Szekciók

P01

Acute and long-lasting immunological changes in prostate cancer patients treated with three different radiotherapy protocols

Katalin Balázs^1,2, Zsolt Jurányi³, Zsuzsa Kocsis S³, Géza Sáfrány¹, Katalin Lumniczky¹

¹ National Public Health Center, Budapest, Hungary

² Doctoral School of Pathological Sciences, Semmelweis University, Budapest, Hungary

³ National Institute of Oncology, Budapest, Hungary

Radiotherapy can modify systemic immune responses of cancer patients. However, little is known about how long these alterations persist in patients successfully cured of cancer. We investigated how prostate cancer and radiotherapy impact the innate and adaptive immune system of cancer patients treated with various radiotherapy protocols in order to mark potential immune-related biomarkers for patient follow-up.

Blood samples were collected from 63 patients treated with three different type of radiotherapy protocols (LDR brachytherapy: n=21; HDR brachytherapy: n=22; LINAC-based teletherapy: n=20 patients) before and at 7 time points after the therapy up to 36 months. Phenotypical changes in peripheral blood mononuclear cells were analysed by multicolor flow cytometry.

Natural killer (NK) cells were among the most strongly altered in all prostate cancer patients. LDR therapy induced very moderate changes within the NK subpopulation, however teletherapy caused a strong and persistent shift toward immature NK cells. The proportion of non-senescent and senescent CD4+ and CD8+ T cells changed very similarly in HDR and teletherapy groups; the balance shifted strongly to the senescent populations during the follow-up. We further investigated the amount of PSA-containing macrophages which level increased several folds in cancer patients before therapy compared to control and found positive correlation between the plasma PSA level and the level of circulating PSA+ macrophages before therapy.

Our studies demonstrate that prostate cancer patients show long-lasting immunological changes. Furthermore different radiotherapy protocols lead to different long-term immune alterations highlighting the importance of deposition kinetics of ionizing radiation energy in modulating systemic immune responses.

This work was supported by a Hungarian research grant funded by the National Research, Development, and Innovation Office (NKFI-124879).

P02

The FINmaj mutation results in hypophosporilation of the sarcomeric titin molecules

Anna Balogh, Andrea Balogh-Molnár, Zsolt Mártonfalvi

Semmelweis University, Faculty of Medicine, Department of Biophysics and Radiation Biology

The giant sarcomeric protein titin has central mechanistic and developmental roles in striated muscle. Mutations in the TTN gene cause a wide spectrum of skeletal and cardiac myopathies. The dominant tibial muscular dystrophy (TMD) and recessive limb griddle muscular dystrophy (LGMD2J) in Finnish patients are caused by the FINmaj mutation, changing four amino acids (EVTW > VKEK) in the most C-terminal Ig-domain M10 of titin, in the sarcomeric M-line, which domain harbors binding sites for obscurin/obscurin-like 1 and myospryn. Overall, the FINmaj induces a pathological cleavage and degradation of a 70–80 kDa portion from the titin C-terminus, soon after its synthesis.

To investigate the effect of the mutation on the sarcomeric structure of titin we isolated titin molecules from FINmaj murine skeletal muscle samples and from skeletal muscle biopsies of TMD/LGMD2J patients homozygous for the FINmaj mutation. The isolated titin molecules were visualized using atomic force microscopy. We found that the isolated titin molecules maintained their oligomeric structures, in which the overlapping C-termini of titins from neighboring sarcomeres remain bound. The oligomers showed a collapsed structure with an M‑band complex that both increased in area and topographical height. Overall, the appearance of the FINmaj oligomers resembled the structure of hypophosphorylated titin molecules.

Our findings suggest that the FINmaj mutation does not destruct the M-band complex in titin oligomers but decreases the level of phosphorylation in the A-band segment of titin.

Acknowledgment

We thank for the FINmaj samples for prof. Udd Bjarne at University of Helsinki

P03

Direct binding of fluorescent vancomycin to MreB

Beáta Longauer¹, Emőke Bódis¹, Zoltán Gazdag⁴, Miklós Nyitrai^1,2,3 and Szilvia Barkó^1,2,3

¹ University of Pécs, Medical School, Department of Biophysics

² MTA-PTE Nuclear-Mitochondrial Interactions Research Group

³ University of Pécs, Szentágothai Research Center

⁴ University of Pécs, Faculty of Sciences, Department of Molecular Biology and Microbiology

The discovery of antibiotics is one of the greatest discoveries in human history. It is also known that bacteria have developed a serious arsenal of weapons to resist antibiotics. As a result, despite the availability of many antibiotics with very different mechanisms of action, the number of antibiotic-resistant bacterial species is increasing. The result is a worldwide crisis which mankind currently seems powerless to tackle.

In our study we describe a novel potential target in bacteria, which is essential for bacterial survival. MreB, which has major role in organising cell wall synthesis and is found in every bacterium, can be inactivated with a MreB-specific drug A22. This target, although it affects a component of the cell wall, is fundamentally different from the antibiotics used so far.

In our studies, we confirmed the binding of Bodipy-vancomycin to the MreB protein by steady-state anisotropy measurements, which showed an affinity on the order of micromolar. The anisotropy further increased upon exposure to native vancomycin, confirming the previous observation that vancomycin molecules can form supercomplexes with each other and with target proteins. Our light-scattering measurements suggest that vancomycin, like other proteins, can induce aggregation of MreB, but this effect is not observed for ATP-bound protein. This supports our previous observations that nucleotide binding plays a crucial role in MreB stabilisation. Our microbiological and fluorescence microscopy results show that A22 promotes the uptake of vancomycin into cells, which may explain why a synergistic effect between A22 and vancomycin in inhibiting E. coli cell division is observed.

All these observations suggest that antimicrobials based on the mechanism of action of A22 could play a key role in the future in the fight against resistant bacterial strains.

P04

Investigation of a red-footed falcon hub in Angola with deep learning

Evelin Berekméri^1,2, Nico Klar,^3,4, Eric Price^4,5, Péter Palatitz⁶, Aamir Ahmad^4,5, Máté Nagy^1,2,7

¹Department of Biological Physics, Eötvös Loránd University, Budapest, Hungary

²MTA-ELTE Lendület Collective Behaviour Research Group, Hungarian Academy of Sciences, Budapest, Hungary

³Center for Solar Energy and Hydrogen Research Baden-Württemberg, Germany

⁴Institute of Flight Mechanics and Controls, University of Stuttgart, Stuttgart, Germany

⁵Max Planck Institute for Intelligent Systems, Tübingen, Germany

⁶MME/BirdLife Hungary, Budapest, Hungary

⁷Max-Planck Institute of Animal Behavior, Konstanz, Germany

Automating the perception of our environment with object detection has an increasing number of applications, not only in everyday life, such as for self-driving cars or in security, but also in scientific research. Object detection is a deep learning-based computer vision technique that involves recognising predefined classes of objects in visual recordings and locating them within the frame of the recording.

In this study, we focus on a recently discovered, yet unpublished migratory bird hub in Angola, where red-footed falcons gather from different points of the world. These hubs play a crucial role in connecting remote locations worldwide, potentially assisting in disease transmission, and acting as indicators of global environmental changes due to the birds' sensitivity to environmental variations.

Our aim is to identify and track birds on video recordings from the hub providing insight into their population size and preparing further collective behaviour studies such as identifying when they are making use of thermals to gain altitude during their flights. We utilise state-of-the-art object detection frameworks and utilize the latest developments in deep learning and computer vision, taking into consideration that we also aim to deploy our method for real-time, on-board detection on drones.

Currently our understanding of the population size of this species, based on traditional counting methods, has multiple orders of magnitude uncertainty. Our approach offers numerous advantages over traditional procedures, being not only faster and more cost-efficient but also can be more accurate and it also offers real-time monitoring. Such approaches have been used to study the population of other species, such as bat colonies.

Acknowledgment

PREPARED WITH THE PROFESSIONAL SUPPORT OF THE DOCTORAL STUDENT SCHOLARSHIP PROGRAM OF THE CO-OPERATIVE DOCTORAL PROGRAM OF THE MINISTRY OF INNOVATION AND TECHNOLOGY FINANCED FROM THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND.

E.B. acknowledges MTA-ELTE Lendület Collective Behaviour Research Group for financial suppport.

This project was partially supported by the National Research, Development and Innovation Office under grant no. K128780.

P05

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements

Ágnes Batta^1,2, Tímea Hajdu¹ and Péter Nagy¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen

² Doctoral School of Molecular Medicine, University of Debrecen

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research. [1]

References

[1] Batta Á, Hajdu T, Nagy P, Cytometry Part A, 2023. DOI: 10.1002/cyto.a.24728

P06

Super-resolution investigation of liposomal nanosystems

Barnabás Bőcskei-Antal, Ádám Orosz, György Török, Balázs Kiss

Semmelweis University, Budapest, Department of Biophysics and Radiation Biology

Dynamic light scattering is a method used for a long time in the study of nano-sized lipid vesicles (~100 nm in diameter). Up until now, no imaging methodology has been used that directly reveals the inner structure of vesicles in this size range. However, the use of a method that makes the structure of nanovesicles visible under physiological conditions would have significant advantages in the planning and development of therapeutic use. Several research teams have already imaged liposomes using confocal microscopy, but this microscopic method does not have a sufficiently high resolution to enable a clear separation of the membrane and the internal fluid space. Our aim was to develop a methodology that the structure of liposomal systems can be visualized in a high-resolution, robust and reproducible manner.

For our work, small unilamellar vesicles produced from DPPC molecules were labeled with fluorescent (Alexa Fluor 594) lipopolysaccharide molecules, and this system was examined by dynamic light scattering and STED microscopy.

The diameter of the liposomes extruded through the 100 nm pore opening was found to be almost the same both during the light scattering measurement and in the confocal reconstruction images (~100‑110 nm). After finding the right DPPC/LPS ratio and finding the optimal STED settings, we managed to record images with a good signal-to-noise ratio. Through the deconvolution processing of the STED images, the internal aqueous phase of the liposomes can also be successfully explored.

Our research group previously dealt with photoreactive damage of the membranes. To test our methodology, we also followed this process using our developed STED approach.

DPPC – Dipalmitoylphosphatidylcholine
LPS – Lipopolysaccharide
STED – Stimulated Emission Depletion

P07

Nanosurgical manipulation of extended von Willebrand factor multimers

Mária Csilla Csányi¹, Dominik Sziklai¹, Tímea Feller², Jolán Hársfalvi¹ and Miklós S.Z. Kellermayer¹

¹ Semmelweis University, Faculty of Medicine, Department of Biophysics and Radiation Biology

² University of Leeds, Leeds Institute of Cardiovascular and Metabolic Medicine, Discovery and Translational Science Department

The von Willebrand factor (VWF) is a varying-length multimeric chain of glycoprotein dimers/protomers with distinct domain structures. Conformational response of domains upon exposure to shear- and elongational forces ensures that VWF is able to mediate platelet adhesion to and aggregation on an injured vessel wall where it is explored or immobilized from the circulation. Human plasma-derived VWF multimers stretched by molecular combing extend through a specific hierarchy of structural intermediates of protomers as we have recently shown by using atomic force microscopy (AFM) [1]. However, the full scope of local domain stabilities and extensibilities remained hidden.

To uncover these cryptic structural details, in the present work, we used an in situ nanosurgical approach to probe whether targeted domains of VWF could be further extended and unfolded. To achieve this, surface-stabilized and pre-extended VWF multimers were manipulated at distinct spatial locations with the tip of the AFM cantilever. By moving the AFM tip in a direction perpendicular to the longitudinal axis of the VWF multimer, protein loops could be pulled out of the chain, the local extension of which was assessed following the acquisition of a subsequent topographic AFM image.

The extension resulted in ruptured and non-ruptured protein loops, with and without the appearance of hairpin-like thin sections and nodules, while the adjacent domains were non-displaced. Ruptures occurred in 27% of the VWF. Extension, which is the ratio of the length of the section following and prior to manipulation was 2.7 vs. 1.6, p= 0.0001 in non-ruptured and ruptured multimers. Nanomanipulation of protomers in which all the 5 nodules are separated, resulted in a mean final length of 345±69 nm, compared to their pre-manipulation length of 168±54 nm. None of the above results correlated with the protomer’s structural hierarchy showing the main role of the adhesion of certain domains to the mica surface. The nanosurgical manipulation used here demonstrates the VWF mechanical properties at the single domain level via extending further the C1-C6 and A1-A2-A3 domains or eventually rupturing them. The observed conformational changes indicate that VWF may have a large conformational force response potential in order to fine-tune the opening up of hidden epitopes for the different functions of the VWF.

Acknowledgment

TKP2021-EGA-23

References

[1] Csányi MC, Salamon P, Feller T, Bozó T, Hársfalvi J, Kellermayer MSZ. (2023) Protein Sci 32(1):e4535.

P08

Regulation of actin cytoskeleton dynamics through adhesion-molecule-mediated mechanotransduction

Éva Gráczer, Laura Harsányi, Antónia Fülöp, Katalin Pászty and Andrea Varga

Semmelweis University, Department of Biophysics and Radiation Biology

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 ICAM-1 or PECAM-1, and is translated to specific cellular responses, such as actin reorganization, in a process called mechanotransduction. To model leukocyte or cancer cell mediated mechanotransduction, we used magnetic beads coated with an antibody against a specific adhesion molecule. After adhesion of the beads, we applied a continuous force (about 40 pN) with a permanent magnet on the endothelial monolayer and followed reorganization of the actin cytoskeleton by confocal microscopy. We have found that during PECAM-1-mediated mechanotransduction adhesion increased peripheral actin ring formation, which was followed by stress fiber formation upon application of force. VE-cadherin junctions were subsequently reorganized, weakening cell-cell junctions. Thus, the force applied by a cancer cell might help its transmigration by weakening endothelial cell-cell junctions. At the same time, the strength of cell-extracellular matrix (ECM) adhesion was increased, which might play a role in the regulation of cell-cell adhesion. During inflammation on the apical side of endothelial cells filopodia-like protrusions are formed, which help not only in the adhesion of immune cells, but also guide them where to pass the endothelial monolayer. We have found that adhesion through ICAM-1 increases the size of these filopodia, and their size is decreased upon force application. We could also visualize filopodia formation during the transmigration step of A375 melanoma cells. The molecular details of the regulation of filopodia size are not known and we propose that the activity of the Yes-associated protein, YAP might play a role. Our integrative biomechanical and cell signaling approach might reveal new targets to inhibit metastasis.

Acknowledgment

The research was supported by NKFIH/OTKA FK-132144 grant (A.V.) and by the Hungarian Academy of Sciences (Bolyai János Fellowship to A.V. and KGYNK 2023-45 Fellowship to É.G.).

P09

Effect of ionizing radiation on the behaviour of miRNA binding proteins

Kinga Győryné Galgand¹, Ilona Barbara Csordás¹, Rita Hargitai¹, Eszter Szarka¹, Katalin Lumniczky¹, Géza Sáfrány¹

¹ National Public Health Center, Department of Radiobiology and Radiohygiene, Unit of Radiation Medicine, 1097 Budapest, Hungary

RNA-binding proteins play an important role in the regulation of miRNAs and affect a number of intracellular processes (e.g. DNA repair). miRNA binding proteins may also affect the regulation of intercellular communication by shaping the content of EVs, which are key players in the transmission of information between cells and are also responsible for mediating the ionizing radiation-induced bystander effect [1-4].

We investigated the effects of ionizing radiation on miRNA-binding proteins. Our aims are to analize the behaviour of proteins that influence the composition of the miRNA content of EVs after exposure and to explore their role in mediating the bystander effect.

