Authors

    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 23rd of August, Tuesday
    • 14:30 – 15:00
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L23

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

    Ágnes Ábrahám1,2, Krisztina Nagy1, Eszter Csákvári1#, László Dér1, Imre Pap1,2, Rebeka Lukács1, Vanda Varga-Zsíros1##, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P44

    Development and applications of bacterial ”Dual Input Mother Machines”

    Imre Pap1,2, Krisztina Nagy1, Ágnes Ábrahám1,2, 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.

    • 22nd of August, Monday
    • 15:15 – 15:45
    • Advances and applications in structural approaches
    • SIOT0032

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P56

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

    Gábor Sipka1, Melinda Magyar1, Parveen Akhtar1,2, Pavel Müller3, Klaus Brettel3, Guangye Han4, Jian-Ren Shen4,6, Stefano Santabarbara5, Petar Lambrev1, Győző Garab1,7

    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 (Fo) to the maximum (Fm) levels; variable chlorophyll-a (Chl-a) fluorescence (FvFm-Fo) upon this transition follows a complex induction kinetics and carries information on the functioning of the photosynthetic machinery. According to the mainstream model, Fo and Fm belong to the open (PSIIO) and closed (PSIIC) 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 PSIIO and PSIIC, this photosystem can assume light-adapted charge-separated state, PSIIL. Formation of PSIIL, via light-induced subtle conformational changes, facilitates the stabilization of the charge-separated state. PSIIL is characterized by distinct features in the energy landscape of trapping/detrapping of excitations in the core-antenna RC complex. The PSIIC–PSIIL transition is responsible for a large part of Fv, 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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 12:15 – 12:30
    • Protein biophysics, molecular spectroscopy II.
    • SIOT0032

    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.
    • 24th of August, Wednesday
    • 12:30 – 12:45
    • Protein biophysics, molecular spectroscopy II.
    • SIOT0032

    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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P62

    Characterization of a novel mutation in Brugada Syndrome

    Tibor G. Szántó1, Szabolcs Gál1,2, 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 (NaV) 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 (INa) 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 NaV1.5 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 NaV1.5 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 NaV1.5 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 INa 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 NaV1.5 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 NaV1.5 channel gating. Moreover, understanding the structure-function relationship of NaV1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P42

    Bacterial evolution of resistance against antibiotics and phages in structured environments

    Krisztina Nagy1, Sarshad Koderi Valappil2, Barbara Dukic1,3, 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.

    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    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).
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P62

    Characterization of a novel mutation in Brugada Syndrome

    Tibor G. Szántó1, Szabolcs Gál1,2, 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 (NaV) 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 (INa) 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 NaV1.5 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 NaV1.5 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 NaV1.5 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 INa 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 NaV1.5 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 NaV1.5 channel gating. Moreover, understanding the structure-function relationship of NaV1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P03

    Calcium-dependent internal dynamics of calmodulin

    Klaudia Onica1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P12

    Mapping the conformational changes of small GTPase Ran

    Janka Czigleczki1, Pedro Túlio de Resende Lara2, Balint Dudas3,4, David Perahia4, Hyunbum Jang5, Ruth Nussinov5,6, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P13

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

    Dániel Tóth1,2, Bálint Dudas2,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P04

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

    Anna Balogh1,2, Kinga Dóra Kovács1,2, 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P62

    Characterization of a novel mutation in Brugada Syndrome

    Tibor G. Szántó1, Szabolcs Gál1,2, 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 (NaV) 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 (INa) 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 NaV1.5 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 NaV1.5 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 NaV1.5 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 INa 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 NaV1.5 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 NaV1.5 channel gating. Moreover, understanding the structure-function relationship of NaV1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L20

    Viscosity measurements using flexible microstructures

    Jana Kubackova1, Cyril Slabý2, Denis Horvath3, Andrej Hovan2, Gergely T. Iványi4,5, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P57

    The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

    Cyril Slabý1, Janka Kubacková2, Denis Horvath3, Andrej Hovan1, Gergely T. Iványi4,5, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P06

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

    Beáta Longauer1,2, András Lukács1,2, Emőke Bódis1, Miklós Nyitrai1,2, Szilvia Barkó1,2

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P63

    The calcium regulated actin remodelling in apoptotic cells

    Alexandra Hencz1,2, 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.

    • 25th of August, Thursday
    • 12:15 – 12:30
    • BioImaging II.
    • SIOT0032

    L55

    Patterned Microfluidics for the Investigation of Plant Root Exudates

    Daniel Patko1, Udara Bimendra Gunatilake1,2, Lionel X. Dupuy3,4, Lourdes Basabe-Desmonts2,4,5,6, Fernando Benito-Lopez1,5,6

    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.
    • 23rd of August, Tuesday
    • 10:15 – 10:45
    • Computer modelling, bioinformatics, systems biology I.
    • SIOT0032

    L08

    Estimation of model and variable complexity and quality

    Bono Lučić1, Viktor Bojović2,3, Antonija Kraljević4,5, 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).

    • 25th of August, Thursday
    • 12:15 – 12:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 25th of August, Thursday
    • 12:15 – 12:30
    • BioImaging II.
    • SIOT0032

    L55

    Patterned Microfluidics for the Investigation of Plant Root Exudates

    Daniel Patko1, Udara Bimendra Gunatilake1,2, Lionel X. Dupuy3,4, Lourdes Basabe-Desmonts2,4,5,6, Fernando Benito-Lopez1,5,6

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 24th of August, Wednesday
    • 15:30 – 15:45
    • Biomedical applications and neuroscience I.
    • SIOT0032

    L43

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

    Aasna Parui1,2, Vandana Mishra3, Subhankar Dutta1, Prasenjit Bhaumik3, Kakoli Bose1,2

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P63

    The calcium regulated actin remodelling in apoptotic cells

    Alexandra Hencz1,2, 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.

    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 23rd of August, Tuesday
    • 17:00 – 17:30
    • Virus biophysics
    • SIOT0032

    L28

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

    Mojca Pavlin1,2, Repas Jernej1, Janžič Larisa3, Maša Bizjak4,Avčin Tadej4,5, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 23rd of August, Tuesday
    • 17:00 – 17:30
    • Virus biophysics
    • SIOT0032

    L28

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

    Mojca Pavlin1,2, Repas Jernej1, Janžič Larisa3, Maša Bizjak4,Avčin Tadej4,5, 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.

    • 23rd of August, Tuesday
    • 12:00 – 12:15
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L12

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

    Višnja Stepanić1, Zlatko Brkljača1,3, 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.

