsolin (GSN) and junctional mediating and regulating Y protein (JMY) play important role in stress response and apoptotic processes. Both protein can bind p53 and actin. We investigated that how the function of p53, GSN and JMY as cytoplasmic or cytoplasmic-nuclear factors can be linked to the cytoskeletal remodelling and cellular motility change in the apoptosis. Especially, how can the cytoplasmic Ca²⁺ level affect the complex formation and dynamics of p53 with actin, GSN and JMY. Here we investigated that micromolar Ca²⁺ activates the GSN, thus helps the continuous rearrangement of actin filaments. The p53 competes with actin on GSN to inhibit p53-JMY complex formation and possibly can prevent the apoptosis. However, the elevated milimolar Ca²⁺ level induces the total activity of GSN thus independently of p53 binding, GSN severing and capping of filamentous actin. High Ca²⁺ level initializes p53 dimerization, the dimer competes with actin on JMY can lead to p53-JMY cotransport into the nucleus thus possibly results apoptosis by the enhanced p53 expression. Here we investigated how the motility and the division rate of HeLa cells change due to low-voltage electroporation of GSN or JMY. We revealed that electroporation alone is able to stimulate the lateral motion of the cells. In conrast, JMY somehow inhibits their motion but it can help cell division. GSN treatment slows down cell division but does not affect cell motility. HeLa cells have fully recovered the gap in 20 hours after the electroporation with JMY then started to release from the glass slides. The cytoplasmic balance of GSN and JMY can play an important role in the stress response which prepares the cells for the apoptosis.

P65

Actin based protrusions between epiblast cells of zebrafish embryos

Katalin Türmer¹, Miklós Nyitrai^1,2, Edina Szabó-Meleg^1,2

¹Department of Biophysics, Medical School, University of Pécs, Pécs, Hungary
²Szentágothai Research Centre, University of Pécs, Pécs, Hungary

Novel cytoskeletal structures, known as tunneling nanotubes (TNTs), are membrane protrusions supported by filamentous actin. Actin remodeling is in the center of the response of cells to external or internal stimuli, which provides a variety of cell projections to form.

While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood [1].

Membrane nanotubes were described both in vitro (in cell cultures) and in vivo (during the embryonic development) [2]. We focused on the in vivo examination of membrane nanotubes in developing zebrafish embryos.

Laser-scanning confocal microscope and transmission electron microscopy were applied to visualize the NTs between the epiblast cells of the embryos.

The characterization of membrane nanotubes of 4-5 hours old embryos shows that their length is approximately identical with the individual cell diameters. In the case of 6-7 hours old embryos, the tubes are considerably longer and their occurrence is pronounced at the animal pole of the embryo. We found relationship between material transport and nanotube thickness: transport processes take place inside thicker nanotubes.

Our data clearly support the in vivo existence of NTs, but further experiments are needed to reveal their possible function in zebrafish embryos.

References

1. Ljubojevic, N., Henderson, J. M., & Zurzolo, C. (2021). The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions. Trends in cell biology, 31(2), 130–142.
2. Caneparo L., Pantazis P., Dempsey W., Fraser S. E. (2011) Intercellular Bridges in Vertebrate Gastrulation. PLoS One 6(5):e20230.

P67

Penetration of the SARS-CoV-2 Spike Protein across the Blood–Brain Barrier, as Revealed by a Combination of a Human Cell Culture Model System and Optical Biosensing

Dániel Petrovszki^1,2, Fruzsina R. Walter¹, Judit P. Vigh^1,3, Anna Kocsis¹, Sándor Valkai¹, Mária A. Deli¹, András Dér¹

¹Institute of Biophysics, Biological Research Centre, ELKH, Temesvári Krt. 62, H-6726 Szeged, Hungary
²Doctoral School of Multidisciplinary Medical Sciences, University of Szeged, Dóm Tér 9, H-6720 Szeged, Hungary
³Doctoral School of Biology, University of Szeged, Közép Fasor 52, H-6726 Szeged, Hungary

Since the outbreak of the global pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), several clinical aspects of the disease have come into attention. Besides its primary route of infection through the respiratory system, SARS-CoV-2 is known to have neuroinvasive capacity, causing multiple neurological symptoms with increased neuroinflammation and blood– brain barrier (BBB) damage. The viral spike protein disseminates via circulation during infection, and when reaching the brain could possibly cross the BBB, which was demonstrated in mice. Therefore, its medical relevance is of high importance.

The aim of this study was to evaluate the barrier penetration of the S1 subunit of spike protein in model systems of human organs highly exposed to the infection. For this purpose, in vitro human BBB and intestinal barrier cell–culture systems were investigated by an optical biosensing method. We found that spike protein crossed the human brain endothelial cell barrier effectively. Additionally, spike protein passage was found in a lower amount for the intestinal barrier cell layer. These observations were corroborated with parallel specific ELISAs.

The findings on the BBB model could provide a further basis for studies focusing on the mechanism and consequences of spike protein penetration across the BBB to the brain.

P69

Determination of correlation between the electrical impedance parameters and the rheological parameters of carrot

Eszter Vozáry, Bíborka Gillay

Hungarian University of Agriculture and Life Sciences, Department of Food Measurements and Control Budapest, Hungary

During the food industrial processes it is often necessary to know the physical properties of food in order to solve various control problem. Online measurement of different physical parameters is often difficult during different processes. Measurement of electrical parameters such as electrical impedance is relatively easy to solve. If we know the relationships between electrical and other physical parameters, we can also give the values of other physical parameters by measuring the electrical impedance.

The aim of the present work is to determine the relationships between rheological and electrical impedance parameters of carrots.

The carrots were purchased from the local store. The carrot was sliced and the electrical impedance spectrum in both the transport and storage tissues was determined on each slice and the relaxation curves were measured at constant deformation. Measurements were made both in the direction of the longitudinal axis of the carrot and in the direction of the radius of the carrot.

The magnitude and the phase angle of the electrical impedance were measured with an HP 4284A LCR analyzer in the frequency range of 30 Hz to 10 MHz at 1 V measuring voltage After open-short correction, the corrected spectra were approximated by the series connection of a distributed element and one resistor with the Solver function of Excel. Resistances, relaxation time, and exponent parameter were determined.

Rheological measurements were performed with a Stable Microsystem TA-XT plus texture analyzer. The relaxation curves were recorded under deformation less than the yield point. For the relaxation curve, the sum of a stretched exponential functions and an equilibrium constant was fitted with the Excel Solver function. The values of the parameters describing the constant and the stretched exponential functions were determined.

A linear relationship was found between the viscosity parameters and the resistances and between the elastic parameters and capacities.

L32

Ultrafast light-induced charge transfer in flavoproteins

Bo Zhuang, Marten H. Vos

LOB, Ecole Polytechnique, Institut polytechnique de Paris, CNRS, INSERM, 91128 Palaiseau, France

Flavins are chromophores that display highly versatile redox properties. Some flavin binding proteins are functionally photoactive [1]. Yet most display light-independent functions, although in these photophysical processes also occur, which could be exploited for photocatalysis or photoswitching applications [2]. This contribution highlights recent studies using ultrafast transient absorption and fluorescence spectroscopy, on short-lived photoproducts in nonphotoactive flavoproteins exploring various redox and ligation states, [3-5] including the discovery of two hitherto unknown photoreactions that occur on the timescale of a few hundred femtoseconds or less: photo-oxidation of anionic flavin radicals by closeby cationic amino acids [4] and photo-isomerization of an inhibitor that is complexed with flavin in a charge-transfer complex [5].

References

1. Sorigué, D.; Hadjidemetriou, K.; Blangy, S.; Gotthard, G.; Bonvalet, A.; Coquelle, N.; Samire, P.; Aleksandrov, A.; Antonucci, L.; Benachir, A., et al., Mechanism and dynamics of fatty acid photodecarboxylase. Science 2021, 372, eabd5687.
2. Zhuang, B.; Liebl, U.; Vos, M. H., Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives. J. Phys. Chem. B 2022, 126, 3199-3207.
3. Zhuang, B.; Seo, D.; Aleksandrov, A.; Vos, M. H., Characterization of Light-Induced Short-Lived Interacting Radicals in the Active Site of Flavoprotein Ferredoxin-NADP+ Oxidoreductase. J. Am. Chem. Soc. 2021, 143 2457-2768.
4. Zhuang, B.; Ramodiharilafy, R.; Liebl, U.; Aleksandrov, A.; Vos, M. H., Ultrafast Photooxidation of Protein-Bound Anionic Flavin Radicals. Proc. Natl. Acad. Sci. U.S.A. 2022, 119, e2118924119.
5. Zhuang, B.; Vos, M. H., Photoswitching Behavior of Flavin–Inhibitor Complex in a Nonphotocatalytic Flavoenzyme. J. Am. Chem. Soc. 2022, 144, 11569–11573.

L33

Mechanism and Dynamics of Fatty Acid Photodecarboxylase

Damien Sorigué¹, Kyprianos Hadjidemetriou², ..., Stéphanie Blangy¹, Catherine Berthomieu¹, Martin Weik², Tatiana Domratcheva³, Klaus Brettel⁴, Marten H. Vos⁵, Ilme Schlichting³, Pascal Arnoux¹, Pavel Müller⁴, Fred Beisson¹

¹Aix-Marseille University, CEA, CNRS, BIAM Cadarache, 13108 St.-Paul-lez-Durance, France
²Université Grenoble Alpes, CEA, CNRS, IBS, 38000 Grenoble, France.
³Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
⁴Université Paris-Saclay, CEA, CNRS, I2BC, 91198 Gif-sur-Yvette, France
⁵LOB, CNRS, INSERM, E. Polytech., Institut Polytechnique de Paris, 91128 Palaiseau, France

Fatty acid photodecarboxylase (FAP) is a recently discovered [1, 2] photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryo-trapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds [3]. High-resolution crystal structures from synchrotron and free electron laser X-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

References

1. D. Sorigué et al., Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway. Plant Physiol. 171, 2393-2405 (2016)
2. D. Sorigué et al., An algal photoenzyme converts fatty acids to hydrocarbons. Science 357, 903-907 (2017)
3. D. Sorigué et al., Mechanism and dynamics of fatty acid photodecarboxylase. Science 372, eabd5687 (2021)

L34

Structural changes of carotenoid echinenone in Orange Carotenoid Protein studied by femtosecond Raman spectroscopy

Miroslav Kloz¹, P. Čubáková¹, T. Friedrich², T. Polivka³, E. Maksimov⁴

¹ELI-Beamlines, Institute of Physics, Praha, Czech Republic
²Technische Universität Berlin, Institute of Chemistry PC14, Berlin, Germany
³Institute of Physics, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic
⁴Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

The orange carotenoid protein (OCP) [1] is a perfect system to study changes in cofactor structure during photoswitching of proteins by Raman techniques. It hosts a single xanthophyll molecule and undergoes well-studied (but not yet fully understood) photocycle that is associated with the loss of vibrational structure in the absorption spectra.

The orange carotenoid protein consists of two subunits that get mutually loose after carotenoid excitation that switches it between the so-called “red” and “orange” states. In that form, it binds to other light-harvesting proteins while greatly increasing their non-radiative decay of excitons. Both the mechanism of OCP photoswitching and its subsequent role as a trigger of non-photochemical quenching is yet to be understood. We studied the wild type and two types of mutants (including utilization of non-canonical amino acids) to understand the role of hydrogen bond formation in the photoswitching mechanism by Stimulated Raman scattering.

References

1. Yaroshevich, I.A., Maksimov, E.G., Sluchanko, N.N. et al. Commun Biol 4, 539 (2021)

L35

Singlet oxygen production by LOV domain

Erik Sedlák

Center for Interdisciplinary Biosciences, Technology and Innovation Park of P. J. Šafárik University in Košice, Slovakia
Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovakia

Photosensitizers are widely used in photodynamic therapy, but it is important to increase their efficiency. Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, ¹O₂. Here, we present our investigation regarding the factors that determine the efficiency of ¹O₂ production. When buried in a protein matrix, the efficiency of FMN to function as a photosensitizer is diminished due to its interactions with surrounding amino acid residues. In some flavoproteins, blue-light irradiation significantly increases ¹O₂ production. We present detailed analysis of the irradiation effects on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced critical cysteine residue, AsLOV2 C450A, which is responsible for the formation of cysteinyl-flavin adduct in the phototropin 1 photocycle. We show that blue light irradiation in both AsLOV2 variants leads to qualitatively similar changes: (i) significantly increased ¹O₂ production, (ii) changes in UV-VIS absorption, circular dichroism, and fluorescence spectra, (iii) oxidative modifications of several amino acids, and (iv) release of flavin cofactor to solvent. However, the extent of the observed changes depends on the AsLOV2 variant. The more efficient FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a new approach of a design of more efficient genetically encoded photosensitizer.

L36

Involvement of dipeptidyl peptidase III in oxidative stress and pain regulation

Sanja Tomić¹, Sara Matić¹, Zrinka Karačić¹, Filip Šupljika², Antonija Tomić¹, Mihaela Matovina¹, Lidija Brkljačić¹, Ana Tomašić Paić¹, Sandra Sobočanec¹, Marija Pinterić¹

¹Ruđer Bošković Institute, Zagreb, Croatia
²Faculty of Food Technology and Biotechnology, Zagreb, Croatia

Dipeptidyl peptidase III is a two-domain zinc dependent exopeptidase and the only member of the M49 peptidase family characterised by two highly conserved motifs HEXXGH and EEXR(K)AE(D), which in human DPP III correspond to ⁴⁵⁰HELLGH⁴⁵⁵ and ⁵⁰⁷EECRAE⁵¹². The histidine residues of the first motif and E508 coordinate the zinc ion, while E451 is involved in proton transfer during peptide hydrolysis.

DPP III is found in almost all living organisms, where it hydrolyzes dipeptides from the unsubstituted N-terminus of its peptide substrates, which are usually three to eight amino acids long. DPP III is common in various mammalian tissues and is preferentially localised in the cytosol, but can also be found on the membrane and in extracellular fluids. Because of its broad substrate specificity, it is thought to be involved in the final steps of normal intracellular protein degradation. It also shows a pronounced affinity for the bioactive peptides angiotensins, and enkephalins, suggesting a role in the regulating their signal transduction.

In addition, it should be noted that DPP III is involved in the regulation of oxidative stress and in the development of some cancers in humans through its binding to the KEAP1 protein, the major oxidative stress sensor in the cell - a case of moonlighting of the protein that has nothing to do with its peptidase activity.

We investigated the interactions between DPP III and KEAP1 and the effects of mutations found in cancer samples on this interaction. We have also screened a number of different neuropeptides as potential substrates and inhibitors of DPP III.

Acknowledgements

This work has been supported by Croatian Science Foundation under the project IP-2018-01-2936.

L37

Magnesium ions reversibly bind to DNA double stranded helix in thin films: an infrared spectroscopy study

Sanja Dolanski Babić^1,2, Kristina Serec^1,2, Silvia Tomić³

¹Department of Physics and Biophysics, School of Medicine, University of Zagreb, Croatia
²Centre of Excellence in Reproductive and Regenerative Medicine, School of Medicine, University of Zagreb, Croatia
³Institute of Physics, Zagreb, Croatia

Effects of magnesium (Mg²⁺) ions on the stability and structural properties of double-stranded DNA are vitally important for DNA folding and functional behavior. We use Fourier transform infrared spectroscopy and band shape analysis to explore in detail the vibrational signatures of DNA-magnesium interaction in the case when DNA charges are neutralized solely by Mg²⁺ cations, hereafter called MgDNA. Ion atmosphere has been controlled by the magnesium to phosphate molar concentration ratio r which varied between 0.0067 and 10. For r=0 we find that spectral features in the base region remain similar as in DNA, whereas changes in the backbone region indicate that the B conformation becomes fully stabilized. With increasing r a pronounced structural reshaping occurs in the phosphate backbone region indicating a blue shift of the asymmetric band, while the symmetric band does not show any displacement in frequency. The band shape analysis of overlapping peaks in the respective phosphate regions demonstrates that the number of constituent modes as well as their positions in frequency do not change, whereas their intensities and bandwidths display disparate changes. The results reflect a variety of local environments at the DNA backbone due to a heterogeneous ion atmosphere with randomly distributed magnesium ions and local patterns of hydrogen bonds which change with increasing r. Remarkably, after crowded r=10 ion atmosphere is depleted, Mg induced spectral changes vanish and structural features of MgDNA (r ≈ 0) are fully restored. Overall results strongly suggest that in MgDNA on highly hydrated thin films the hydrogen-base pairing remains preserved and that Mg²⁺ ions retain their mobility.

L38

Animal type I cryptochromes from migrating and non-migrating species. Are they alike and can they function as photomagnetoreceptors?

Nataliya Archipowa¹, Roger Jan Kutta²

¹ University of Regensburg, Institute for Biophysics and Physical Biochemistry, Germany
² University of Regensburg, Institute of Physical and Theoretical Chemistry, Germany

Cryptochromes (CRY) can be found mainly across the plant and animal kingdoms most of which are flavoprotein blue-light photosensors that regulate the circadian clock throughout nature, growth, and development in plants, and are suggested as the candidate photomagnetoreceptor in animals. They are closely structurally related to photolyases, the ancient light-dependent DNA repair enzymes, but have an additional C-terminal tail (CTT) which is responsible for mediating phototransduction.

 Insect type I CRY from Drosophila melanogaster (DmCRY) functions as the primary blue-light receptor that mediates circadian photo-entrainment. The mode of action starts with the absorption of a photon leading to reduction of the protein-bound FAD via consecutive electron transfer along a conserved tryptophan (Trp) tetrad forming a long-lived radical pair stabilised by charge separation over a long distance. As we have recently shown, the photoredox processes cause the known irreversible CTT release, Trp decomposition, and finally FAD dissociation from the protein pocket that subsequently induce signalling cascades. [1] Since detailed mechanistic studies were essentially performed on the model type I CRY from Drosophila, here, we investigate the generality of the mechanism for other type I CRY by studying two further representatives of this family, i.e. the mosquito Anopheles gambiae (AgCRY) and the migrating monarch butterfly Danaus plexippus (DpCRY), by means of time-resolved and stationary absorption spectroscopy combined with quantum chemical and molecular dynamics calculations. This study finds similarities and differences between migrating and non-migrating insects using type I CRY. As the monarch butterfly responds to magnetic fields [2] and as CRY are the suggested photomagnetoreceptors, their ability to undergo the widely believed radical pair mechanism [3-5] is discussed.