We studied the behaviour of miRNA binding proteins in mouse bone marrow cells, which are highly sensitive to ionizing radiation. The experiments were performed in a control (0 Gy) and two X-ray irradiated groups (0.1 Gy and 3 Gy) 24 hours after exposure. Changes in the expression level of miRNA binding proteins were examined by Real-Time PCR, and intracellular protein levels by Western blot. we compared the results to the post-irradiation data of miRNAs detected by them and also present in EVs.

The results show significant differences in the expression levels of 4 out of 17 proteins tested after 3 Gy irradiation. Based on a combined interpretation of the results and the miRNA measurement data, it can be concluded that the abundance of certain proteins and the EV abundance of their bound miRNAs show a similar trend upon exposure to radiation. According to this information, it can be concluded that certain miRNA binding proteins are able to modulate the miRNA composition of extracellular vesicles in response to radiation, and thus are likely to play an important role in the bystander effect.

References

1. Han, X., et al., Single-cell mechanistic studies of radiation-mediated bystander effects. Front Immunol, 2022. 13: p. 849341.

2. Kis, D., et al., The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles.Br J Radiol, 2020. 93(1115): p. 20200319.

3. Kis, D., et al., Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow. Cells, 2022. 11(1).

4. Fabbiano, F., et al., RNA packaging into extracellular vesicles: An orchestra of RNA-binding proteins? J Extracell Vesicles, 2020. 10(2): p. e12043.

P10

A comprehensive review of various metal-containing nanoparticles in terms of their antibacterial effect and cytotoxic properties

Sarolta Halmóczki, Angéla Jedlovszky-Hajdú

Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University

One of the main reasons, why the healing of chronic wounds is highly challenging is the emergence of multidrug-resistant bacteria. These wounds usually cannot be treated effectively with conventional antibiotics, therefore, alternative methods and agents are investigated to replace them. [1] [2] In this regard, metal nanoparticles (MeNPs) could be promising substances, because they possess significant antimicrobial activity, with the advantage that bacteria are unlikely to develop resistance against them. [3] On the other hand, application of MeNPs may be challenging, since their toxic effect against microorganisms is not selective, and they additionally carry cytotoxic effect. [2] Numerous research articles have been published, which examine the potential of using different MeNPs for wound healing applications. However, it would be invaluable to have a review article that compares the antimicrobial and cytotoxic properties of the most promising MeNPs.

More than 50 scientific articles have been studied, regarding the wound healing potential of silver, gold, copper-, iron- and zinc containing nanoparticles. In the comparison, the main focus was on the antimicrobial activity and cytotoxic effect. Numerous types of Gram-positive and Gram-negative bacteria was treated with metal nanoparticles. In most of the cases, MeNPs were not on their own, rather in a hydrogel, or along with other agents, such as traditional antibiotics. Cytotoxicity was tested on various malignant and normal cell lines. Several cytotoxicity-reducing methods were exhibited, but in general, the effect of the MeNPs is dose-, shape- and size-dependent, but exposition time and the exact cell line used are also not negligible factors.

Acknowledgment

This research was supported by NKFIH FK 137749

References

[1] C. Liao, Y. Li, S.C. Tjong, Bactericidal and cytotoxic properties of silver nanoparticles, Int. J. Mol. Sci. 20 (2019). https://doi.org/10.3390/ijms20020449.

[2] N. Naderi, D. Karponis, A. Mosahebi, A.M. Seifalian, Nanoparticles in wound healing; from hope to promise, from promise to routine, Front. Biosci. - Landmark. 23 (2018) 1038–1059. https://doi.org/10.2741/4632.

[3] L. Wang, C. Hu, L. Shao, The antimicrobial activity of nanoparticles: Present situation and prospects for the future, Int. J. Nanomedicine. 12 (2017) 1227–1249. https://doi.org/10.2147/IJN.S121956.

P11

Biophysical Characterization of Clot Retraction in Platelet Rich Plasma of Patients with Primary Anti-phospholipid Syndrome

Jolán Hársfalvi¹_, Tímea Feller¹, György Domjan³, Klára Gadó³, Katalin Várnai², Eszter Barabás², Miklós Kellermayer¹

¹Department of Biophysical and Radiation Biology, ²Department of Laboratory Medicine and ³Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary

Anti-phospholipid syndrome (APS) is an autoimmune process leading to thrombotic disorders [1], but with poorly understood mechanisms.

By using atomic force microscopy (AFM)-based nano-thrombelastography (nTEG) [2], we investigated the biophysical characteristics of the fibrin network during clot formation and degradation in platelet-rich plasma (PRP) of 38 APS patients compared with 18 controls. Patients with APS and venous thromboembolism (VTE) were selected as case and control, respectively.

Citrated blood was centrifuged at room temperature (150 g, 10 min) to obtain PRP, so that platelet count was set to 50G/L. An AFM cantilever was submerged in a 0.3-mL sample and cyclically moved up and down with an amplitude of 1 μm and a speed of 1 μm/s. In addition to PRP the sample was completed with 10mM Ca²⁺, and clotting was initiated with thrombin at a final activity of 1 IU/ml. As the sample clotted, the cantilever deflected progressively during its vertical travel, reflecting the onset and increase in the elastic and viscous properties of the clot. The onset of clot formation was determined by measuring the time delay until the first non-zero force signal appeared. Clot contractility was assessed by measuring the rate of force increase. Finally, the viscoelastic response of the clot was obtained by measuring the force hysteresis area and the peak force difference in the datasets collected in the opposite cantilever directions (up versus down). The median parameter values of the APS and control samples were compared.

We found that in the APS group, the delay until the first force signal increased 2-3-fold [sec]; the slope of the force generation decreased to about 1/2 [nN/sec]; and the maximal force difference in the mechanical cycles decreased to about 1/3 [nN]. These results compare well with recent observations in which macroscale methods were used [3].

In sum, we were able to characterize quantitatively the nanoscale changes in the viscoelastic properties during platelet contractility and fibrin network formation in human PRP in a distinct pathology. We expect that the rich dataset provided by the AFM-based measurement employed here will provide insights into the molecular mechanisms associated with the pathology of APS.

References

1. Vreede AP, Bockenstedt PL, McCune WJ, Knight JS (2019) Curr Opin Rheumatol. 31(3):231-240.

2. Feller T, Kellermayer MS, Kiss B. (2014) Journal of Structural Biology 462-71

3. Le Minh, G., A.D. Peshkova, I.A. Andrianova, T.B. Sibgatullin, A.N. Maksudova, J.W. Weisel, and R. Litvinov, (2018) Clinical Science 132: 243-254

P12

Crystallographic and molecular dynamics simulations shed light on the self-inactivated conformation of the Venezuelan equine encephalitis virus (VEEV) protease

Gyula Hoffka^1,2, George T. Lountos³, József Tőzsér¹ and János András Mótyán¹

¹ University of Debrecen, Faculty of Medicine, Department of Biochemistry and Molecular Biology, Laboratory of Retroviral Biochemistry

² University of Debrecen, Doctoral School of Molecular Cell and Immune Biology

³ Frederick National Laboratory for Cancer Research, Basic Science Program

The application of computational simulations in molecular biology allows us to examine a wide range of properties of enzymes, which would otherwise be challenging using experimental methods. Molecular dynamics are exceptionally useful to study conformational landscapes, substrate binding patterns, as well as interaction networks.

The Venezuelan equine encephalitis virus (VEEV) is responsible for causing mild to severe disease in both humans and livestock. The non-structural protein 2 protease (nsP2pro) of VEEV is considered as a drug target due to its crucial role in the viral life-cycle. Therefore, investigation of this viral protein may provide valuable information for structure-based drug design.

We aimed to study the structure of VEEV nsP2pro using both in vitro and in silico approaches. The structure, containing the wild-type N475 N-terminal residue at the active site, was determined experimentally at high resolution (1.46 Å), using X-ray crystallography [1]. The protein exhibited an unexpected conformation wherein the N-terminus mimics substrate binding. This self-inactivated conformation was previously observed only for a N475A mutant enzyme.

To compare the active and inactive conformers, we have used the Amber16 software for molecular dynamics simulations. We investigated the dynamic hydrogen bond networks at the active site, and compared the differences between the dynamic properties of the active and self-inactivated conformers as well as between the N475 and A475 containing variants. The comparison sheds light on the interactions that are crucial to the stabilization of the conformers.

Project no. TKP2021-EGA-20 (Biotechnology) has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme. We acknowledge KIFÜ for awarding us access to resource based in Hungary.

[1] Hoffka G, Lounto GT, Needle D, Wlodawer A, Waugh DS, Tőzsér J, Mótyán JA (2023) J Mol Biol 435:168012

P13

Preliminary Experiments for the Rheological Study of Oleogels

Szabolcs Homolya¹, Katalin Badakné Kerti¹, Eszter Vozáry², Tímea Kaszab² and Anikó Lambertné Meretei¹

^1,2 Hungarian University of Agriculture and Life Science, Institute of Food Science and Technology

¹Department of Grain and Industrial Crops

²Department of Food Industrial measurements and Control

Due to deforestation to expand oil palm plantations and the many negative health effects of palm oil, the food industry needs other alternative sources of fats to replace palm oil. In our research, we investigated blends of fully hydrogenated rapeseed oil and non-hydrogenated high oleic sunflower oil, as well as beeswax and high oleic sunflower oil as possible oleogel alternatives. Oleogels are gelled forms of vegetable oils. Gelling agents can be waxes or proteins. The rheological properties of these oleogels were compared with the rheological properties of palm oil and a commercially available confectionery fat.

The rheological measurements were performed on the following materials: Unfractionated palm oil, Chocofill confectionery fat, Blends of fully hydrogenated rapeseed oil and non-hydrogenated high oleic sunflower oil: 25 m/m% rapeseed oil + 75 m/m% sunflower oil; 30 m/m% rapeseed oil + 70 m/m% sunflower oil; 35 m/m% rapeseed oil + 65 m/m% sunflower oil and Beeswax and high oleic sunflower oil mixture: 85 m/m% sunflower oil + 15 m/m% beeswax.

The flow curve in the shear rate range 0,1 to 100 1/s, as well storage (G’) and loss (G”) modules of oleogels, palm oil and confectionery fat were determined with an Anton-Paar MCR302 oscillatory rheometer. The flow curves were approximated with the Hershel-Bulkley and Casson model using the Excel Solver program. The linear viscoelastic limit was determined from the curves obtained by amplitude sweeping.

The confectionary fat showed rheological similarity with palm oil, however, this type of sample was the furthest from the rapeseed-sunflower and beeswax-sunflower blends in rheological properties. Based on the data analyses and the characteristics of the substitute blends, the sample containing 35% fully hydrogenated rapeseed oil and beeswax oleogel might be suitable as palm oil substitutes, but further studies are needed to determine this statement.

P14

Comparative Analyses of the Gelsolin Homology Domains of Gelsolin and Flightless-I

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

¹University of Pécs, Medical School, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary

²Linnaeus University, Department of Chemistry and Biomedical Sciences, SE-39182, Kalmar, Sweden

³Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee

Flightless-I is a unique member of the gelsolin (GSN) superfamily alloying six gelsolin homology (GH) domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton. However, its biochemical activities in actin dynamics regulation are still largely elusive. To better understand its biological functioning, we performed a comparative analysis of GSN and Flightless-I by in vitro fluorescence spectroscopy and single filament TIRF microscopy approaches. We found that Flightless-I can 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.

Acknowledgment

Activities of Flightless I revealed by acto-myosin based in vitro motility techniques, 2021-4.1.2-NEMZ_KI-2022-00025 (TH); New National Excellence Program of the Ministry for Innovation and Technology ÚNKP-21-3-II-PTE-997 (PG). We thank József Mihály (Institute of Genetics, Biological Research Centre) for the Drosophila Flightless-I plasmids, Tomohito Higashi (Fukushima Medical University) for the human Flightless-I plasmids and Robert C. Robinson (Okoyama University) for the human GSN plasmid.

P15

Co-assemblies of cationic antimicrobial peptides with anionic small molecules: unique thermophoretic behaviour

Tünde Juhász¹, Mayra Quemé-Peña¹, Tamás Beke-Somfai¹

¹ Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences

Peptidic supramolecules formed via self- and co-assembly show emerging applications from materials chemistry to biomedicine. Recently we have demonstrated for a couple of cationic, amphiphilic antimicrobial peptides (AMPs) the ability to form co-assemblies with various anionic, aromatic small molecule (SM) binding agents. Their interaction results in modulating their activity in vitro. Herein we addressed interaction systems of four selected AMPs (CM15, Dhvar4, LL-37, and FK-16, coupled to carboxyfluorescein) with four selected SMs (suramin, tartrazine, biliverdin, and bilirubin ditaurate) exploiting the sensitivity of microscale thermophoresis (MST). MST is a solution phase method utilizing the phenomenon of induced thermophoresis where movement of the molecules is monitored via the fluorescence of the target partner. Besides substantial similarities, various scenarios of thermophoretic responses were revealed. Interestingly, negative thermophoresis was frequently observed, and maximal MST responses were typically found not at the highest SM concentrations applied. Findings suggest substantial contributions of charge and hydration effects. Variations in the thermophoretic behaviour could also be attributed to the oligomeric state of the individual components, and the dynamic nature of the association process. Results demonstrate that the MST method is an excellent additional technique for identifying and studying peptidic assemblies.

Acknowledgment

The authors thank for support from LP2016-2, NVKP_16-1-2016-0007, TKP2021-EGA-31, KKP22 144180, SA-87/2021, KEP-5/2021, and 2020-1-1-2-PIACI-KFI_2020-00021.

P16

Conformational Dynamics of Human Calmodulin at Low Ca²⁺ Saturations

Gusztáv Schay¹, Klaudia Onica², Arian Jafari¹, Franci Merzel³, Miklós Kellermayer¹, Erika Balog¹, and Károly Liliom¹

¹Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

²Pázmány Péter Catholic University, Budapest, Hungary

³National Institute of Chemistry, Ljubljana, Slovenia

Calmodulin is an extremely conservative key protein of intracellular signal transduction. It can bind four Ca²⁺-ions by two EF-hand motifs in its N- and C-terminal domains. During Ca²⁺-binding hidden hydrophobic surfaces become available, which take part in recognition of a broad range of target proteins. These observations led to the idea of calmodulin being a molecular switch, activating its partners when Ca²⁺-saturated at elevated intracellular calcium levels. It is known that calmodulin can adopt numerous conformational states, however, the change of conformers along Ca²⁺-binding is not known. Importantly, considering the intracellular calmodulin and Ca²⁺-levels, and the Ca²⁺-affinities of calmodulin EF-hands, it is unlikely that calmodulin gets Ca²⁺-saturated in physiologic signaling conditions. What is then the mechanism of target protein selection? The Ca²⁺-binding of calmodulin displays cooperativity which can be described by a Perutz binding model to explain Ca²⁺-saturation experiments in vitro. The function/conformation of calmodulin at low Ca²⁺-loads has not been explored yet in details, whereas all physiologic Ca²⁺-signaling happens at low Ca²⁺/calmodulin concentration ratio. Compartmentalization of target proteins and canonic binding modes of Ca²⁺-saturated calmodulin cannot account for why the spatiotemporal information content of intracellular Ca²⁺-signaling is not lost during calmodulin signal transduction.

Our experiments uncovered an increased conformational diversity at low Ca²⁺/calmodulin concentration ratio, and that the binding of model peptide melittin to calmodulin happened much before the Ca²⁺-saturation of the protein. We carried out molecular dynamics simulations of calmodulin with adding two Ca²⁺-ions to the C-terminal EF-hands of apocalmodulin and also by removing two Ca²⁺-ions from the N-terminal EF-hands of holocalmodulin. We observed striking differences in the structural dynamics of the N-terminal apo–C-terminal holo calmodulin, depending on the initial state from which we have reached this state. Based on our experiments and molecular dynamics calculations we hypothesize that the spatiotemporal changes in intracellular Ca²⁺-concentrations can be coded into the structural/conformational dynamics of calmodulin.

