Összes szerző

Varga Zoltán

az alábbi absztraktok szerzői között szerepel:

Biró-Gyuris Katinka
Role of the hHv1 proton channel in vascular smooth muscle cells

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

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.

Bóta Attila
Nanoerythrosome-based promising drug delivery system

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

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.

Domingos Geraldo Jorge
Identification of inhibitors of the human hv1 proton channel

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

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.

Farkasné Bebesi Tímea
Spectroscopic study of extracellular vesicles using plasmonic gold nanoparticles

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

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.

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

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

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).

Tasvilla Sonallya
Systematic investigation and classification of host defence and cell penetrating peptides based on their affinity for interaction with extracellular vesicles

Aug 30 - szerda

09:55 – 10:10

Membránok és membránfehérjék biofizikája

E24

Systematic investigation and classification of host defence and cell penetrating peptides based on their affinity for interaction with extracellular vesicles.

Tasvilla Sonallya^1,2, Imola Cs. Szigyártó¹ , Tünde Juhász¹, Edit I. Buzas^4,5,6 Delaram Khamari ⁴,Kinga Ilyes^2,3 Zoltán Varga³, and Tamás Beke-Somfai^1*

¹Institute of Materials and Environmental Chemistry, Biomolecular Self-assembly Research Group, Research Centre for Natural Sciences, Budapest H-1117, Magyar tudósok körútja 2, Hungary,

²Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest H-1117, Pázmány Péter sétány 1/A, Hungary.

³Institute of Materials and Environmental Chemistry, Biological Nanochemistry Research Group, Research Centre for Natural Sciences, Budapest, H-1117, Magyar tudósok körútja 2, Hungary.

⁴Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary

⁵HCEMM Extracellular Vesicle Research Group, Semmelweis University, Budapest, Hungary

⁶ELKH-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary.

Host defence peptides (HDP) are promising biomaterials with antimicrobial and anticancer applications. By disturbing or lysing the cell membrane, they carry out their biological role. These peptides show numerous types of membrane interaction mechanisms i.e., carpet, toroidal pore, and barrel stave. Cell penetrating peptide find application in cargo loading and uptake of small molecules and nanoparticles. The interactive mechanism of these peptides has been studied widely with model membranes however our knowledge with extracellular vesicles (EV) is scarce. There are several aspects where EV – HDP interactions could be relevant, ranging from cooperative presence on infection sites functions to EV cargo loading. Hence, based on their in-depth investigation using biophysical techniques, the binding affinity with extracellular vesicles was studied and categorised as low binding affinity, medium binding affinity and high affinity. This initial categorisation gives further insight into its specific interactive mechanism.

Acknowledgment:

This work was funded by the Ministry of Innovation and Technology of Hungary through the National Research, Development and Innovation Office, financed under the TKP2021-EGA-31, the 2020-1-1-2-PIACI-KFI_2020-00021, 2019-2.1.11-TÉT-2019-00091 and KKP_22 project no. 144180. Support from Eötvös Loránd Research Network, grant no. SA-87/2021 and KEP-5/2021 is also acknowledged. Z.V. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

References

1. Singh, P. et al. Removal and identification of external protein corona members from RBC‐derived extracellular vesicles by surface manipulating antimicrobial peptides. J. Extracell. Biol. 2, (2023).

2. Singh, P. et al. Membrane Active Peptides Remove Surface Adsorbed Protein Corona From Extracellular Vesicles of Red Blood Cells. Front. Chem. 8, (2020).

Varga Zoltán
Hollow Organosilica Beads: A Novel Reference Material for the Flow Cytometry Analysis of Extracellular Vesicles

Aug 30 - szerda

14:36 – 14:54

Sejtanalitika biofizikai megközelítéssel

E34

Hollow Organosilica Beads: A Novel Reference Material for the Flow Cytometry Analysis of Extracellular Vesicles

Anikó Gaál, Zoltán Varga

¹ Biological Nanochemistry Research Group, Research Centre for Natural Sciences

Extracellular vesicles (EVs) are increasingly recognized as key biomarkers with significant potential in diagnostic and therapeutic applications. Their precise concentration measurement in body fluids is crucial, for which flow cytometry is currently the clinically most applicable method. This technique often uses solid polystyrene reference beads for calibration, but the disparity in refractive indices between these beads and EVs can distort accurate size determination. In response to this challenge, this study aims to prepare, characterize, and test hollow organosilica beads (HOBs) with different diameters as reference beads to set EV size gates in flow cytometry investigations.