References

1. R. J. Kutta, N. Archipowa, and N. S. Scrutton, PCCP, 2018, 20, 28767-28776.
2. P.A. Guerra, R.J. Gegear and S.M. Reppert, Nat. Com., 2014, 5(1), 4164.
3. K. Schulten, C. E. Swenberg, and A. Weller, Z. für Phys. Chem., 1978, 111, 1-5.
4. T. Ritz, S. Adem, and K. Schulten, Biophys. J., 2000, 78(2), 707-718.
5. P. J. Hore and H. Mouritsen, Annu. Rev. Biophys., 2016, 45(1), 299-344.

L39

Role of molecular oxygen in the photochemical mechanism of the B₁₂-dependent photoreceptor protein CarH

Roger Jan Kutta¹, Nataliya Archipowa²

¹University of Regensburg, Institute of Physical and Theoretical Chemistry, Germany
²University of Regensburg, Institute for Biophysics and Physical Biochemistry, Germany

The coenzyme B₁₂-dependent protein, CarH, is one of two bacterial photoreceptors involved in the pathway of light-dependent carotenogenesis protecting the organism against highly reactive species such as singlet oxygen at high levels of light exposure. [1] On binding of coenzyme B₁₂ via one histidine the monomeric apoprotein of CarH forms tetramers in the dark that have a high affinity for binding to the operator DNA repressing transcription. [2] Upon illumination the CarH tetramer dissociates and releases from DNA, which enables transcription. [2] In the presence of molecular oxygen the mechanism upon photon absorption proceeds via a heterolytic cleavage of the sugar adenosyl moiety of B₁₂ resulting in several cob(III)alamin intermediates ending in a stable bis-histidine adduct. [3-4] It is postulated that bis-histidine adduct formation triggers tetramer dissociation. The quantum yield for this process is ca. 8.4% consistent with the need for carotenoid biosynthesis only under high light intensity conditions, when the bacteria that express CarH are likely to experience significant photo-oxidative stress. [2] Here, we expand the mechanistic picture by demonstrating that the reaction pathway is strongly dependent on the presence of molecular oxygen as the initially heterolytically cleaved cob(III)alamin intermediate undergoes formation of a cob(I)alamin ending in a cob(II)alamin and, thus, no bis-histidine species is observed. Our data suggest that CarH activation requires molecular oxygen and excess of light. We discuss the necessity of a bis-histidine adduct formation as a trigger for tetramer dissociation. This work provides a deeper mechanistic understanding of the emerging field of B₁₂ photobiology and will further guide the development of a new class of optogenetic tools for the control of gene expression.

References

1. M. Galbis-Martínez, S. Padmanabhan, F. Murillo, M. Elías-Arnanz, Journal of Bacteriology, 2012, 194(6), 1427-1436.
2. J. M. Ortiz-Guerrero, M. C. Polanco, F. J. Murillo, S. Padmanabhan, M. Elás-Arnanz, PNAS, 2011, 108(18), 7565-7570.
3. R. J. Kutta, S. J. O. Hardman, L. O. Johannissen, B. Bellina, H. L. Messiha, J. M. Ortiz-Guerrero, M. Elias-Arnanz, Padmanabhan, P. Barran, N. S. Scrutton, A. R. Jones, Nat. Com., 2015, 6(7907).
4. M. Jost, J. Fernandez-Zapata, M. C. Polanco, J. M. Ortiz-Guerrero, P. Y.-T. Chen, G. Kang, S. Padmanabhan, M. Elias-Arnanz, L. Drennan, Nature, 2015, 526, 536-541.

L40

Dynamic Single Molecule Analysis to assess biomolecular interactions across scales

Roman Renger, Nicholas Luzzietti

LUMICKS, Paalbergweg 3, 1105 AG Amsterdam

Biological processes occur across various time and length scales, connecting molecular details with collective, emerging phenomena. Detailed insights into the most fundamental building blocks of life such as individual proteins and nucleic acids provide essential information for understanding the fundamentals of intra- and extracellular organization. Direct, real-time observations of biomolecular interactions are required to validate and complete the current biological and biophysical models.

Single-molecule technologies offer an exciting opportunity to meet these challenges and enable researchers to study molecular function and activity in real-time. Here, we present our efforts towards further enabling discoveries in the field of biomolecular interactions by correlating optical tweezers and confocal fluorescence microscopy. We present several examples in which our technologies enhanced the understanding of membrane-less compartments, genome organization and biomolecular folding. Furthermore, we show how advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that has the ability to open up new avenues for breakthrough discoveries in biology and biophysics.

L41

Multiscale view on calcium phosphate formation – a route for rationalizing the design of novel bioimplant materials

Maja Dutour Sikirić

Ruđer Bošković Institute, Division of Physical Chemistry, Zagreb, HR

Calcium phosphates (CaPs) attract attention due to their role in biological and pathological mineralization, as well as in different industrial processes. At conditions close to physiological, the formation of calcium phosphates usually proceeds in two steps, the first of which is the formation of an amorphous precursor (ACP). The formation of ACP can be envisioned as a series of aggregation steps, Posners clusters aggregate to form primary particles that in turn grow and aggregate into chain-like aggregates that in contact with mother liquor grow and transform into crystalline phase. [1] Dynamic light scattering and laser scattering studies have revealed that the aggregation of ACP particles is a process which from the earliest stages simultaneously takes place at wide length scales, from nanometres to micrometres, resulting in a highly polydisperse precipitation system. [2]

Such behaviour offers the possibility to regulate ACP, and consequently crystalline CaP, formation by controlling ACP formation at the scale of interest. A possible way to achieve this is to precipitate CaPs in the presence of nanostructured materials.

Motivated by this, we investigated the influence of different materials, surfactant micelles [3], liposomes [4], TiO₂ nanomaterials of different dimensionality [5,6] and silver nanoparticles on CaP’s precipitation. It was shown that nanomaterials can exert a variety of the effects on the formation of CaPs and in certain cases the effect on the precipitation process observed at the microscale could be the result of different pathways at the nanoscale. As some of nanomaterials exhibit biological activities, this opens a pathway for biomimetic preparation of multifunctional bioimplant materials.

Acknowledgments

Financial support from Croatian Science Foundation, Grant HRZZ- IP-2018-01-1493 and Marie Sklodowska-Curie Grant Agreement No. 861138 is greatly acknowledged.

References

1. L. Brečević et al. Colloids Surf., 1987, 28, 301.
2. V. Čadež et al., Crystals, 2018, 8, 254.
3. A. Selmani et al. CrystEngComm, 2015, 17, 8529.
4. I. Erceg et al., Minerals, 2022, 12, 208.
5. I. Erceg et al., Colloids and Surfaces A, 2020, 593, 124615
6. I. Erceg et al., Nanomaterials, 2021, 11, 1523.

L42

An innate immune receptor sentinelling in synapses from development into aging

Maja Djurisic

Depts. Biology, Neurobiology, and Bio-X, Stanford University, Stanford, California, USA

Experience modulates synaptic strength throughout life via processes like Hebbian plasticity, meta-plasticity, and homeostatic plasticity. Molecular mechanisms underlying these processes are shared between different life stages, in spite of well-known decrease in capacity for plasticity and learning from young to old. Here we discuss an innate immune receptor, Paired Immunoglobulin-like receptor B (PirB; human LilrB2), a negative regulator of synaptic strength that operates in changing synaptic environments from development to aging. PirB is found in postsynaptic compartment of central excitatory synapses. In juvenile animals, in the wake of synapse overproduction throughout cortex, PirB mediates activity-dependent synaptic pruning that establishes mature synaptic density and synaptic strength. In young adults, PirB opposes excessive strengthening of excitatory synapses by recruiting NMDARs and endocannabinoid system, thus maintaining bi-directional Hebbian plasticity, and capacity for synaptic strengthening. Congruently, genetic removal of PirB results in loss of LTD, larger LTP, ~50% increase in synaptic density, and faster learning, in both juvenile and adult mice. With aging, levels of PirB/LilrB2 go up in the brain parenchyma. In addition, non-cognate PirB ligands increase in concentration with age and in disease, including Aß oligomer. High-affinity PirB-Aß interaction mediates loss of LTP in aged hippocampus, and is hypothesized to contribute to excessive weakening of synapses and eventual synaptic loss in Alzheimer’s disease by overactivation of PirB-dependent signaling. A picture emerges in which activation of PirB/LilrB2 cascade opposes synaptic strengthening, but to a degree and with outcomes shifting as available ligands change in the synaptic environment over the course of life.

L43

Inter-subunit Crosstalk Synergistically Regulates Allosteric Activation of Proapoptotic Serine Protease HtrA2

Aasna Parui^1,2, Vandana Mishra³, Subhankar Dutta¹, Prasenjit Bhaumik³, Kakoli Bose^1,2

¹Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Sector 22, Navi Mumbai – 410210, India
²Homi Bhabha National Institute, Anushaktinagar, Mumbai-400094, India
³Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India

High-temperature requirement protease A2 (HtrA2) is a complex trimeric mitochondrial serine protease that primarily acts as a key player in apoptosis. It belongs to a large family of multi-domain serine proteases (S1, chymotrypsin family) that is found to be conserved from prokaryotes to humans. Deregulation of this trimeric protease is associated with various diseases including neurodegenerative disorders and cancer thus making it an important therapeutic target. Despite the availability of structural details, the reports on HtrA2’s mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound on the role of regulatory PDZ domains in promoting synergistic coordination between HtrA2 subunits, we generated heterotrimeric HtrA2 variants comprising different numbers of PDZs and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual activity in a way that proffered a hypothesis advocating intermolecular allosteric crosstalk via PDZ domains in trimeric HtrA2 that has been established through an array of studies including fluorescence-labeled enzyme kinetics, protein engineering, and biochemical assays. Furthermore, structural (x-ray crystallography) and computational snapshots affirmed the role of PDZs in secondary structural element formation and coordinated reorganization of the N-terminal region and regulatory loops. Therefore, apart from providing cues for devising structure-guided therapeutic strategies, this study establishes a working model of complex allosteric regulation through a multifaceted trans-mediated cooperatively-shared energy landscape.

S01

Opening and short introduction of Medtech4 Europe project

Zoltán János Haász, Zsolt Pálmai

Abstract not available

S02

Human diagnostic and therapeutic treatment – the medtech SME action plan

Ákos Szépvölgyi

Abstract not available

S03

The medtech showroom of University of Pécs

András Lukács

Abstract not available

S04

Innovations and international relations in medical technology at the Semmelweiss University

tba

Abstract not available

S05

Questions and answers

Abstract not available

L44

Humoral factors of immunity as biophysical markers in personalized medicine of ALS

Pavle Andjus

University of Belgrade- Faculty of Biology, Institute for Physiology and Biochemistry “Jean Giaja”, Center for laser microscopy, Belgrade, Serbia

Our research was initially inspired by the experiments with ALS IgGs of Stanley Appel and Jozsef Engelhardt (e.g. PNAS 1991 88:647). Our studies with ALS IgGs comprised of diverse physiological phenomena in vitro: a) a rise in the frequency of postsynaptic currents of hippocampal neurons in culture (Andjus et al., 1996,1997); b) intracellular calcium mobilization in response to ALS IgGs on neurons and astroglia (Milošević et al.,2013); c) acute free radical release in the BV2 microglial cell line (Milošević et al., 2017); and d) an increase in the mobility of acidic vesicles (mostly endosomes and lysosomes) in primary cortical astrocytes (Stenovec et al., 2011).

Novel results with ALS IgGs will be presented demonstrating: a) Fc-fragment dependence of the Ca²⁺ response in astrocytes, b) the physicochemical/metabolic alterations in astrocytes vs microglia upon ALS IgG – treatment (FTIR using a synchrotron light source), c) ultrastructural changes in astrocytes (SEM and AFM studies).

Finally, we will present the rationale based on our results of ALS IgG effects on cell biophysics and the state of the art of an automated microfluidic lab-on-a-chip that offers better diagnostics and personalized patient stratification for ALS and other neurodegenerative diseases.

Acknowledgements

Supported by the EC H2020 grant #778405 and the Innovation Fund Republic of Serbia grant #5360.

L45

Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

Linda Waldherr¹, Verena Handl^1,2, Theresia Arbring Sjöström³, Tobias Abrahamsson³, Maria Seitanidou³, Marie Jakešová⁴, Sabine Erschen¹, Sophie Honeder⁵, Tamara Tomin⁵, Ruth Birner-Grünberger⁵, Nassim Ghaffari Tabrizi-Wizsy⁶, Stefan Ropele⁷, Muammer Üçal², Ute Schäfer², Silke Patz², Daniel Simon³, Rainer Schindl¹

¹Gottfried Schatz Research Center – Biophysics, Med. Univ. Graz
²Experimental Neurotraumatology, University Clinic of Neurosurgery, Med. Univ. Graz
³Laboratory of Organic Electronics, Linköping University
⁴CEITEC - Central European Institute of Technology, Brno University of Technology
⁵Institute of Chemical Technologies and Analytics, TU Wien
⁶Otto Loewi Research Center - Immunology and Pathophysiology, Med. Univ. Graz
⁷Division of General Neurology, Med. Univ. Graz

Poor delivery and systemic toxicity of many chemotherapeutic agents limit their therapeutic success in cancer treatment. Local chemotherapy approaches offer a new path to efficiently interfere with cancer growth and reduce tumor size, especially in the case of brain tumors.

We present miniature devices for iontronic drug delivery able to administer chemotherapeutics via electric control with high spatiotemporal precision.¹ Incorporated in these devices are anionic hyperbranched polyglycerol membranes (AHPGs), forming an ion selective matrix of multiple fixed negative charges.² Through this polymeric ion exchange membrane, drugs electromigrate in an electric field towards a target of choice. These bioelectronic devices, called chemotherapeutic ion pumps (chemoIPs) used for the delivery of chemotherapeutics and their performance were characterized and tested in different brain tumor models with increasing complexity (cell culture and different in vivo models). Treatment efficiency is analyzed based on cell death, tumor suppression and pharmacokinetics.

AHPG ion exchange membranes enable drug delivery with pmol*min^-1 delivery precision at currents in the nano-ampere range. The further application of this electrical and temporal control was shown in brain tumor cell culture, triggering the disintegration of targeted tumor spheroids among chemoIP treatment. Gem furthermore triggers cellular effects suitable for the application in the brain: it effectively kills brain tumor cells and is at the same time harmless to neurons and astrocytes. Additionally, we show that chemoIP treatment significantly reduces tumor growth and induces apoptotic tumor cell death in brain tumors grown on the chick chorioallantoic membrane (CAM) model.

The here exemplified electrically-driven drug delivery via chemoIPs is a drug administration method that can serve as basis for further implant development, which has the potential to increase the efficacy of chemotherapy due to highly-targeted and locally-controlled drug delivery.

References

1. Waldherr, L. & Seitanidou, M. Targeted Chemotherapy of Glioblastoma Spheroids with an Iontronic Pump. Adv. Mater. Technol. 2021, 6, 2001302.
2. Abrahamsson, T. Formation of Monolithic Ion-Selective Transport Media Based on Click Cross-Linked Hyperbranched Polyglycerol. Front Chem. 2019

L46

Intercellular nanomechanics in brain metastasis formation

Attila Gergely Végh¹, Katalin Csonti^1,2,3, Csilla Fazakas¹, Kinga Molnár¹, Imola Wilhelm¹, István A. Krizbai¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²Semilab Semiconductor Physics Laboratory Co. Ltd., Budapest, Hungary
³Doctoral School of Physics, University of Szeged, Szeged, Hungary

Brain metastasis formation is a complex and not entirely understood process, with the worst prognosis and the most feared complications. Proper homeostasis of the central nervous system relies on the structural and functional integrity of the neurovascular unit, which is formed by cerebral microvascular endothelium together with pericytes, astrocytes, neurons and the extracellular matrix. Orchestrated connections between all members are essential to sustain the physiological function of the neurovascular unit, namely formation and maintenance of the blood-brain barrier. Due to the lack of classical lymphatic drainage, the haematogenous route for invasion is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling tumor cells and the tightly connected layer of the endothelium. Pericytes are positioned at the duplication of the basement membrane of capillaries and are in intimate proximity with the endothelium, astrocytes and neurons, therefore they can be regarded as a second active defense line of the central nervous system against different solute and cellular elements circulating in the blood stream. The active and important role of pericytes in mechanobiology of the neurovascular unit is above discussion, however, mechanical roles and functions are not entirely elucidated.

Hereby we present our latest results on the mechanical properties of living brain endothelial cells and pericytes when they come into direct contact with breast adenocarcinoma cells. Nanomechanical monitoring of the interaction of living breast adenocarcinoma cells to endothelium and pericytes were compared and analysed by means of single cell force spectroscopy. Exploring the mechanobiology of endothelium and pericytes could not only lead to a better understanding of their function in the neurovascular unit but could also help to identify novel targets for the improvement of its barrier function.

Acknowledgements

This work was supported by the National Science Fund of Hungary OTKA FK128654. C. F. was supported by the János Bolyai Fellowship of the Hungarian Academy of Sciences BO/00213/19/8.

L47

Ruler or Stabilizer? The role of nebulin in thin filament length regulation.

Balázs Kiss

Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary

Nebulin is a giant, modular actin-binding protein that binds to the thin filament of skeletal muscle. Mutations affecting nebulin are the main contributors of nemaline myopathy, a progressive skeletal muscle disease characterized by a dramatic decrease in muscle strength. Nebulin consists of numerous tandem copies of 38.5 nm long “super repeats”. Exon deletion affecting just a part of super repeat #9 causes severe nemaline myopathy in humans, yet little is known about the interaction between nebulin and the thin filament.

We recently developed two novel mouse models in which nebulin super-repeats 9-11 are deleted or duplicated, respectively. Surprisingly, homozygous mice are viable, show Mendelian inheritance, and develop without significant muscle mass deficits. Our structural studies revealed that the length of thin filaments correlates with the length of nebulin and the degree of correlation depends on the rate of contraction of the muscle. In fast-twitch fibers, only a very short distal thin filament segment extends beyond the N-terminus of nebulin, whereas in slow-twitch fibers, the nebulin-free distal thin filament segment is much longer. We identified that the length of the distal segment is co-regulated by nebulin and leiomodin-2 (Lmod2). We propose that nebulin is responsible for providing short thin filaments required for rapid muscle contraction, whereas in slow fibers, nebulin contributes to economic muscle function together with Lmod2 by allowing longer thin filaments to appear.

