Book of Abstracts

Schabussova Irma
Institute of Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna,
*irma.schabussova@meduniwien.ac.at

Bacterial extracellular vesicles (bEVs) are increasingly recognised as central players in cross-talk between the microbiota and the mammalian immune system. In my talk, I will highlight how EVs from probiotic bacteria integrate environmental cues and deliver complex molecular cargo to shape mucosal immune responses.

Using the Gram-negative probiotic Escherichia coli O83 (EcO83) as a model, we showed that its outer membrane vesicles (OMVs) are nanosized, protein- and LPS-rich particles that activate multiple pattern-recognition receptors (TLR2/4/5, NOD1/2), induce both pro- and anti-inflammatory cytokines in innate immune cells, and, when administered intranasally, prevent experimental allergic airway inflammation in mice by reducing airway hyperresponsiveness, eosinophilia and Th2 cytokines.

In follow-up work, we performed in-depth characterization of EcO83-EVs according to MISEV guidelines and dissected their interaction with the respiratory tract. EcO83-EVs modulated the proteome of primary human nasal epithelial cells, upregulating pathways linked to oxidative stress and inflammatory responses, and in vivo they targeted nasal-associated lymphoid tissue, were internalised by airway macrophages, recruited neutrophils to the lung, and activated NF-κB–dependent nitric oxide production. These data support EcO83-EVs as a rational postbiotic platform for mucosal immunomodulation.

Complementing this Gram-negative model, our recent multi-platform analysis of EVs from the Gram-positive probiotic Lactiplantibacillus plantarum demonstrates that environmental stressors dynamically reprogram EV biogenesis and cargo. Condition-specific EV signatures captured by nanoparticle analysis, spectroscopy and omics approaches reveal that EVs act as sensitive indicators of bacterial stress responses while concurrently exporting metabolites and proteins with potential immunomodulatory functions.

Together, these studies position bacterial EVs as both sensors and effectors at the host–microbe interface, with immediate implications for the development of safe intranasal postbiotics, next-generation mucosal vaccines and EV-based biomarkers of microbiota–host interactions.

Michael Rappolt*
School of Food Science and Nutrition, University of Leeds, Woodhouse Lane, United Kingdom
*m.rappolt@leeds.ac.uk

After an introduction into the history of membrane model fitting, applying both small angle X-ray and neutron scattering, I will focus on the interplay between the lipid bilayer matrix and interfacial water. Despite extensive knowledge on membrane nanostructure and its mechanical properties under various environmental conditions, there is still some lack of understanding on the role membrane hydration. In my presentation, the nature of the confined water between two adjacent bilayers is described in the light of the recently proposed Three-Water Layer model (TWL) (1), discerning (i) ‘headgroup water’, (ii) ‘perturbed water’ near the membrane/water interface, and (iii) a core region of ‘free’, unperturbed water. The interaction of water with lipid leaflets in all three regions is examined in relation to factors such as head-group type and orientation, membrane rigidity, temperature, ion concentration, and membrane curvature. The model will be presented for both, lamellar phases and inverse hexagonal phases. Multilamellar liposomes have been studied under the influence of cholesterol concentration and the addition of different chloride salts (Na+, K+, Li+, Ca2+ and Mg2+), and the hydration of the inverse hexagonal phase was studied under different conditions of curvature frustration (2) as well as for testing best hydrational conditions for mRNA delivery (3). Finally, the TWL model is discussed in the context of spectroscopic observations of water within soft confinement, and I will point out how varying water dynamics affect the interacting forces between adjacent membranes.

References:
(1) Vancuylenberg, G., Sadeghpour, A., Tyler, A.I.I., Rappolt, M. Soft Matter, 19: 5179 (2023).
(2) Vancuylenberg, G., Sadeghpour, A., Tyler, A.I.I., Rappolt, M. Soft Matter, 19: 8519 (2023).
(3) Philipp, J., Sudarsan, A., Kostyurina, E., Meklesh, V., Berglund, M., Rappolt, M., Westergren, J., Lindfors, L., Schwierz, N., Rädler, J.O. Soft Matter, 21: 8049 (2025).

Böde K.*, University of Ostrava, Czech Republic
Dlouhý O., University of Ostrava, Czech Republic
Karlický V., University of Ostrava, Czech Republic
Špunda V., University of Ostrava, Czech Republic
Garab G., University of Ostrava, Czech Republic / Biological Research Centre, HUN-REN, Szeged, Hungary

*kinga.bode@osu.cz

Non-bilayer lipids are essential constituents of all biological energy-converting membranes, including the inner mitochondrial membranes (IMMs) and plant thylakoid membranes (TMs) (1). These lipids promote structural plasticity to the membrane organization through the formation of non-bilayer lipid phases, however, their functional significance is far from being understood.

In plant TMs, the major lipid species, monogalactosyldiacylglycerol (MGDG), is a non-bilayer lipid. It has been broadly documented, mainly by ³¹P-NMR spectroscopy, that all distinct structural domains of TMs show pronounced lipid polymorphism (2): non-bilayer lipid phases are present in the granum, the stroma lamellae and the highly curved marginal regions. The consistent presence suggests that this feature must be a fundamental organizing principle that might have profound effect on the overall functioning of these membranes.

Over the past years, functional roles have been proposed for specific non-bilayer phases: the H_II phase has been associated with lipids interacting with stromal-side proteins, while isotropic lipid phases have been implicated in the regulation of violaxanthin de-epoxidase activity. Using BBY membrane sheets, we have further shown that an isotropic lipid phase, with well discernible physico-chemical features, plays key role in the purely lipid-mediated membrane fusion (3-4). Recent molecular dynamics simulations have also demonstrated that MGDG enhances membrane dynamics, induces fluctuations in membrane thickness, and promotes the spontaneous formation of non-bilayer structures in stacked membrane systems (5). These simulations also indicate that the stability of such phases depends strongly on both the MGDG concentration and membrane hydration state.