    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P08

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

    Kinga Böde1,2, Ottó Zsiros1, Ondřej Dlouhý3, Uroš Javornik4, Avratanu Biswas1,2, Primož Šket4, Janez Plavec4,5,6, Vladimír Špunda3, Petar H Lambrev1, Bettina Ughy1, Győző Garab1,3

    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 (HII) 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 HII 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 HII phases and does not perturb the structure and function of the photosynthetic machinery. Surprisingly, Photosystem II (BBY) membrane particles displayed no lamellar and HII 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
    • 22nd of August, Monday
    • 16:15 – 16:45
    • Advances and applications in structural approaches
    • SIOT0032

    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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P06

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

    Beáta Longauer1,2, András Lukács1,2, Emőke Bódis1, Miklós Nyitrai1,2, Szilvia Barkó1,2

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P26

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

    János Horváth1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P43

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

    János Horváth1,2, 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.

    • 23rd of August, Tuesday
    • 10:15 – 10:45
    • Computer modelling, bioinformatics, systems biology I.
    • SIOT0032

    L08

    Estimation of model and variable complexity and quality

    Bono Lučić1, Viktor Bojović2,3, Antonija Kraljević4,5, 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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P39

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

    Barbara Majoros1, Beatrix Péter1, Imre Boldizsár2,3, Szilvia Bősze4,5, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P45

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

    Beatrix Péter1, Inna Székács1, Szilvia Bősze2,3, Imre Boldizsár4,5, Gábor M. Kovács4,6, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P37

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

    Beatrix Magyaródi1, Kinga Dóra Kovács1,2, Attila Bonyár3, Ildikó Szabó4,5, Szilvia Bősze4,5, 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
    5
    National 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)
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P42

    Bacterial evolution of resistance against antibiotics and phages in structured environments

    Krisztina Nagy1, Sarshad Koderi Valappil2, Barbara Dukic1,3, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P37

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

    Beatrix Magyaródi1, Kinga Dóra Kovács1,2, Attila Bonyár3, Ildikó Szabó4,5, Szilvia Bősze4,5, 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
    5
    National 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)
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P39

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

    Barbara Majoros1, Beatrix Péter1, Imre Boldizsár2,3, Szilvia Bősze4,5, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P45

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

    Beatrix Péter1, Inna Székács1, Szilvia Bősze2,3, Imre Boldizsár4,5, Gábor M. Kovács4,6, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P09

    Study of plant growth and development by biophysical and mathematical tools: a case study of sunflower

    Maamar Boukabcha

    Faculty of natural sciences and life, Hassiba Benbouali University of Chlef, Chlef, Algeria
    Email: m.boukabcha@univ-chlef.dz

    Plant growth and development is a very important natural and biological phenomenon among the natural and biological systems of living complexes. To study the phenomenon of plant growth and development, we use many tools and methods, including those of biophysics, mathematics, informatics and biological. The results of the growth and development of plants, especially the sunflower plant, which we obtained through field experiments, where many tools and methods related to biophysics, mathematics, informatics and biology, were used to analyse the results of our field experiments, as well as some results of previous studies of the same type. The estimation and calculation of some parameters with the aforementioned methods and tools used for this study plays an important objective of studying the growth and development of plants, especially the sunflower plant as a special case of our study.

    Keywords: Plant growth, Sunflower, Biophysical tools, Mathematical tools, Biological system.

    • 23rd of August, Tuesday
    • 17:30 – 18:00
    • Virus biophysics
    • SIOT0032

    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.

    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P56

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

    Gábor Sipka1, Melinda Magyar1, Parveen Akhtar1,2, Pavel Müller3, Klaus Brettel3, Guangye Han4, Jian-Ren Shen4,6, Stefano Santabarbara5, Petar Lambrev1, Győző Garab1,7

    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 (Fo) to the maximum (Fm) levels; variable chlorophyll-a (Chl-a) fluorescence (FvFm-Fo) upon this transition follows a complex induction kinetics and carries information on the functioning of the photosynthetic machinery. According to the mainstream model, Fo and Fm belong to the open (PSIIO) and closed (PSIIC) 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 PSIIO and PSIIC, this photosystem can assume light-adapted charge-separated state, PSIIL. Formation of PSIIL, via light-induced subtle conformational changes, facilitates the stabilization of the charge-separated state. PSIIL is characterized by distinct features in the energy landscape of trapping/detrapping of excitations in the core-antenna RC complex. The PSIIC–PSIIL transition is responsible for a large part of Fv, 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.
    • 23rd of August, Tuesday
    • 12:00 – 12:15
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L12

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

    Višnja Stepanić1, Zlatko Brkljača1,3, 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.

    • 24th of August, Wednesday
    • 11:15 – 11:45
    • Protein biophysics, molecular spectroscopy II.
    • SIOT0032

    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.

    • 23rd of August, Tuesday
    • 15:00 – 15:15
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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)

    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 25th of August, Thursday
    • 11:45 – 12:00
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 (PLpro) and a 3C‑like protease (3CLpro), form the virus replication and transcription machinery. Structure of 3CLpro 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 3CLpro 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 KP = 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.

    • 23rd of August, Tuesday
    • 12:45 – 13:00
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L22

    Structural and Conformational Dynamics of a Disordered Protein Motif

    Mónika Ágnes Tóth1, Péter Gaszler1, 2, Andrea Teréz Vig1, Veronika Takács-Kollár1, Rauan Sakenov1, Réka Pintér1, Beáta Bugyi1, 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.
    • 25th of August, Thursday
    • 16:30 – 17:00
    • Summer school - advanced optical microscopy II.
    • SIOT0033

    L69

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

    Beáta Bugyi1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P10

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

    Péter Bukovics1, Roland Gábor Vékony1,3, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P27

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

    Tamás Huber1,3, Péter Gaszler1,3, Péter Bukovics1, Réka Pintér1, Rauan Sakenov1, Andrea Teréz Vig1, Mónika Ágnes Tóth1, Veronika Takács-Kollár1, Venukumar Vemula2, Marko Ušaj2, Alf Månsson2, Beáta Bugyi1,3

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 Bugyi1,2

    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 10-30oC 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

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P10

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

    Péter Bukovics1, Roland Gábor Vékony1,3, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P27

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

    Tamás Huber1,3, Péter Gaszler1,3, Péter Bukovics1, Réka Pintér1, Rauan Sakenov1, Andrea Teréz Vig1, Mónika Ágnes Tóth1, Veronika Takács-Kollár1, Venukumar Vemula2, Marko Ušaj2, Alf Månsson2, Beáta Bugyi1,3

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 Bugyi1,2

    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 10-30oC 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

    • 23rd of August, Tuesday
    • 11:45 – 12:00
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L11

    Boundary scattering and the spatial distribution of confined active matter

    András Búzás1, Pál Ormos1, Roberto Di Leonardo2,3, Gaszton Vizsnyiczai1,4

    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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P33

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

    Tamás Gerecsei1,*, Tamás Visnovitz2,3,*, Kinga Dóra Kovács1, Beatrix Peter1, Sándor Kurunczi1, Anna Koncz2, Krisztina Németh2, Dorina Lenzinger2, Krisztina V. Vukman2, Péter Lőrincz4, Inna Székács1, Edit I. Buzás2,5,6,**, Robert Horvath1,**

    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.