The viability of genetically modified mice predicts the human applicability of exon-skipping in the therapy of nemaline myopathy. Thin filament stabilizers can contribute to higher levels of thin filament activation that gives rise to the higher speeds and force levels. The increased active force might be beneficial in the treatment of muscle weakness observed in (cardio)myopathies.

L48

Combined characterization of protein stability, size and aggregation

Pawel Kania

NanoTemper Technologies

In NanoTemper we are constantly working on improving and extending the capabilities of our innovative solutions. Over the past few years, the Prometheus system, based on nanoDSF technology, has become the new gold standard in protein stability assessment, proving its specificity, sensitivity, accuracy and ease of use.  

Now, in response to the growing demands of the research community, we are pleased to present our newest release - Prometheus Panta. For the first time parallel particle sizing, thermal unfolding, and aggregation results collected throughout an entire thermal ramp will give a researchers completely new perspective about stability attributes at the domain level of analyzed protein.

Multi-parameter stability characterization is what Prometheus Panta is all about. With addition of innovative DLS optics, this unique solution allows for simultaneous measurement of T_m, r_H PDI and many more crucial parameters, bringing new insights into your stability characterization. With this outstanding improvement, Prometheus keeps the status of gold standard for easy, rapid and accurate analysis and characterization protein, with applications in membrane protein research, protein engineering, formulation development and quality control.

P02

Trimeric photosystem I facilitates efficient energy transfer from the phycobilisome antenna in cyanobacteria

Parveen Akhtar, Avratanu Biswas, Fanny Balog-Vig, László Kovács, Petar H. Lambrev

Institute of Plant Biology, Biological Research Centre, Szeged, Hungary

The light reactions of photosynthesis are carried out by protein complexes in the thylakoid membranes, such as the two photosystems. Plants and eukaryotic algae have specialized membrane-bound light-harvesting antenna complexes that increase the absorption cross-section of the photosystems. In cyanobacteria, the main light-harvesting function is carried out by the phycobilisomes - large water-soluble protein complexes attached peripherally to the thylakoid membrane, containing pigment-binding phycobiliproteins such as phycocyanin and allophycocyanin. Unlike its eukaryotic counterpart, photosystem I (PSI) is trimeric in many cyanobacterial species, and the physiological significance of this is not well understood. Here we compared the composition and light-harvesting function of phycobilisomes in cells of Synechocystis sp. PCC 6803 (WT), which has primarily trimeric PSI, and two mutant strains, ΔpsaL and ΔFIJL, which contain only monomeric PSI. Both strains with monomeric PSI accumulated significantly more allophycocyanin per chlorophyll, indicating higher abundance of phycobilisomes. On the other hand, a higher phycocyanin:allophycocyanin ratio in WT suggests larger phycobilisomes or the presence of phycobilisomes without allophycocyanin (CpcL-type), that are not assembled in cells with monomeric PSI. Steady-state and time-resolved fluorescence spectroscopy at room temperature and 77 K revealed that PSII receives more energy from the phycobilisomes at the expense of PSI in cells with monomeric PSI, regardless of the presence of PsaF. Taken together, these results show that the oligomeric state of PSI has an impact on the excitation energy flow in Synechocystis, which might be one physiological and evolutionary advantage of trimeric PSI in cyanobacteria. More details can be found in the article published in Plant Physiolgy, doi:10.1093/plphys/kiac130.

P04

Label-free tracking of cell adhesion kinetics as a function of cell surface density

Anna Balogh^1,2, Kinga Dóra Kovács^1,2, Inna Székács¹, Robert Horvath¹

¹Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research
²Eötvös Lóránd University, Department of Biological Physics, Budapest, Hungary

Most tissue cells can not survive for more than a few hours without adherence - adhesion is also an important factor  in solid malignancies. Thus, if we were able to prevent their adhesion, metastasis could be prevented and these tumor cells would die. Understanding and modelling cell adhesion mechanics is an important tool in our hands to inhibit these processes. [1, 2]

In the present work, we investigated the extent to which the adhesion kinetics of cells are affected by the surface density of the cells, which display the integrin specific RGD (arginine–glycine–aspartic acid) tripeptide motif. The high resolution kinetic data used for analysis was recorded by a surface sensitive, label-free, resonant waveguide grating based optical biosensor. Using the recorded overall signals of 50-9000 cells, and then normalizing with the cell number we were able to obtain the signal characteristics of the averaged cell.

Based on the obtained kinetic data we determined the association and dissociation rates of the integrins and their ligands, the recruitment rate of the integrins to the active zone, the maximum possible surface concentration of the integrins in the adhesion zone, and the two dimensional kinetic dissociation constant. [2]

As a conclusion, our experimental results and data analysis demonstrated that when cells are present at an ideal surface concentration, they help each other to adhere: contact enhancement can be observed. The kinetic data obtained during the analysis also support these results. Our results potentially open the way for further analysis of the kinetic data obtained from the adhering cells.

Acknowledgements

This work was supported by the Lendület (HAS) research program, the National Research, Development and Innovation Office of Hungary (VEKOP, ELKH topic-fund, Élvonal KKP_19 and KH grants, TKP2022-EGA-04 program financed from the NRDI Fund).

References

1. Sudhakar A. History of Cancer, Ancient and Modern Treatment Methods. J Cancer Sci Ther. 2009 Dec 1;1(2):1-4. doi: 10.4172/1948-5956.100000e2.
2. Harvey Lodish, Arnold Berk, Paul Matsudaira, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, Lawrence Zipursky, James Darnell. Molecular Cell Biology.
3. Kanyo, N., Kovacs, K.D., Saftics, A. et al. Glycocalyx regulates the strength and kinetics of cancer cell adhesion revealed by biophysical models based on high resolution label-free optical data. Sci Rep 10, 22422 (2020). https://doi.org/10.1038/s41598-020-80033-6

P06

Purification and characterization of bacterial actin Thermotoga maritima MreB: a novel approach

Beáta Longauer^1,2, András Lukács^1,2, Emőke Bódis¹, Miklós Nyitrai^1,2, Szilvia Barkó^1,2

¹Department of Biophysics, Medical School, University of Pécs, Hungary
²Nuclear Mitochondrial Interactions Research Group, Eötvös Loránd Research Network (ELKH), University of Pécs, Hungary

Most bacteria have homologue of the major eukaryotic cytoskeletal protein actin, called MreB. It is a key player in organization of elongation of cells, formation of bacterial cell wall and partitioning of DNA. MreB is present in almost all rod-shaped bacteria and fulfils an important role in maintenance of cell shape and viability. MreB appears to be essential in all bacteria studied so far.

One of the major problems of in vitro description of MreB is the weak solubility and functionality of isolated MreB protein. As a novel approach we have established a new method for expression and purification of soluble and fully functioning MreB. In our assays ArcticExpress (DE3) competent cells were used to express MreB from Thermotoga maritima (Tm-MreB). This system intrinsically contains bacterial chaperone proteins which can help to achieve native conformation of MreB avoiding insolubility of protein and formation of inclusion bodies. As compared with other bacterial expression systems this method can increase the amount of native Tm-MreB with approximately 95%.

As a result it became possible to fulfil various spectroscopic investigation assays on Tm-MreB which is almost unique in the in vitro MreB literature. We have described the heat stability of Tm-MreB using the signal of its tryptophan residue.

Using a fluorescent non-hydrolysing ATP analog TNP-ATP we have described the binding of nucleotide to Tm-MreB. It was also concluded that type of nucleotide affects the polymerization rate but not the critical concentration of MreB. Our phosphate release assay showed that MreB hydrolyses ATP already in salt-free environment and as compared to eukaryotic actin the rate of hydrolysis is elevated significantly.

Therefore, it can be concluded that this purification method makes possible to investigate prokaryotic actin from other species in a molecular level and gives opportunity to describe working mechanisms of bacterial cell wall complexes.

P08

Origin of the isotropic lipid phases in plant thylakoid and Photosystem II membranes

Kinga Böde^1,2, Ottó Zsiros¹, Ondřej Dlouhý³, Uroš Javornik⁴, Avratanu Biswas^1,2, Primož Šket⁴, Janez Plavec^4,5,6, Vladimír Špunda³, Petar H Lambrev¹, Bettina Ughy¹, Győző Garab^1,3

¹Biological Research Centre, Szeged, Hungary
²Doctoral School of Biology, University of Szeged, Szeged, Hungary
³Faculty of Science, University of Ostrava, Ostrava, Czech Republic
⁴National Institute of Chemistry, Ljubljana, Slovenia
⁵EN-FIST Center of Excellence, Ljubljana, Slovenia
⁶Faculty of University of Ljubljana, Ljubljana, Slovenia

Functional plant thylakoid membranes (TMs), in addition to the bilayer, contain two isotropic lipid phases and an inverted hexagonal (H_II) phase. The non-bilayer propensity of bulk TM lipids have been proposed to safe-guard the lipid homeostasis of TMs; further, an isotropic phase has been shown to arise from VDE:lipid assemblies (VDE is a luminal photoprotective enzyme) [1]. Effects of proteases and lipases on the lipid polymorphism of TMs have revealed that the H_II phase originates from lipids encapsulating stroma-side proteins or polypeptides, and suggested that the isotropic phases are to be found in domains outside the protein-rich regions of TM vesicles; they might be involved in the fusion of membranes and thus the self-assembly of the highly organized TM network [2].

The aims of the present study are (i) to substantiate the notion concerning the role of (an) the isotropic lipid phase(s) in the fret formation of TMs, and (ii) to scrutinize the conditions of their lipid homeostasis. We capitalize on the fact that wheat-germ lipase (WGL) selectively eliminates the ³¹P-NMR-spectroscopy detectable isotropic phases while exerting no effect on the bilayer and H_II phases and does not perturb the structure and function of the photosynthetic machinery. Surprisingly, Photosystem II (BBY) membrane particles displayed no lamellar and H_II phases; nevertheless, the WGL-susceptibility of BBY was similar to TMs. Our currently available data, obtained from sucrose gradient centrifugation experiments and spectroscopic measurements (³¹P-NMR, linear and circular dichroism, FTIR, fast chlorophyll fluorescence transients) strongly suggest that (i) WGL is capable of disintegrating intact TMs and the large sheets of BBY membranes, and (ii) TMs operate at the percolation threshold of their bulk lipid phase, which may have consequences on the membrane energization and the utilization of the proton-motive force.

References

1. Garab G. et al. 2022 Progr Lipid Res
2. Dlouhý et al. 2022 RBC2022

P10

Investigation of the role of the neuropeptide PACAP in the regulation of the cytoskeletal system

Péter Bukovics¹, Roland Gábor Vékony^1,3, Andrea Tamás², Beáta Bugyi¹

¹University of Pécs, Medical School, Department of Biophysics, Pécs, Hungary
²University of Pécs, Medical School, Department of Anatomy, Pécs, Hungary
³University of Pécs, Faculty of Pharmacy, TDK Student, Pécs, Hungary

The behaviour of actin-regulating proteins and the pattern of their presence can be characteristic in various clinical diseases. Modification of the quality and quantity of regulatory proteins can have a significant effect on the cytoskeletal and regenerative regulation underlying pathological changes. Pituitary adenylate cyclase activating polypeptide (PACAP) is a cytoprotective neuropeptide that is abundant in the nervous system and endocrine organs and plays an important role in the regulation of differentiation and migration of neurons, caused by actin. Our goal is to identify the cytoskeletal role of PACAP as a potential actin-regulating polypeptide, to investigate its effect on the incorporation of actin monomers into filaments, and to understand the underlying regulatory mechanisms, dynamics and interactions.

In our experiments, we examined two biologically active forms of the peptide (PACAP27 and PACAP38) and another fragment (PACAP6-38). The binding capabilities of peptides to actin monomers was measured by fluorescence spectroscopic methods and by steady-state anisotropy measurements. Besides, functional polymerization tests were also performed to explore the coordination of the underlying structure-function coordination.

In our studies, it was demonstrated that, in contrast to PACAP27, PACAP38 and 6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, resulting in almost identical dissociation equilibrium constants. We found that PACAP27 binds weakly to G-actin, while a robust actin-PACAP interaction was observed in case of the studied two other forms. Our functional results showed that PACAP27 did not affect actin polymerization, while PACAP38 and 6-38 significantly accelerated the kinetics of actin incorporation.

In our study, we demonstrated that PACAP38 and 6-38 bind strongly to G-actin and play an important role in the polymerization of actin molecules.

P12

Mapping the conformational changes of small GTPase Ran

Janka Czigleczki¹, Pedro Túlio de Resende Lara², Balint Dudas^3,4, David Perahia⁴, Hyunbum Jang⁵, Ruth Nussinov^5,6, Erika Balog¹

¹Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
²Federal University of ABC, Laboratório de Biologia Computacional e Bioinformática, São Paulo Brasil
³Inserm U1268 MCTR, CiTCoM UMR 8038 CNRS - University of Paris, Paris, France
⁴Ecole Normale Supérieure Paris-Saclay, Laboratoire de Biologie et Pharmacologie Appliquée, France
⁵Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD, United States
⁶Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Ran (RAs-related nuclear) belongs to the Ras superfamily of small GTPases. It is the main regulator of nucleo-cytoplasmic import and export through the nuclear pore complex (NPC) and controls cell cycle progression by the regulation of microtubule polymerization and mitotic spindle formation. Like other small GTPases, it operates as a molecular switch by cycling between GDP-bound cytosolic inactive- and GTP-bound nucleus-located active state. Since deregulation of Ran is linked to numerous cancers from the stage of cancer initiation to metastasis, understanding the complexity of its interaction, especially the regulatory mechanism, is critical for drug discovery.

The full-length structure of RanGDP, is composed of a G-domain (GTP binding domain) and a C-terminus which – unlike other GTPases – terminates in a unique acidic (DEDDDL) tail.

• the G-domain – as in other GTPases – contains the phospathe-binding loop (P-loop) that, together with the Mg²⁺ ion, stabilizes the nucleotide binding; and two critical motifs, switch I and II, which upon the nucleotide exchange undergo a major conformational change allowing to interact with the downstream partners.
• crystal structures show that in the RanGDP form, the C-terminal is wrapped around the G-domain, but the standalone structure of RanGTP hasn’t been determined.

It is hypothesized that upon GTP binding not only switch I and II undergo a major conformational change, but also ‘the C-terminal switch’. Experimentally this hypothesis could not have been tested, since the full-length RanGTP structure could not have been determined.

Starting from the experimentally determined structures and using different methods of all-atom simulations: Molecular Dynamics with excited Normal Modes (MDeNM - which proved to be capable of mapping large-scale conformational changes) and Molecular Dynamics (MD) we present the dynamical behaviour of the inactive and active form of Ran, and the role of the C-terminal switch in the activation process.

P14

A Novel Approach in Heart-Rate-Variability Analysis Based on Modified Poincaré Plots

András Búzás¹, Tamás Horváth², András Dér¹

¹Biological Research Centre, Institute of Biophysics, 6726 Szeged, Hungary
²Centre of Cardiology, Medical Faculty, University of Szeged, 6725 Szeged, Hungary

Heart-rate variability (HRV), measured by the fluctuation of beat-to-beat intervals, has been growingly considered the most important hallmark of heart rate (HR) time series. The HRV can be characterized by various statistical measures both in the time and frequency domains, or by nonlinear methods. During the past decades, an overwhelming amount of HRV data has been piled up in the research community, but the individual results are difficult to reconcile due to the different measuring conditions and the usually HR-dependent statistical HRV-parameters applied. Moreover, the precise HR-dependence of HRV parameters is not known.

Using data gathered by a wearable sensor of combined heart-rate and actigraphy modalities, here, we introduce a novel descriptor of HRV, based on a modified Poincaré plot of 24-h RR-recordings. We show that there exists a biexponential HRV versus HR ”master” curve (”M-curve”) that is highly conserved for a healthy individual on short and medium terms (on the hours to months scale). At the same time, we reveal how this curve is related to age in the case of healthy people, and establish alterations of the M-curves of heart-attack patients. A stochastic neuron model accounting for the observed phenomena is also elaborated, in order to facilitate physiological interpretation of HRV data.

Our novel evaluation procedure applied on the time series of interbeat intervals allows the description of the HRV(HR) function with unprecedented precision. To utilize the full strength of the method, we suggest a 24-hour-long registration period under natural, daily-routine circumstances (i.e., no special measuring conditions are required). By establishing a patient’s M-curve, it is possible to monitor the development of his/her status over an extended period of time.

 On these grounds, the new method is suggested to be used as a competent tool in future HRV analyses for both clinical and training applications, as well as for everyday health promotion.

P16

HaloTag technology in directed evolution of haloalkane dehalogenase

Veronika Dzurillová¹, Ľuboš Ambro², Peter Artimovič¹, Kristína Fecková¹, Erik Sedlák²

¹Department of Biophysics, Faculty of Science, P.J. Šafárik University, Košice, Slovakia
²Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University, Košice, Slovakia

Haloalkane dehalogenases (HLDs) represent a group of hydrolases enabling to cleave off carbon-halogen bond by which facilitate the conversion of toxic halogenated hydrocarbons to non-hazardous alcohols. The dehalogenation catalysed by these enzymes is a reaction of great industrial importance. HLDs are used in a wide variety of applications in biocatalysis, decontamination, biosensing or cellular imaging. Its considerable application potential is highlighted in the field of bioremediation of eminent environmental pollutants such as 1,2-dichloroethane or 1,2,3-trichloropropan. However, the practical use of HLDs has several limitations, such as limited stability, specificity and insufficient catalytic efficiency of natural HLDs. We believe that the latter two properties of enzymes can be modified/improved by using approach of directed protein evolution such as ribosome display.

The ribosome display enables to perform robust selection from protein libraries containing up to 10¹² members per selection round. In order to obtain enrichment of improved HLD, we implemented the HaloTag technology for capturing of protein library against immobilized biotinylated chloroalkane. The stable covalent bond between displayed enzyme and substrate (HaloTag Ligand) was mediated through formation of alkyl-enzyme intermediate based on underlying principle of HaloTag technology. DhaA variants from randomised libraries were successfully selected upon several consecutive rounds of ribosome display. Based on following sequence analysis, we were able to identify several hot-spot mutations of which the influence on the enzyme activity are presently analyzed. Our results suggest the feasibility using ribosome display in combination with HaloTag technology in evolution-directed engineering of HLDs.