This work was supported by the grants FK-135517, TKP2021-EGA-23, and STIA-KFI-0012.

P17

Single amino acid mutation decouples photochemistry of the BLUF domain from the enzymatic function of OaPAC and drives the enzyme to a switched-on state 

Emőke Bódis¹, Jinnette Tolentino Collado², Mihály Szűcs¹, Zsuzsanna Fekete¹, Elek Telek¹, Kinga Pozsonyi¹, Sofia M. Kapetanaki¹, Greg Greetham³, Peter J. Tonge², Stephen R. Meech⁴, and András Lukács¹

¹ Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary

² Department of Chemistry, Stony Brook University, New York, 11794, United States.

³ Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, U.K

 ⁴ School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, U.K.

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation [1-3]. The light-induced changes in the BLUF domain are transduced via a mechanism that has not been established yet to the adenylate cyclase domain. One critical residue in the vicinity of the flavin in BLUF proteins is the glutamine amino acid close to the N5 of the flavin[4-6]. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we have applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation decelerated the primary electron transfer process but switched the enzyme in to a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the wild-type OaPAC. Differential scanning calorimetry measurements pointed out to a less compact structure for the mutant. These findings provide insight into the important elements in PACs and how their fine tuning may result to the design of optogenetic devices.

Acknowledgment

A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021 E.T was supported by PTE ÁOK-KA-2022-09.

References

[1] Iseki M, Park SY. Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications. Adv Exp Med Biol. 2021;1293:129-39.

[2] Ohki M, Sugiyama K, Kawai F, Tanaka H, Nihei Y, Unzai S, et al. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium. Proceedings of the National Academy of Sciences of the United States of America. 2016;113:6659-64.

[3] Collado J, Iuliano J, Pirisi K, Jewlikar S, Adamczyk K, Greetham G, et al. Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis. Acs Chemical Biology. 2022;17:2643-54.

[4] Udvarhelyi A, Domratcheva T. Glutamine rotamers in BLUF photoreceptors: a mechanistic reappraisal. J Phys Chem B. 2013;117:2888-97.

[5] Domratcheva T, Hartmann E, Schlichting I, Kottke T. Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry. Sci Rep. 2016;6:22669.

[6] Hontani Y, Mehlhorn J, Domratcheva T, Beck S, Kloz M, Hegemann P, et al. Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction. J Am Chem Soc. 2023;145:1040-52.

P18

How nanoparticles affect aggregation and cytotoxicity of the amyloid-beta peptide?

Éva Moussong¹, Márton Péter Nyiri¹, Nikoletta Murvai¹, Judit Kun^1,2, Attila Kovács^1,2, Tamás Molnár¹, András Micsonai^1,2, József Kardos^1,2

¹Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest H-1117, Hungary

²ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest H-1117, Hungary

Using nanoparticles is becoming common in medicine and biotechnology. Additionally, nanoparticles are also present in our environment as nano-pollution which might enter living organisms. Proteins which get into contact with nanoparticles, might undergo structural changes. Aggregation and amyloid formation of proteins and peptides can also be affected by nanoparticles. Our group is interested in investigating the aggregation and amyloid formation of the amyloid-beta (Aβ) peptide which is known for creating amyloid fibrils in the brain in Alzheimer’s disease. We used in-house expressed recombinant Aβ(1-42) to study the structure, aggregation kinetics, and cytotoxicity of the peptide. We characterized nanoparticles of different materials (CaF₂, silica, polystyrene) and studied their effects on Aβ. Based on thioflavin T fluorescence, we found that nanoparticles typically reduce lag time of the aggregation. Polystyrene nanoparticles bind peptide monomers up to a saturation point and let only excess peptides to form fibrils. CaF₂ and silica do not show an effect similar to this, but they influence the secondary structure of the fibrils. We used circular dichroism (CD) spectroscopy and the BeStSel method to determine secondary structure composition. Aβ fibrils formed alone have a high content of parallel β-sheets. Nanoparticles, depending on their type, affect the secondary structure composition of Aβ aggregates altering the fractions of the different type of β-sheet structures. Toxicity measurements on mouse hippocampal cells suggest that Aβ is highly cytotoxic, but nanoparticles might reduce cytotoxicity of Aβ if applied in appropriate concentrations.

Acknowledgement

This work was supported by the National Research, Development and Innovation Office of Hungary (grants PD135510, K138937, 2019-2.1.6-NEMZ_KI-2019-00012, and 2019-2.1.11-TÉT-2020-00101), the National Brain Research Program NAP 3.0 of the Hungarian Academy of Sciences (NAP2022-I-3/2022), and the Gedeon Richter Talentum Foundation (grant ’Richter Gedeon Kiválósági PhD Ösztöndíj’).

P19

Investigation of antibody-mediated adhesion force of individual immune cells using computer-controlled micropipette and fluidic force microscopy

S. Novák¹, Z. Szittner¹, I. Sallai¹, I. Székács¹, R. Horvath¹

¹ Nanobiosensorics Laboratory, Centre of Energy Research, Eötvös Loránd Research Network, Budapest, Hungary

Cell adhesion is a fundamental process that plays a critical role in various biological phenomena, such as the immune response. To understand the mechanisms of cell adhesion and develop new therapies, it is necessary to measure the forces involved.Computer-controlled micropipettes (CCMP) and Fluidic Force Microscopy (FluidFM) techniques have become effective instruments for the accurate determination of cell adhesion in recent years.

A CCMP is a tool that can be used to probe single cell interactions with specific macromolecules and surfaces. The micropipette is mounted onto a micromanipulator on a normal inverted microscope and is controlled by a computer. The adhesion force of surface-attached cells can be accurately probed by repeating a pick-up process on the examined cells while increasing a vacuum applied through a pump system in the pipette positioned above the cell. Using this methodology, high number of cells adhered to specific macromolecules, treated surfaces can be measured one by one in a short period of time. Additionally, the probed single cells can be easily picked up and separated for further examinations by other techniques. This is a definite advantage of the CCPM.[1]

FluidFM, on the other hand, uses a hollow cantilever with a small opening at the tip that can be filled with liquid using a fluid reservoir that is attached to a pressure control system, allowing for precise fluid dispensing and manipulation at the nanoscale. This technology enables the precise measurement of adhesion forces between cells and their targets and makes it possible to record the entire cell detachment process quickly and accurately. [2]

The combination of these two technologies provides high accuracy and resolution in the measurement of cell adhesion forces and in addition to measuring adhesion forces. We used these techniques to study the adhesion strength of immune cells on different surfaces.

Acknowledgment

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 134195 for Z.Sz, ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] R. Salánki et al., “Single Cell Adhesion Assay Using Computer Controlled Micropipette,” PLoS One, vol. 9, no. 10, pp. e111450-, Oct. 2014, [Online]. Available: https://doi.org/10.1371/journal.pone.0111450

[2] Á. G. Nagy, I. Székács, A. Bonyár, and R. Horvath, “Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy,” Eur J Cell Biol, vol. 101, no. 4, Sep. 2022, doi: 10.1016/j.ejcb.2022.151273.

P20

Salt loaded fibrous meshes from polysuccinimide for medical applications

Veronika Pálos¹, Judit Domokos², Dóra Szabó², Ákos Zsembery³, Rita Pázmány¹, Krisztina S.-Nagy¹, Angéla Jedlovszky-Hajdú¹

¹ Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University

² Institute of Medical Microbiology, Semmelweis University

³ Institute of Oralbiology, Semmelweis University

Silver is increasingly being pushed out of medicine, and other ions with antibacterial effect are coming to the forefront. This is because since silver ions have huge environmental burden.

The aim of my research is to create a bicomponent polymer network by electrospinning, which contains zinc and strontium salts in addition to the polymer, which according to the literature, have antibacterial properties. If such a new type of biocompatible wound dressing could be created, not only would be a mechanical barrier to infection but also have antibacterial activity against microorganisms.

The first step was to synthesize the polymer, then mix it with the selected inorganic salts in dimethylformamide, and then optimize the electrostatic fiber formation parameters. The inorganic salts used in the experiments were Zn(O₂CCH₃)₂ and Sr(NO₃)₂. Next, the chemical and mechanical properties of the complete polymer networks were investigated by FTIR spectroscopy, SEM images, and their mechanical behavior through specific load capacity. In the next step, I investigated the antibacterial effect of polymer networks containing inorganic salts on four different bacterial species.

Based on FTIR spectroscopy, the salts formed a physical bond with polysuccinimide, but there was no chemical bonding between them. SEM of a polymer mesh containing inorganic salts showed a big difference between the fiber diameters in the presence of the salts. However, no difference was found in their specific load capacity. In antibacterial experiments, a more significant clearance zone appeared for the polymer containing Zn(O₂CCH₃)₂, and a lesser inhibition zone for those containing Sr(NO₃)₂ salts. The next step was to find out if the polymer meshes with the salts have cytotoxic activity against human tumor and fibroblast cells, because these fibrous structures are going to use as wound dressing, so they must interact with the human cells.

Acknowledgment

The work was supported by the National Research, Development, and Innovation Office FK137749 and TKP 2021-EGA-23.

P21

Nanoparticle uptake of living cells with digested glycocalyx

Beatrix Peter^a, Nicolett Kanyo^a, Kinga Dora Kovacs^a,b, Viktor Kovács^a, Inna Szekacs^a, Béla Pécz^c, Kinga Molnár^d,Hideyuki Nakanishi^e, Istvan Lagzi^,,f,g, Robert Horvath^a

^a Nanobiosensorics laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege út 29-33, H-1121 Budapest, Hungary

^b Department of Biological Physics, Eötvös University, Budapest, Hungary

^c Thin Films Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege út 29-33, H-1120 Budapest, Hungary

^d Department of Anatomy, Cell and Developmental Biology, ELTE, Eötvös Loránd University, Pázmány Péter stny. 1/C, Budapest, H-1117, Hungary

^e Department of Macromolecular Science and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Kyoto 606-8585, Japan

^f Department of Physics, Institute of Physics, Budapest University of Technology and Economics, Műegyetem rkp. 3, Budapest H-1111, Hungary

^g ELKH BME Condensed Matter Research Group, Műegyetem rkp. 3, Budapest H-1111, Hungary

In biomedical imaging and targeted drug delivery, functionalized nanoparticles are widely used due to their penetration into living cells. The glycocalyx is a surface sugar layer of the cells, which presumably plays an essential role in any uptake process. However, its exact function in nanoparticle uptake is still uncovered. We in situ monitored the penetration of positively charged gold nanoparticles into adhered cancer cells with or without preliminary glycocalyx digestion. During the experiments, the components of glycocalyx of HeLa cells were digested by chondroitinase ABC enzyme. The measurements were performed by applying a high-throughput label-free resonant waveguide grating biosensor. The positively charged gold nanoparticles were used with different sizes (S, M, L). Negatively charged citrate-capped tannic acid nanoparticles, and other types of glycocalyx digesting enzymes were also applied in control experiments. The biosensor data confirmed the cellular uptake of the functionalized nanoparticles with an active process, which was verified by transmission electron microscopy [1,2]. Based on the findings we conclude that the components of gylcocalyx control the uptake process in size- and charge-dependent manner, and the possible roles of various glycocalyx components were highlighted.

Acknowledgements

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 131543 for B.P., ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] B. Peter, N. Kanyo, K. D. Kovacs, V. Kovács, I. Szekacs, B. Pécz, K. Molnár, H. Nakanishi, I. Lagzi, R. Horvath. Glycocalyx components detune the cellular uptake of gold nanoparticles in a size- and charge-dependent manner. ACS Applied Bio Materials, 2023.

[2] B. Peter, I. Lagzi, S. Teraji, H. Nakanishi, L. Cervenak, D. Zámbó, A. Deák, K. Molnár, M. Truszka, I. Szekacs, R. Horvath. Interaction of positively charged gold nanoparticles with cancer cells monitored by an in situ label-free optical biosensor and transmission electron microscopy. ACS Applied Materials & Interfaces, 2018.

P22

Mathematical modelling of low dose hyper-radiosensitivity and induced radioresistance

Szabolcs Polgár^1,2, Balázs Madas^2,3

¹ Doctoral School of Physics, ELTE Eötvös Loránd University, Budapest, Hungary

² Environmental Physics Department, Centre for Energy Research, Budapest, Hungary

³ Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, Budapest, Hungary

The surviving fraction of cells decreases exponentially with the increase of the absorbed dose at high doses. At low doses however, experiments show that in many different cell lines the surviving fraction differs from this, due to the effects of hyper-radiosensitivity and induced radioresistance [1]. The result is a function that starts steeper and after a local minimum starts to increase to a local maximum as the dose increases before following the exponential decrease at higher doses.

The aim of this study was to test if the principle of minimum mutation load [see e.g., 2] can describe both hyper-radiosensitivity and induced radioresistance at low doses. In this case the principle means that the most damaged cells in a vicinity use apoptosis to reduce the mutation rate in the tissue.

To test this hypothesis a mathematical model was developed and an experimental database constructed from published articles for validation.

For the model validation a database was used, consisting of various experimental data featuring low dose hyper-radiosensitivity [3]. The model has been developed in Python. A total of 600 cells are placed randomly in a circle with a given radius. The cells are able to communicate their DNA damage by a signal, its concentration following normal distribution, centered on the cell. The DNA damage of the cells follow Poisson distribution for any given dose.

The fit parameters for the model were acquired by fitting with the Nelder-Mead method to the experimental data in each case. The parameter calculation consisted first a preliminary calculation (using the starting slope and the local minimum of each dataset) to acquire the initial parameters, then a two dimension fit with the Nelder-Mead method was calculated to reach the best values for them. The results then were evaluated and compared to the induced repair model where possible (where there was a fit in the original article).

Acknowledgment

This study is part of the RadoNorm project which has received funding from the Euratom research and training programme 2019-2020 under grant agreement No. 900009. The study was also supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (bo-37-2021) and the ÚNKP-22-5 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund (ÚNKP-22-5-BME-299).

References

[1] Marples, B., Collis, S.J., 2008. Low-Dose Hyper-Radiosensitivity: Past, Present, and Future. International Journal of Radiation Oncology*Biology*Physics 70, 1310–1318. https://doi.org/10.1016/j.ijrobp.2007.11.071

[2] Derényi, I., Szöllősi, G.J., 2017. Hierarchical tissue organization as a general mechanism to limit the accumulation of somatic mutations. Nature Communications 8, 14545. https://doi.org/10.1038/ncomms14545

[3] Polgár, S., Schofield, P.N., Madas, B.G., 2022. Datasets of in vitro clonogenic assays showing low dose hyper-radiosensitivity and induced radioresistance. Sci Data 9, 555.

https://doi.org/10.1038/s41597-022-01653-3

P23

Single-cell adhesion measurements using fluidic force microscopy

Imola Rajmon^1,2, Anna Balogh^1,2, Kinga Dóra Kovács^1,2, Inna Szekács² , Robert Horvath²

¹ELTE Eötvös Loránd University, Department of Biological Physics, Budapest, Hungary

²Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary

Nowadays single-cell techniques are becoming valuable tools in the field of biology and biophysics. By investigating at a cellular level, we can better understand the cellular heterogeneity and the possible subpopulations in a tissue. This knowledge can bring novel applications and solutions to health and medicine. Fluidic Force Microscopy (FluidFM) is similar to atomic force microscopy (AFM), but uses a hollow, microfabricated cantilevers connected to a liquid reservoir and pressure controller system [1]. Through its precise force control, the cantilever is ideal to approach individual cells gently and reproducibly. Depending on the applications, the end of the cantilever can be different. For cell adhesion measurements 2-8 μm circular openings are ideal [2], while for live cell sampling and injections [3][4], so-called nanosyringes are used. These cantilevers have pyramidal tips with a 600 nm opening at their side. The cantilevers used for cell adhesion measurements can have different spring constants, which also determine some applications of the device. The short lecture will introduce a robotic version of the technique by presenting experimental results on the adhesive and mechanical properties of both cancerous and healthy cell types.