    • 26th of August, Friday
    • 10:05 – 10:20
    • Young investigators session
    • SIOT0032

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P29

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

    Alba Espina1, S. Sanchez-Cortes2,3, 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.

    • 23rd of August, Tuesday
    • 9:45 – 10:15
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L15

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

    Beatrice Senigagliesi1,2, Pietro Parisse1,3, 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.

    • 25th of August, Thursday
    • 11:45 – 12:00
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 (PLpro) and a 3C‑like protease (3CLpro), form the virus replication and transcription machinery. Structure of 3CLpro 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 3CLpro 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 KP = 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 (Yt), 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 Yt values, PVA (0.5-1.5 GPa) and PSI (0.5-3.5 GPa) were more rigid than PCL (0.1-0.5 GPa). Yt 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. Yt 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 12:00 – 12:15
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L12

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

    Višnja Stepanić1, Zlatko Brkljača1,3, 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.

    • 23rd of August, Tuesday
    • 14:30 – 15:00
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L23

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

    Ágnes Ábrahám1,2, Krisztina Nagy1, Eszter Csákvári1#, László Dér1, Imre Pap1,2, Rebeka Lukács1, Vanda Varga-Zsíros1##, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P62

    Characterization of a novel mutation in Brugada Syndrome

    Tibor G. Szántó1, Szabolcs Gál1,2, 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 (NaV) 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 (INa) 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 NaV1.5 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 NaV1.5 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 NaV1.5 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 INa 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 NaV1.5 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 NaV1.5 channel gating. Moreover, understanding the structure-function relationship of NaV1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P11

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

    Marianna Csaplár1, Árpád Szöőr1, György Vereb1,2

    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 1st (.z) 2nd (CD28.z, 41BB.z) and 3rd (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. 1st, 2nd and 3rd 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.

    • 23rd of August, Tuesday
    • 10:30 – 10:45
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L17

    Imaging the infection cycle of T7 at the single virion level

    Bálint Kiss1,2, Luca Annamária Kiss1, Zsombor Dávid Lohinai1, Dorottya Mudra1, Hedvig Tordai1, Levente Herényi1, Gabriella Csík1, Miklós Kellermayer1,2

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 17:30 – 17:45
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L46

    Intercellular nanomechanics in brain metastasis formation

    Attila Gergely Végh1, Katalin Csonti1,2,3, 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.

    • 25th of August, Thursday
    • 9:45 – 10:15
    • BioImaging I.
    • SIOT0032

    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.

    • 25th of August, Thursday
    • 17:00 – 17:30
    • Summer school - advanced optical microscopy II.
    • SIOT0033

    L65

    Which is the best microscope?

    Gábor Csúcs

    Abstract not available

    • 24th of August, Wednesday
    • 10:00 – 10:30
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P12

    Mapping the conformational changes of small GTPase Ran

    Janka Czigleczki1, Pedro Túlio de Resende Lara2, Balint Dudas3,4, David Perahia4, Hyunbum Jang5, Ruth Nussinov5,6, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P63

    The calcium regulated actin remodelling in apoptotic cells

    Alexandra Hencz1,2, 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.

    • 23rd of August, Tuesday
    • 15:00 – 15:15
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    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
    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 26th of August, Friday
    • 9:20 – 9:35
    • Young investigators session
    • SIOT0032

    L72

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

    Tamás Fekete1,2, 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P32

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

    András Kincses1, Ana R. Santa-Maria1, Fruzsina R. Walter1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 Petrovszki1,2, Fruzsina R. Walter1, Judit P. Vigh1,3, 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.

    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P24

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

    János Horváth1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P26

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

    János Horváth1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P32

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

    András Kincses1, Ana R. Santa-Maria1, Fruzsina R. Walter1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P43

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

    János Horváth1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 Petrovszki1,2, Fruzsina R. Walter1, Judit P. Vigh1,3, 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.

    • 23rd of August, Tuesday
    • 14:30 – 15:00
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L23

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

    Ágnes Ábrahám1,2, Krisztina Nagy1, Eszter Csákvári1#, László Dér1, Imre Pap1,2, Rebeka Lukács1, Vanda Varga-Zsíros1##, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P32

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

    András Kincses1, Ana R. Santa-Maria1, Fruzsina R. Walter1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P42

    Bacterial evolution of resistance against antibiotics and phages in structured environments

    Krisztina Nagy1, Sarshad Koderi Valappil2, Barbara Dukic1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P44

    Development and applications of bacterial ”Dual Input Mother Machines”

    Imre Pap1,2, Krisztina Nagy1, Ágnes Ábrahám1,2, 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.

    • 26th of August, Friday
    • 9:35 – 9:50
    • Young investigators session
    • SIOT0032

    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.

    • 26th of August, Friday
    • 9:50 – 10:05
    • Young investigators session
    • SIOT0032

    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.
    • 26th of August, Friday
    • 9:50 – 10:05
    • Young investigators session
    • SIOT0032

    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.
    • 24th of August, Wednesday
    • 15:00 – 15:30
    • Biomedical applications and neuroscience I.
    • SIOT0032

    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.

    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P08

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

    Kinga Böde1,2, Ottó Zsiros1, Ondřej Dlouhý3, Uroš Javornik4, Avratanu Biswas1,2, Primož Šket4, Janez Plavec4,5,6, Vladimír Špunda3, Petar H Lambrev1, Bettina Ughy1, Győző Garab1,3

    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 (HII) 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 HII 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 HII phases and does not perturb the structure and function of the photosynthetic machinery. Surprisingly, Photosystem II (BBY) membrane particles displayed no lamellar and HII 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
    • 22nd of August, Monday
    • 15:15 – 15:45
    • Advances and applications in structural approaches
    • SIOT0032

    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.

    • 24th of August, Wednesday
    • 11:45 – 12:15
    • Protein biophysics, molecular spectroscopy II.
    • SIOT0032

    L37

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

    Sanja Dolanski Babić1,2, Kristina Serec1,2, 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.

    • 23rd of August, Tuesday
    • 11:30 – 11:45
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L10

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

    Marko Gosak1,2, Jurij Dolenšek1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 25th of August, Thursday
    • 12:15 – 12:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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.