P18

Designing of LOV2 protein into efficient photosensitizer

Kristína Felčíková¹, Veronika Dzurillová¹, Andrej Hovan¹, Gregor Bánó¹, Tibor Kožár², Erik Sedlák²

¹Department of Biophysics, Faculty of Science, P.J. Šafárik University, Jesenná 5, 040 01 Košice, Slovakia
²Center for Interdisciplinary Biosciences, Technology and Innovation Park, P.J. Šafárik University, Jesenná 5, 040 01 Košice, Slovakia

Photodynamic therapy is a treatment that requires interactions between three independent factors: photosensitizer, oxygen and light. Photosensitizer is a chemical compound that can be promoted to an excited state upon absorption of light and undergoes intersystem crossing with oxygen is producing singlet oxygen (¹O₂). Molecule of ¹O₂ is highly cytotoxic, rapidly attacking any organic compounds it encounters.

Currently, large effort has been invested into a design of protein-based photosensitizer containing flavin mononucleotide (FMN). FMN belongs to the group of effective photosensitizers with high value of quantum yield of ¹O₂ production. However, triplet excited state of FMN encapsulated in protein is efficiently quenched by surrounding protein matrix, diminishing thus ¹O₂ production. Light-Oxygen-Voltage (LOV) domain 2 from Avena sativa (AsLOV2) belongs to the flavoproteins, which are intensively studied as potential efficient photosensitizers. The general approach to reach this goal relies on a weakening interaction of FMN with the protein matrix.

We propose different approach that relies on the FMN dissociation caused by irradiation-induced oxidation of amino acids at the binding site. The important part was to suggest such mutation that upon protein irradiation by light and subsequent oxidation of the mutated amino acid would increase its volume and triggers the cofactor dissociation from the protein without destabilization of the protein native structure. Molecular dynamics simulations of suggested mutants were verified experimentally and they indeed indicate increased efficiency of a production of ¹O₂ by certain mutants, demonstrated by ¹O₂ phosphorescence.

P20

The impact of fluorophore conjugation on the functions of antibodies

Tímea Hajdu, Gábor Mocsár, Péter Nagy

Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Antibodies conjugated with fluorescent dye molecules are frequently used in molecular biological investigations. In most cases the fluorophores are attached to the lysine side chains of the IgG in a nonspecific manner. The presence of the fluorescent dye has been proved to influence the epitope-binding efficacy of the antibody. We aimed to reveal how the functions of the IgG and its certain domains are altered depending on the amount of dye molecules present on its surface.

Degree of labeling (DOL) represents the average number of dye molecules on an antibody molecule. Trastuzumab, an anti-HER2 antibody and W6/32, a mouse IgG against MHC-I were examined after labeling them with AlexaFluor-546 or -647 dyes. We performed flow cytometric binding experiments to study the function of the epitope-binding domain and by applying Fc-specific capture antibodies we could investigate the antigenicity as a function of the DOL. Binding antibodies to Protein-G characterizes CH1, while binding to Protein-A and Fc receptors characterizes the function of the linker region between CH2 and CH3. Time resolved anisotropy measurements were carried out to study the impact of the DOL on the motion of the heavy and light chain.

We found that not only the epitope-binding domain, but also the functions of other antibody domains are influenced by fluorophore conjugation. Antibodies with high degree of labeling bound less with their Fc regions to the examined antigens, suggesting that fluorophore conjugation leads to a functional weakening spread to the whole IgG. Time resolved anisotropy measurements pointed out that the speed of the wagging motion of the Fab domain increases as a function of the DOL. This finding could partly explain the observed weakening in antigen binding, which requires a more rigid antibody conformation.

P22

Laser emission of dye stained biomolecules for the detection of specific molecular structures linked with genetic and neurodegenerative diseases

Piotr Hanczyc

Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland

To successfully treat genetic and neurodegenerative diseases there is an emerging need for the early detection of disease signs long before the cognitive symptoms occur [1].

In the case of Alzheimers disease, the main perpetrators are believed to be small mobile protein aggregate forms called amyloid oligomers but statistical data suggests also that the propensity for disease development rises significantly due to the genetic burden. It indicates that degenerative processes occur at the sub-cellular level during the protein and DNA interactions when their polymorphic structure is changing over time.

 A common and widespread method to detect specific biomolecular structures is fluorescence. For that purpose, biomolecules are stained with organic dyes. However, fluorescence has limitations, for example in detecting transient states of early-stage protein aggregates or local distortion of DNA helix [1, 2].

To boost the fluorescence sensitivity one can amplify light in the process of stimulated emission. Lasing in dye-stained amyloid proteins and DNA G-quadruplexes has orders higher detection sensitivity than standard fluorescence, thus it helps to reveal critical molecular structures involved in disease development.

Lasing was detected in various DNA structures, in different protein aggregates, in tissues including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant was seeded with the patient’s fluid [3]. By monitoring laser emission a remarkable recognition sensitivity to early forms of biomolecules linked with diseases can be achieved. Thus, in contrast to fluorescence, lasing can be used to detect and differentiate specific biomolecular structures and evaluate the risk levels of neurodegenerative diseases in potential patients before the clinical symptoms occur so that patients can receive rapid information about their health condition and have a better outcome in the therapy.

References

1. Hanczyc, P., & Fita, P. (2021). Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase. ACS Photonics, 8(9), 2598-2609.
2. Hanczyc, P., Rajchel-Mieldzioć, P., Feng, B., & Fita, P. (2021). Identification of thioflavin T binding modes to DNA: a structure-specific molecular probe for lasing applications. The Journal of Physical Chemistry Letters, 12(22), 5436-5442.
3. Hanczyc, P., Słota, P., Radzewicz, C., & Fita, P. (2022). Two-photon excited lasing for detection of amyloids in brain tissue. Journal of Photochemistry and Photobiology B: Biology, 112392.

P24

Investigation of the structural ensemble and distributed kinetics of NADH cofactor in different solvation environments: a simulation study

János Horváth^1,2, András Dér¹, Zoltán Násztor¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²Doctoral School of Physics, University of Szeged, Szeged, Hungary

Nicotinamide Adenine Dinucleotide (NAD) and its reduced form, NADH are important cofactors of central roles in the metabolism of living cells. NAD and NADH participate in electron transfer reactions. For instance, the phosphorylation of adenosine diphosphate (ADP) in oxidative phosphorylation is coupled to the oxidation of the principal electron donor, NADH. Thus, insight into the kinetics and protein interactions of NADH is fundamental to the understanding of cellular energy metabolism. However, in spite of the importance of these cofactors, answers for some basic questions, such as whether their fluorescent decay shows distributed kinetics or not, are still disputed. In the present work, we address this question utilizing simulation tools.

The parameterization of the NADH molecule was done using Gaussian09 and the AMBER package, utilizing the hybrid density functional B3LYP method in combination with the aug-cc-PVDZ basis set. Aside to the pure water system, a 20/80 v/v-percent water-methanol mixture was also considered on the course of molecular dynamics (MD) runs. Starting from the final structure of the energy minimization, 1-μs long NPT simulations were performed.

In order to characterize the open and closed structures of the NADH cofactor, the distance between the centers of mass (COMs) of the nicotinamide ring and the adenine ring was calculated. In the next step, a normalization with the total number of frames was done, providing a probability distribution. The obtained distributions suggest that distributed kinetics is a viable description for NADH. Moreover, in accordance with experimental facts, the presence of methanol molecules in the solution dramatically changes the structural ensemble of NADH. Increasing the methanol concentration of the mixture, closed structures turn out to be less frequent, but conformations with greater ring-distances become significant. The findings are interpreted in the framework of the theory of Hofmeister effects.

P26

Examination of the interaction of graphene oxide sheets with human serum albumin, utilizing computational tools

János Horváth^1,2, Ferenc Bogár³, András Dér¹, Zoltán Násztor¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²Doctoral School of Physics, University of Szeged, Szeged, Hungary
³MTA-SZTE Biomimetic Systems Research Group, University of Szeged, Szeged, Hungary

Carbon-based functional nanomaterials have attracted immense scientific interest due to their extraordinary physical and chemical properties offering a huge potential in a diverse range of applications such as energy storage, nanoelectronic devices, and biomedicine, including antibacterial materials, drug delivery and tissue engineering. Graphene Oxide (GO) is one of the most important chemical derivatives of graphene-based nanomaterials. The interactions of GO, a 2-dimensional nanomaterial of large, flat hydrophobic basal surface and hydrophilic edges, with biological macromolecules, are of key importance for the development of novel nanomaterials for biomedical applications. The molecular interactions of GO with plasma proteins, in particular with bovine and human serum albumin (HSA), have been studied previously with experimental tools, in respect to their dependence on pH, ionic strength, temperature etc.

In order to rationalize the experimental results, we utilized Protein Swarm Optimization (PSO) based calculations to model the binding of HSA in its partially unfolded and intact form to GO, corresponding to the low- and high-ionic-strength cases, respectively. In our study, we used this method to „dock” the HSA in its native conformation, as well as the coupled D1-D2 and the separated D3 domains to the GO surface. The binding poses obtained this way were refined by molecular dynamics simulations, and the binding affinity was estimated with the MM/GBSA method.

The PSO algorithm together with classical molecular dynamics can be applied to address as large systems as HSA and a correspondingly large GO sheet, providing atomic-level insight to the details of interactions between GO and HSA. Experimental calorimetry data was supported by these molecular modelling calculations, implying different modes of GO interaction with albumin at low ionic strength, while strongly suggesting that GO does not exhibit toxic effect via albumin in the blood flow.

P28

Differences in the ordering of the phospholipid bilayer of the cytoplasmic membrane and in the transmembrane potential induced by the resistance of the yeast Candida auris

Juraj Jacko^1,2, M. Morvová Jr¹, N. Toth-Hervay², Y. Gbelská², L. Šikurová¹

¹Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynská dolina F1, 84248 Bratislava, Slovakia
²Faculty of Natural sciences, Comenius University, Ilkovičova 6, 84248 Bratislava, Slovakia

The growing level of yeast resistance to azole antifungals is a worldwide medical problem. At present, however, the increasing number of yeast strains resistant to azole antifungals present complications in the treatment of yeast infections. Candida infections are the most common cause of human opportunistic yeast infections worldwide. Candida auris is the 3rd most common cause of the yeast disease in the world [1].

The plasma membrane is important cell part, and it plays a notable role in drug resistance mechanisms. The plasma membrane is the first line of cell defence against changes in external environment, thus its integrity and functionality are of utmost importance [2]. Changes in membrane composition leads to changes in azole antifungals susceptibility [3]. The aim of this work is to study the difference in the ordering of the phospholipid bilayer of the cytoplasmic membrane and transmembrane potential between sensitive and resistant Candida auris yeast.

Differences in membrane fluidity was studied by measuring of fluorescence anisotropy of fluorescent probes DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (4-trimethyl-amino-1,6-diphenyl-1,3,5-hexatriene). Differences in yeast transmembrane potential was studied by monitoring shift in the position of emission maximum (∆λmax) in the fluorescence spectrum of the DiS-C3(3) probe in cells.

The results of our experiments show that there is significant difference in membrane fluidity in hydrophobic part of cytoplasmatic membrane (0,269 ± 0,01 for sensitive and 0,293 ± 0,001 for resistant strain), but no difference in hydrophilic part (0,356 ± 0,002 for sensitive and 0,357 ± 0,001 for resistant strain). Significant difference is on the ∆λmax value (2,35 ± 0,21 nm for sensitive and 6,25 ± 0,3 nm for resistant strain), that shows that the plasma membrane of the sensitive strain is depolarized compared to the resistant strain.

Acknowledgement

This study was supported by grants APVV-SK-BY-RD-19-0019, KEGA 041UK-4/2020 and UK/126/2022.

References

1. https://www.cdc.gov/fungal/candida-auris/candida-auris-qanda.html, online, [17.3.2022]
2. Toth Hervay, N. et al, 2015. Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces lactis. Canadian journal of microbiology. 61(4), 273-279
3. VAN DEN HAZEL, H. et al, 1999. J. of Biological Chemistry. 274(4), 1934-1941.

P30

Elastic microtools for the optical manipulation of single cells

Lóránd Kelemen¹, Gaszton Vizsnyiczai¹, Tamás Gergely Iványi¹, Botond Nemes¹, Jana Kubackova², Gregor Bánó³, Zoltán Tomori²

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²Slovakian Academy of Sciences, Institute of Experimental Physics, Kosice, Slovakia
³Pavol Jozef Šafárik University, Institute of Physics, Kosice, Slovakia

Investigation of single cells often requires their manipulation with micrometer accuracy and precise temporal control. The recent years witnessed the evolution of microtools designed for specific tasks performed on single cells such as translation, rotation, deformation or even culturing. Laser microfabrication is a preferred method for the preparation of 3D microtools; this method is capable of producing structures with sub-micrometer features and size up to hundreds of micrometers. The possibility of being able to change the shape of these microtools at will can substantially extend the range of tasks they can perform compared to the only rigid ones.

We introduce a family of deformable microtools made of the photopolymer Ormocomp to be used in microfluidic environment. These tools are deformed with optical tweezers via the elastic elements are introduced into the otherwise rigid structure. It is their elasticity that unnecessitates any chemical modification for taking hold of single cells and being able to release them at any time. The elastic force is also sufficient to keep the cells and the structures together without the need of the optical tweezers. The diversity of the achievable cell manipulation schemes are demonstrated with three types of elastic structures. First, a mobile cage is presented that can engulf and carry cells without applying a squeezing force on them; this tool is ideal for collecting selected cells from a mixture. The second tool grabs the cells firmly minimizing their fluctuation and enabling their precise microscopic observation from any preferred directions. The third is a pair of tools that allows for the realization of cell-to-cell interaction; one tool mounts a cell to the substrate while the other carries another cell and attaches it to the mounted one.

Acknowledgements

This work was supported by the joint project of Slovak and Hungarian Academies of Sciences (no. NKM-53/2021) and by the Slovak Research and Development Agency (grant no. APVV-21-0333).

P32

Determination of Surface Charge Properties of Cell Monolayers Using a Lab-on-a-Chip Tool

András Kincses¹, Ana R. Santa-Maria¹, Fruzsina R. Walter^1,2, László Dér¹, Judit Vígh¹, Sándor Valkai¹, Mária A. Deli¹, András Dér¹

¹Institute of Biophysics, Biological Research Centre, Szeged, Hungary
²Department of Cell Biology and Molecular Medicine, University of Szeged, Hungary

Lab-on-a-chip (LOC) devices became popular tools for modelling biological barriers in the last decade. Controlled conditions, integrated electrodes and the possibility of the fluid flow in the microfluidic channels provide ideal circumstances for the investigation of physiological functions, transport mechanisms and pathologies. The measurement of the physical and physicochemical parameters, the trans-endothelial/epithelial electric resistance, the electrical impedance and the passive permeability, give information about the barrier integrity.

In the case of barrier forming cells, the luminal surface has a high surface charge density, the blood-brain barrier has the highest among all the barriers. Unfortunately, these studies were performed on cell suspensions via laser-Doppler velocimetry (LDv). There were no reported methods to measure the zeta potential of confluent cell monolayers, so the exact effects of the surface charge on the barrier integrity are unclear. We decided to upgrade our versatile LOC device to be capable of the measurement of a transient streaming potential signal. We proved with model simulations and control experiments by LDv that the amplitude of the transient signal is proportional to the zeta potential of the cell monolayer.

P34

Single-molecule mechanics of porphyrin-DNA binding

Balázs Kretzer, Gabriella Csík, Levente Herényi, Bálint Kiss, Hedvig Tordai, Miklós Kellermayer

Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary

Porphyrins and their derivatives have been the subject of numerous studies due to their role in photodynamic therapy. Cationic derivatives — such as tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP), the subject of the current research — have, in addition, a broad spectrum of antimicrobial activity. TMPYP has strong affinity for DNA, and it has also been investigated for its properties to interact with G-quadruplexes, which may increase its role in cancer treatment. It is highly likely that the mechanical status of DNA has a significant influence on TMPYP binding (eg., intercalation or groove binding). Here we explored TMPYP-DNA binding at the single molecule level while adjusting the conditions that may have an impact on the binding process: TMPYP concentration, ionic strength, DNA stretch and stretch rate. Thus, conclusions may be drawn regarding both the structural changes of DNA and the dynamics of TMPYP binding.

Experiments were carried out on λ-phage DNA by using an optical tweezers instrument combined with microfluidics. Measurements were performed at 3 different NaCl concentrations and 3 DNA stretching rates at several TMPYP concentrations. More than 500 different DNA molecules were characterized. Force-distance curves showed major structural changes in DNA due to TMPYP binding. Varying the measurement conditions caused different alterations in DNA structure and in the dynamics of TMPYP binding. We developed a model to mathematically describe the force-distance curve of the DNA, therefore we were able categorize the effects that TMPYP had on λ-DNA within the experimental parameter space. The results of our research on DNA-TMPYP interaction provide a good ground for understanding the binding process and its impact on the structure of DNA. Furthermore, it can serve as a basis for the development of additional conjugates for medical therapies.

Acknowledgements

Funding: ÚNKP-21-3-II-SE-37, NKFIH (NRDIO), ITM; SE250+; TKP2021-EGA-23.

P36

The effect of cyclophosphamide treatment on actin

Dénes Lőrinczy, Dávid Szatmári

Department of Biophysics, Medical School, University of Pécs, H-7624 Pécs Szigeti str. 12, Hungary

The continuous management of actin filaments within cytoskeletal system in eukaryotic cells is the key machinery of their plasticity which based on the different complexes of actin with divalent cations (Ca²⁺ or Mg²⁺) and nucleotides (ATP, ADP). We carried out DSC scans to investigate the direct effect of cyclophosphamide (CP) on the structural thermodynamics of actin. It has been demonstrated that run of DSC curves, melting temperature (T_ms ) together with the enthalpy change (ΔH_cal) exhibit clear CP effect. In case of Ca²⁺ G-actin it is manifested in a well separated second high denaturing temperature as a consequence of CP binding into the cleft. This way the nucleotide binding cleft with subdomain 1 and 3 seems less flexible, indicating clear sensitivity to CP treatment. In case of F-actin the main peak represents the thermal denaturation of subdomain 1 and 3, and the increased calorimetric enthalpy administrating Ca²⁺ as well as CP refers to a more rigid structure. These alterations can be the molecular background in the malfunction of muscle in case of polyneuropathy after CP treatment. We have analysed the thermal stability modifier act of interdomain links in actin, monitored by DSC, with the concept of that how did the nucleotide binding cleft between the two main domains of actin affect the activation energy of domains if it was blocked by CP binding. CP treatment of Ca²⁺ F-actin lacks the structural integrity of a more flexible polymer and shows same stability as CP bound monomers. However, Mg²⁺ F-actin did not show any kinetic response to the CP treatment. We can assume that the CP binding blocks the interdomain linkers of actin which originally reduced the stability of domains thus resulted a more reactive and variable structure with the thermodynamic advantage in the development of multidomain proteins. Of interest, even a single dose of CP modifies the whole structural dynamics of each subdomain in actin monomers.