HOBs were produced through the hard template sol-gel method, followed by a thorough analysis of their morphology, size, and colloidal stability. Their suitability as reference particles was then examined using flow cytometry. Findings indicated that HOBs exhibit a uniform size distribution and shell thickness. Further, two-angle light scattering measurements showed that HOBs scatter far less light than comparable solid silica beads due to differences in refractive indices. The scattering intensity of HOBs aligns with that expected from EVs of similar sizes.

Moreover, the study demonstrated that HOBs can be used to standardize EV concentration measurements, independent of the light scattering collection angles of the flow cytometer. Concentration measurements of platelet-derived EVs, conducted using size gates established by HOBs, showed the smallest percentage difference relative to the mean concentration, pointing to HOBs' superiority over solid beads.

In conclusion, HOBs, due to their similar structure and light scattering properties to EVs, can set size gates in nanometers, regardless of the flow cytometer's optical configuration.

Varga Zoltán
In vivo Biodistribution of Extracellular Vesicles: Developing Efficient Radiolabeling Techniques

Aug 29 - kedd

14:00 – 14:20

Orvosi biofizika és sugárbiológia

E14

In vivo Biodistribution of Extracellular Vesicles: Developing Efficient Radiolabeling Techniques

Zoltán Varga¹, Kinga Ilyés¹, Dávid Szöllősi², Ildikó Horváth², Domokos Máthé^2,3, Krisztina Németh^4,5, Viola Tamási^4,*, Edit I Buzás^4,5,6, Krisztián Szigeti²

¹ Biological Nanochemistry Research Group, Research Centre for Natural Sciences

² Department of Biophysics and Radiation Biology, Semmelweis University

³ HCMM-SE In Vivo Imaging Advanced Core Facility

⁴ Department of Genetics, Cell- and Immunobiology, Semmelweis University

⁵ ELKH-SE Translational Extracellular Vesicle Research Group

⁶ HCMM-SE Extracellular Vesicle Research Group

^*current affiliation: Department of Molecular Biology, Semmelweis University

The understanding of extracellular vesicle (EV) biodistribution plays an important role in advancing circulating biomarker research. Nuclear imaging techniques like single-photon emission computed tomography (SPECT) hold potential, but the literature on radioisotope labeling of EVs for in vivo studies remains scarce. This presentation explores the evolution of novel radiolabeling methods, focusing on the development and comparative evaluation of various Tc99m radiolabeling strategies.

Our initial method involved the radioisotope labeling of erythrocyte-derived EVs using the Tc99m-tricarbonyl complex [1]. In vivo SPECT/CT biodistribution studies in mice showed that intravenously administered Tc99m-labeled EVs primarily accumulated in the liver and spleen. Our observations suggested good in vivo stability, with a minor fraction of the radioactive label detaching from the EVs. Next, we explored an alternative approach using Tc99m-HYNIC-Duramycin to label cell-derived EVs [2]. Duramycin, a membrane-active peptide, specifically labels EVs, resulting in higher labeling efficiency. Following previous observations, significant uptake of EVs in the liver and the spleen was observed.

The latest experimental focus is on the use of recombinant proteins with His-tag in conjunction with the Tc99m-tricarbonyl complex to label EVs. Preliminary investigations indicate promising superior performance with this method compared to the previous techniques.

This presentation will provide an in-depth comparison of these methods, emphasizing their development process and potential implications for advancing in vivo EV imaging studies. Through a critical evaluation of their advantages and potential limitations, we aim to foster a greater understanding of efficient tracking of EV biodistribution for future research.

References

[1] Varga Z et al. (2016) Cancer Biother. Radiopharm. 31: 168-173.

[2] Németh K et al. (2021) Cell Mol Life Sci 78: 7589–7604