    • 25th of August, Thursday
    • 12:00 – 12:15
    • BioImaging II.
    • SIOT0032

    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 Peric4,5, Aleksandar Krmpot1

    1Institute of Physics Belgrade, University of Belgrade, Serbia
    2Institute for Medical Research, University of Belgrade, Serbia
    3Karolinska Institute
    t, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P35

    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 Peric4,5, Aleksandar Krmpot1

    1Institute of Physics Belgrade, University of Belgrade, Serbia
    2Institute for Medical Research, University of Belgrade, Serbia
    3Karolinska Institute
    t, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P12

    Mapping the conformational changes of small GTPase Ran

    Janka Czigleczki1, Pedro Túlio de Resende Lara2, Balint Dudas3,4, David Perahia4, Hyunbum Jang5, Ruth Nussinov5,6, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P13

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

    Dániel Tóth1,2, Bálint Dudas2,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P42

    Bacterial evolution of resistance against antibiotics and phages in structured environments

    Krisztina Nagy1, Sarshad Koderi Valappil2, Barbara Dukic1,3, 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.

    • 25th of August, Thursday
    • 11:00 – 11:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 Nagy3,4, 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).

    • 25th of August, Thursday
    • 12:15 – 12:30
    • BioImaging II.
    • SIOT0032

    L55

    Patterned Microfluidics for the Investigation of Plant Root Exudates

    Daniel Patko1, Udara Bimendra Gunatilake1,2, Lionel X. Dupuy3,4, Lourdes Basabe-Desmonts2,4,5,6, Fernando Benito-Lopez1,5,6

    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.
    • 24th of August, Wednesday
    • 15:30 – 15:45
    • Biomedical applications and neuroscience I.
    • SIOT0032

    L43

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

    Aasna Parui1,2, Vandana Mishra3, Subhankar Dutta1, Prasenjit Bhaumik3, Kakoli Bose1,2

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 12:00 – 12:15
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L12

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

    Višnja Stepanić1, Zlatko Brkljača1,3, 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.

    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P29

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

    Alba Espina1, S. Sanchez-Cortes2,3, 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.

    • 23rd of August, Tuesday
    • 18:00 – 18:15
    • Virus biophysics
    • SIOT0032

    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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 24th of August, Wednesday
    • 17:30 – 17:45
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L46

    Intercellular nanomechanics in brain metastasis formation

    Attila Gergely Végh1, Katalin Csonti1,2,3, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 26th of August, Friday
    • 9:20 – 9:35
    • Young investigators session
    • SIOT0032

    L72

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

    Tamás Fekete1,2, 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 25th of August, Thursday
    • 12:15 – 12:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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.

    • 24th of August, Wednesday
    • 10:00 – 10:30
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P62

    Characterization of a novel mutation in Brugada Syndrome

    Tibor G. Szántó1, Szabolcs Gál1,2, 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 (NaV) 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 (INa) 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 NaV1.5 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 NaV1.5 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 NaV1.5 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 INa 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 NaV1.5 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 NaV1.5 channel gating. Moreover, understanding the structure-function relationship of NaV1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

    • 23rd of August, Tuesday
    • 14:30 – 15:00
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L23

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

    Ágnes Ábrahám1,2, Krisztina Nagy1, Eszter Csákvári1#, László Dér1, Imre Pap1,2, Rebeka Lukács1, Vanda Varga-Zsíros1##, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P42

    Bacterial evolution of resistance against antibiotics and phages in structured environments

    Krisztina Nagy1, Sarshad Koderi Valappil2, Barbara Dukic1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P44

    Development and applications of bacterial ”Dual Input Mother Machines”

    Imre Pap1,2, Krisztina Nagy1, Ágnes Ábrahám1,2, 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.

    • 23rd of August, Tuesday
    • 10:15 – 10:30
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L16

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

    María Pedrosa1,2, Pablo Graván-Jiménez2,3, Jesús Peña-Martín2,3, Julia Maldonado-Valderrama1,2, Matej Kanduč4, Arturo Moncho-Jordá1,5, María José Gálvez-Ruiz1,2

    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.
    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 25th of August, Thursday
    • 11:00 – 11:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 Nagy3,4, 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).

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P08

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

    Kinga Böde1,2, Ottó Zsiros1, Ondřej Dlouhý3, Uroš Javornik4, Avratanu Biswas1,2, Primož Šket4, Janez Plavec4,5,6, Vladimír Špunda3, Petar H Lambrev1, Bettina Ughy1, Győző Garab1,3

    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 (HII) 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 HII 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 HII phases and does not perturb the structure and function of the photosynthetic machinery. Surprisingly, Photosystem II (BBY) membrane particles displayed no lamellar and HII 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P56

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

    Gábor Sipka1, Melinda Magyar1, Parveen Akhtar1,2, Pavel Müller3, Klaus Brettel3, Guangye Han4, Jian-Ren Shen4,6, Stefano Santabarbara5, Petar Lambrev1, Győző Garab1,7

    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 (Fo) to the maximum (Fm) levels; variable chlorophyll-a (Chl-a) fluorescence (FvFm-Fo) upon this transition follows a complex induction kinetics and carries information on the functioning of the photosynthetic machinery. According to the mainstream model, Fo and Fm belong to the open (PSIIO) and closed (PSIIC) 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 PSIIO and PSIIC, this photosystem can assume light-adapted charge-separated state, PSIIL. Formation of PSIIL, via light-induced subtle conformational changes, facilitates the stabilization of the charge-separated state. PSIIL is characterized by distinct features in the energy landscape of trapping/detrapping of excitations in the core-antenna RC complex. The PSIIC–PSIIL transition is responsible for a large part of Fv, 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.
    • 23rd of August, Tuesday
    • 15:00 – 15:30
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L24

    Aptamer-functionalized surfaces and nanomotors as potential platforms for diagnostics

    Veronika Subjakova1, Zuzana Garaiova1, Joseph Wang2 , Tibor Hianik1

    1Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska Dolina F1, 842 48 Bratislava, Slovakia
    2
    Department of Nanoengineering, University of California San Diego
    La Jolla, California, 92093, United States

    Early diagnosis of diseases is crucial for prevention as well as for therapy. Rapid and accurate identification of diseases such as cancer, infectious or others may decrease mortality and reduce health complications. In the same way, it helps to provide the appropriate and effective treatment at earlier stages. Conventional methods of diagnostics such as polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemical method and fluorescence in situ hybridization are typically used to detect markers in various diseases. These methods provide precise and sensitive detection but are time-consuming and require expensive equipment. Therefore, low cost, faster and highly specific methods are required for early diagnostics. Biosensor technology can offer these requirements. A biosensor consists of a receptor that recognize target molecules and a transducer that converts typically chemical signal to a physical value that can be analyzed. Among receptors the DNA aptamers are of substantial interest in biosensor development. Aptamers are single stranded nucleic acids, DNA or RNA, and have several advantages over antibodies. They are synthesized in vitro and are more stable than antibodies. In solution aptamers fold into 3D structure creating specific binding site for target molecule. They can be chemically modified which allows their immobilization on the different surfaces or labeling by probes [1]. Nanomaterials are often used as immobilization platform in biosensors to enhance signal, reduce volume as well as for miniaturization of device [2]. Nanomotors are nanostructures capable of converting energy from different sources (chemical, light, magnetic, ultrasound) to motion. The functionalization of their surface by bioreceptors allows their usage for biosensing, diagnostics, therapy, or targeted drug delivery [3,4]. In this work we will present an overview of application of aptamer-functionalized surfaces and nanomotors for detection of cancer markers.