P38

Effect of CO₂ on membrane potential of Chara Globularis and its link to negative voltage at the cell wall/water interface

Manohara Mahadeva, Sebastian Niestępski, Magdalena Kowacz

Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Department of Reproductive Immunology and Pathology, Olsztyn, Poland

An increase in the amount of CO₂ in the atmosphere has an impact on photosynthetic organisms that may include changes in their electrical properties. We study the effect of CO₂ on the membrane potential of green algae Chara globularis by microelectrode technique. The cells were exposed to (i) the externally supplied CO₂ as well as to (ii) dark/light (D/L) conditions inducing physiological changes in intracellular concentration of CO₂. Main cells show hyperpolarization (from -180±15 mV up to -210±5 mV) in the D and depolarization (back to -180±15 mV) in the L. Cortex cells follow the same trend, but with lower amplitude between the D (from -85±10mV up to -95±2 mV) and L (back to -85±10 mV). Algae response to the external CO₂ is always depolarization (by -22±5 mV in the main cell and -12±3 mV in the cortex) irrespective of the D/L conditions.

External CO₂ has to pass through the cell wall in order to reach the main cell. Our measurements show that at the cell wall/water interface there is a negative voltage of -18±2 mV. It increases in magnitude in response to the external CO₂ (up to -35±3 mV) and shows no response to the D/L cycle. We observed this phenomenon for the first time in C. globularis. Depolarization of the cell (measured as electric potential difference between intra and extracellular environment) induced by CO₂, in fact, corresponds to changes in negativity of this near-wall (extracellular) zone. The mechanism behind negative voltage development within the zone can be inferred from our experiments on gels (mimicking cell wall). It results from separation of charges coming from CO₂ dissolution with H⁺ diffusing within the gel (cell wall) and HCO₃^- being left in solution. Similar mechanism can potentially contribute also to algae response to intracellular changes of CO₂.

Depolarization of algal cells as predicted response to increased atmospheric CO₂ would affect electric force acting across cell membrane, therefore impacting e.g. passive ion transport.

Acknowledgement

This work was supported by the National Science Centre (Poland) [grant No.2020/38/E/NZ3/00039]

P40

Membrane induced dynamical changes of K-Ras as a trigger of protein activity

Neli Sedej¹, Erika Balog², Franci Merzel¹

¹Theory Department, National Institute of Chemistry, Ljubljana, Slovenia
²Institute of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

K-Ras is a small GTPase that acts as a regulator of cellular signal transduction. By switching between the active GTP-bound state and the inactive GDP-bound state, it acts as a molecular switch that regulates the intensity of activation of cellular signaling pathways. Mutations affecting the regulation of GTP - GDP exchange in K-Ras are commonly associated with cancer.

To investigate the role of membrane binding in K-Ras activity we performed all-atom molecular dynamics simulations with a total duration of ~15ms. Using variants of principal component analysis (PCA), we observed changes in structural stiffness and entropy as a function of ligand and mutation. We found that the stiffness of the core domain (CD) of the protein is positively correlated with the tightness of the contact with the membrane.

Our study suggests that the membrane plays a role in K-Ras activity that goes beyond geometric constraints by modulating the internal dynamics of the CD. This reveals an additional level of complexity in the K-Ras-membrane interaction and supports strategies in the development of K-Ras inhibitors, that involve disruption of the common K-Ras-membrane interaction.

P42

Bacterial evolution of resistance against antibiotics and phages in structured environments

Krisztina Nagy¹, Sarshad Koderi Valappil², Barbara Dukic^1,3, Julia Bos⁴, László Dér¹, Gábor Rákhely², Robert H. Austin⁵, Péter Galajda¹

¹Biological Research Centre, Institute of Biophysics, Szeged, Hungary
²University of Szeged, Department of Biochemistry, Szeged, Hungary
³Biological Research Centre, Institute of Biochemistry, Szeged, Hungary
⁴Pasteur Institute, Department of Genomes and Genetics, Paris, France
⁵Princeton University, Department of Physics, Princeton, NJ, United States

Bacteria in their natural habitats are surrounded by various environmental factors, some of these can have crucial affect on the survival of a population. The distribution of different stress factors is often heterogeneous. Some studies suggest that such inhomogeneities in the selection pressure might accelerate bacterial evolution.

In our laboratory we study the effect of spatial structure and chemical heterogeneity on the evolution of resistance against antibiotics and bacteriophage viruses. Microfluidics offers great tools to model the microstructure of natural environments. In our experiments we use two different microfluidic devices: 1) an elaborate chamber and channel network, which is suitable to create a complex stress landscape; 2) a device to create a simple linear chemical concentration gradient across a microchannel. Motile bacteria, e.g. E. coli, can move around and explore these precisely controlled landscapes. The growth and distribution of a population can be monitored by fluorescence time-lapse microscopy for several days.

We studied the effect of chemical concentration gradients of antibiotics with different mode of actions on E. coli. We observed characteristic spatial distributions along the gradient, and the emergence of fast-growing populations within 10-12 hours. Biofilms formed in regions with sub-inhibitory concentrations of antibiotics, which quickly expanded into the high antibiotic regions.

In case of T4r bacteriophage gradients, we observed the formation of biofilms at different points of the stress landscape after about 24 hours. From these loci bacteria spread to other parts of the device.

At the end of the experiments the devices were opened and bacteria were collected for further analysis. We measured the level of resistance of single clones and performed whole genome sequencing to identify mutations that could be responsible for the observed higher resistance.

P44

Development and applications of bacterial ”Dual Input Mother Machines”

Imre Pap^1,2, Krisztina Nagy¹, Ágnes Ábrahám^1,2, László Dér¹, Péter Galajda¹

¹Institute of Biophysics, Biological Research Centre, Szeged, Hungary
²Doctoral School of Multidisciplinary Medical Science, University of Szeged, Szeged, Hungary

A large part of our current understanding of cellular biology has been gained through population level studies. This method is essential, although population-averaging methods mask cell-to-cell differences. In recent decades, single-cell characterization has become the focus of research in many different fields of biology. The shift from population to single-cell analysis is facilitated by the expansion of microfluidics and fluorescent time-lapse microscopy. Microfluidics offers precise spatiotemporal control of the environment and the possibility of the collecting of high-throughput single cell level data. One of the most popular microfluidic device is the so called Mother Machine (MM) device. This device lacking the capability of rapidly change bacterial environment due to a single inlet-outlet, thus changing media takes time and may cause fluctuations in the media flow.

The Dual Input Mother Machine (DIMM) is an advanced Mother Machine design which solves this problem and also opens new realm of possibilities in applications and measurements. Via the alteration of the inlet flow rates, the device is capable of switching between two media rapidly and at the same time precisely change the ratio of the two medium thus the concentration which gets to the bacteria. In this poster we describe the principle of operation, optimization, fabrication and applications of Dual Input Mother Machine microfluidic device.

P46

Methodological advancement in production of giant unilamellar vesicles for lipid mixtures with very high cholesterol content

Marija Raguz, Zvonimir Boban, Ivan Mardešić

University of Split School of Medicine, Department of Medical Physics and Biophysics, Split, Croatia

The lipid bilayer portion of lens fiber cell plasma membrane forms significant barrier to oxygen transport into the lens interior and helps protecting against cataract formation. It has unique lipid composition. One of the major properties is that the cholesterol content is extremely high, increases with age, and is higher in the nucleus as compared with the cortex. It strongly affects organization and properties of the lipid bilayer portion of fiber cell plasma membranes. Investigations of membrane properties performed for cholesterol concentrations surpassing the phospholipid bilayer saturation threshold are immensely important. In our research we use giant unilamellar vesicles as model membranes. These vesicles have size comparable to eukaryotic cells. The method most commonly used for their production is electroformation. Due to a large number of parameters required their values have to be carefully adjusted during preparation especially for membranes with high cholesterol contents. We produced artificial membranes from lipid mixtures that resemble composition of the lipid bilayer portion of lens fiber cell plasma membrane (phosphatidylcholine/sphingomyelin/cholesterol). Fluorescent and atomic force microscopy were utilized to find the optimal values of electroformation parameters. We investigated effect of electrical parameters, lipid film thickness and cholesterol concentration on vesicle formation. Increasing the cholesterol concentration decreases the vesicle size and quality, but this effect can be reduced if appropriate electroformation parameters are chosen. The optimal lipid film thickness was found to be around 30 nm. The best frequency-voltage combinations were in the ranges of 2-6 V and 10-100 Hz.

P48

Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy: improved accuracy for samples with spatially varied autofluorescence to signal ratio

István Rebenku^1,2, Cameron B. Lloyd^1,3, János Szöllősi^1,2, György Vereb^1,2

¹Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
²MTA-DE Cell Biology and Signaling Research Group, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
³Department of Oncology and The Milner Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK

The importance of interaction between signaling species in cellular processes often precedes that of expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions since it is highly sensitive to proximity in the range of 1-10 nm. Intensity-based, spectral spillover-corrected FRET is a cost effective and versatile approach which can be applied in flow cytometry and various modalities of fluorescence microscopy, but may be hampered by varying levels of cellular autofluorescence. Here, we have implemented pixel by pixel autofluorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofluorescence that allow the correct determination of all spectral spillover factors, and present an ImageJ/FIJI plugin for manual and automatic creation of quantitative FRET efficiency maps. For validation, we have used bead and cell-based FRET models covering a range of signal to autofluorescence ratios and FRET efficiencies and compared the approach with conventional average autofluorescence/background correction. The pixel-by-pixel autofluorescence correction is easy to implement and is superior in the reliability of results, particularly for samples with spatially varying autofluorescence and low fluorescence to autofluorescence ratios, the latter often being the case for physiological expression levels.

P50

Lipid bilayer cavitation and adhesion energy using molecular dynamics simulations

Marin Šako, Matej Kanduč

Jožef Stefan Institute, Ljubljana, Slovenia

Liquids under tension are found in many systems around us in nature as well as in technology. Examples include lithotripsy and sonoporation of cell membranes [1,2], octopus suckers [3], catapulting mechanisms of fern spores [4,5], and the hydraulic system in plants [6,7]. Such systems under these metastable conditions are vulnerable to cavitation. Lipid membranes, as part of cell membranes, are found in almost every biological system. The study of cavity formation in lipid membranes under tension plays an important role in the research of biological systems. In this context, lipid-lipid adhesion energy, as well as adhesion energy between lipids and other surfaces, is a crucial physical property seeing that it tells us a lot about the strength of interaction between lipids and other matter.

In this talk, I will present the adhesion energies of several systems obtained from molecular dynamics simulations. More specifically, I will discuss how the lipid-lipid adhesion energy of a bilayer depends on the DLPC to DPPC ratio in each layer. Additionally, I will show how cavitation of alkane liquid and lipid bilayers looks like.

References

1. Andrew J Coleman and John E Saunders. A survey of the acoustic output of commercial extracorporeal shock wave lithotripters. Ultrasound Med. Biol, 15(3):213–227, 1989.
2. Claus-Dieter Ohl, Manish Arora, Roy Ikink, Nico De Jong, Michel Versluis, Michael Delius, and Detlef Lohse. Sonoporation from jetting cavitation bubbles. Biophys. J., 91(11):4285–4295, 2006.
3. Andrew M Smith. Negative pressure generated by octopus suckers: a study of the tensile strength of water in nature. J. Exp. Bot., 157(1):257–271, 1991.
4. KT Ritman and J A Milburn. The acoustic detection of cavitation in fern sporangia. J. Exp. Bot., 41(9):1157–1160, 1990.
5. Xavier Noblin, NO Rojas, J Westbrook, Clement Llorens, M Argentina, and J Dumais. The fern sporangium: a unique catapult. Science, 335(6074):1322–1322, 2012.
6. Abraham D Stroock, Vinay V Pagay, Maciej A Zwieniecki, and N Michele Holbrook. The physicochemical hydrodynamics of vascular plants. Annu. Rev. Fluid Mech., 46:615–642, 2014.
7. Alexandre Ponomarenko, Olivier Vincent, Amoury Pietriga, Herve Cochard, E Badel, and Philippe Marmottant. Ultrasonic emissions reveal individual cavitation bubbles in water-stressed wood. J. Royal Soc. Interface, 11(99):20140480, 2014.

P52

Conformational plasticity of calmodulin under steady state calcium loads

Gusztáv Schay¹, Klaudia Onica², Judit Somkuti¹, László Smeller¹, Tünde Juhász³, J. Michael Klopf⁴, Erika Balog¹, Miklós Kellermayer¹, Károly Liliom¹

¹Semmelweis University, Dept. of Biophysics and Radiation Biology, Budapest, Hungary
²Pázmány Péter Catholic University, Budapest, Hungary
³Institute of Materials and Environmental Chemistry, RCNS, Budapest
⁴Helmholtz Zentrum Dresden-Rossendorf , Rossendorf, Germany

An essential question in the function of calmodulin is how the protein selects the appropriate ones from a large number of potential binding partners during calcium-ion signaling. We assume, that the conformational space of calmodulin serves as an information-storage space. The conformationally coded information about the upstream signal sources will then drive calmodulin to select among its downstream partners in the information flow process. It is well known that calmodulin can adopt a variety of conformations in the apo form (as confirmed by NMR studies), and crystal structures show profoundly more rigid structures in the calcium saturated form. There is, however, little knowledge about the partial saturation states of calmodulin, though this may play a key role if one considers the temporal profile variations of calcium signals. We hypothesize that as calmodulin traverses gradually through the calcium loaded states, distinct conformational states get enriched in the population. As a preliminary test, we have measured the FTIR absorbance in the near and far IR, as well as the Tyr fluorescence lifetime of calmodulin under various calcium loads, and find that none of the signals follow a classical titration curve, indicating that multiple conformational transitions may take place. We have also identified possible vibrational modes related to FTIR absorption regions.

P54

Membrane-induced dynamical changes of K-Ras as a trigger of protein activity

Neli Sedej¹, Erika Balog², Franci Merzel¹

¹Theory Department, National Institute of Chemistry, Ljubljana, Slovenia
²Institute of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

K-Ras is a small GTPase that acts as a regulator of cellular signal transduction. By switching between the active GTP-bound state and the inactive GDP-bound state, it acts as a molecular switch that regulates the intensity of activation of cellular signaling pathways. Mutations affecting the regulation of GTP - GDP exchange in K-Ras are commonly associated with cancer.

To investigate the role of membrane binding in K-Ras activity we performed all-atom molecular dynamics simulations with a total duration of ~15μs. Using variants of principal component analysis (PCA), we observed changes in structural stiffness and entropy as a function of ligand and mutation. We found that the stiffness of the core domain (CD) of the protein is positively correlated with the tightness of the contact with the membrane.

Our study suggests that the membrane plays a role in K-Ras activity that goes beyond geometric constraints by modulating the internal dynamics of the CD. This reveals an additional level of complexity in the K-Ras-membrane interaction and supports strategies in the development of K-Ras inhibitors, that involve disruption of the common K-Ras-membrane interaction.

P56

Light-adapted charge-separated state of Photosystem II. Structural and functional dynamics of the closed reaction center

Gábor Sipka¹, Melinda Magyar¹, Parveen Akhtar^1,2, Pavel Müller³, Klaus Brettel³, Guangye Han⁴, Jian-Ren Shen^4,6, Stefano Santabarbara⁵, Petar Lambrev¹, Győző Garab^1,7

¹Biological Research Centre, Eötvös Loránd Research Network, Szeged, Hungary
²ELI-ALPS, ELI-HU Nonprofit Ltd., Szeged, Hungary
³Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France
⁴Photosynthesis Research Center, Chinese Academy of Sciences, Beijing, China
⁵Photosynthetic Research Unit, Institute of Biophysics, National Research Council of Italy, Milano, Italy
⁶Photosynthesis Research Center, Okayama University, Okayama, Japan
⁷Faculty of Science, University of Ostrava, Ostrava, Czech Republic

Monitoring the activity of Photosystem II (PSII) upon dark-to-light transition is routinely performed by recording the rise of fluorescence intensity from the minimum (F_o) to the maximum (F_m) levels; variable chlorophyll-a (Chl-a) fluorescence (F_vF_m-F_o) upon this transition follows a complex induction kinetics and carries information on the functioning of the photosynthetic machinery. According to the mainstream model, F_o and F_m belong to the open (PSII_O) and closed (PSII_C) states of the reaction center (RC) states, which, respectively, are ready and incapable of utilizing the absorbed light for stable charge separation. Although Chl-a fluorescence measurements have provided a wealth of information on the mechanisms of photosynthetic light-energy conversion, the mainstream model is not free of controversies [1, 2]. We explain the peculiar features of Chl-a fluorescence induction kinetics and show that in addition to PSII_O and PSII_C, this photosystem can assume light-adapted charge-separated state, PSII_L. Formation of PSII_L, via light-induced subtle conformational changes, facilitates the stabilization of the charge-separated state. PSII_L is characterized by distinct features in the energy landscape of trapping/detrapping of excitations in the core-antenna RC complex. The PSII_C–PSII_L transition is responsible for a large part of F_v, which thus appears to reflect the structural dynamics of PSII [3], which also depends on the lipid matrix of the RC complex [4]. Our data suggest key roles of strong local stationary and transient electric fields and dielectric relaxation processes during the operation of PSII.

References

1. Magyar M et al. (2018) Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction. Sci Rep 8 (1):2755.
2. Sipka G et al. (2019) Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus. Physiol Plant 166 (1):22-32.
3. Sipka G et al. (2021) Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center. Plant Cell 33 (4):1286-1302.
4. Magyar M et al. (2022) Dependence of the rate-limiting steps in the dark-to-light transition of photosystem II on the lipidic environment of the reaction center. Photosynthetica 60 (1):147-156.

P58

Stability and stabilization of viral G-quadruplexes 

Judit Somkuti, Orsolya R. Molnár, Anna Grád, Miklós Cervenak, László Smeller

Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

G-Quadruplex (GQ) is a non-canonical structure of the nucleic acids. This four stranded motif is formed by guanine-rich sequences of the genome. Potentially GQ-forming sequences were found in crucial loci of the human genome, where formation of GQs can take part in the regulation of important processes like cell proliferation and cell death. Their appearance in the oncogene promoter regions made GQs an attractive target of the cancer research.