Acknowledgements

„Supported by the ÚNKP-22-2 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.”

References

[1]: Li, M., Liu, L. & Zambelli, T. FluidFM for single-cell biophysics. Nano Res. 15, 773–786 (2022). https://doi.org/10.1007/s12274-021-3573-y

[2]: Sztilkovics, M., Gerecsei, T., Peter, B. et al. 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

[3]: Chen, W., Guillaume-Gentil, O., Rainer, P.Y. et al. Live-seq enables temporal transcriptomic recording of single cells. Nature 608, 733–740 (2022). https://doi.org/10.1038/s41586-022-05046-9

[4]: Robert Horvath,Single-cell temporal transcriptomics from tiny cytoplasmic biopsies, Cell Reports Methods,Volume 2, Issue 10, 2022, 100319, ISSN 2667-2375, https://doi.org/10.1016/j.crmeth.2022.100319.

P24

G-quadruplexes: Thermodynamic characterization, identifying the physicochemical parameters influencing the stability

László Smeller¹, Miklós Cervenák¹, András Végh², Orsolya R. Molnár^1, Anna Grád¹, Judit Somkuti¹

¹ Semmelweis University, Budapest, Department of Biophysics and Radiation Biology

² Semmelweis University, Budapest, Department of Ophthalmology

Besides of the well-known double-helical structures nucleic acids can form non-canonical motifs as well. The most important one is the G-Quadruplex (GQ), which 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.

Here we report the results we obtained on two oligos, both DNA and RNA ones [1-3]. They were chosen from the genome of the hepatitis B and SARS-CoV-2 viruses. We selected a short oligo that was suggested by computer analysis of the genome to form GQ with high probability.

Infrared and fluorescence spectroscopic methods have been used to determine the conformation of the GQs. Förster resonance energy transfer allowed following the unfolding of the oligos.

Since GQs were considered as targets of the cancer therapy, several ligands were developed for stabilization of the human GQs. We investigated three of these ligands, whether they can stabilize the viral GQs as well.

As conclusion, we have proven that both HepB (DNA) and C19/1 (RNA) viral oligos form G-quadruplex structure however with different temperature stability. The unfolding mid-points were 53 and 37 °C respectively.

All the ligands we investigated (TMPyP4, BRACO19, PhenDC3) were proven to bind and stabilize these oligos (except BRACO19 which did not bind C19/1).

These results might have important impact in the fighting against viruses, since stabilization of the GQs will influence the proliferation of the virus.

Acknowledgment

The authors thank for the support from NFKI K-124697 and TKP2021-EGA-23 grants.

References

[1] Molnár, OR., et al.,(2021) Scientific Reports (2021) 11:23243
[2] Somkuti, J., et al.,(2021) Biology 10:1173;
[3] Smeller, L., (2023) Int. J. Mol. Sci. 24:1803

P25

Variation of Electrical Impedance Parameters of Dry Crops during Soaking

Tamás Somogyi, Eszter Vozáry and Viktória Zsomné Muha

Hungarian University of Agriculture and Life Science, Institute of Food Science and Technology

Department of Food Industrial Measurements and Control

When processing nutritionally important dry pulses such as beans, soaking is one of the most important preparatory steps. As this process is relatively time-consuming. However, this long soaking time can be reduced by ultrasound.

The experiments were performed on red kidney beans (Phaseolus vulgaris cv. Rampart). Prior to the actual sample preparation, beans were manually sorted into four groups according to their size (S - small ≤ 13 mm; M - medium = 13 - 15 mm; L – large = 15 - 17 mm; X - extra-large ≥ 17 mm).

For the ultrasound treatments we used an ultrasonic bath (HBM Machines, The Netherlands). The treatments applied were performed at 40 kHz 300 W at 20 °C.

The magnitude and phase angle of the impedance of the beans were measured in the frequency range of 30 Hz - 30 MHz with an HP4284A and HP4285A precision LCR meters at a measuring voltage of 1 V in an HP16451 B test fixture. To achieve a good electrical contact, an ECG electrically conductive gel was placed between the bean shell and the electrode.

As the soaking time increased, the magnitude of the impedance decreased, which may indicate that the increased water content reduced the viscosity and thus increased the mobility of the charges, resulting in a decrease in resistance and impedance.

This reduction is also clearly visible in the ultrasonic treatments compared to the control samples.

The phase angle spectrum shifted towards lower frequencies, which in turn may be a result of the destructive effect of ultrasound. The effect of the treatments shifts the minimum point around 31 kHz towards lower frequencies. The longer the soaking time, the lower the impedance value and the more the ultrasound-induced structure damage was well detected. These suggest that it may be possible to qualify the structural condition of the soaked dry structures.

P26

Anisotropy imaging using RCM

Gábor Steinbach^1,2, Dávid Nagy³, Gábor Sipka^2,4 , Ábel Garab² , Győző Garab^2,4 and László Zimányi³

¹ ELKH, Biological Research Centre, Szeged, C ellular Imaging Laboratory

² Biofotonika R&D Ltd.

³ ELKH, Biological Research Centre, Szeged, Institute of Biophysics

⁴ ELKH, Biological Research Centre, Szeged, Institute of Plant Biology

Both the differential polarization attachments for LSMs and Re-scan Confocal Microscopy (RCM) facilitate revealing structures at the molecular level [1]. For subcellular structures, increasing the available resolution can be fundamental. The re-scan confocal microscopy (RCM) provides 4 times better signal to noise ratio (compared to conventional PMTs) using an sCMOS camera, moreover, due to the second scanner, the image resolution increases by the factor of 1.4, while the axial resolution is identical with the LSMs [2].

With additional polarization elements, the RCM enables the 2D and 3D microscopic mapping of the anisotropy of samples via measuring fluorescence-detected linear dichroism (FDLD). The usage of the liquid crystal modulator for the RCM and the high frequency modulation (photoelastic modulator – PEM) are synchronised with the imaging. The DP-LSMs and the pRCM (RCM equipped with polarization attachment) are suitable for obtaining unique structural information on the anisotropic molecular organization of biological samples and intelligent materials [3, 4] in 2D and 3D.

References

[1] Steinbach et al. (2019) European Biophysics Journal 48: 457-463., doi: 10.1007/s00249-019-01365-4

[2] De Luca GMR et al. (2017) Methods Appl Fluoresc 5:015002., doi: 10.1088/2050-6120/5/1/015002

[3] Simonović Radosavljević J et al. (2021) Int. J. Mol. Sci. 22: 7661, doi: 10.3390/ijms22147661

[4] Pleckaitis M et al. (2022) Nano Research, 15: 5527-5537., doi: 10.1007/s12274-021-4048-x

P27

Fibrillin-1 microfibrils in Marfan syndrome: nanoscale structural characterization using atomic force microscopy

Cristina M. Șulea^1,2,3, Zsolt Mártonfalvi¹, Csilla Csányi¹, Dóra Haluszka¹, Miklós Pólos^2,3, Kálmán Benke^2,3, Zoltán Szabolcs^2,3 and Miklós S. Z. Kellermayer¹

¹ Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary

² Heart and Vascular Center, Semmelweis University, 1122 Budapest, Hungary

³ Hungarian Marfan Foundation, 1122 Budapest, Hungary

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation in microfibrillar function and, conceivably, alterations in the microfibrils’ supramolecular structure.

The aim of the study was to perform a nanoscale structural characterization of fibrillin-1 microfibrils isolated from human aortic samples with different FBN1 gene mutations and to compare them with microfibrillar assemblies purified from non-MFS human aortic tissue.

Aortic wall samples were obtained from patients undergoing specific cardiovascular surgical interventions. Fibrillin-rich microfibrils were extracted by bacterial collagenase digestion and purified by size-exclusion chromatography. Atomic force microscopy was employed to visualize and study the microfibrillar assemblies.

Fibrillin-1 microfibrils displayed a characteristic “beads-on-a-string” appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50–52 nm among samples.

In conclusion, the data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-specific aortic symptomatology.

Acknowledgment

Funding sources: NRDI Office (ÚNKP-22-3-I-SE-49 to C.M.Ș.; K135360 to M.S.Z.K.; TKP2021-EGA-23), European Union (RRF-2.3.1-21-2022-00003 – National Cardiovascular Laboratory).

P28

Automated single cell isolation on a microscope

Bálint Szabó

CellSorter Kft.

Current robots for single cell manipulations can only handle surface attached cells limiting the field of applications. CellSorter developed a computer vision-based robot to automatically recognize and gently isolate intact individual cells (for subsequent analysis, e.g., DNA/RNA sequencing) from suspension without immobilizing cells on the surface of the Petri dish. We used a motorized microscope and robotic micropipette controlled by the computer vision of cells. Combination of 1 µm positioning precision, adaptive cell targeting and < 1 nl liquid handling precision resulted in an unprecedented accuracy in robotic cell isolation. Single cells could be picked up in a volume of 1.4 ±0.6 nl without removing neighboring cells. Sorting process did not affect the viability of cells. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. Our straightforward technique needs minimal sample preparation and can be applied for virtually any tissue cell type. We expect new applications in circulating tumor cell (CTC) detection and isolation for molecular characterization and personalized treatment.

P29

Finite element modelling and analysis of fluid dynamic phenomena in two-phase droplet based microfluidic systems

Zsombor Szomor^1,2, Eszter L. Tóth ¹, Péter Fürjes ¹

¹ Microsystems Lab., Inst. of Technical Physics and Materials Science, Centre for Energy Research, ELKH, Budapest, Hungary

² Óbuda University Doctoral School on Materials Sciences and Technologies, Budapest, Hungary

Recently the development and application of Lab-on-a-Chip and microfluidic devices have been spreading, enabling not only the extension of biological sample preparation and sensing solutions, but the comprehensive analysis and understanding of microscale fluid dynamic phenomena. The high performance finite element modelling (FEM) environments combined with experimental methods are enable solid description of microfluidic processes and reliable prediction the behaviours of novel architectures. However, the modelling of multiple-phase flows in microscale geometries is still a challenge.

Finite element Modelling code – COMSOL Multiphysics – was applied to analyse the process of droplet formation in 2D and 3D two-phase models to achieve more accurate comprehension of the behaviour of microfluidic systems depending on geometries and fluid parameters [1]. The analysis is based on the numerical solution of the governing Navier-Stokes and continuity equations. The fluidic environment and boundary conditions in these specific capillary systems can be characterised by the Capillary-number, which describes the relationship between the surface tension and the viscous forces. In this work the influence of channel geometries, the volume flow ratio at the inlets, the viscosity of the fluids, and the interface tension were studied in successive parametric sweep simulations. For handling multiphase fluids in 2D and 3D models a robust Level Set calculation method was used [2].

The droplet generation phenomena was studied by experimental methods, to verify FEM results, proving that the droplet formation processes as well as droplet sizes and generation frequencies are significantly affected accordingly can be effectively controlled by the geometric and flow parameters. The microfluidic systems were manufactured by soft lithography techniques in Polydimethylsiloxane (PDMS) polymer and tested using water / oil two phase fluid system.

Acknowledgement

The work was supported by the National Research, Development and Innovation Fund (NKFIA) via INBIOM TKP2021-EGA-04 grant.

References

• [1] K. J. Donovan. „Computational fluid dynamics modeling of two - dimensional and three - dimensional segmented flow in microfluidic chips”, San Jose State University, 2014. doi:10.31979/etd.3tzd-y6xm.
• [2] H. A. Akhlaghi Amiri, A. A. Hamouda, Evaluation of level set and phase field methods in modeling two phase flow with viscosity contrast through dual-permeability porous medium, International Journal of Multiphase Flow 52, 22-34, 2013

P30

Structural features of a disordered protein motif

Mónika Ágnes Tóth¹, Péter Gaszler^{1, 3}, Andrea Vig¹, Veronika Takács-Kollár¹, Illés Csonka², Tamás Huber^{1, 3}, Rauan Sakenov¹, Réka Pintér¹, Beáta Bugyi^{1, 3}

¹University of Pécs, Medical School, Department of Biophysics, Pécs Szigeti str 12. H-7624

²Lajos Nagy High School of the Cistercian Order

³Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee, Pécs, Hungary

SALS (sarcomere length short) is a Drosophila-specific sarcomeric protein that regulates sarcomere length and organization. Lack of SALS is lethal in embryonic age, possibly due to the shortening of sarcomeric actin filament length or the disruption of their order.

Our bioinformatics analysis suggests that SALS is an intrinsically disordered protein (IDP). IDPs are biologically active proteins, however, do not have a well-defined three-dimensional structure. They possess specific physicochemical properties different from those characteristics for ordered proteins (e.g., amino acid composition, thermal stability, electrophoretic mobility). There is growing attention to studying IDPs for their key roles in diseases or cellular processes. SALS contains two intrinsically disordered protein regions (IDRs), the Wiscott-Aldrich syndrome homology 2 (WH2) domains, composed of a few tens of amino acids. Proteins containing WH2 domains possess actin-binding properties and can exhibit multifunctional character depending on the number and sequence of WH2 domains.

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. The structural properties and conformational dynamics of SALS-WH2 have not yet been described. Therefore, we further aim to characterise these features using in silico and experimental approaches. Our prediction-based results were experimentally verified by fluorescence spectroscopy and thermal analysis.

Acknowledgements

This work was supported by ÚNKP-21-3-II-PTE-997 (PG), University of Pécs, Medical School (MÁT), 2021-4.1.2-NEMZ_KI-2022-00025 (TH). We thank József Mihály (Institute of Genetics, Biological Research Centre, Szeged) for the SALS plasmid.

P31

Investigation of actin polymerization: methodology and case study

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

¹ University of Pécs, Medical School, Department of Biophysics

² Regional Committee of The Hungarian Academy of Sciences at Pécs, The Expert Committee of Physics and Astronomy, Spectroscopy Committee, Pécs, Hungary

Actin is one of the most important components of the cytoskeleton that plays a crucial role in many cellular processes. The organization of the actin cytoskeleton is under the control of actin binding proteins (ABPs). Total internal reflection microscopy (TIRFM) is a proper technique for the observation and characterization of actin dynamics from single molecules to a complex system in vitro, it works as a bridge between in vitro and in vivo actin polymerization assays. Furthermore, it is suitable to investigate the role of ABPs in modifying the structural and dynamic properties of actin. Flightless-I (Fli-I) is a member of gelsolin superfamily. It has a unique structure including gelsolin-like domains (GH) and leucine-rich repeats (LRR). Fli-I can interact with actin, however, its biochemical activities in actin dynamics regulation are largely elusive.

The aim of our study is to provide an overview of actin polymerization experiments by fluorescence spectroscopy and TIRFM from basic principles to data/image analysis. We test the applicability of different fluorophores and specify the advantages and limitations of each approach. As a case study, we investigate the effect of wild-type Fli-I and its disease-causing mutations and truncations on actin dynamics. Pyrene-actin-based fluorescence spectroscopy reveals a biphasic effect of Fli-I on actin assembly kinetics, complementary TIRFM assays enlightens the molecular mechanism underlying the activities of Fli-I and its disease-related variants at the single filament level.

Acknowledgment

The research in Hungary was funded by NKFIH within the framework of the project TKP2021-EGA-17 and 2021-4.1.2-NEMZ_KI-2022-00025 ” Activities of Flightless I revealed by acto-myosin based in vitro motility techniques” (TH). We thanks to József Mihály (SZBK, Institute of Genetics, Biological Research Centre) for the Fli-I plasmids.