    Acknowledgements

    This work was funded under European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No 101007299 as well as by Science Grant Agency VEGA, project No. 1/0419/20

    References

    1. V. Subjakova, V. Oravczova, M. Tatarko, T. Hianik, Electrochimica Acta 389 (2021) 138724.
    2. M. Holzinger, A. Le Goff, S. Cosnier, Front. Chem. 2 (2014) 1–10.
    3. V. Subjakova, V. Oravczova, T. Hianik, Polymers, 13 (2021) 1-4.
    4. M. Beltrán-Gastélum, B. Esteban-Fernández de Ávila, H. Gong, P. Lekshmy Venugopalan, T. Hianik, J. Wang, V. Subjakova, ChemPhysChem. 20 (2019) 3177-3180.
    • 23rd of August, Tuesday
    • 15:00 – 15:15
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    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
    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 23rd of August, Tuesday
    • 12:45 – 13:00
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L22

    Structural and Conformational Dynamics of a Disordered Protein Motif

    Mónika Ágnes Tóth1, Péter Gaszler1, 2, Andrea Teréz Vig1, Veronika Takács-Kollár1, Rauan Sakenov1, Réka Pintér1, Beáta Bugyi1, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P27

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

    Tamás Huber1,3, Péter Gaszler1,3, Péter Bukovics1, Réka Pintér1, Rauan Sakenov1, Andrea Teréz Vig1, Mónika Ágnes Tóth1, Veronika Takács-Kollár1, Venukumar Vemula2, Marko Ušaj2, Alf Månsson2, Beáta Bugyi1,3

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 Bugyi1,2

    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 10-30oC 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

    • 26th of August, Friday
    • 9:35 – 9:50
    • Young investigators session
    • SIOT0032

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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 Jacko1,2, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P33

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

    Tamás Gerecsei1,*, Tamás Visnovitz2,3,*, Kinga Dóra Kovács1, Beatrix Peter1, Sándor Kurunczi1, Anna Koncz2, Krisztina Németh2, Dorina Lenzinger2, Krisztina V. Vukman2, Péter Lőrincz4, Inna Székács1, Edit I. Buzás2,5,6,**, Robert Horvath1,**

    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.

    • 22nd of August, Monday
    • 15:45 – 16:15
    • Advances and applications in structural approaches
    • SIOT0032

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 23rd of August, Tuesday
    • 11:30 – 11:45
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    L10

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

    Marko Gosak1,2, Jurij Dolenšek1,2, 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.

    • 23rd of August, Tuesday
    • 10:15 – 10:30
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L16

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

    María Pedrosa1,2, Pablo Graván-Jiménez2,3, Jesús Peña-Martín2,3, Julia Maldonado-Valderrama1,2, Matej Kanduč4, Arturo Moncho-Jordá1,5, María José Gálvez-Ruiz1,2

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 25th of August, Thursday
    • 12:15 – 12:30
    • BioImaging II.
    • SIOT0032

    L55

    Patterned Microfluidics for the Investigation of Plant Root Exudates

    Daniel Patko1, Udara Bimendra Gunatilake1,2, Lionel X. Dupuy3,4, Lourdes Basabe-Desmonts2,4,5,6, Fernando Benito-Lopez1,5,6

    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.
    • 24th of August, Wednesday
    • 9:30 – 10:00
    • Protein biophysics, molecular spectroscopy I.
    • SIOT0032

    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)
    • 25th of August, Thursday
    • 9:45 – 10:15
    • Membrane and ion channel biophysics, cell mechanics I.
    • SIOT0033

    L57

    Membrane distribution of ion channels in T cells

    Orsolya Vörös1, György Panyi1, Péter Hajdu1,2

    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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 25th of August, Thursday
    • 10:15 – 10:30
    • BioImaging I.
    • SIOT0032

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P56

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

    Gábor Sipka1, Melinda Magyar1, Parveen Akhtar1,2, Pavel Müller3, Klaus Brettel3, Guangye Han4, Jian-Ren Shen4,6, Stefano Santabarbara5, Petar Lambrev1, Győző Garab1,7

    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 (Fo) to the maximum (Fm) levels; variable chlorophyll-a (Chl-a) fluorescence (FvFm-Fo) upon this transition follows a complex induction kinetics and carries information on the functioning of the photosynthetic machinery. According to the mainstream model, Fo and Fm belong to the open (PSIIO) and closed (PSIIC) 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 PSIIO and PSIIC, this photosystem can assume light-adapted charge-separated state, PSIIL. Formation of PSIIL, via light-induced subtle conformational changes, facilitates the stabilization of the charge-separated state. PSIIL is characterized by distinct features in the energy landscape of trapping/detrapping of excitations in the core-antenna RC complex. The PSIIC–PSIIL transition is responsible for a large part of Fv, 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.
    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Computer modelling, bioinformatics, systems biology II.
    • SIOT0032

    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).
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.
    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P60

    Collection, prediction, and publication of ABC transmembrane protein structures

    Erzsébet Suhajda1,2, Hedvig Tordai1, Ian Sillitoe3, Sreenath Nair4, Mihály Váradi4, Tamás Hegedűs1,5

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 (Yt), 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 Yt values, PVA (0.5-1.5 GPa) and PSI (0.5-3.5 GPa) were more rigid than PCL (0.1-0.5 GPa). Yt 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. Yt 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.

    • 25th of August, Thursday
    • 11:00 – 11:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 Nagy3,4, 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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P63

    The calcium regulated actin remodelling in apoptotic cells

    Alexandra Hencz1,2, 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.

    • 23rd of August, Tuesday
    • 10:30 – 10:45
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L17

    Imaging the infection cycle of T7 at the single virion level

    Bálint Kiss1,2, Luca Annamária Kiss1, Zsombor Dávid Lohinai1, Dorottya Mudra1, Hedvig Tordai1, Levente Herényi1, Gabriella Csík1, Miklós Kellermayer1,2

    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.