We investigated several viral sequences which might potentially form GQ. Here we report mainly the results we obtained on three oligos taken from the genome of the hepatitis B, whose infection is among the ten leading causes of death.

Infrared spectroscopy, fluorescence (FRET) spectroscopy were applied combined with the high pressure diamond cell technique.

Our experiments clearly prove the existence of GQ structure in all three oligos. We determined the volumetric parameters of the unfolding in case or all the three mentioned oligos (called HepB1-3). Pressure stabilized the oligos HepB1 and HepB3, while slight destabilization was observed in case of HepB2. The dTm/dp values are 28 -10, and 8 °C/GPa respectively. This gives a volume change comparable to the volume of one water molecule in case of HepB1 [1-3].

We also tried to stabilize the folded GQ structure with ligands that were originally developed for human GQs. Different, but pronounced stabilization was found for TMPyP4, BRACO19 and PhenDC3. 

Acknowledgements

This work was funded by the National Research, Development and Innovation Office of Hungary, NKFI K-124697.

References

1. Molnár, OR., Végh, A., Somkuti, J., Smeller, L., Scientific Reports 11 (2021) 23243; https://doi.org/10.1038/s41598-021-02689-y
2. Smeller, L., J. Mol. Sci. 23 (2022) 5761. https://doi.org/10.3390/ijms23105761
3. Somkuti, J., Molnár, OR., Grád, A., Smeller, L., Biology 10 (2021) 1173; https://doi.org/10.3390/biology10111173

P60

Collection, prediction, and publication of ABC transmembrane protein structures

Erzsébet Suhajda^1,2, Hedvig Tordai¹, Ian Sillitoe³, Sreenath Nair⁴, Mihály Váradi⁴, Tamás Hegedűs^1,5

¹Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
²Budapest University of Technology and Economics, Faculty of Electrical Engineering and Informatics, Budapest, Hungary
³University College London, Institute of Structural and Molecular Biology, London, UK
⁴European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK
⁵Eötvös Loránd Research Network, ELKH-SE Biophysical Virology Research Group, Budapest, Hungary

The number of experimentally solved unique transmembrane (TM) protein structures has doubled in the last four years thanks to the revolution of cryo-electron microscopy. In addition, AlphaFold2 provided a reliable computational approach to protein structure prediction, thus contributing to the growing number of known protein structures. However, if a specific protein family is the subject of a study, collecting the structures of the family members is a highly challenging task. Here, we demonstrate this through the ABC protein superfamily, assessing the applicability and usability of an automatic collection method and presenting the collected protein structures. Our pipeline identifies and classifies transmembrane ABC protein structures using PFAM search and determines their conformational states based on special geometric measures, conftors. Since the AlphaFold database contains structure predictions only for single-chain polypeptides, we performed AlphaFold-Multimer predictions for human ABC half transporters functioning as dimers. In order to facilitate further studies, we exposed the collected ABC protein structures via a web application (http://abc3d.hegelab.org), where we also made our novel human ABC protein dimer structure predictions available.

Acknowledgements

This work was supported by funds from NRDIO/NKFIH: K127961, K137610 and 2020-2.1.1-ED-2021-00179; and from the Cystic Fibrosis Foundation (CFF): HEGEDU20I0. We thank the Wigner Scientific Computing Laboratory (WSCLAB, the former Wigner GPU Laboratory) for computational resources.

P62

Characterization of a novel mutation in Brugada Syndrome

Tibor G. Szántó¹, Szabolcs Gál^1,2, Beáta Arnódi-Mészáros¹, István Balogh³, Bálint L. Bálint⁴, Zoltán Csanádi², György Panyi¹

¹University of Debrecen, Department of Biophysics and Cell Biology, Debrecen, Hungary
²University of Debrecen, Department of Cardiology and Cardiac Surgery, Debrecen, Hungary
³University of Debrecen, Department of Human Genetics, Debrecen, Hungary
⁴University of Debrecen. Faculty of Medicine. Department of Biochemistry and Molecular Biology. Genomic Medicine and Bioinformatic Core Facility, Debrecen, Hungary

Voltage-gated sodium channels (Na_V) play a key role in the initiation and propagation of cardiac action potential essential for the rhythmic beating of the heart. Therefore, alterations of the sodium current (I_Na) in cardiomyocites can lead to diseases responsible for cardiac arrhythmias, such as Brugada Syndrome (BrS). BrS is characterized by an ST elevation in ECG and an increased risk for sudden cardiac death due to ventricular fibrillation. The major disease gene for BrS is SCN5A encoding the primary alpha-subunit of the cardiac Na_V1.5 channel. Exploring SCN5A mutations in patients with inherited arrhythmogenic syndromes is critical for understanding the pathogenesis of arrhythmias.

Accordingly, we aimed to fully characterize the biophysical properties of Na_V1.5 channels containing a novel heterozygous mutation of R893C localized in the P-loop of domain II identified in a patient with BrS. We subsequently compared the main gating parameters of R893C channels to wild-type Na_V1.5 channels (WT). The channels were transiently expressed in CHO cells and sodium currents were measured using the standard whole cell patch-clamp technique.

We found that the peak current density is substantially reduced by the R893C mutation with respect to WT channels. We also observed slower activation kinetics of I_Na current in R893C channels, although the mutation had no significant effect on the steady-state activation. All observations confirmed the loss-of-function of R893C channels. Pharmacological studies revealed that DTT might restore the normal function of Na_V1.5 containing R893C by reducing the cysteine bridges that may be responsible for the loss of conduction.

Our findings may facilitate the understanding of arrhythmogenesis mechanisms of BrS highlighting the importance of S5-S6 loop of DII in Na_V1.5 channel gating. Moreover, understanding the structure-function relationship of Na_V1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

P64

Evolution of staphylokinase´s binding affinity by ribosome display technology

Maria Tomková¹, Veronika Dzurillová², Erik Sedlák¹

¹P. J. Šafárik University, Center for Interdisciplinary Biosciences, Technology and Innovation Park, Košice, Slovakia
²P. J. Šafárik University, Department of Biophysics, Faculty of Science, Košice, Slovakia

Ischemic stroke and myocardial infarct are leading causes of death and disability world-wide. Currently available thrombolytics (plasminogen activators) are not optimal, thus there is a need to develop more effective treatment. Staphylokinase (SAK), a single chain extracellular protein secreted by Staphylococcus aureus, is a promising new thrombolytic agent. However, inefficient binding to plasmin is the main factor limiting overall SAK effectivity.

Directed evolution is a powerful approach to tailor protein properties toward new or enhanced functions. We employed ribosome display, in vitro selection and evolution method, to improve SAK affinity for plasmin. Human plasmin was used as an antigen, against which SAK variants were selected.

We have successfully implemented selection of SAK variants by ribosome display and after first round of display we proved that it is feasible to evolve SAK by this technology. Evolution techniques usually require several consecutive rounds, therefore we accomplished second and third round of the display. Sequencing of individual clones after 3rd round of ribosome display revealed some identical amino acid substitutions, that were found in several different mutants, indicating that such residues might be involved in SAK-plasmin interaction and its replacement might change SAK property towards higher plasmin affinity.

Enhancement of SAKs affinity for plasmin is a highly desirable objective. Preliminary data obtained from SAK engineered by ribosome display showed that the evolution approach has high potential to improve the properties of SAK. However, to prove this statement we need to perform further rounds of ribosome display followed by detailed biophysical characterisation of selected mutants.

P66

Vertical value chain: from new knowledge to commercially exploitable technologies

Iztok Urbančič¹, Aleksandar Sebastijanović^1,2, Tilen Koklič^1,2, Janez Štrancar^1,2

¹Jožef Stefan Institute, Ljubljana, Slovenia
²Infinite LLC, Maribor, Slovenia

In the Laboratory of Biophysics at the Jožef Stefan Institute we investigate molecular mechanisms and biophysical interactions that lead from the first contact of our bodies with external impactors, such as nanoparticles from polluted environment, to the development of disease symptoms. To this end, we use live-cell imaging with advanced optical microscopy methods (such as STED imaging), quantitative analysis approaches, and modelling of the observed processes, to highlight the events that can be used for prediction of adverse outcomes. We have recently shown that we can predict the onset of chronic lung inflammation based on early structural changes within the cell ahead of time (Kokot et al., Advanced Materials, 2020). We now further investigate similar events that can be used to predict environmentally-induced cardiovascular and neurodegenerative disorders, as well as immune response to medical implants.

To transfer the new knowledge towards everyday practice as quickly as possible, we have started a spin-out company Infinite LLC, dedicated to the development of technologies for safety testing of materials, chemicals, vaccines, and drugs, using cellular processors – as the alternative to expensive, slow, and often unreliable animal assays. With the tissue-on-chip and microscope-on-chip technologies, we aim to provide more resource-efficient safety testing of materials and chemicals for industrial producers and regulators. The long-term partnership between the research laboratory and the company thus represents a vertical value chain, which directs the findings from the basic research towards applied marketable solutions.

P68

Fatty acid binding to ovalbumin studied by EPR

Ana Vesković, Đura Nakarada, Ana Popović Bijelić

University of Belgrade - Faculty of Physical Chemistry, Belgrade, Serbia

Serum albumins (SAs) act as main carriers of various endogenous and exogenous compounds, including non-esterified fatty acids (FAs), in the blood plasma of mammals. Avian SAs exhibit certain similarities and moderate sequence identity to mammalian SAs, i.e. human and bovine (HSA and BSA). Chicken SA (α-livetin) was found to transport FAs like the mammalian, although with lower affinity towards long-chain FAs. This protein can be found in the hens blood serum and the egg yolk, but not in the egg white.

Ovalbumin (OVA), the albumin from hen egg white, accounts for more than half of the total albumen protein content, while in the egg yolk appears only in its dephosphorylated form, presumably serving as the nutrition source for the growing embryo. OVA belongs to the serpin superfamily, despite lacking any protease inhibitory activity. Although it is the first protein isolated in pure form from egg white, its physiological function has not yet fully been resolved. It is mainly proposed that OVA serves as a transport and storage protein for metal ions and amino acids.

The aim of this study was to determine whether OVA binds long-chain FAs, and to compare it with HSA and BSA, using electron paramagnetic resonance spectroscopy (EPR) spin labeling, a technique well established in the characterization of SA binding properties. The proteins were labeled with 5-, and 16-doxyl-stearic acids (5-DS and 16-DS). The EPR spectral analysis revealed strong binding of 5-DS and 16-DS to both HSA and BSA, and only slight immobilisation in a 1:1 molar ratio solution with OVA. Most probably, the observed restricted motion of FAs is the consequence of their surface interactions with OVA residues, rather than specific binding to the protein, as observed for SAs. These results indicate that OVA lacks a FA binding site, unlike SAs which contain multiple high-affinity FA binding sites. Moreover, it may be concluded that FA binding and transport is not a primary physiological function of OVA.

Acknowledgment

This research was funded by the Science Fund of the Republic of Serbia, PROMIS, #6062285, PHYCAT.

P70

Investigating the competitive regulation of Las17 through Agent-Based modelling

Lewis Hancock, Kathryn Ayscough, Mike Williamson

The University of Sheffield, School of Biosciences, Sheffield, United Kingdom

Clathrin-mediated endocytosis (CME) is a mechanism used by eukaryotic cells to move membrane-associated elements into the cytosol. Proteins are recruited to form a patch that is later invaginated and pinched off to produce a vesicle [1]. A key step in the progression of new patches involves the de novo nucleation of actin filaments. Las17, a largely natively unstructured actin-binding protein, is thought to regulate this process, although it is not known how [2].
The central region of Las17 has recently been shown to bind and nucleate actin via a series of largely uncharacterised polyproline binding sites which are also known to weakly bind the SH3 domains of several patch-associated proteins [3,4]. Human WASP family proteins (homologues of Las17) have been linked to several neurodegenerative diseases [5,6]. These domains can be thought of as a ‘cloud’ of SH3s which changes in composition as endocytosis progresses and more domains are recruited. Some proteins even possess multiple SH3 domains [7]. A pure experimental characterisation of these interactions would prove challenging due to the high number of binding sites and proteins involved.
Agent-based modelling can provide a valuable insight into (i) how the combined effect of weakly binding SH3 domains can deliver robust endocytic control via their joint effect on Las17 activity and (ii) why some of these proteins interact through a tandem of SH3 domains. Here, we built a parallel coded, agent-based computational model within the FLAMEGPU modelling environment [8] which, through its modular nature, allows for an in-depth investigation of this regulation. The program is being supplemented by key binding data such as affinities obtained using Microscale thermophoresis (MST) and Biolayer interferometry (BLI).

References

1. Goode, B. L., Eskin, J. A. & Wendland, B. Actin and endocytosis in budding yeast. Genetics 199, 315–58 (2015).
2. Urbanek, A. N., Smith, A. P., Allwood, E. G., Booth, W. I. & Ayscough, K. R. A novel actin-binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3. Curr Biol 23, 196–203 (2013).
3. Tyler, J. J., Allwood, E. G. & Ayscough, K. R. WASP family proteins, more than Arp2/3 activators. Biochem Soc Trans 44, 1339–1345 (2016).
4. Feliciano, D., Tolsma, T. O., Farrell, K. B., Aradi, A. & di Pietro, S. M. A second Las17 monomeric actin-binding motif functions in Arp2/3-dependent actin polymerization during endocytosis. Traffic 16, 379–97 (2015).
5. Kitamura, Y. et al. Possible involvement of Wiskott–Aldrich syndrome protein family in aberrant neuronal sprouting in Alzheimer’s disease. Neuroscience Letters 346, 149–152 (2003).
6. Kumar, S. et al. Compound heterozygous variants in Wiskott-Aldrich syndrome like (WASL) gene segregating in a family with early onset Parkinson’s disease. Parkinsonism & Related Disorders 84, 61–67 (2021).
7. Tong, A. H. Y. et al. A Combined Experimental and Computational Strategy to Define Protein Interaction Networks for Peptide Recognition Modules. Science (1979) 295, 321–324 (2002).
8. Paul Richmond, Dawn Walker, Simon Coakley & Daniela Romano. High performance cellular level agent-based simulation with FLAME for the GPU. Briefings in Bioinformatics 11, 334–347 (2010).

L49

Three-dimensional microscopy and lithography with sub-diffractional resolution for mimicking blood vessels

Boris Buchroithner¹, Sandra Mayr¹, Fabian Hauser¹, Eleni Priglinger⁴, Ana Raquel Santa-Maria³, Mária A. Deli³, András Dér³, Thomas A. Klar², Markus Axmann¹, Dmitry Sivun¹, Mario Mairhofer¹, Jaroslaw Jacak¹

¹University of Applied Sciences Upper Austria, School of Applied Health and Social Sciences, Garnisonstr. 21, 4020 Linz
²Johannes Kepler University, Department of Applied Physics, Altenberger Straße 69, 4040 Linz
³Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary
⁴Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstraße 13, 1200 Vienna, Austria

Tissue engineering is a rapidly growing scientific field. As cells need structural support and guidance for growth, we fabricated polymeric bio-compatible scaffolds by multi-photon lithography (MPL). In MPL, a femtosecond-pulsed laser focused into a photosensitive resin solution initializes polymerization solely within the focal volume. Hence, sub-micrometer resolution can be achieved in three dimensions (lateral/axial resolution <200 nm and around 500 nm). Hence, its flexible additive manufacturing performance makes MPL a well-suited technique for 3D-structuring of materials for tissue scaffolds. The challenge is still the development of a photoresist that is biocompatible, mechanically stable and can be structured at a high writing speed.

We present 2D and 3D biocompatible scaffolds structured onto cell culture membranes combined with microfluidics. The scaffolds were seeded with cells for biocompatibility testing. In order to promote cell adhesion, we functionalized the scaffolds with antibodies, DNA-linkers or RGD-peptides. Human endothelial cells were used to model a blood vessel wall within a microfluidic device. Its design allowed for high-resolution (down to single-molecule sensitive) imaging using a high numerical aperture objective with a short working distance. Our dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles. We plan to address molecular processes like transportation of macromolecules with our platform.

References

1. Buchroithner, B. et al. Dual Channel Microfluidics for Mimicking the Blood-Brain Barrier. ACS Nano (2021).
2. Mayr, S. et al. Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model. PLOS Comput. Biol. 16, e1007902 (2020).
3. Hauser, F et al. Real-time 3D single-molecule localization microscopy analysis using lookup tables. Biomed. Opt. Express 12, 4955–4968 (2021).

L50

Quantitative phase imaging using a holo-tomographic system

Sung Sik Lee, Gábor Csúcs

ScopeM, ETH Zurich, Zurich, Switzerland

Quantitative Phase Imaging (QPI) is an emerging field of label-free light microscopy methods. In contrast to the well-known phase contrast method it can provide quantitative information about the phase shift caused by the sample and hence can be used not only to establish contrast but also for characterization of material or physical/biophysical properties. Over the past couple of years, various implementations of QPI have been introduced. In my talk I focusing on the one that is probably the best suited to investigate 3D samples – holo-tomography. Recently our platform has acquired such a system and I am reporting about our first experiences and further plans and present potential fields of application.

L51

Study of transport processes mediated by membrane nanotubes

Henriett Halász¹, Viktória Tárnai², Tamás Madarász¹, Miklós Nyitrai¹, János Matkó³, Edina Szabó-Meleg¹

¹University of Pécs, Medical School, Department of Biophysics, Pécs, Hungary
²University of Pécs, Faculty of Natural Sciences, Institute of Biology, Pécs, Hungary
³Eötvös Loránd University, Department of Immunology, Budapest, Hungary

Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells over long distances. These membrane projections are heterogeneous, thin and fragile structures existing both in vitro and in vivo. NTs have role in material transport processes (e.g. delivery of cell components), in the development of resistance against chemotherapeutic agents, in the intercellular spread of pathogens and in the deterioration of some neurological disorders (e.g. Alzheimer’s disease).

NTs of B cells are less studied compared to NTs of other immune cells. Consequently, our aims were to characterize some transport processes of B cell NTs and to identify their mediators with specific inhibitors and gene silencing techniques using confocal and structured illumination microscopy.