P32

Adaptive changes in the A-band region of the giant protein titin in diseased human cardiac sarcomere

György Török¹, Iliza Ramazanova¹, Péter Dániel¹, András Jámbor¹, Dalma Kellermayer^1,2, Cristina M. Șulea^1,2, Zoltán Szabolcs², Miklós S. Z. Kellermayer¹ and Balázs Kiss¹

¹Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

²Heart and Vascular Center, Semmelweis University, Budapest, Hungary

Titin, the largest protein known, spans the half-sarcomere, the contractile unit of skeletal and cardiac muscle through its Z-disk to M-line and interacts with thin and thick filaments in the I- and A-band of the muscle sarcomere, respectively. Titin’s A-band segment is not well understood but is shown to be orders of magnitude less extensible than the I-band region of the molecule. Heterozygous truncating mutations (TTNtv) affecting A-band titin are often associated with dilated cardiomyopathy (DCM). Marfan syndrome (MFS), a connective tissue disorder caused by mutations of the matrix glycoprotein fibrillin is also associated with impaired cardiac contractility but its exact pathomechanism is largely unknown.

Here, we performed STED super-resolution microscopy on sections of stretched and fixed demembranated human cardiac myofibrils carrying heterozygous TTNtv mutations or on samples originated from MFS patients. Sequence-specific anti-titin antibodies included the 1) MIR, which labels titin at the ends of the thick filaments, 2) A170, labeling titin close to the M-line (this epitope is missing in TTNtv+). Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres. Our experiments indicate that truncated titin is able to integrate into the cardiac sarcomere. We propose that the truncated titin cannot precisely register the ends of the thick filaments, and this can ultimately lead to the manifestation of DCM by disrupting the overlapping of thin and thick filaments. In MFS sarcomeres a pronounced, ~30 nm shift away from the M-line was found in the case of the A170 titin epitope suggesting that alterations in the M-band ultrastructure might be important contributors of the impaired cardiac contractility of MFS patients.

P33

The effects of contrast agents on renal cell lines and on the actin cytoskeleton

Szilvia Barkó¹, Elek Telek¹, Kinga Ujfalusi-Pozsonyi¹ Gábor Hild^1,2 and Zoltán Ujfalusi¹

¹ Department of Biophysics, Medical School, University of Pécs; Pécs, Hungary

² Department of Medical Imaging, Clinical Centre, University of Pécs, Pécs, Hungary

Medical images may show some degree of contrast loss in most imaging techniques. In such cases contrast materials are the best tools to enhance the density and intensity of the given area. Nowadays, radiologists can choose from a wide range of contrast agents. The active material of these contrast media penetrates in cells and because of their limited ability of depletion these molecules can accumulate in cells of different tissues. In many cases, contrast agents are used in relatively high volumes, which places a heavy burden on kidney function. A few years ago, contrast agent treatment was identified as the third leading cause of hospital-acquired acute kidney injury (after surgery and hypotension), accounting for 12% of all cases. Today, ~5% of hospitalized patients who develop acute renal failure have normal renal function before contrast administration. We believe that contrast agents exert a significant proportion of their cell-damaging effects by affecting the actin cytoskeleton. The overview of the corresponding literature clarifies that the effects of the clinically applied contrast materials expressed directly on the actin cytoskeleton and its detailed molecular mechanisms are unknown. Our aim is to investigate all possible effects contrast materials can express on human renal cells and the actin protein inside, especially the dynamic organization/rearrangement of the actin network.

Our results clearly show that the applied contrast materials greatly affect the polymerization properties of actin. The examined contrast compounds changed other parameters of actin too and caused dramatic changes on the examined cell lines as well. The DSC results show significantly decreased thermal stability for the treated actin filaments.

P34

Effect of Weight Loss on the Electrical Impedance Parameters of Lettuce and Iceberg Lettuce Stored at Room Temperature

Eszter Vozáry, Bíborka Gillay

Hungarian University of Agriculture and Life Science, Institute of Food Science and Technology

Department of Food Industrial Measurements and Control

We bought the lettuce and iceberg lettuce at the local market. The leaves were stored at room temperature. The weight loss of the leaves was measured with a scale. The electrical impedance spectra were determined with HP4284A and 4285A precision LCR meters in the frequency range of 30 Hz to 30 MHz. At a measuring voltage of 1 V, the magnitude and phase angle of the electrical impedance were measured. The measured spectra were corrected with the stray inductance and capacitance values. A homemade needle electrode and ECG electrodes were used for the measurement. The initial moisture content of the leaves was determined from samples dried to constant weight in an oven at 110°C. Both the impedance measurement and the weight measurement were performed at room temperature for each leaf for 10 minutes over 4-5 hours until the leaf completely withered. The measured impedance spectra were approximated with three distributed elements connected in series. We determined the resistance and capacity of the elements of the model circuit, as well as the relaxation time. The change in the obtained parameters followed well the weight loss, i.e. the change in the water content of the leaf.

P35

Label-free tracking of cell adhesion kinetics as a function of various parameters

Anna Balogh^1,2, Kinga Dora Kovacs^1,2, Imola Rajmon^1,2, Inna Szekacs²,

Beatrix Peter², Robert Horvath²

¹Eötvös Lóránd University, Department of Biological Physics, Budapest, Hungary

² Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research

Most tissue cells cannot survive for more than a few hours without adherence, if we were able to prevent the adhesion of malignant cells, metastasis could be prevented [1] [2]. The data used for analysis was recorded by a surface sensitive, label-free, resonant waveguide grating based optical biosensor. Due to the force calibration of the optical signal provided by the sensor for individual cells, this technique is suitable for determining the force curves of a large number of cells [3]. To analyse the adhesion signals at the molecular level, we applied and developed different kinetic models. These models take into account relevant molecular parameters, such as dissociation and association rates, the two dimensional kinetic dissociation constant which describes the integrin-ligand binding strength [4].

We investigated the extent to which the adhesion kinetics of cells are affected by the surface density of the cells, the density of echistatin, and the effect of gold nanoparticles on cell adhesion. Echistatin is a potent inhibitor of αIIβ3, αvβ3 and α5β1 receptors [5]. Functionalized nanoparticles can penetrate into living cells, Peter et. al. demonstrated from the recorded kinetic adhesion data that the uptake of the functionalized nanoparticles is an active process [6]. Peter et. al. also investigated the role of glycocalyx components in the cellular uptake of nanoparticles [7]. Subjecting these results to further analysis, we developed procedures and codes that enable the study of molecular parameters influencing adhesion in living cells, in their real environment, without isolating the relevant molecules. Our results potentially open the way for further analysis of the kinetic data obtained from the adhering cells.

Acknowledgment

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 134195 for Z.Sz, PD 131543 for B.P., ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] Sudhakar A. History of Cancer, Ancient and Modern Treatment Methods. J Cancer Sci Ther. 1, 2 (2009). https://doi.org/10.4172/1948-5956.100000e2

[2] Lodish, H., Berk, A., Matsudaira, P., et al. Molecular Cell Biology. (2003)

[3] Sztilkovics, M., Gerecsei, T., Peter, B. et al. 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

[4] 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

[5] Szekacs, I., Orgovan, N., Peter, B., et. al. Receptor specific adhesion assay for the quantification of integrin–ligand interactions in intact cells using a microplate based, label-free optical biosensor. Sensors and Actuators B: Chemical, 256 (2018). https://doi.org/10.1016/j.snb.2017.09.208

[6] Peter, B., Lagzi, I., Teraji, S., et. al. Interaction of Positively Charged Gold Nanoparticles with Cancer Cells Monitored by an in Situ Label-Free Optical Biosensor and Transmission Electron Microscopy. ACS Appl. Mater. Interfaces 10, 32 (2018). https://doi.org/10.1021/acsami.8b01546

[7] Peter, B., Kanyo, N., Kovacs, K.D., et. al Glycocalyx Components Detune the Cellular Uptake of Gold Nanoparticles in a Size- and Charge-Dependent Manner. ACS Appl. Bio Mater. 6, 1 (2022). https://doi.org/10.1021/acsabm.2c00595

P36

Spectroscopic study of extracellular vesicles using plasmonic gold nanoparticles

Tímea Bebesi Farkasné^1,2, Marcell Pálmai¹, Imola Csilla Szigyártó¹, Anikó Gaál¹, Orsolya Bálint-Hakkel³, Attila Bóta¹, Zoltán Varga¹, Judith Mihály¹

¹ Research Centre for Natural Sciences, Institute if Material and Environmental Sciences

² Eötvös Lóránd University, Hevesy György PhD School of Chemistry

³ Centre for Energy Research, Institute of Technical Physics and Material Sciences

Extracellular vesicles (EVs), spontaneously released by cells, play an important role in intercellular communication. Due to their special size and composition (lipid bilayer-bounded nanosystems, usually smaller than 200 nm, containing both proteins and RNA), they play diagnostic, prognostic and therapeutic roles, for example, they can be "new generation" biomarkers of various diseases.

IR spectroscopy, especially attenuated total reflection (ATR), is rapidly emerging as a label-free promising tool for molecular profiling of EVs. However, the relative low number of extracellular vesicles (~10¹⁰ particle/mL) and possible impurities (protein aggregates, lipoproteins, buffer molecules, etc.) present in EV samples might result in poor signal-to-noise (S/N) ratio. The plasmonic properties of gold nanoparticles (AuNPs) are used in many characterization techniques, inclusive characterization and testing of EVs. Surface-enhanced infrared spectroscopy (SEIRA – Surface-enhanced IR absorption) using plasmonic nanoparticle, however, is still an unexploited method.

Nanosized gold nanoparticles and tailored nanostructures with confined electromagnetic near-fields were prepared, characterized and tested with model-EVs (EV-like liposomes) and red blood cell derived EVs. A concentration dependent interaction was established between the citrate-stabilized gold nanoparticles and the lipid bilayers, which strongly affected both the plasmonic behaviour of AuNPs and the bilayers lipid organization. At appropriate extracellular vesicle – gold nanoparticle ratio a 6-fold maximum enhancement was obtained in the lipid spectral signatures. Exploiting the fine details of EV – gold nanoparticles interaction, further surface modifications of gold nanoobjects are planned, enhancing the sensitivity and specificity of EV detection enabling a strong platform for IR spectroscopic investigations of EVs.

Acknowledgment

This work was funded by ÚNKP-22-3-II-ELTE-507 and NKFIH K-131657, K131594, 2020-1-1-2- PIACI-KFI_2020-00021, TKP2021-EGA and KKP_22-144180 grants. ZV and MP are supported by the János Bolyai Research Scholarship of the HAS.

P37

Role of the hHv1 proton channel in vascular smooth muscle cells

Katinka Gyuris¹, Geraldo Domingos¹, Éva Korpos² and Zoltán Varga¹

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

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

The hHv1 voltage-dependent proton channel is a passive transporter that selectively transfers protons across the membrane, and thus plays an important role in pH regulation of many cell types. Vascular smooth muscle cells (VSMCs) found in the arterial wall have a resting or contractile phenotype under physiological conditions. When the vessel wall is damaged, the cells switch to a synthetic, migratory and proliferative phenotype, which allows tissue regeneration. Failure of migrating/proliferating cells to switch back to a contractile phenotype induces pathogenic vascular remodelling leading to atherosclerosis.

In our work, we aim to demonstrate the presence of hHv1 on vascular smooth muscle cells and to elucidate the role of the channel in normal and pathological cellular activities through the regulation of intracellular pH. We investigate the role of hHv1 in VSMC survival, differentiation and matrix production.

If hHv1 plays a key role in the pathological activity of VSMCs during atherosclerosis but not in normal function, the channel may become an important pharmacological target to inhibit the pathological activity of VSMCs in atherosclerosis.

To confirm this hypothesis, we performed viability assays using MTT assay, motility experiments using scratch assay method and impedance measurement, PCR and Western blot experiments to detect hHv1, alizarin red staining to visualize the effect on calcified extracellular matrix production, and microscopic studies.

Our results support the hypothesis that hHv1 is expressed in VSMCs and plays a role in their ability to migrate and influences their viability. This conclusion may provide a basis for further experiments to quantify the difference in hHv1 expression between contractile and differentiated proliferative cells and to identify related functional differences.

P38

Nanoerythrosome-based promising drug delivery systems

Attila Bóta¹, Judith Mihály¹, Kinga Ilyés¹, Bence Fehér², Tünde Juhász³, András Wacha¹, Heinz Amenitsch⁴ and Zoltán Varga¹

¹Research Centre for Natural Sciences, Biological Nanochemistry Research Group, Budapest

²Laboratory of Self-Organizing Soft Matter, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven, The Netherlands

³Research Centre for Natural Sciences, Biomolecular Self-assembly Research Group, Budapest

⁴Austrian SAXS beamline@ELETTRA, Are Science Park, Basovizza TS, Trieste, Italy and Inorganic Chemistry, Graz University of Technology, Graz, Austria

Nanoerythrosomes are artificial vesicle-like objects formed from erythrocyte-membranes, named ghosts, by physical processes, such as extrusion or sonication. Phosphatidylcholines (PCs) and sphingomyelins (SMs) are outer membrane constituents, while phosphatidylserines (PSs) and phosphatidylethanolamine (PEs) generally take place on the inner side of the membrane-bilayer. By addition of different artificial lipids, very different size-ranges of nanoerythrosomes can be achieved, therefore proper reference materials and drug delivery systems with adequate surface chemical behaviour can be prepared [1]. The presence of dipalmitoyl-phosphatidylethanolamine (DPPE) results in the formation of larger nanoerythrosomes, while the addition of dipalmitoylphosphatidylcholine (DPPC) induces the formation in a middle-range (140 -160 nm). The presence of the mixture of DPPC - LPC (lysophosphatidylcholine) causes bicelle – micelle type nanoparticles [2]. Here we show that in the complex physic-chemical study, among the different experimental methods (transmission electron-microscopy combined with freeze-fracture (FF-TEM), Microfluidic Resistive Pulse Sensing (MRPS), dynamic light scattering (DLS), the small-angle X-ray scattering (SAXS) turned out to be a powerful tool in the complex physic-chemical study of this drug delivery system.

Acknowledgment

The project was supported by the National Research, Development and Innovation Office of Hungary under grants K131657 (A. Bóta) and K131594 (J. Mihály) and 2018-1.2.1-NKP-2018-00005 under the 2018-1.2.1-NKP funding scheme (A. Bóta, Z. Varga). Z Varga and A. Wacha are supported by the János Bolyai Research Scholarship of the HAS.

References

[1] Deák R, Mihály J, Szigyártó ICS, Beke-Somfai T, Turiák L, Drahos L, Wacha A, Bóta A and Varga Z (2020)  Mat. Sci. and Eng. C 109:110428-110437.

[2] Bóta A, Fehér B, Wacha A, Juhász T, Szabó D, Turiák L, Gaál A, Varga Z, Amenitsch H and Mihály J(2023) J Mol. Liq. 369: 120791-120800.

P39

Functionally important C-terminus of small GTPase Ran: exploring its nucleotide-specific conformational surface

Janka Czigleczki¹, Pedro Tulio de Resende Lara², Balint Dudas^1,3,4, Hyunbum Jang⁵, David Perahia⁴, Ruth Nussinov ^5,6and Erika Balog¹

¹Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary

²Department of Medical Genetics and Genomic Medicine, School of Medical Sciences, University of Campinas—UNICAMP, Campinas, Brazil 

³Inserm U1268 MCTR, CiTCoM UMR 8038 CNRS—Université Paris Cité, Paris, France

⁴Laboratoire et Biologie et Pharmacologie Appliquée, Ecole Normale Supérieure Paris-Saclay, Gif-sur-Yvette, France

⁵Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Cancer Innovation Laboratory, National Cancer Institute, Frederick, MD, United States

⁶Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

As a member of the Ras superfamily of small GTPases, Ran (Ras-related Nuclear protein) is the main regulator of the nucleo-cytoplasmic transport through the nuclear core complex. It functions as a molecular switch cycling between the GDP-bound inactive or “off” and GTP-bound active or “on” 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.