    • 23rd of August, Tuesday
    • 16:30 – 17:00
    • Virus biophysics
    • SIOT0032

    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 Kis2,3

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 15:00 – 15:30
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    L24

    Aptamer-functionalized surfaces and nanomotors as potential platforms for diagnostics

    Veronika Subjakova1, Zuzana Garaiova1, Joseph Wang2 , Tibor Hianik1

    1Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska Dolina F1, 842 48 Bratislava, Slovakia
    2
    Department of Nanoengineering, University of California San Diego
    La Jolla, California, 92093, United States

    Early diagnosis of diseases is crucial for prevention as well as for therapy. Rapid and accurate identification of diseases such as cancer, infectious or others may decrease mortality and reduce health complications. In the same way, it helps to provide the appropriate and effective treatment at earlier stages. Conventional methods of diagnostics such as polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemical method and fluorescence in situ hybridization are typically used to detect markers in various diseases. These methods provide precise and sensitive detection but are time-consuming and require expensive equipment. Therefore, low cost, faster and highly specific methods are required for early diagnostics. Biosensor technology can offer these requirements. A biosensor consists of a receptor that recognize target molecules and a transducer that converts typically chemical signal to a physical value that can be analyzed. Among receptors the DNA aptamers are of substantial interest in biosensor development. Aptamers are single stranded nucleic acids, DNA or RNA, and have several advantages over antibodies. They are synthesized in vitro and are more stable than antibodies. In solution aptamers fold into 3D structure creating specific binding site for target molecule. They can be chemically modified which allows their immobilization on the different surfaces or labeling by probes [1]. Nanomaterials are often used as immobilization platform in biosensors to enhance signal, reduce volume as well as for miniaturization of device [2]. Nanomotors are nanostructures capable of converting energy from different sources (chemical, light, magnetic, ultrasound) to motion. The functionalization of their surface by bioreceptors allows their usage for biosensing, diagnostics, therapy, or targeted drug delivery [3,4]. In this work we will present an overview of application of aptamer-functionalized surfaces and nanomotors for detection of cancer markers.

    Acknowledgements

    This work was funded under European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No 101007299 as well as by Science Grant Agency VEGA, project No. 1/0419/20

    References

    1. V. Subjakova, V. Oravczova, M. Tatarko, T. Hianik, Electrochimica Acta 389 (2021) 138724.
    2. M. Holzinger, A. Le Goff, S. Cosnier, Front. Chem. 2 (2014) 1–10.
    3. V. Subjakova, V. Oravczova, T. Hianik, Polymers, 13 (2021) 1-4.
    4. M. Beltrán-Gastélum, B. Esteban-Fernández de Ávila, H. Gong, P. Lekshmy Venugopalan, T. Hianik, J. Wang, V. Subjakova, ChemPhysChem. 20 (2019) 3177-3180.
    • 23rd of August, Tuesday
    • 15:00 – 15:15
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    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
    • 23rd of August, Tuesday
    • 18:15 – 18:30
    • Virus biophysics
    • SIOT0032

    L31

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

    Elek Telek1,†, Zoltán Ujfalusi1,†, Gábor Kemenesi2,3,4, Brigitta Zana2,3,4, Ferenc Jakab2,3,4, 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.

    • 23rd of August, Tuesday
    • 18:15 – 18:30
    • Virus biophysics
    • SIOT0032

    L31

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

    Elek Telek1,†, Zoltán Ujfalusi1,†, Gábor Kemenesi2,3,4, Brigitta Zana2,3,4, Ferenc Jakab2,3,4, 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.

    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L20

    Viscosity measurements using flexible microstructures

    Jana Kubackova1, Cyril Slabý2, Denis Horvath3, Andrej Hovan2, Gergely T. Iványi4,5, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P57

    The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

    Cyril Slabý1, Janka Kubacková2, Denis Horvath3, Andrej Hovan1, Gergely T. Iványi4,5, 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.
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P04

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

    Anna Balogh1,2, Kinga Dóra Kovács1,2, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P17

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

    Eniko Farkas1, Robert Tarr1,2, Tamás Gerecsei1,3, Andras Saftics1, Kinga Dóra Kovács1,3, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P33

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

    Tamás Gerecsei1,*, Tamás Visnovitz2,3,*, Kinga Dóra Kovács1, Beatrix Peter1, Sándor Kurunczi1, Anna Koncz2, Krisztina Németh2, Dorina Lenzinger2, Krisztina V. Vukman2, Péter Lőrincz4, Inna Székács1, Edit I. Buzás2,5,6,**, Robert Horvath1,**

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P37

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

    Beatrix Magyaródi1, Kinga Dóra Kovács1,2, Attila Bonyár3, Ildikó Szabó4,5, Szilvia Bősze4,5, 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
    5
    National 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)
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P39

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

    Barbara Majoros1, Beatrix Péter1, Imre Boldizsár2,3, Szilvia Bősze4,5, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P45

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

    Beatrix Péter1, Inna Székács1, Szilvia Bősze2,3, Imre Boldizsár4,5, Gábor M. Kovács4,6, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P47

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

    Imola Rajmon1,2, Kinga Dóra Kovács1,2, 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
    • 25th of August, Thursday
    • 12:15 – 12:30
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P24

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

    János Horváth1,2, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P26

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

    János Horváth1,2, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P43

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

    János Horváth1,2, 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.

    • 25th of August, Thursday
    • 11:30 – 12:00
    • BioImaging II.
    • SIOT0032

    L53

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

    Péter Horváth1,2

    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.

    • 25th of August, Thursday
    • 14:00 – 14:30
    • Summer school - advanced optical microscopy I.
    • SIOT0033

    L70

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

    Péter Horváth

    Abstract not available

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L20

    Viscosity measurements using flexible microstructures

    Jana Kubackova1, Cyril Slabý2, Denis Horvath3, Andrej Hovan2, Gergely T. Iványi4,5, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P57

    The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

    Cyril Slabý1, Janka Kubacková2, Denis Horvath3, Andrej Hovan1, Gergely T. Iványi4,5, 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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P27

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

    Tamás Huber1,3, Péter Gaszler1,3, Péter Bukovics1, Réka Pintér1, Rauan Sakenov1, Andrea Teréz Vig1, Mónika Ágnes Tóth1, Veronika Takács-Kollár1, Venukumar Vemula2, Marko Ušaj2, Alf Månsson2, Beáta Bugyi1,3

    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.

    • 25th of August, Thursday
    • 11:00 – 11:30
    • BioImaging II.
    • SIOT0032

    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.
    • 25th of August, Thursday
    • 16:00 – 16:30
    • Summer school - advanced optical microscopy II.
    • SIOT0033

    L68

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

    Veronika Huntošová

    Abstract not available

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.
    • 22nd of August, Monday
    • 15:45 – 16:15
    • Advances and applications in structural approaches
    • SIOT0032

    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.