We showed the functional importance of B cell NTs, since as active channels, NTs can mediate intensive, bidirectional transport of mitochondria and microvesicles. The delivery of mitochondria is promoted by the cooperation of two different cytoskeletal motorproteins, while vesicular transport seems to be a simpler process. We revealed that actin is a key element in the induction of B cell NTs, and microtubules are responsible for their stability.

Our results may contribute to better understand the molecular background of some pathological conditions and to develop new therapeutic approaches targeting disorders associated with mitochondrial dysfunction.

Acknowledgements

This work is supported by the ÚNKP-21-3-II New National Excellence program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation, the GINOP-2.3.2-15-2016-00036, the EFOP-3.6.1-16-2016-00004 and Dr. János Szolcsányi research funds.

L56

From liposomes to cells: filling the gap between biophysical and microbiological studies of the activity and selectivity of host‐defense peptides

Lorenzo Stella

University of Rome Tor Vergata, Department of Chemical Science and Technologies, Rome, IT

Host-defense peptides (HDPs) kill bacteria mainly by perturbing their cell membranes, with low toxicity towards host cells, and are promising compounds to fight drug-resistant microbes. However, rational design of new molecules with the same activity as HDPs, but improved pharmacological properties, has proven difficult. This issue is due to the fact that multiple phenomena modulate the properties of HDPs, such as conformational transitions, aggregation processes, water-membrane partition equilibria, and association with different cell components. While these aspects can be studied by biophysical approaches in model systems, they are often not addressed in microbiological experiments with bacteria. By combining experiments on liposomes and live cells, we clarified several aspects of HDP behavior. For instance, we showed that peptide conformation and aggregation propensity can be exploited to optimize HDP selectivity, we determined the minimum number of peptide molecules that must bind to a single bacterium to kill it, we characterized the cell-density dependence of HDP activity and selectivity, and measured the differential peptide affinity for various cell components. Overall, these studies provide several novel insights into the behavior of HDPs, question the reliability of current microbiological assays, and give useful indications for the rational design of new antibiotic molecules.

References

1. PNAS 2021 118:e2014364118; BBA 2020 862:183291; “Antimicrobial Peptides: Basics for Clinical Application” Springer 2019; Chem Comm 2018 54:4943; Pept Sci 2018 10:e24041; ACS Chem Biol 2017 12:52; BBA 2015 1848:581; ACS Chem Biol 2014 :2003; J.Pept.Sci 2013 19:758; BBA 2013 1828: 1013.

L57

Membrane distribution of ion channels in T cells

Orsolya Vörös¹, György Panyi¹, Péter Hajdu^1,2

¹University of Debrecen, Department of Biophysics and Cell Biology, Debrecen, Hungary
²University of Debrecen, Department of Dental Biochemistry, Debrecen, Hungary

Ion channels play vital role in regulation in Ca²⁺-dependent T lymphocyte functions. In vivo activation, which is a crucial step in initiation of the adaptive immune response, happens in the immunological synapse (IS) formed between an antigen presenting cell and a T cell.  Previously, it was described that main ion channel species of T cells accumulate (Kv1.3, KCa3.1 and CRAC: formed by the ion-conducting Orai1 and the ER-resident STIM1) in the immunological synapse.  However, a little is known about the molecular background of ion channels’ redistribution, IS-anchoring and physiological relevance of ion channels IS-accumulation.
The presentation highlights the kinetics of Orai1 redistribution into the IS, its molecular background and consequence on the Ca²⁺-response of T cells. We could show that binding of SAP97 to the Orai1 N-terminus could be responsible for the removal of Orai1 from the IS: knockdown of SAP97 or deletion of the Orai1 N-terminal tail – the region where SAP97 binds to Orai1- resulted in a sustained residency of Orai1 in the IS. The prolonged IS-dwelling of Orai1 modified the Ca²⁺ response of IS-engaged T cells: number of Ca²⁺-oscillations increased. Based on the results we suppose that short residence time of Orai1 in the IS is essential to prevent T cell hyperactivity via Ca²⁺-dependent pathway.

Acknowledgements

This work was supported by NKFIH K128525 K119417, GINOP-2.3.2-15-2016-00044, EFOP-3.6.3-VEKOP-16-2017-00009, and GINOP-2.3.2-15-2016-00020.

L58

V-ATPases – Nature’s most versatile proton pumps

Tibor Páli

Institute of Biophysics, Biological Research Centre, Szeged, Hungary

The transport protein complex that is responsible for the acidification of the internal compartments and the extracellular space of eukaryotic cells is Nature's most universal and most versatile proton pump, the vacuolar proton-ATPase (V-ATPase). It is a membrane-attached molecular rotary engine, which energises the rotation of the rotor domain from the chemical energy of ATP hydrolysis, and the rotation leads to a trans-membrane proton pumping through the interface between the stator and rotor domains. Little is known about the precise regulation of the coupled catalysis-rotation-transport mechanism of this nano-motor despite it is being a key factor in several normal or pathological cellular processes. The so-called c-ring proteins of the rotor domain of V-ATPase, along with that of the related ATP synthase, belong to a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices, called Ductins. The active forms of the Ductins are ring-shaped oligomeric assemblies in certain biomembranes, and they provide various pore and channel functions and assist in membrane fusion processes.

In my talk I present published and ongoing research of the Membrane Biophysics Research Group contributing the following key aspects of the rotary mechanism, c-ring proteins and regulation of V-ATPase: (i) measurement of the activity and rate of rotation in a native V-ATPase; (ii) non-enzymatic functions and common features of Ductin proteins; and (iii) regulation of V-ATPase in the lysosome.

L52

Time-resolved detection of oxidative stress level in cancer cells

Veronika Huntošová

Pavol Jozef Šafárik University in Košice, Center for Interdisciplinary Biosciences, Košice, Slovakia

Oxidative stress in cells is normally maintained at levels necessary for the basic organisation of intercellular reaction. Reactive oxygen species (ROS) and antioxidant enzymes constantly maintain the balance to prevent cell death. The main organelles responsible for the production of ROS are the mitochondria. For this reason, they are often the target of cancer therapies. In our laboratory, we have investigated the time-resolved fluorescence microscopy (FLIM) approach of mitochondrial fluorescence probes to detect oxidative stress changes in cancer cells and spheroids. We found that this method is more sensitive than intensity-based techniques that detect low levels of ROS production. In addition, the low level of phototoxicity produced by the system allows repeated detection of ROS in the same cells over longer incubation periods. We propose FLIM as a complementary method for characterising the intracellular environment.

Acknowledgements

This work was supported with EU H2020-WIDESPREAD-05-2020 grant No. 952333, CasProt (Fostering high scientific quality in protein science in Eastern Slovakia) and Slovak grant agency APVV-20-0340.

L53

Life beyond the pixels: single-cell analysis using deep learning and image analysis methods

Péter Horváth^1,2

¹Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
²Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, Finland

In this talk I will give an overview of the computational steps in the analysis of a single cell-based large-scale microscopy experiments. First, I will present a novel microscopic image correction method designed to eliminate illumination and uneven background effects which, left uncorrected, corrupt intensity-based measurements. New single-cell image segmentation methods will be presented using differential geometry, energy minimization and deep learning methods (www.nucleAIzer.org). I will discuss the Advanced Cell Classifier (ACC) (www.cellclassifier.org), a machine learning software tool capable of identifying cellular phenotypes based on features extracted from the image. It provides an interface for a user to efficiently train machine learning methods to predict various phenotypes. For cases where discrete cell-based decisions are not suitable, we propose a method to use multi-parametric regression to analyze continuous biological phenomena. To improve the learning speed and accuracy, we propose an active learning scheme that selects the most informative cell samples.

Our recently developed single-cell isolation methods, based on laser-microcapturing and patch clamping, utilize the selection and extraction of specific cell(s) using the above machine learning models. I will show that we successfully performed DNA and RNA sequencing, proteomics, lipidomics and targeted electrophysiology measurements on the selected cells.

L54

Ultrashort laser pulses interaction with hemoglobin: micro-patterning and label-free imaging

Mihajlo Radmilovic¹, Ivana Drvenica², Mihailo Rabasovic¹, Vesna Ilic², Danica Pavlovic¹, Sho Oasa³, Mina Peric^4,5, Aleksandar Krmpot¹

¹Institute of Physics Belgrade, University of Belgrade, Serbia
²Institute for Medical Research, University of Belgrade, Serbia
³Karolinska Institutet, Stockholm, Sweden
⁴Faculty of Biology, University of Belgrade, Serbia
⁵Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia

Label-free imaging of red blood cells using single-photon excitation microscopy (confocal and/or epifluorescent) is practically impossible because the hemoglobin (Hb) fluorescence is hardly detectable due to fast non-radiative decay which is dominant over spontaneous emission from the singlet state. On the other hand, large two photon absorptivity of Hb and the two photon excited fluorescence (TPEF) of a newly formed photoproduct upon the interaction of ultrashort laser pulses with Hb were reported. TPEF microscopy was successfully used in number of studies for label-free hemoglobin entities and erythrocytes imaging.

In this work, we present the photophysical properties of formed photoproduct and its possible applications. We created fluorescent photoproduct patterns with a spatiotemporal control on the thin Hb film using femtosecond laser writing (micro-patterning). The photoproduct formed on thin Hb films using ultrashort laser pulses is characterized using different (micro)spectroscopic techniques: emission upon two photon absorption, UV-VIS single photon absorption, and spectral imaging. Spectroscopic properties of Protoporphyrin IX (PpIX) are considered as well, as a PpIX is a structural part of H band and potentially can be the precursor for the photoproduct formation. Moreover, we treated Hb solution with hydrogen peroxide to test the hypothesis of similarity in optical response between chemically induced degradation products of Hb and photoproduct, formed by the interaction of ultrashort laser pulses with Hb.

Photo stability of Hb photoproduct was sustained in the time range of several months. Based on the photoproduct formation that can be controlled spatiotemporally, photo-labelling of individual RBCs and tracing of their movement in the whole blood was successfully performed.

L55

Patterned Microfluidics for the Investigation of Plant Root Exudates

Daniel Patko¹, Udara Bimendra Gunatilake^1,2, Lionel X. Dupuy^3,4, Lourdes Basabe-Desmonts^2,4,5,6, Fernando Benito-Lopez^1,5,6

¹Microfluidics Cluster UPV/EHU, Analytical Microsystems & Materials for Lab-on-a-Chip Group, Analytical Chemistry Department, University of the Basque Country UPV/EHU, Spain
²Microfluidics Cluster UPV/EHU, BIOMICs microfluidics Group, Lascaray Research Center, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain
³NEIKER, Derio, Spain
⁴IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
⁵Bioaraba Health Research Institute, Microfluidics Cluster UPV/EHU, Vitoria-Gasteiz, Spain
⁶BCMaterials, Basque Centre for Materials, Micro and Nanodevices, UPV/EHU Science Park, Leioa, Spain

Modern agriculture is made possible by the intensive usage of fertilisers and agro chemicals. The application of traditional fertilizers is easy, but due to leaching, this technology can significantly damage the natural environment [1]. Using microbes as biological fertilizers is ecologically more sustainable, however, it is still challenging to maintain a strong and lasting interaction between the roots and the microbes [2]. Roots exude a wide range of biomolecules to attract beneficial microorganisms, but we still lack of essential knowledge to investigate and understand this process deeply.
Traditionally, the use of hydroponic cultures makes possible the extraction of exuded molecules from the root [3], but this process is insufficient to reveal the nature of the root-microbe communication. Powerful microscopic techniques like fluorescent light-sheet microscopy or confocal microscopy can explore the bacterial activity around the root [4], but they provide little information about the chemicals involved in this relationship.
To be able to advance in the state of the art, we propose a novel microfluidic based approach to overcome the above described limitations. We applied a combined, cutting-edge, paper-polymer based advanced technology that provides a cost effective, easy to use system to observe and control the root microenvironment. Combined with microscopy the developed microfluidic devices can reveal the root exudation pattern spatially and temporally and thus the microbial activity around the root could be revealed.

Acknowledgments

This work was supported by the European Commission’s EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions program, RhizoSheet MSCAIF, grant agreement number: 101028242, the MaMi project, funded by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 766007 and the support from “Ministerio de Ciencia y Educación de España” under grant PID2020-120313GB-I00 / AIE / 10.13039/501100011033.

References

1. S. Delin, M. Stenberg, Effect of nitrogen fertilization on nitrate leaching in relation to grain yield response on loamy sand in Sweden, European Journal of Agronomy. 52 (2014) 291–296. https://doi.org/10.1016/j.eja.2013.08.007.
2. A. Carminati, M. Zarebanadkouki, E. Kroener, M.A. Ahmed, M. Holz, Biophysical rhizosphere processes affecting root water uptake, Annals of Botany. 118 (2016) 561–571. https://doi.org/10.1093/aob/mcw113.
3. A.F. Galloway, J. Akhtar, S.E. Marcus, N. Fletcher, K. Field, P. Knox, Cereal root exudates contain highly structurally complex polysaccharides with soil‐binding properties, The Plant Journal. (2020). https://doi.org/10.1111/tpj.14852.
4. Y. Liu, D. Patko, I. Engelhardt, T.S. George, N. Stanley-Wall, V. Ladmiral, B. Ameduri, T.J. Daniell, N. Holden, M.P. MacDonald, L.X. Dupuy, Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere, Proc Natl Acad Sci U S A. 118 (2021) e2109176118. https://doi.org/https://doi.org/10.1073/pnas.2109176118.

L77

Presentation of the István Ábrahám Nano-Bio-Imaging Center

András Lukács

Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary

The laboratories of István Ábrahám Nano-Bio-Imaging Core Facility are situated in the new theoretical building at the campus of the Medical School of University of Pecs (Szigeti út 12, H-7624 Pécs, Hungary).

The primary but not exclusive purpose of the Core Facility to increase the number of high-quality publications of the university’s research groups by providing access to super-resolution and other advanced fluorescence microscopy techniques. Besides this, the Core Facility is open for any national or international cooperation. We are able to carry out imaging tasks as well as to participate in complex studies.

In case you are interested in using our microscopes or there is any opportunity of cooperating, please visit our website at medschool.pte.hu/nbicf.

We will organize visits to the Facility on Thursday, from 15:00 to 17:00. Please inquire at the Reception Desk.

L59

Ultrastructural and biophysical studies on plastid membranes under salt and drought stress

Roumaissa Ounoki¹, Richard Hembrom¹, Helga Fanni Schubert¹, Adél Sóti¹, Renáta Ünnep², Márton Markó², Gergely Nagy^3,4, Ottó Zsiros⁵, Gábor Sipka⁵, Emilja Dukic⁶, Cornelia Spetea⁶, Győző Garab⁵, Katalin Solymosi¹

¹ELTE Eötvös Loránd University, Department of Plant Anatomy, Budapest, Hungary
²ELRN Centre for Energy Research, Neutron Spectroscopy Department, Budapest, Hungary
³Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, US
⁴ELRN Wigner Research Centre for Physics, Complex Fluids Department, Budapest, Hungary
⁵ELRN Biological Research Centre, Institute of Plant Biology, Szeged, Hungary
⁶University of Gothenburg, Department of Biological and Environmental Sciences, Gothenburg, Sweden

Climate change increases the length and frequency of high temperature and drought periods in Hungary. Along with improper irrigation and land cultivation practices, these factors may also lead to high soil salinity. All these stressors strongly influence plastid structure and function (e.g., photosynthesis), and thus plant growth and crop production. Therefore, basic questions about how different stress factors influence plastid structure and function are of great significance for agriculture.

We will present and critically compare ultrastructural data on plastid membranes obtained with conventional, chemical fixation transmission electron microscopy (TEM) with noninvasive methods like small-angle neutron scattering (SANS). We also investigated the molecular background and light-dependence of the salt or osmotic or drought stress-induced swelling of the intrathylakoidal space using different plants, different plastid types as well as wild type and mutant plants lacking thylakoid ion transport components. Our data show that chloroplasts of fully developed green leaves are less sensitive to salt stress than etioplasts of dark-grown leaves or young, cotyledonal chloroplasts. Determination of the thylakoid-membrane repeat distance (RD) values of grana with SANS and TEM provide similar results: a decrease in RD values is observed both under moderate salt and drought stresses. While dehydration only induced slowly progressing changes in the RD values, rewatering of drought-stressed plants resulted in a fast recovery. It must be noted, however, that long and strong stress conditions may result in the irreversible loss of granum regularity and photosynthetic activity. We have also shown that thylakoid-located ion transporters/channels (K⁺/H⁺ antiporter KEA3, the Cl⁻ channel/transporter CLCe and the voltage-dependent Cl⁻ channel VCCN1) play a role in the thylakoid membrane swelling often observed in salt-stressed plants.

Acknowledgement

This work was supported by NKFIH (OTKA FK124748).

L61

Bistability and switching in the model of membrane voltage dynamics in glia

Predrag Janjic, Dimitar Solev, Ljupco Kocarev

Macedonian Academy of Sciences and Arts, Research Centre for Computer Science and Information Technologies, Skopje, North Macedonia

Despite the molecular evidence that close to linear steady-state I-V relationship in mammalian astrocytes reflects current resulting from more than one differently regulated K⁺ conductances, a detailed ODE model of membrane voltage V_m incorporating multiple conductances is still lacking. Repeated experimental results of deregulated expressions of major K⁺ channels in glia, Kir4.1 in models of neurodegenerative disease, as well as their altered rectification when assembling heteromeric Kir4.1/Kir5.1 channels have motivated us to attempt a detailed model incorporating the weaker K2P-TREK1 K⁺ current, in addition to Kir4.1 and study the stability of the resting state V_r, expecting more complex whole-cell voltage responses than mimicking a potassium electrode that follows the altered E_K. Using a minimal 2-dimensional model of the astrocytic membrane near V_r we show that different alterations of Kir4.1 channel function may result in multistability of astrocytic V_m if the model incorporates the typically observed K⁺ currents - Kir, K2P and non-potassium leak. Depending on the scenario, a decrease or loss of inward/outward Kir4.1 conductance, introduce instability of the resting membrane potential, near E_K, through robustly observed fold bifurcation giving birth to a much more depolarized second, stable resting state V_dr > -10 mV. Transient perturbations by currents through the gap-junctions or from the local field potentials lead to depolarization of the astrocyte and switching of V_m between the two stable states, in a DOWN-state – UP-state manner. Realistic time-series were used to perturb the modeled rodent hippocampal astrocyte, from recordings adjacent to the astrocytic membrane during electrographic seizures in whole hippocampus preparation. Prolonged depolarization at V_dr, if proved experimentally plausible, is a catastrophic event impacting all aspects of the glial function, from metabolic support to membrane transport and practically all neuromodulatory roles assigned to glia.