 Ran consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. The crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD), whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, by using molecular dynamics (MD) and MDeNM (Molecular Dynamics with excited Normal Modes) simulation methods, we present how rigid the C-terminal region is in the inactive RanGDP form and for the first time in the literature, we were able to follow its conformational flexibility in the GTP-bound form. This conformational mapping allows us to envisage how the C-terminus can embrace RanBDs during the function of Ran.

The simulations were carried out by JC, and were analyzed and interpreted by JC, PR, BD, HJ, RN, DP, and EB.

P40

Identification of inhibitors of the human h_v1 proton channel

Geraldo Domingos¹, Adam Feher¹, Eva Korpos ^1,2, Tibor G. Szanto¹, Martina Piga³, Tihomir Tomasic³, Nace Zidar³, Adrienn Gyongyosi⁴, Judit Kallai⁴, Arpad Lanyi⁴, Ferenc Papp¹, Katinka Gyuris¹, Zoltan Varga¹

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

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

3 Department of Pharmaceutical Chemistry, University of Ljubljana, Slovenia, 4 Department of Immunology, Faculty of Medicine, University of Debrecen, Hungary

The human voltage-gated proton channel (hH_V1) plays an important role in immune system and cancer cells being involved in functions such as proliferation, migration, and oxidative burst. H_V1 does not have a conventional ion-conducting pore, the conduction occurs through the voltage-sensing domain. This difference may be the reason for the lack of selective hH_V1 inhibitors. Currently, 5-chloro-2-guanidinobenzimidazole (ClGBI) is the most widely used inhibitor of H_V1 but it has low selectivity for the channel. This could lead to misinterpretation of functional assays addressing the role of H_V1 with the use of ClGBI. Thus, our aim was to find potent and more selective inhibitors for hH_V1, which could be useful research tools and serve as lead molecules for the development of drug molecules targeting H_V1.

We used patch-clamp to test the affinity and selectivity of potential inhibitors of H_V1 on CHO and HEK cells expressing hH_V1 and other channels. Seven “hit” molecules were identified among the NZ family of compounds of which NZ-13 has the best selectivity profile.

The widely-used HV1 inhibitor ClGBI blocks various ion channels and therefore is not a selective HV1 blocker. This must be considered in functional tests investigating the role of HV1 in healthy and pathological conditions.

We have identified a new family of hHV1 inhibitors, which have comparable affinities for the channel to ClGBI.

Most NZ molecules have low selectivity for hHV1, but NZ-13, the one with the highest selectivity, may be better suited for functional tests than ClGBI as it inhibits T cell proliferation less.

P41

Controlling Live Cell Adhesion through Characterization of Biofunctionalized Surfaces using Label-Free Biosensors

Eniko Farkas¹, Kinga Dóra Kovács^1,3, Beatrix Peter¹, Attila Bonyár², Sandor Kurunczi¹, Inna Szekacs¹, and Robert Horvath¹

¹ Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, Centre for Energy Research, Budapest, Hungary

² Department of Electronics Technology, Faculty of Electrical Engineering and Informatics, Budapest University of Technology and Economics, Budapest, Hungary

Biomaterial coatings that possess cell-repellent or cell-adhesive properties have a significant interest in medical and biotechnological applications [1-4]. However, conventional approaches lack in-depth analysis and quantitative comparison of these coatings for regulating adhesion, particularly for bacterial cell adhesion. Label-free Optical Waveguide Lightmode Spectroscopy (OWLS) can offer a solution for the detailed analysis of biomaterial coatings. OWLS biosensors detect the optical properties of the adhesive surface using evanescent waves with a penetration depth of 100-150 nm [5-7]. This surface-sensitive technique enables a thorough evaluation of biomaterial coatings for regulating adhesion. Uniquely, OWLS enables the in situ measurement of both the coating process and subsequent cell adhesion.

The present study utilizes the OWLS method for in-depth characterization of biomaterial surfaces with regard to bacterial adhesion. Initially, adhesion blocking biomaterials, namely bovine serum albumin, I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P), and PLL-g-PEG, with varying coating temperatures, were screened. PAcrAM-P exhibited the best blocking capability against bacterial concentrations up to 10⁷ cells/mL. Subsequently, different immobilization methods, such as Mix&Go (AnteoBind) films, protein A, avidin-biotin based surface chemistries, and simple physisorption, were employed to captureEscherichia coli specific antibodies. Bacterial cell adhesion was then tested on immobilized antibodies with various blocking agents. The OWLS analysis allowed for the determination of the parameters of the applied agents by considering the kinetic data of adhesion, the surface mass density, and the protein orientation. Based on the experimental results, surfaces were created and tested for controlling both bacterial and mammalian cell adhesion. [8]

Acknowledgment

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] Frutiger A, et. al. (2021) Chem Rev 121: 8095–8160.

[2] Rigo S, et. al. (2018) Adv Sci 5: 1700892.

[3] Castillo-Henríquez L, et. al. (2020) Sensors 20: 6926.

[4] D’Agata R, et. al. (2021) Polymers 13:1929.

[5] Vörös J, et. al. (2002) Biomaterials 23: 3699–3710.

[6] Tiefenthaler K, et. al. (1989) J Opt Soc Am. B 6: 209–220.

[7] Saftics A, et. al. (2021) Adv Colloid Interface Sci 294: 102431–102433.

[8] Farkas E, et. al. (2022) Biosensors 12: 56.

P42

Role of ion channels in CAR T-cell

Ghofrane Medyouni¹, Vivien Jusztus¹, Orsolya Vörös¹, Maria Eduarda Lima^1,2, György Panyi¹, Péter B. Hajdu^{1, 2}

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

² University of Debrecen, Faculty of Dentistry, Division of Dental Biochemistry

Cancer immunotherapy partly relies on the reprogramming of host immune cells to eliminate cancer cells. Genetic modification of T cells to express chimeric antigen receptors (CARs) is utilized in the treatment of hematological malignancies. Despite its success, many challenges remain to improve the efficacy and safety of this therapy. Ion channels in T-cells participate in the regulation of Ca²⁺-dependent activation pathway and play a role in various effector functions inevitable for target cell abolition. Hence, modification of ion channels’ function can contribute to successful immune therapy. However, no study has been reported about functional role of CAR T-cell ion channels yet.

We established a 3^rd-generation CAR expressing cell line (CD19-CAR cells) from Jurkat cells. We used the whole-cell patch-clamp technique and FURA-2-based Ca²⁺-imaging to determine the biophysical properties of Kv1.3 and Ca²⁺-response of CD19-CAR cells, respectively. We adapted a Calcein Red based killing assay to test CD19-CAR cells’ target cell killing capacity. We assessed the localization of Kv1.3 in standalone and in the CAR-synapse engaged CD19-CAR cells.

We showed that Kv1.3 activation and inactivation kinetics are the same in non-transfected and CD19-CAR cells, while voltage-dependence of activation was different. Thapsigargin-induced Ca²⁺-response of CD19-CAR cells was lower as compared to the control. We showed that Kv1.3 channels are co-localized with CARs in standalone CD19-CAR cells, and they redistribute to the contact region between a CD19-CAR cell and a target cell (Raji B cell). Upon Vm24 addition (specific Kv1.3 inhibitor, 1 nM) the target cell ability of CD19-CAR cells was impaired. Based on these results, we suppose that ion channels can affect the outcome of immunotherapy, and further experiments are needed to clarify their functional role.

Acknowledgment

This work was supported by Stipendium Hungaricum Scholarship to M.G.t and NKFIH (K128525, P.H.).

P43

Ion channel expression of CD8⁺ T cells in ovarian cancer

Vivien Jusztus¹, Ghofrane Medyouni¹, Orsolya Vörös¹, Zsolt Szabó¹, Rudolf Lampé³, György Panyi¹, Orsolya Matolay³, Eszter Maka³, Zoárd Krasznai³, Péter Hajdu^1,2

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

² University of Debrecen, Faculty of Dentistry, Division of Dental Biochemistry

³ University of Debrecen, Faculty of Medicine, Department of Gynecology and Obstetrics

The ion channels of T lymphocytes have an important role in effector functions such as activation, cytokine production and tumor cell elimination. T cells recognise and kill cancer cells during continuous monitoring. Although tumor infiltrating lymphocytes (TILs) are able to penetrate the tumor, they cannot fulfil their effector function due to the suppressive nature of the tumour microenvironment. K⁺ channels, such as Kv1.3 and KCa3.1, stabilize the negative membrane potential of T cells to control Ca²⁺-influx through CRAC channels and Ca²⁺-dependent signaling. In the present study, we determined the expression of T cell ion channels from peripheral blood of untreated ovarian cancer patients and healthy donors.

PBMCs were isolated from blood of ovarian patients and healthy donors using Ficoll-Paque density gradient method. Cells were activated with CD3/CD28 antibodies. Whole-cell current was measured in activated CD8⁺ T cells using patch-clamp technique. Ca²⁺ response of CD8⁺ cells was evaluated with FURA-2 Ca²⁺-imaging method.

KCa3.1 expression level in blood CD8⁺ cells from malignant tumor patients were lower than in healthy and benign tumor groups. Contrary, the Kv1.3 conductance of CD8⁺ T cells were significantly higher in malignant tumor patients as compared to other two groups. To asses Ca²⁺ response of cells, we determined the quotient of FURA-2 ratios measured in 2 mM Ca²⁺ and 0 mM Ca²⁺ after thapsigargin addition: there was no differences between the groups.

In summary, we suppose that down-regulation of KCa3.1 expression and Kv1.3 level upregulation in blood CD8⁺ cells could be a reporter on the presence of malignancy, as we reported before for head and neck cancer patients [1].

Acknowledgment

This work was supported by Stipendium Hungaricum Scholarship to M.G. and NKFIH (K128525, P.H.).

References

[1] Chimote AA, Balajthy A, Arnold MJ, Newton HS, Hajdu P, Qualtieri J, et al. (2018) Sci Signal. 11(527): 1–12

P44

Isolation of novel peptide toxins from the venom of the scorpion Centruroides bonito which blocks Kv1.2 ion channel with picomolar affinity 

Kashmala Shakeel¹, Muhammad Umair Naseem¹, Lourival D Possani², Gyorgy Panyi¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary

² Departamento de Medicina Molecular y Bioprocesos, Universidad Nacional Autónoma de México, Mexico

Seven new peptide toxins named as CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito by liquid chromatography. The primary structure of these peptides were determined by Edman degradation. Mass spectrometry analysis was used to determine the molecular weights which range between 3760.4 Da to 4357.9 Da, comprising 32 to 39 amino acid residues cross-linked with three tightly folded disulfide-bridges. The amino acid sequence alignment with known potassium scorpion toxins (KTx) and phylogenetic tree analysis unveiled that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to α-KTx 10 subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), CboK7 (α-KTx 2.24) bears more than 95% amino acid similarity with the members of α-KTx 2 subfamily, and CboK5 is 100% identical with previously described Ce3 toxin (α-KTx 2.10). The electrophysiological assays (whole-cell patch clamp) revealed that except CboK1, all other six peptide toxins blocked the voltage-gated potassium channel Kv1.2 with high affinity, having Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 ion channel with comparatively less potency (Kd values between 20-171 nM). Moreover, CboK2 and CboK3 inhibited ~10% and CboK7 inhibited ~50% of Kv1.1 currents at 100 nM concentration. Among all CboK7 (α-KTx 2.24) has the highest affinity for Kv1.2 ion channel with Kd value of 24 pM, and reasonable selectivity over Kv1.3 (~1000-fold) and Kv1.1 (~6000-fold) ion channels. These distinguishable characteristics of the CboK7 toxin may provide a framework for developing tools to treat Kv1.2-related gain of function channelopathies.

P45

Light energy harvesting by photosystem I in cyanobacterial cells

Petar Lambrev¹, Parveen Akhtar¹, Avratanu Biswas¹, Ivo van Stokkum²

¹ Biological Research Centre, Szeged, Institute of Plant Biology

² Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands

Photosystem I (PSI) is a crucial component of the light-dependent reactions of oxygenic photosynthesis occurring in cyanobacteria, algae and plants. It is a multi-subunit pigment-protein complex binding more than a hundred pigment – chlorophylls (Chls) and carotenoids, as well as cofactors carrying out photoinduced electron transport with a quantum yield of near unity. Whereas PSI in plants and eukaryotic algae is attached to peripheral membrane-intrinsic light-harvesting complexes, cyanobacteria utilize the membrane extrinsic phycobilisomes (PBS) as the main light-harvesting antenna complex¹. While the structural and energetic interaction between the PBS and photosystem II (PSII) is well established, less is known about the connectivity of PBS and PSI and the ability of the PBS to transfer energy directly to PSI is debated. We investigated the pathways and dynamics of energy transfer from PBS to the photosystems in Synechocystis sp. PCC 6803. The excitation kinetics of PBS and PSI were followed by picosecond time-resolved fluorescence spectroscopy in the wild-type strain and in a mutant devoid of PSII². We found that PBS are capable of directly transferring energy to PSI in the PSII-deficient mutant, in a time scale of about 20 ps at room temperature. Based on an earlier model of energy transfer in Synechocystis sp. PCC 6803³ and simultaneous fitting to the measured data of isolated complexes and intact cells, a detailed model of energy transfer between different PBS, PSI and PSII chromophore groups was obtained.

Many cyanobacterial species, when exposed to iron limitation conditions, produce a specialized pigment-protein complex, IsiA, that is known to associate into rings around PSI⁴. The physiological function of IsiA is not fully understood. In isolated PSI-IsiA complexes IsiA efficiently transfers absorbed photon energy to the PSI core⁵, which can extend the absorption cross-section of the photosystem and help reduce the number of iron-rich PSI core complexes in the cells. However, IsiA has also been proposed to have a photoprotective, energy-dissipating role or to serve as a Chl depot⁶. To find more about the light-harvesting role of IsiA in vivo, we followed the cellular content of IsiA in cells of Synechocystis sp. PCC 6803 under iron limitation and investigated the energy transfer from IsiA to PSI by time-resolved spectroscopy. IsiA formed PSI-IsiA supercomplexes in vivo having similar energy transfer characteristics as isolated supercomplexes – confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a significant fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Moreover, we found that Synechocystis mutants containing only monomeric PSI contained far fewer IsiA units per PSI compared to the wild-type strain. We conclude that the trimeric organization of PSI in wild-type Synechocystishas role both in the accumulation and the energy transfer capabilities of IsiA under iron stress.

References

1. Blankenship, R. E., Molecular mechanisms of photosynthesis. John Wiley & Sons: 2021.

2. Bittersmann, E.; Vermaas, W., Fluorescence lifetime studies of cyanobacterial photosystem II mutants. Biochim. Biophys. Acta 1991, 1098 (1), 105–116.

3. van Stokkum, I. H.; Gwizdala, M.; Tian, L.; Snellenburg, J. J.; van Grondelle, R.; van Amerongen, H.; Berera, R., A functional compartmental model of the Synechocystis PCC 6803 phycobilisome. Photosynth. Res. 2018, 135 (1-3), 87–102.

4. Toporik, H.; Li, J.; Williams, D.; Chiu, P.-L.; Mazor, Y., The structure of the stress-induced photosystem I–IsiA antenna supercomplex. Nat. Struct. Mol. Biol. 2019, 26 (6), 443–449.