    • 26th of August, Friday
    • 9:50 – 10:05
    • Young investigators session
    • SIOT0032

    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.
    • 25th of August, Thursday
    • 12:00 – 12:15
    • BioImaging II.
    • SIOT0032

    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 Peric4,5, Aleksandar Krmpot1

    1Institute of Physics Belgrade, University of Belgrade, Serbia
    2Institute for Medical Research, University of Belgrade, Serbia
    3Karolinska Institute
    t, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P35

    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 Peric4,5, Aleksandar Krmpot1

    1Institute of Physics Belgrade, University of Belgrade, Serbia
    2Institute for Medical Research, University of Belgrade, Serbia
    3Karolinska Institute
    t, 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.

    • 23rd of August, Tuesday
    • 15:00 – 15:15
    • Nanoscale biophysics, nanobiotechnology, material sciences III.
    • SIOT0032

    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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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)

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L20

    Viscosity measurements using flexible microstructures

    Jana Kubackova1, Cyril Slabý2, Denis Horvath3, Andrej Hovan2, Gergely T. Iványi4,5, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P57

    The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

    Cyril Slabý1, Janka Kubacková2, Denis Horvath3, Andrej Hovan1, Gergely T. Iványi4,5, 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.
    • 25th of August, Thursday
    • 9:00 – 9:45
    • BioImaging I.
    • SIOT0032

    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).
    • 25th of August, Thursday
    • 14:30 – 15:00
    • Summer school - advanced optical microscopy I.
    • SIOT0033

    L66

    Superresolution optical microscopy

    Jaroslaw Jacak

    Abstract not available

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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 Jacko1,2, 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.
    • 23rd of August, Tuesday
    • 18:15 – 18:30
    • Virus biophysics
    • SIOT0032

    L31

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

    Elek Telek1,†, Zoltán Ujfalusi1,†, Gábor Kemenesi2,3,4, Brigitta Zana2,3,4, Ferenc Jakab2,3,4, 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.

    • 24th of August, Wednesday
    • 17:00 – 17:30
    • Biomedical applications and neuroscience II.
    • SIOT0032

    L45

    Bioelectronic Chemo Drug Delivery for Brain Tumor Treatment

    Linda Waldherr1, Verena Handl1,2, 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
    • 26th of August, Friday
    • 9:35 – 9:50
    • Young investigators session
    • SIOT0032

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P12

    Mapping the conformational changes of small GTPase Ran

    Janka Czigleczki1, Pedro Túlio de Resende Lara2, Balint Dudas3,4, David Perahia4, Hyunbum Jang5, Ruth Nussinov5,6, 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.

    • 25th of August, Thursday
    • 11:30 – 11:45
    • Membrane and ion channel biophysics, cell mechanics II.
    • SIOT0033

    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 Vm 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 Vr, expecting more complex whole-cell voltage responses than mimicking a potassium electrode that follows the altered EK. Using a minimal 2-dimensional model of the astrocytic membrane near Vr we show that different alterations of Kir4.1 channel function may result in multistability of astrocytic Vm 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 EK, through robustly observed fold bifurcation giving birth to a much more depolarized second, stable resting state Vdr > -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 Vm 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 Vdr, 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.

    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P08

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

    Kinga Böde1,2, Ottó Zsiros1, Ondřej Dlouhý3, Uroš Javornik4, Avratanu Biswas1,2, Primož Šket4, Janez Plavec4,5,6, Vladimír Špunda3, Petar H Lambrev1, Bettina Ughy1, Győző Garab1,3

    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 (HII) 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 HII 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 HII phases and does not perturb the structure and function of the photosynthetic machinery. Surprisingly, Photosystem II (BBY) membrane particles displayed no lamellar and HII 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
    • 22nd of August, Monday
    • 15:45 – 16:15
    • Advances and applications in structural approaches
    • SIOT0032

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 (Yt), 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 Yt values, PVA (0.5-1.5 GPa) and PSI (0.5-3.5 GPa) were more rigid than PCL (0.1-0.5 GPa). Yt 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. Yt 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.

    • 23rd of August, Tuesday
    • 17:00 – 17:30
    • Virus biophysics
    • SIOT0032

    L28

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

    Mojca Pavlin1,2, Repas Jernej1, Janžič Larisa3, Maša Bizjak4,Avčin Tadej4,5, 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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P53

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

    Hana Majaron1,2, Boštjan Kokot1,3, Aleksandar Sebastijanović1,2, Carola Voss4, Rok Podlipec1,5, Patrycja Zawilska1, Trine Berthing6, Carolina Ballester López4, Pernille Høgh Danielsen6, Claudia Contini7, Mikhail Ivanov8, Ana Krišelj1, Petra Čotar1,9, Qiaoxia Zhou4,10, Jessica Ponti11, Vadim Zhernovkov12, Matthew Schneemilch7, Zahra Manel Doumandji14, Mojca Pušnik13, Polona Umek1, Stane Pajk1,13, Olivier Joubert14, Otmar Schmid4, Iztok Urbančič1, Martin Irmler15, Johannes Beckers15,16,17, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P29

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

    Alba Espina1, S. Sanchez-Cortes2,3, 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.

    • 24th of August, Wednesday
    • 15:30 – 15:45
    • Biomedical applications and neuroscience I.
    • SIOT0032

    L43

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

    Aasna Parui1,2, Vandana Mishra3, Subhankar Dutta1, Prasenjit Bhaumik3, Kakoli Bose1,2

    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.

    • 23rd of August, Tuesday
    • 10:15 – 10:30
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L16

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

    María Pedrosa1,2, Pablo Graván-Jiménez2,3, Jesús Peña-Martín2,3, Julia Maldonado-Valderrama1,2, Matej Kanduč4, Arturo Moncho-Jordá1,5, María José Gálvez-Ruiz1,2

    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.
    • 23rd of August, Tuesday
    • 11:45 – 12:15
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.
    • 24th of August, Wednesday
    • 18:00 – 18:15
    • Biomedical applications and neuroscience II.
    • SIOT0032

    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 Tm, rH 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.

    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P47

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

    Imola Rajmon1,2, Kinga Dóra Kovács1,2, 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
    • 22nd of August, Monday
    • 16:15 – 16:45
    • Advances and applications in structural approaches
    • SIOT0032

    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.
    • 24th of August, Wednesday
    • 11:15 – 11:45
    • Protein biophysics, molecular spectroscopy II.
    • SIOT0032

    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.

    • 22nd of August, Monday
    • 16:45 – 17:15
    • Advances and applications in structural approaches
    • SIOT0032

    L04

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

    Ondřej Dlouhý1, Václav Karlický1,2, Uroš Javornik3, Irena Kurasová1, Ottó Zsiros4, Primož Šket3, Divya Kanna4, Kristýna Večeřová2, Kinga Böde4, Otmar Urban2, Edward S. Gasanoff5,6, Janez Plavec3,7,8, Vladimír Špunda1,2, Bettina Ughy4, Győző Garab1,4

    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 HII 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
    • 23rd of August, Tuesday
    • 12:15 – 12:30
    • Nanoscale biophysics, nanobiotechnology, material sciences II.
    • SIOT0033

    L20

    Viscosity measurements using flexible microstructures

    Jana Kubackova1, Cyril Slabý2, Denis Horvath3, Andrej Hovan2, Gergely T. Iványi4,5, 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.