L62

Drug-membrane interaction of potential SARS-CoV-2 antiviral

Mária Klacsová¹, Adriána Čelková¹, Alexander Búcsi¹, Juan Carlos Martínez², Daniela Uhríková¹

¹Comenius University Bratislava, Faculty of Pharmacy, Department of Physical Chemistry of Drugs, Bratislava, Slovakia
²ALBA synchrotron, Barcelona, Spain

SARS-CoV-2 is an enveloped (+)ssRNA virus belonging to the β‑lineage of coronaviruses. Polyproteins cleaved by viral proteases, a papain-like protease (PL^pro) and a 3C‑like protease (3CL^pro), form the virus replication and transcription machinery. Structure of 3CL^pro is highly conserved among CoVs, therefore it is studied as a potential pharmacological target for specific antiviral drugs. GC376, a bisulfide prodrug of GC373, was discovered to strongly inhibit the 3CL^pro of coronaviruses including SARS-CoV-2.
The drug must pass across one or more phospholipid bilayers to reach the intracellular targets and elicit a response to their pharmacological action. Knowledge of drug-lipid interactions is thus inevitable. We studied partitioning and effect of GC376 on model membranes prepared from synthetic phospholipids. We found that GC376 partitions into lipid bilayer at the level of lipid head‑groups, close to the polar/hydrophobic interface. The lipid/water partition coefficient of GC376 is low, reaching K_P = 46.8. Structural and thermodynamic properties strongly depend on GC376/lipid mole ratio. Above 0.03 mol/mol formation of domains with different GC376 content, resulting in the lateral phase separation and changes in both, main transition temperature and enthalpy, were detected. We attribute observed changes to the response of the system on the increased lateral stresses induced by partitioning of GC376.

Acknowledgements

This project was supported by VEGA 1/0223/20, APVV 17-0239 and APVV-PP-COVID 20-0010 grants, and JINR project 04-4-1142-2021/202. SAXS/WAXS experiments were performed at BL11-NCD beamline (proposal 2021025016) at Alba Synchrotron with the collaboration of Alba staff.

L63

Mechanics of a multilayer epithelium instruct tumor architecture and function

V.F. Fiore¹, M. Krajnc^2,3, F. Garcia Quiroz¹, J. Levorse¹, H. A. Pasolli⁴, S.Y. Shvartsman^2,5,6, E. Fuchs¹

¹Howard Hughes Medical Institute, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA
²Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
³Jozef Stefan Institute, Ljubljana, Slovenia
⁴Electron Microscopy Resource Center, The Rockefeller University, New York, NY, USA
⁵Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA
⁶Department of Molecular Biology, Princeton University, Princeton, NJ, USA

Structural changes of epithelial tissues are typically indicative of oncogenic transformations. Using genetic manipulations, biophysical measurements, and computational modeling, we study morphologies of cancerous tissues in premalignant basal and squamous cell carcinomas—cancers that are associated with considerably different prognoses. We find that the two tumor types cause distinct morphological changes of the epidermis. These differences are mainly caused by distinct mechanical properties of the extracellular matrix, secreted by the respective mutated basal cells. In particular, softening and enhanced remodelling of the basement membrane promote tumor budding, while stiffening of the basement membrane promotes folding. Our findings suggest that mechanical forces function to shape premalignant tumour architectures and influence tumor progression.

L64

Reliable and straightforward cardiac safety liability and proarrhythmic assessment using automated patch clamp

András Horváth¹, Ravi Vaidyanathan², Cara Rieger², Alison Obergrussberger¹, Niels Fertig¹, Sonja Stoelzle-Feix¹, Elena Dragicevic¹, Nadine Becker¹

¹Nanion Technologies, Technologies, Technologies, Munich, Germany
²FUJIFILM Cellular Dynamics, Inc., Madison, WI, USA

Automated patch clamp (APC) devices became important, higher throughput alternatives to manual patch clamp for cardiac safety testing and for studying ion channel mutations and pharmacology.  There is growing interest to use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on those platforms, triggering the development of optimized tools and assays to enable action potential (AP) recordings in addition to the classical voltage clamp recordings. Here, we developed a range of APC assays for commercially available hiPSC-CM lines.

Recordings were performed in voltage clamp or current clamp mode combined with dynamic clamp to obtain reliable AP pharmacology recordings on APC. We studied the effects of known calcium, sodium, late sodium and hERG channel modulators on AP parameters. hERG pharmacology experiments were performed at room temperature and at 37°C.

Class 1/C blocker flecainide effectively inhibited the sodium current and accordingly reduced the AP amplitude (APA) of hiPSC-CMs in a concentration-dependent manner; Class 1/B blocker mexiletine also showed the expected concentration-response curve (IC₅₀: 5.6 µM). The late sodium channel inhibitor ranolazine significantly reduced the APA (14%), upstroke velocity (24%) and AP duration (APD₉₀) at high concentrations. Increased pacing rate from 0.5 Hz to 3Hz resulted in more pronounced effects on APA, as expected. Selective hERG blocker dofetilide prolonged the APD₉₀ and increased the short-term variability of the APs. L-type calcium channel showed sensitivity to blockers (nifedipine and diltiazem), while channel activator BayK 8644 prolonged APD₉₀ in a concentration-dependent manner, which could be reversed by nifedipine.

Our data shows that cardiac ion channel pharmacology can be recorded using hiPSC-CMs in APC, providing a reliable tool for cardiac safety screening and the study of cardiac ion channel diseases in a model system closer to in vivo physiology than heterologous expression systems.

L70

Life beyond the pixels: single-cell analysis using deep learning and image analysis methods

Péter Horváth

Abstract not available

L66

Superresolution optical microscopy

Jaroslaw Jacak

Abstract not available

L67

Fluorescence correlation spectroscopy and its applications in cell biology

György Vámosi

Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary

Fluorescence correlation spectroscopy (FCS) is a fluorescence microscopy technique based on the analysis of intensity fluctuations due to diffusion of fluorescent particles across a subfemtoliter detection volume (typically the focus of a confocal microscope) and photophysical processes. From the fluorescence intensity, the temporal autocorrelation function is generated, which provides information about molecular mobility (molecular mass, free or bound state), the mechanism of diffusion (free, hindered, directed), the local concentration down to the picomolar range and the aggregation state of the molecules. Its unique feature is the increase of the autocorrelation amplitude with decreasing concentration lending the technique single molecule sensitivity. Its two-color version, fluorescence cross-correlation spectroscopy (FCCS) can detect the co-mobility of distinctly labeled molecules indicating their stable association e.g. in protein complexes. Point detection can be extended to 2D mapping of diffusion properties by applying e.g. light sheet or TIRF illumination and photon counting EMCCD cameras, and can be complemented with the simultaneous measurement of molecular proximities by FRET (SPIM-FRET-FCCS). Biological applications for the study of membrane protein complexes and transcription regulatory complexes will be presented.

L68

Intravital imaging as a tool for photodiagnostics and prerequisites for photodynamic therapy

Veronika Huntošová

Abstract not available

L69

Total internal reflection fluorescence microscopy; basics and its applications in biological sciences

Beáta Bugyi^1,2, Veronika Takács-Kollár¹

¹University of Pécs, Medical School, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary
²Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee, Pécs, Hungary

Total internal reflection fluorescence microscopy (TIRFM) imaging is based on the use of an evanescent wave produced at the boundary of two optical media with appropriate refractive indices upon total internal reflection. TIRFM can be considered as one of the earliest super-resolution imaging techniques that significantly overcomes the diffraction limit, by providing axial resolution of ~100 nm. Due to the unique properties of the excitatory evanescent field, TIRFM is emerged as a powerful tool to investigate the surface and/or near-surface features of the structure and dynamics of biomolecules or their assemblies. The combination of TIRFM with other techniques, including microfluidics and surface micropatterning, as well as the improvement of the methodology, provides novel tools for biochemical kinetics investigations and super-resolution single molecule detection approaches. The talk will introduce the basic physical principles and the experimental setup for TIRFM and discusses its classic and novel applications in protein biochemistry, cell biology, and other areas of biological sciences.

L65

Which is the best microscope?

Gábor Csúcs

Abstract not available

L71

National and international young academies: what are the benefits and who are the beneficiaries?

Katalin Solymosi^1,2,3

¹ELTE Eötvös Loránd University, Department of Plant Anatomy, Budapest, Hungary
²Hungarian Young Academy
³Young Academy of Europe

The first Young Academy was die Junge Akademie founded in Germany 22 years ago. Since then, several similar bottom-up initiatives have gathered together early-career researchers at the national or international level to facilitate inter- and transdisciplinary networking among their members, to increase their involvement in science outreach, science policy, science advice mechanisms and decision-making, as well as to provide advocacy for early-career researchers. In a Nature paper, we have briefly summarized six reasons to launch a young academy (Bálint et al. 2021).

In this talk, as founding member and current co-chair of the Hungarian Young Academy, I’ll briefly present how this organization was founded in 2019 with the help of the Hungarian Academy of Sciences, and will summarize its major activities and achievements at the national level since then. I’ll also mention international young academies, with emphasis on the Young Academy of Europe (YAE) (for more details about this organization celebrating its 10^th anniversary this year please see: Modinos et al. 2022). As member and recruitment vice-chair of YAE, I am among others responsible for widening European participation and increasing geographical and other diversity within this organization. YAE is also actively taking part in the core writing group of the European Research Assessment Reform. Since May 2022, the Young Academies Science Advice Structure formed by national and international young academies is officially involved in SAPEA+, i.e. Science Advice for Policy by European Academies. Increasing diversity – e.g. also by improving the participation of early- and mid-career researchers – benefits everybody from working groups, to national societies, institutions as well as society at large.

References

1. Bálint E, ... Solymosi K (2021) Nature 594, 599-601.
2. Modinos G, Solymosi K, Kuppuswamy A (2022): https://network.febs.org/posts/the-young-academy-of-europe-10-years-supporting-science-advice-and-policy

L72

Optically manipulated microtools to measure adhesion of the nanoparticle-targeting ligand glutathione to brain endothelial cells

Tamás Fekete^1,2, Mária Mészáros¹, Gaszton Vizsnyiczai¹, Mária Deli¹, Zsolt Szegletes¹, László Zimányi¹, Szilvia Veszelka¹, Lóránd Kelemen¹

¹Institute of Biophysics, Biological Research Centre, ELKH, Szeged, Hungary
²Doctoral School in Multidisciplinary Medicine, University of Szeged

In the presented research we elaborated a method that is capable of measuring pico-Newton adhesion forces between optically manipulated functionalized microtools and endothelial cell (EC) surfaces. ECs form the Blood Brain Barrier (BBB) that inhibits chemical substances such as pharmacons to easily reach the central nervous system. A promising way to still increase pharmacon uptake through the BBB is to encapsulate them into vesicles. The functionalization of the vesicles with the ligands of solute carrier transporters (SLC), found on the surface of the ECs offer an even more efficient way to deliver pharmacons through the BBB. The tripeptide glutathione (GSH) was shown to be one such successful BBB targeting ligand in the recent years [1].

Our goal was to characterize the binding of GSH-targeted vesicles to endothelial cells by measuring the adhesion force between a surface coated with GSH and that of BBB-forming living endothelial cells. To achieve this, we microfabricated purpose-designed manipulators that can be actuated by optical tweezers [2] and is equipped with a well-defined contact surface. GSH was covalently immobilized on their surface with PEG linkers. The cells were cultured on vertical supporting walls and the adhesion force was obtained by first pushing the micromanipulators against the cells and then retracting them in the lateral direction. The forces were determined on two types of endothelial cells using two different retraction speeds. The measurements were validated with atomic force microscopy which corresponded to the optical tweezers-based results.

Our method can be easily adapted to various ligands of interest owing to a wide spectrum of available PEG-linkers. The assessment of the adhesion force for other ligands or even ligand mixtures can help target BBB-forming cells in a more potent way [3].

References

1. Mészáros M, Porkoláb G, Kiss L et al. Eur J Pharm Sci, 123 (2018) 228–240
2. Aekbote BL, Fekete T, Jacak J, Vizsnyiczai G, Ormos P, and Kelemen L, Biomed Optics Express, 7 (2016) 45-56
3. Fekete T, Mészáros M, Szegletes Zs, Vizsnyiczai G, Zimányi L, Deli MA, Veszelka Sz, Kelemen L, ACS Appl Mater Interf 13 (2021) 39018-39029

L73

Light stimulation of organic electrolytic photocapacitive devices induces ion channel gating and action potentials in neurons

Tony Schmidt¹, Marie Jakešová², Vedran Đerek³, Linda Waldherr¹, Marta Nowakowska⁴, Karin Kornmueller¹, Muammer Üçal⁴, Silke Patz⁴, Theresa Rienmüller⁵, Eric Daniel Głowacki², Rainer Schindl¹

¹Medical University of Graz, Chair of Biophysics, Graz, Austria
²Brno University of Technology, CEITEC, Brno, Czech Republic
³University of Zagreb, Department of Physics, Zagreb, Croatia
⁴Medical University of Graz, Department of Neurosurgery, Graz, Austria
⁵Graz University of Technology, Institute of Health Care Engineering, Graz, Austria

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. Herein we have benchmarked the performance of organic electrolytic photocapacitors (OEPCs) at the level of single mammalian cells. These optoelectronic devices use nontoxic organic pigments that form a planar semiconductor on top of ITO and act as an extracellular stimulation electrode driven by deep red light.

Light stimulation and signal propagation require close contacts between cell membranes and pigments. We could biochemically prove cell viability and show with SEM imaging that cell culture cell lines adhere to the surface and neuronal networks establish and exhibit neurite outgrowth.

Our electrophysiological recordings show that millisecond light-stimulation of OEPCs shifted heterologous expressed voltage-gated K⁺ channel activation by ~ 30 mV. We further demonstrate a time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation and compared our experimental findings with a mathematical model of this bioelectronic-cell system.

In a further step we cultured primary hippocampal neurons on OEPCs and found that millisecond optical stimuli trigger repetitive action potentials in these neurons. Our findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with high precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire new in vivo applications for the regeneration of axonal sprouting in damaged neuronal tissues.

L74

Biophysical modeling and analysis of the predictors of the COVID-19 transmission and clinical severity

Andjela Rodic¹, Igor Salom², Sofija Markovic¹, Ognjen Milicevic³, Dusan Zigic², Bojana Ilic², Magdalena Djordjevic², Marko Djordjevic¹

¹University of Belgrade, Faculty of Biology, Belgrade, Serbia
²University of Belgrade, Institute of Physics, Belgrade, Serbia
³University of Belgrade, Faculty of Medicine, Belgrade, Serbia

During the first wave of the COVID-19 pandemic, the SARS-CoV-2 virus spread according to its inherent transmissibility in a given population, best described by the Basic Reproduction Number (R₀), and the effectiveness of the introduced mitigation measures. We constructed a dynamic compartmental model describing the infection propagation in a population and derived the dependence of R₀ on the slope of the case growth curve in the initial exponential phase on a semi-logarithmic scale.

To identify direct predictors of R₀, determined for 118 countries, among potentially relevant meteorological and sociodemographic factors, characterized by significant mutual correlatedness, we applied the Principal Component Analysis to these two variable sets. [1] Next, we searched for robust R₀ predictors among the obtained, non-correlated Principal Components (PCs) using independently four different methods: the custom multiple regression analysis, the Stepwise regression, the Lasso, and the Elastic net, where the last two methods utilize both regularization and variable selection. To interpret the resulting predictor PCs, we analyzed their correlations with the starting variables and obtained that the countrys prosperity level (probably a proxy for the extent of long-distance contacts), indoor crowdedness, the delay of the epidemic onset, and unhealthy lifestyle and environment appear as the most robust, direct predictors of the SARS-CoV-2 transmissibility [1].

Using a similar methodology on the example of US states, we identified the age, chronic diseases, race, long-term pollution, and population density as direct predictors of a proposed COVID-19 severity measure, independent from R₀. [2]

References

1. Djordjevic M. et al. Inferring the Main Drivers of SARS-CoV-2 Global Transmissibility by Feature Selection Methods. GeoHealth 2021;5(9):e2021GH000432.
2. Markovic S. et al. COVID-19 severity determinants inferred through ecological and epidemiological modeling. One Health 2021;13:100355.

L75

Investigating host-pathogen interaction in disease model for drug development and building a screening platform

Arpita Roy¹, Sylvester Byrne², Nirod K. Sarangi¹, Paul V. Murphy², Tia Keyes¹

¹School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland
²School of Chemistry, NUI Galway, University Road, Galway, Ireland

Seasonal periodic pandemics, and epidemics caused by Influenza A viruses (IAVs) are associated with high morbidity and mortality worldwide. [1] They are frequent and unpredictable in severity so there is a need biophysical platform that can be used to provide both mechanistic insights in influenza virulence and its potential treatment by anti-IAV agents. Host membrane viral association of through the glycoprotein hemagglutinins (HA) of influenza virus is one of the primary steps in influenza viral infection. [2] HA is thus a potential target for drug development against influenza. Deconvolution of the multivalent interactions of HA at the interfaces of host cell membrane can help unravel potential therapeutic targets. Here, we reported on the interaction of a multivalent HA glycoprotein at a microcavity supported lipid bilayer (MSLB) array to investigate the association of artificial host membrane interfaces where different parameters such as membrane resistance, capacitance and membrane diffusivity can be evaluated. We then investigated the inhibition of the influenza HA glycoprotein association at the host cell surface using label-free electrochemical impedance spectroscopy by natural and synthetic sialic acid-based inhibitors (e.g., Sia2,3-Gal, FB127, 3-silyl lactose). The inhibitory activity against influenza HA membrane binding was evaluated. Overall, the data suggest that MSLBs provide a useful label-free screening platform to test potential anti-IAV therapeutics and may they help evaluate potential future therapeutics in an effective, and affordable cell free approach before moving to testing in more expensive cell-based platforms.

 

References

1. Li, Y.; Liu, D.; Wang, Y.; Su, W.; Liu, G.; Dong, W.; Peeples, M. The Importance of Glycans of Viral and Host Proteins in Enveloped Virus Infection. Front. Immunol., 2021, 12, 1–12.
2. Du, R.; Cui, Q.; Rong, L. Competitive Cooperation of Hemagglutinin and Neuraminidase during Influenza a Virus Entry. Viruses 2019, 11, 1–13.