5. Andrizhiyevskaya, E. G.; Frolov, D.; van Grondelle, R.; Dekker, J. P., Energy transfer and trapping in the photosystem I complex of Synechococcus PCC 7942 and in its supercomplex with IsiA. Biochim. Biophys. Acta 2004, 1656 (2–3), 104–113.

6. Jia, A.; Zheng, Y.; Chen, H.; Wang, Q., Regulation and functional complexity of the chlorophyll-binding protein IsiA. Frontiers in Microbiology 2021, 12, 774107.

P46

Role of glycocalyx in cancer cell adhesion: kinetics of interactions from label-free optical biosensor measurements

Beatrix Magyaródi¹, Boglárka Kovács¹, Inna Székács¹, Robert Horvath¹

¹ Nanobiosensorics Laboratory, Research Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege u 29-33, 1120 Budapest, Hungary

The glycocalyx is a sugar rich layer covering the surface of the cells.[1] It is composed of glycoproteins and proteoglycans. The cellular glycocalyx plays an important, but not yet understood, role in cellular signaling and metabolism, its disorders generate pathological process.[2] Interestingly, the thickness of the glycocalyx layer of cancer cells is significantly larger compared to that of healthy cells. This fact further highlights the importance of glycocalyx in tumor progression and treatment. In an earlier work, a regulatory mechanism of cellular glycocalyx in cancer adhesion was revealed using label-free optical biosensor, fluorescent microscopy, and cell surface charge measurements. [3]

The primary goal of our work is to study the role of glycocalyx components in cellular adhesion by employing various types of digesting methods. In these initial measurements we use a label-free, high-throughput, resonant waveguide grating-based optical biosensor. The instrument is well suited for monitoring of cellular adhesion kinetics in real-time, even at the single-cell level.[4]

Acknowledgment

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 134195 for Z.Sz, PD 131543 for B.P., ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants)

References

[1] M. J. Paszek et al., “The cancer glycocalyx mechanically primes integrin-mediated growth and survival,” Nature, vol. 511, no. 7509, pp. 319–325, 2014, doi: 10.1038/nature13535.

[2] E. R. Cruz-Chu, A. Malafeev, T. Pajarskas, I. V. Pivkin, and P. Koumoutsakos, “Structure and response to flow of the glycocalyx layer,” Biophys. J., 2014, doi: 10.1016/j.bpj.2013.09.060.

[3] N. Kanyo 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., 2020, doi: 10.1038/s41598-020-80033-6.

[4] M. Sztilkovics et al., “Single-cell adhesion force kinetics of cell populations from combined label-free optical biosensor and robotic fluidic force microscopy,” Sci. Rep., 2020, doi: 10.1038/s41598-019-56898-7.

P47

The effect of light and age of the leaves on plastid differentiation and essential oil composition of spearmint (Mentha spicata L.)

Adrienn Dobi¹, Anna Skribanek², Bernadett Szögi-Tatár³, Andrea Böszörményi³, Imre Boldizsár¹, and Katalin Solymosi¹

¹Eötvös Loránd University, Budapest, Hungary

²Eötvös Loránd University, Savaria University Centre, Szombathely, Hungary

³Institute of Pharmacognosy, Semmelweis University, Budapest, Hungary

Spearmint (Mentha spicata L.) is a widely used spice, aromatic and medicinal plant with a characteristic fragrance. Its valuable active substances are monoterpene and sesquiterpene essential oil components produced by exogenous secretory structures, i.e. the various glandular hairs located on the shoot, especially on the leaves. The biosynthesis of the terpenoid essential oils requires isoprenoid biosynthesis of peculiar plastids present in the glandular hairs.

In this work, we have investigated 1) whether the prolamellar body-like membrane structures observed in the neck cells of peltate glandular hairs of spearmint can be considered as homologues with the prolamellar bodies present in etioplasts of dark-grown plants, 2) whether the presence and the activity (essential oil production) of the glandular hairs depends on the developmental stage of the leaves, 3) whether light conditions (e.g. light or dark development) affect plastid differentiation and the essential oil production of the leaves.

We have described in detail the various plastids and the organization of their inner membranes in the different cells of the exogenous secretory structures of spearmint, i.e. in capitate and peltate glandular hairs. We found clear structural differences between the prolamellar bodies of spearmint etioplasts of dark-grown leaves and of the similar structures found in secretory cells, which rather ressembled regularly arranged clusters of plastoglobuli, and not tubuloreticular membranes. Dark-growth did not influence much the structure of the plastids in the secretory cells, and our data obtained with solid-phase microextraction followed by gas chromatography and mass spectrometry (GC/MS) confirmed that not the illumination conditions but rather the developmental stage of the leaves influences the essential oil composition of spearmint.

Acknowledgments

The work was supported by the ÚNKP-22-5 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund (to K.S.) and by the Bolyai János Research Scholarship of the Hungarian Academy of Sciences (to K.S.).

P48

Cm39 (α-KTx 4.8): A novel scorpion toxin that inhibits voltage-gated K⁺ channel Kv1.2 and small- and intermediate-conductance Ca²⁺-activated K⁺ channels KCa2.2 and KCa3.1

Muhammad Umair Naseem¹, Georgina Gurrola-Briones², Margarita R. Romero-Imbachi³, Jesus Borrego¹, Edson Carcamo-Noriega², José Beltrán-Vidal³, Fernando Z. Zamudio², Kashmala Shakeel¹, Lourival D. Possani², Gyorgy Panyi¹

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology, Hungary

² Departamento de Medicina Molecular y Bioprocesos, Universidad Nacional Autónoma de México, Mexico

³ Departamento de Biología, Facultad de Ciencias Naturales, Universidad del Cauca, Colombia

A novel peptide toxin, Cm39, was identified in the venom of the Colombian scorpion Centruroides margaritatus. It is composed of 37 amino acid residues with a MW of 3980.2 Da and folded by three disulfide bonds. The Cm39 sequence also contains the Lys-Tyr (KY) functional dyad required to block voltage-gated K⁺ (Kv) channel. Amino acid sequence comparison with previously known K⁺ channel inhibitor scorpion toxins (KTx) and phylogenetic analysis revealed that Cm39 is a new member of α-KTx 4 family and registered with systematic number of α-KTx4.8. The full chemical synthesis and proper folding of Cm39 was obtained. The pharmacological properties of the synthetic peptide were determined using patch-clamp electrophysiology. Cm39 inhibits the voltage-gated K⁺ channel hKv1.2 with high affinity (K_d = 65 nM). The conductance-voltage relationship of Kv1.2 was not altered in the presence of Cm39, the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel, and therefore the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca²⁺-activated KCa2.2 and KCa3.1 channels, with K_d = 575 nM, and K_d = 59 nM, respectively, however, the peptide does not inhibit hKv1.1, hKv1.3, hKv1.4, hKv1.5, hKv1.6, hKv11.1, mKCa1.1 potassium channels or the hNav1.5 and hNav1.4 sodium channels at 1 µM concentration. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels [1].

References

[1] Naseem MU, Gurrola-Briones G, Romero-Imbachi MR, Borrego J, Carcamo-Noriega E, Beltrán-Vidal J, Zamudio FZ, Shakeel K, Possani LD and Panyi G, (2023) Toxins 15(1), p.41.

P49

A synthetic flavonoid derivate modulates the fluorescent signal of voltage-gated proton channels

Zoltán Pethő^1,2, Gilman E. S. Toombes, Dávid Pajtás¹, Martina Piga³, Zsuzsanna Magyar⁴, Nace Zidar³, György Panyi¹, Zoltán Varga¹ and Ferenc Papp¹

¹ Department of Biophysics and Cell Biology,Faculty of Medicine, University of Debrecen,

² Institut für Physiologie II, Münster, Germany

³ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana

⁴ Department of Physiology, Faculty of Medicine, University of Debrecen

The voltage-sensing domain (VSD) of voltage-gated proton channel (Hv1) serves as a pore for protons as well as a voltage sensor, which makes this channel unique among voltage-gated channels. Natural flavonoids, which are widely distributed and act as chemical messengers and physiological regulators in plants, modulate the function of some voltage-gated ion channels (EAG1, HCN2. etc.) in animal cells. We have designed synthetic flavonoid derivatives to inhibit the current of Hv1. We produced and tested tens of flavonoid derivatives on Ciona intestinalis Hv1 using the voltage-clamp fluorometry (VCF). The most potent compound, molecule #109, changed the originally negative VCF signal to positive and altered the biphasic VCF signal shape to monophasic. Also, this molecule caused a rightward shift in the conductance-voltage relationship in a concentration dependent manner. This flavonoid derivative quenched the TAMRA-MTS fluorescence in cuvette and on frog oocytes decreasing the baseline fluorescence, independently the oocyte expressed Hv1 or not. Furthermore, #109 could stain the membrane of HEK cells. These results indicate that #109 binds not directly to CiHv1 but to the cell membrane and in this way, it indirectly modifies the gating of Hv1 and the VCF signal, as a strong quenching molecule in close vicinity of the fluorophore. The latter was confirmed by our model calculations, in which we assumed that molecule #109, as a strong quencher, is embedded in the cell membrane close to TAMRA and during the conformational change due to depolarization, TAMRA is continuously moving away from this strong quencher molecule. Therefore, the VCF signal becomes a continuously increasing fluorescence change from the originally complex shape, which was overall a decreasing fluorescence change.

Acknowledgment

This work was supported by OTKA Bridging Fund 1G3DBKB0BFPF247 (FP); by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00355/21/8) (FP); by the ÚNKP-21-5-DE-460 New National Excellence Program of the Ministry for Innovation and Technology (FP); OTKA 132906 (ZV). This study was also funded by the Slovenian Research Agency (Grant No. P1-0208) (NZ).

P50

Remote detection of life through full-Stokes spectropolarimetry

C.H. Lucas Patty¹, Mathilda Fatton², Urs A. Schroffenegger¹, Saskia Bindschedler², Pilar Junier², Brice-Olivier Demory¹

¹Center for Space and Habitability (CSH), Universität Bern, Switzerland

²Institute of Biology, University of Neuchâtel, Switzerland

Homochirality is a generic and unique property of all biochemical life as we know it. There is a growing concensus that homochirality is a universal prerequisite of life and therefore as a biosignature free from the assumptions made by using terrestrial life as a benchmark [1]. Spectropolarimetry and, in particular, the detection of non-zero signatures in circular polarization is an indicator of the homochiral nature of molecular and supramolecular organic matter and is thus a direct and intuitive proxy for the remote detection of life using unpolarized incident light such as from the Sun [2]. We will describe the ongoing effort to characterize and quantify the nature of these chiroptical signals resulting from living organisms. While various studies have been performed to this end on eukaryotic photosynthetic organisms, in both the laboratory and in the field, including aerial observations (see e.g. [3][4]), relatively little systematic observations have been made of prokaryotic life. The results gathered so far on microbial mats and pure cultures show a remarkable variety in terms of both polarimetric spectral shape and magnitude [5]. Within the framework of the SenseLife project, we aim to further characterize and quantify the nature of these signals including the polarimetric signal response to external factors and physiology. We will further present novel results demonstrating the potential of characterization aerobiology using spectropolarimetry in the visible. Within this context we will describe the performance of FlyPol [2], a fast and sensitive spectropolarimetric instrument dedicated to the remote detection of linear and circular polarizance. In addition, we will present the results of ongoing aerial and field measurement campaigns probing the polarizance resulting from natural habitats, providing an outlook on the endeavor of measuring especially circular polarizance from space using Earth as a benchmark.

1. Glavin, D. P., Burton, A. S., Elsila, J. E., Aponte, J. C., & Dworkin, J. P. (2019). The search for chiral asymmetry as a potential biosignature in our solar system. Chemical reviews, 120(11), 4660-4689.

2. Patty, C. H. L., Ten Kate, I. L., Sparks, W. B., & Snik, F. (2018). Remote sensing of homochirality: a proxy for the detection of extraterrestrial life. In Chiral Analysis (pp. 29-69). Elsevier.

3. Patty, C. H. L., Pommerol, A., Kühn, J. G., Demory, B. O., & Thomas, N. (2022). Directional aspects of vegetation linear and circular polarization biosignatures. Astrobiology, 22(9), 1034-1046.

4. Patty, C. H. L., Kühn, J. G., Lambrev, P. H., Spadaccia, S., Hoeijmakers, H. J., Keller, C., ... & Demory, B. O. (2021). Biosignatures of the Earth-I. Airborne spectropolarimetric detection of photosynthetic life. Astronomy & Astrophysics, 651, A68.

5. Sparks, W. B., Parenteau, M. N., Blankenship, R. E., Germer, T. A., Patty, C. H. L., Bott, K. M., ... & Meadows, V. S. (2021).

P51

Assessing the effect of photobleaching on morphometric parameters of different cell types in the central nervous system

Tamás F. Polgár^1,2, Krisztina Spisák^1,2, Zalán Kádár¹, Nora Alodah³, László Siklós¹ and Roland Patai¹

¹ Institute of Biophysics, Biological Research Centre, Szeged, Hungary

² Theoretical Medicine Doctorate School, University of Szeged, Szeged, Hungary

³ College of Medicine, Alfaisal University, Kingdom of Saud Arabia

The sensitivity of immunofluorescence to the illumination time and intensity is well known; however, its effects on morphological parameters are less considered. Morphometric evaluation of different cell types could indicate the condition of the region of interest; for example, thickness and number of branches in microglia can imply their activation state.

Fluorescent detection using immunohistochemistry (IHC) targeting microglia/macrophages (fine structures) and neurons (bulk structures) was performed on neural tissues using different types of fluorophores while IHC with photostable diamonibenzidine was served as standard. Time series (0.5, 1, 2, 5, 10 and 15 minutes of illumination after taking the first image) were acquired from each spinal cord sections with the initial microscope settings. After standard mean intensity measurements, morphological parameters were extracted. Dynamic and relative total area microglial density were measured with a macro developed in our lab used routinely for quantifying glial activation. Fractal geometrical parameters were measured with the help of the Fiji plugin FracLac, and changes were examined between the initial image and the images containing faded structures.

Standard mean intensity measurements show different fading properties of different fluorophores. Dynamic and relative total area density comparisons show that in some cases all structures can fade to the point of becoming non-detectable after 2 minutes of illumination, while in some fractal geometrical parameters, more than 35 % differences can be observed.

Our results suggest that while fluorescent detection using IHC is an excellent method for localization and co-localization, for proper fine structure morphological measurements a photostable staining method is essential.

Acknowledgment

This work was partially supported by the Ministry for National Economy of Hungary through the GINOP-2.3.2-15-2016-00034 program. K.S. was supported from the UNKP-21-3-SZTE-73 New National Excellence Program of the Hungarian Ministry of Human Capacities. T.F.P. was supported by the EFOP 3.6.3-VEKOP-16-2017-00009 with the financial aid of the Ministry of Finance. R.P. was supported by the “National Talent Programme” with the financial aid of the Ministry of Human Capacities (NTP-NFTÖ-21-B-0203).

P52

Label-free immune cell analysis using optical biosensor

I. Sallai¹, Z. Szittner¹, Sz. Novák¹, I. Székács¹ and R. Horvath¹

¹ Nanobiosensorics Laboratory, Center of Energy Research, Eötvös Loránd Research Network, Budapest, Hungary

Understanding of activation processes at the single-cell level in response to different stimuli is essential for the diagnosis of certain diseases.

External stimuli induced differences are reflected in the dynamic changes of cell biophysical parameters, such as cell motility, shape, spreading and adhesion properties [1]. Novel highly sensitive optical biosensors allow the monitoring of changes in these parameters in a label-free manner. The advantage of label-free detection is that cells can be examined in an intact/organism-specific manner without modification.