    • 26th of August, Friday
    • 9:20 – 9:35
    • Young investigators session
    • SIOT0032

    L72

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

    Tamás Fekete1,2, 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
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P01

    Emergence of Phenotypic Heterogeneity in Bacteria Studied by Microfluidic Devices

    Ágnes Ábrahám1,2, Krisztina Nagy1, László Dér1, Imre Pap1,2, Eszter Csákvári1,3, 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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).

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P57

    The viscoelastic behaviour of photopolymer nanowires – experiments and modeling

    Cyril Slabý1, Janka Kubacková2, Denis Horvath3, Andrej Hovan1, Gergely T. Iványi4,5, 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.
    • 23rd of August, Tuesday
    • 10:30 – 10:45
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L17

    Imaging the infection cycle of T7 at the single virion level

    Bálint Kiss1,2, Luca Annamária Kiss1, Zsombor Dávid Lohinai1, Dorottya Mudra1, Hedvig Tordai1, Levente Herényi1, Gabriella Csík1, Miklós Kellermayer1,2

    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.

    • 23rd of August, Tuesday
    • 16:30 – 17:00
    • Virus biophysics
    • SIOT0032

    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 Kis2,3

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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.

    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    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 (Yt), 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 Yt values, PVA (0.5-1.5 GPa) and PSI (0.5-3.5 GPa) were more rigid than PCL (0.1-0.5 GPa). Yt 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. Yt 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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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.

    • 23rd of August, Tuesday
    • 18:15 – 18:30
    • Virus biophysics
    • SIOT0032

    L31

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

    Elek Telek1,†, Zoltán Ujfalusi1,†, Gábor Kemenesi2,3,4, Brigitta Zana2,3,4, Ferenc Jakab2,3,4, 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.

    • 22nd of August, Monday
    • 16:15 – 16:45
    • Advances and applications in structural approaches
    • SIOT0032

    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.
    • 26th of August, Friday
    • 10:05 – 10:20
    • Young investigators session
    • SIOT0032

    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.
    • 23rd of August, Tuesday
    • 18:45 – 22:00
    • Poster session, exhibition I.

    P31

    Dielectric Response of a Novel LC Resonant Metamaterial Architecture using THz Impedance Spectroscopy for Application in Bio-Sensing

    Heena Khand, Rudrarup Sengupta, Gabby Sarusi

    Department of Photonics and Electro-Optics Engineering, School of Electrical and Computer Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel

    With recent advances in terahertz (THz) spectroscopy, metamaterial nanostructures are used to detect/determine dielectric properties of viruses, bacteria, and fungi, using the concept of inductor-capacitor ( ) resonance-based particle detection [1]. The nanostructure is designed as an  element, with fundamental resonant frequency,  (  is dependent on the effective dielectric constant ( ) of the material inside the capacitor gap). A change in  of can be brought by any foreign substance deposited in the capacitive gap; changing the  and the capacitance, resulting in redshift of  ( ) with respect to the pristine  circuit in the array.

    We have created a new  resonant split-ring metamaterial design, with the intention of maximizing the capacitor-gap-area, aimed towards bio-sensing. We placed the capacitor gaps at both geometric diagonals of a square inductor, which enables to achieve greater effective capacitance and polarization independence for biomolecule detection. We have effectively increased the sensitivity, by increasing the probability of biomolecules falling inside the capacitive gap and yet maintaining a sub-micron capacitor gap, thereby enabling higher resonance frequency shifts even in lower concentrations. We have achieved 5-fold increase in sensitivity to detect biomolecules compared to other metamaterials for bio-sensing [2]. Our results are supported with system level CST simulations and THz impedance spectroscopy with nanoparticles, Bovine Serum Albumin (BSA) solutions and viruses.

    To realize a kit for bio-sensing, we have also designed a plastic enclosure (radome) for the metamaterial chip, which serves as a protective enclosure and an anti-reflective coating for the chip, thereby improving the THz transmission. Since our initial results have shown nearly 90% accuracy to detect coronaviruses in breath-test [3], we further aim to detect various viruses/bacteria from breath/swab of individuals with higher accuracy, with our designed kit.

    References

    1. Kang, J.-H.; Kim, D.-S.; Seo, M. Terahertz Wave Interaction with Metallic Nanostructures. Nanophotonics 2018, 7 (5), 763–793. https://doi.org/doi:10.1515/nanoph-2017-0093
    2. Park, S. J.; Cha, S. H.; Shin, G. A.; Ahn, Y. H. Sensing Viruses Using Terahertz Nano-Gap Metamaterials. Biomed. Opt. Express 2017, 8 (8), 3551. https://doi.org/10.1364/boe.8.003551
    3. Rudrarup Sengupta, Heena Khand, and Gabby Sarusi; Terahertz Impedance Spectroscopy of Biological Nanoparticles by a Resonant Metamaterial Chip for Breathalyzer-Based COVID-19 Prompt Tests; ACS Applied Nano Materials, 2022, 5 (4), 5803-5812, DOI: 10.1021/acsanm.2c00954
    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    P32

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

    András Kincses1, Ana R. Santa-Maria1, Fruzsina R. Walter1,2, 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.

    • 23rd of August, Tuesday
    • 16:30 – 17:00
    • Virus biophysics
    • SIOT0032

    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 Kis2,3

    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.

    • 23rd of August, Tuesday
    • 10:30 – 10:45
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L17

    Imaging the infection cycle of T7 at the single virion level

    Bálint Kiss1,2, Luca Annamária Kiss1, Zsombor Dávid Lohinai1, Dorottya Mudra1, Hedvig Tordai1, Levente Herényi1, Gabriella Csík1, Miklós Kellermayer1,2

    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.

    • 24th of August, Wednesday
    • 17:45 – 18:00
    • Biomedical applications and neuroscience II.
    • SIOT0032

    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.

    • 23rd of August, Tuesday
    • 10:30 – 10:45
    • Nanoscale biophysics, nanobiotechnology, material sciences I.
    • SIOT0033

    L17

    Imaging the infection cycle of T7 at the single virion level

    Bálint Kiss1,2, Luca Annamária Kiss1, Zsombor Dávid Lohinai1, Dorottya Mudra1, Hedvig Tordai1, Levente Herényi1, Gabriella Csík1, Miklós Kellermayer1,2

    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.

    • 23rd of August, Tuesday
    • 16:30 – 17:00
    • Virus biophysics
    • SIOT0032

    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 Kis2,3

    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.

    • 24th of August, Wednesday
    • 18:30 – 22:00
    • Poster session, exhibition II.

    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 mathematicall