The above parameters can be studied using microplate-based, high-throughput systems [2]. Surface functionalisation provide the opportunity to create an organism-specific environment [3]. In classical measurements, such as microscopy, dye-conjugated antibody labelling is essential and can be combined with label-free data [4].

Our aim is to interpret the activation mechanisms of various cell types and their function triggered by different stimuli and compare the results with conventional testing methods.

Acknowledgment

This work was supported by the National Research, Development, and Innovation Office (Grant Numbers: PD 134195 for Z.Sz, ELKH topic-fund, "Élvonal" KKP_19 TKP2022-EGA-04 grants).

References

[1] Z. Szittner, B. Péter, S. Kurunczi, I. Székács, and R. Horvath, “Functional blood cell analysis by label-free biosensors and single-cell technologies,” Advances in Colloid and Interface Science, vol. 308. Elsevier B.V., Oct. 01, 2022. doi: 10.1016/j.cis.2022.102727.

[2] M. Sztilkovics et al., “Single-cell adhesion force kinetics of cell populations from combined label free optical biosensor and robotic fluidic force microscopy,” Sci Rep, vol. 10, no. 1, Dec. 2020, doi: 10.1038/s41598-019-56898-7.

[3] N. Orgovan et al., “In-situ and label-free optical monitoring of the adhesion and spreading of primary monocytes isolated from human blood: Dependence on serum concentration levels,” Biosens Bioelectron, vol. 54, pp. 339–344, Apr. 2014, doi: 10.1016/j.bios.2013.10.076.

[4] S. Zheng, J. C. H. Lin, H. L. Kasdan, and Y. C. Tai, “Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples,” Sens Actuators B Chem, vol. 132, no. 2, pp. 558–567, Jun. 2008, doi: 10.1016/j.snb.2007.11.031.

P53

Effect of salt stress on etioplast and chloroplast membranes of thylakoid transporter mutants of Arabidopsis thaliana

Helga Fanni Schubert ¹, Adél Sóti ¹, Richard Hembrom ¹, Roumaissa Ounoki ¹, Enkhjin Enkhbileg¹, Emilija Dukic ², Cornelia Spetea ², and Katalin Solymosi ¹

¹ Eötvös Loránd University, Budapest

² University of Gothenburg, Gothenburg, Sweden

Soil salinity is an increasing problem for agriculture worldwide. Salinity has a complex effect on plants and influences the structure of plastids in different ways. Most often the effect of salt stress is studied in leaf chloroplasts, and in several cases swelling of the intrathylakoidal space of chloroplast inner membranes is reported under such conditions. However, it is yet unclear what causes the swelling of these membranes, and whether it has any relation to ion transport processes across these membranes. In this work, plastid ultrastructure was compared in the cotyledons and leaves of Arabidopsis thaliana plants of different developmental stages and grown under different light regimes under control conditions as well as under salt stress (30 min treatment with 200 or 300 mM NaCl or 600 mM NaCl:KCl, 1:1). In addition to the wild-type (WT) plants, we also analysed the thylakoid membrane structure and photosynthetic activity in single, double and triple mutants of the thylakoid-located voltage-gated chloride ion channel VCCN1, chloride ion channel CLCe and potassium proton exchanger KEA3. The above salt treatment did not affect the structure of the photosynthetic apparatus of mature chloroplasts in old leaves, however, it influenced the structure of chloroplasts in cotyledons in various ways, indicating the sensitivity of the young seedlings to the stress, and the potential presence of protective mechanisms that stabilize chloroplast structure at later developmental stages even under the above stress conditions. Salt stress also had an effect on the etioplasts of both WT and some mutant plants.

Acknowledgment

The work was funded by the grant OTKA FK124748, and supported by the ÚNKP-22-5 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund (to K.S.) and by the Bolyai János Research Scholarship of the Hungarian Academy of Sciences (to K.S.).

P54

Effect of rational modification of disordered domains of the epidermal growth factor receptor on its biophysical characteristics

Tímea Szatmári¹, Péter Nagy¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen

The epidermal growth factor receptor (EGFR) belongs to the ErbB receptor tyrosine kinase family. The extracellular domain of EGFR consists of four subdomains (I-IV). Upon ligand binding, the extracellular (EC) domain of the receptor is rearranged, resulting in exposition of the dimerization arm of subunit II providing an opportunity to form homodimers or heterodimers. The 3D structure determined by the amino acid sequence affects protein function. Proteins in which the formation of an ordered structure is not complete are called intrinsically disordered proteins. Recently, an algorithm (FuzPred) was developed that predicts the tendency of proteins to form disordered regions and their structural changes upon interacting with other proteins. The amino acid sequence of the EGFR was analyzed by FuzPred, which determines the tendency of distinct sequence regions to constitute globular (assuming ordered conformations), disordered (intrinsically disordered adopting ordered conformation upon binding), or fuzzy regions (remaining disordered even in their bound state). The following mutations were designed based on the software prediction that alter the fuzziness and the molecular interactions of EGFR domain II. 1. Mutation T274G is predicted to result in increased dynamics and destabilization of the dimerization site. It still forms a dimer, but the interaction is weak. 2. Mutation Q276F is expected to decrease dynamics of the dimerization arm and create a rigid binding site. As a result, it is unable to form a dimer due to lack of flexibility. 3. Mutation K284Q is expected to decrease dynamics while strengthening the binding site. This mutation promotes dimerization. We successfully generated all three mutants (dark and EGFP tagged on the C terminal of the proteins) by site-directed mutagenesis and stably transfected CHO cells expressing the EGFP-tagged mutant EGFRs. We investigated the ligand binding and the cooperativity of the wild-type EGFR and its mutant variants. CHO cells transfected with the wild-type or the mutant EGFRs were labeled with a concentration series of fluorescently labeled EGF (TAMRA-EGF). Our results suggest that those mutants that were predicted to be less prone to dimerization bind EGF less cooperatively and with a slightly lower affinity. In our further experiment we would like to examine the dimerization process (FRET, N&B) itself and consequent transmembrane signaling.

P55

Analysis of unilateral Walker A and A-loop mutants indicate that a single active catalytic site is sufficient to promote transport in ABCB1

Zsuzsanna Ritter^1,2, Szabolcs Tarapcsák¹, Zsuzsanna Gyöngy^1,2, Orsolya Bársony¹, Nimrah Ghaffar^1,2 Thomas Stockner³, Gergely Szakács⁴, and Katalin Goda¹

¹Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
²Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, Debrecen, Hungary
³Institute of Pharmacology, Center for Physiology and Pharmacology,
⁴Institute of Cancer Research, Medical University of Vienna, Vienna, Austria

The human ABCB1 is a full transporter with two nucleotide binding domains (NBDs) and two pseudo-symmetric transmembrane domains (TMDs). The two NBDs form two symmetrically arranged composite nucleotide binding sites (NBSs). Each NBS is formed by the A-loop, H-loop, Walker A, Walker B and Q-loop of one NBD, and the X-loop and signature sequence of the other NBD. The conserved tyrosine of the A-loop aligns the adenine ring of the bound ATP, contributing to nucleotide binding affinity through stacking interactions. The Walker A lysine interacts with the α and β phosphate of ATP. The two nucleotide binding domains were shown to be functionally equivalent, and the integrity of both catalytic centers is generally believed to be needed for transport. Consistently with the widely accepted models predicting that the two NBDs hydrolyze ATP in a strictly alternating order, unilateral mutation of these residues have been described to disrupt ATP hydrolysis and even affect ATP binding.

Here we demonstrate that while ABCB1 variants carrying bilateral A-loop or Walker A mutations are completely inactive, the unilateral exchange of the A-loop tyrosine to alanine or the unilateral mutation of the Walker A lysine to methionine is compatible with both ATP hydrolytic activity and transport function. Characterization of the single mutants revealed the significant (about 10-fold) reduction of the apparent ATP binding affinity compared to wild-type ABCB1. Stabilization of the post-hydrolytic complex by phosphate mimicking anions, such as vanadate or BeFx also occurred at higher ATP concentrations compared to wild-type, supporting that the mutated site probably has an effect on the overall conformation of the NBD dimer. Although the basal catalytic activity was strongly reduced in accordance with the decreased ATP binding affinity of the single mutants, the degree of ATPase stimulation by verapamil was almost identical to that of the wild-type, showing that drug-stimulation of the ATPase activity is preserved in the single mutants. Location of the mutation in the N or C terminal NBD did not affect the extent of ATPase stimulation by verapamil. Taken together, our data indicate that, in contrast to prevailing views, single-site NBD mutant ABCB1

P56

Evaluation of peptide carrier candidates using tissue barrier models

Júlia Tárnoki-Zách¹, Bence Stipsicz^2,3, Előd Méhes¹, Ildikó Szabó^2,4, Kata Horváti⁴, Bernadett Pályi⁵, Zoltán Kis⁵, Szilvia Bősze^3,5 András Czirók¹

1 Department of Biological Physics, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117, Budapest, Hungary

2 ELKH-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117, Budapest, Hungary

3 Doctoral School of Biology, Eötvös Loránd University, Pázmány Péter

sétány 1/C, H-1117, Budapest, Hungary

4 MTA-TTK Lendület Peptide-Based Vaccines Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest H-1117, Hungary.

5 National Public Health Center, Albert Flórián út 2-6, Budapest, 1097, Hungary

Targeting peptides represent a promising approach to improve uptake and efficiency of pharmacological compounds. In vitro barrier models are valuable screening tools to evaluate peptide transport, uptake and toxicity. Here we characterize a number of readily available lung and kidney epithelial cell lines in a transwell barrier model. After forming a monolayer and a subsequent maturation phase of cell-type specific duration, the epithelial cells develop tight junctions on the surface of polycarbonate inserts as evidenced by beta-catenin and ZO1 immunolabeling as well as delayed transport of targeting peptides. Daily monitoring of transepithelial electrical resistance (TEER) values reveal a cell line specific, characteristic time course. Thus, the TEER method with this calibration data offers a non-destructive and agent-free procedure to time pharmacological transport measurements, and to evaluate cytotoxicity of the transported agents. To characterize peptide targeting efficiency, a detector cell layer was cultured in the basolateral compartment of the barrier tissue model. Uptake of fluorescein-labelled peptides was evaluated by flow cytometry. We demonstrate that a derivative of the well-known peptide penetratin that also contains the neuropilin-binding sequence from tuftsin, a naturally occurring tetrapeptide produced by enzymatic cleavage from immunoglobulin G, is better able to pass through barrier layers that expresses its receptor, NRP-1.

P57

Molecular characterization of pathological and tissue-specific TRPM2 cation channel variants

Ádám V. Tóth¹, Ádám Bartók¹ and László Csanády¹

¹ Department of Biochemistry, Semmelweis University, Budapest, Hungary

TRPM2 is a temperature-sensitive, Ca2+-permeable, non-selective cation channel, showing high level of expression in cells of the central nervous system, bone marrow, granulocytes and pancreatic β-cells. Activation of the channel requires the simultaneous intracellular presence of adenosine diphosphate ribose (ADPR), Ca2+ ions and phosphatidylinositol 4,5-bisphosphate (PIP2). In these cells, TRPM2 contributes to Ca2+ influx resulting in important physiological and pathological functions, such as body temperature regulation, cytokine production, oxidative stress response, inflammation or controlled cell death. Moreover, certain TRPM2 point mutations show close genetic connection with bipolar disorder (D543E, R755C) [1] or amyotrophic lateral sclerosis and Parkinson's dementia (P1018L) [2]. Interestingly, alternative splice products were isolated from healthy neutrophil granulocytes (ΔC-TRPM2) [3] and from striatum (SSF-TRPM2) [4], which presumably modify the ligand specificity and function of the channel in a cell-specific manner.

Until now, the mentioned variants have only been investigated using fluorescent imaging techniques or whole-cell electrophysiological methods providing limited opportunities to study the ligands acting intracellularly. Our aim is to examine the listed ion channel variants in molecular details. To this end, expression vector encoding TRPM2 variants have been produced and expressed transiently in HEK cells. Functional measurements are performed by inside-out patch clamp configuration enabling reliable recordings of micro- and macroscopic currents and fast exchange of intracellular ligands. With our method, it is possible to map crucial biological and biophysical parameters of the channel variants: ADPR and Ca2+ sensitivity, gating parameters, inactivation kinetics, temperature dependence. This detailed knowledge is essential for a comprehensive understanding of the role of these mutants in pathomechanisms and tissue-specific variant functions.

References

[1] A. McQuillin et al. Mol Psychiatry 11, 134-142 (2006)

[2] M. C. Hermosura et al. Proc Natl Acad Sci USA 105, 18029-34 (2008)

[3] E. Wehage et al. J Biol Chem 277, 23150-6 (2002)

[4] T. Uemura et al. Biochem Biophys Res Commun 328, 1232-43 (2005)

P58

Substrate selectivity of Sulfotransferase Isoenzymes, results based on Molecular Dynamics and Ensemble Docking

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

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

Sulfotransferase enzymes (SULTs) are 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, their substrates vary considerably in size and complexity. The aim of our project is to better understand the reasons of selectivity between the different SULT isoenzymes, by comparing the broad targeting hepatic detoxifier SULT1A1, and the ileum located, dopamine selective SULT1A3.

Based on our previous results and Molecular Dynamics (MD) and Molecular Dynamics with excited Normal Modes (MDeNM), an extended conformational space of the PAPS-bound SULT1A1 was explored. Further developments of our method utilising ensemble docking with categorised ligands, a method known as Virtual Screening was achieved. Moreover, we have broadened our scope to use the same approach for the SULT1A3.

Based on our new results, we identified the key differences, that are responsible for changing the protein dynamics and binding mechanisms, 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 helpful in the future to develop an algorithm for machine learning, that could differentiate and even predict new substrates of the different isoforms, thus helping in the development of ADME-Tox profiling of novel drug candidates and xenobiotics.

Acknowledgment

TKP2021 EGA 23, Ministry for Innovation and Technology in Hungary,

2021-0143339, Ministére francais de l’Europe et des Affaires Etrangéres

Tét-Balaton project „drug-drug interactions of sulfotransferases” ( 2019-2.1.11-TÉT-2020-00096 )

P59

Examinations of cellular uptake of cell penetrating peptides in vitro and in vivo

Gabriella Tóth¹, Gyula Batta^1,2, Levente Kárpáti³, Árpád Szöőr¹, István Mándity³, Péter Nagy¹

¹University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

²University of Debrecen, Faculty of Science and Technology, Institute of Biotechnology, Department of Genetics and Applied Microbiology

³Semmelweis University, Faculty of Pharmacy, Institute of Organic Chemistry

Cell-penetrating peptides (CPPs) are peptides that enter cells by endocytosis and/or directly through the cell membrane. CPPs in general have been considered potential carriers of molecules that have difficulties entering cells. This is the feature that we would like to exploit and thereby establishing the opportunity for CPPs to have therapeutic applications in the long term. Our previously published results have shown that we can increase the cellular uptake and endosomal release of CPPs with statins. Our goal was to modify them and test if it is possible to make them enter the cells more efficiently. We also aimed to test the biodistribution of CPPs in mice after intravenous administration. We examined the cellular uptake and endosomal release by flow cytometry and confocal microscopy in SKBR-3 and MDA-MB-231 cell lines, while for the in vivo experiments a mouse model was applied. Fluorescently-labeled CPPs were used both in the in vivo and in vitro experiments. We compared the differences in the biophysical properties of the original and the modified CPPs, and we found that the cellular uptake of the modified version is more effective. There is a difference between the enhancement in the uptake of CPPs labeled by the pH-sensitive naphthofluorescein or Alexa Fluor 532. In the case of in vivo experiments, we found that peptides enter the mouse organs, including the liver, for which we have shown that CPPs is present in the intracellular space of hepatocytes. CPPs hold promise for increasing the efficiency and specificity of drug delivery to cells.