Together, experimental observations and computational insights suggest that lipid polymorphism and hydration states collectively shape the structural and energetic landscape of TMs, providing a plausible physical basis for efficient and spatially organized photosynthetic energy conversion.

References:

1. G. Garab et al., Structural and functional roles of non-bilayer lipid phases of chloroplast thylakoid membranes and mitochondrial inner membranes. Progress in Lipid Research 86, 101163 (2022).
2. G. Garab et al., Lipid polymorphism of plant thylakoid membranes. The dynamic exchange model – facts and hypotheses. Physiologia Plantarum 177, e70230 (2025).
3. K. Böde et al., Role of isotropic lipid phase in the fusion of photosystem II membranes. Photosynthesis Research 161, 127-140 (2024).
4. K. Böde et al., Lipid phase behaviour of the curvature region of thylakoid membranes of Spinacia oleracea. Physiologia Plantarum 177, e70289 (2025).
5. B. Fehér et al., Molecular level insight into non-bilayer structure formation in thylakoid membranes: a molecular dynamics study. Photosynthesis Research 163:36 (2025).

This study reveals a fundamental constraint on biomembrane structure: the spontaneous curvature of biomembrane leaflets limits the size of hydrophobic inclusions. We demonstrate in experimental observations and theoretical model that inclusions exceeding a critical size are spontaneously expelled from the membrane, self-assembling into distinct micellar structures. Our analysis reveals that this critical size is predominantly determined by the monolayer’s spontaneous curvature, exhibiting only a weak dependence on its bending rigidity and compression-expansion moduli. Specifically, we find that the small positive curvatures characteristic of the plasma membrane, largely due to an abundance of phosphatidylcholine, favor the incorporation of nanoinclusions with sizes comparable to the bilayer thickness. These findings elucidate a novel, purely physical mechanism for guiding membrane trafficking. The intrinsic curvature mismatch between membrane leaflets is sufficient to power both the engulfment of nanoinclusions and subsequent vesicle scission, obviating the need for ATP or other metabolic energy.

Osella, S. – Centre of New Technologies, Warsaw, Poland
Bacalum, M. – Horia Hulubei National Institute Physics and Nuclear Engineering, Romania
Aisenbrey, C.; De, K.; Bechinger, B. – Institut de Chimie de Strasbourg, France
Ameloot, M. – Biomedical Research Institute, Hasselt University, Belgium
Knippenberg, S.* – Theory Lab, Hasselt University, Belgium
* stefan.knippenberg@uhasselt.be

Light is a powerful tool to probe the structure and dynamics of biomolecules and biological systems. In most cases, this cannot be done directly with visible light because of the absence of absorption by those biomolecules. In this talk, organic fluorophores are embedded in lipid bilayers and are described by a multiscale computational approach. Combining different length and time scales, a full description of the probe might lead to novel insight into the effect of the environments.
A study on the Laurdan probe is presented, sketching how a multiscale approach based on extended molecular dynamics and hybrid quantum mechanics-molecular mechanics frameworks can predict probes’ fluorescence properties, including spectra, lifetime and time resolved anisotropy. We show not only how computer simulations can explain particular confocal experiments by analysing the localization and orientation of probes in different membrane phases, but also how computation can uncover novel functionalities of well-used probes.
Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. We present the first direct measurements of the two conformers which followed upon our theoretical prediction.
Molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for different lipid bilayer membranes at various temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. It is seen that for Conf-I embedded in a DPPC membrane, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the corresponding surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. We conclude that multiscale modelling can assess a priori novel probes’ optical properties and guide the analysis and interpretation of experimental data.

Osella, S.; Murugan, N. A.; Jena, N. K.; Knippenberg, S. J. Chem. Theory Comput. 12 (2016), 6169.
Osella, S.; Smisdom, N.; Ameloot, M.; Knippenberg, S. Langmuir, 35 (2019), 11471.
Osella, S.; Knippenberg, S. ACS Appl. Bio. Mater. 2 (2019), 5769.
Osella, S.; Knippenberg, S. BBA-Biomembranes 1863 (2021), 183494.
Bacalum, M.; Radu, M.; Osella, S.; Knippenberg, S.; Ameloot, M. J. Photochem. Photobiol. B 250 (2024), 112833.
Knippenberg, S.; De, K.; Aisenbrey, C.; Bechinger, B.; Osella, S. Cells 13 (2024), 1232.
Osella, S.; Knippenberg, S. Acc. Chem. Res. 57 (2024), 2245.

Kralj S. *, Jožef Stefan Institute, Condensed Matter Physics Department, 1000 Ljubljana, Slovenia
Kralj-Iglič V., University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, 1000 Ljubljana, Slovenia
Iglič A., University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, 1000 Ljubljana, Slovenia
*samo.kralj@ijs.si

We consider domain-type patterns in biological membranes that possess an in-plane membrane order. Domains are inseparable linked to topological defects and many features related to them could be guessed based on universal topological arguments. However, much more complex membrane patterns are typically observed. As possible generators of such configurations we analyze two relatively simple and universal phenomena. Both are based on continuous symmetry breaking (CSB) that manifests ubiquitously in all branches of physics. Firstly, we present the Imry-Ma argument (1) which in addition to CSB requests presence of uncorrelated random-field-type disorder. Next, we discuss the Kibble-Zurek mechanism (2). In addition to CSB it considers dynamical slowing down on crossing a relevant phase transition. These approaches were originally introduced in magnetism (1) and cosmology (3), respectively. We adapt them to effectively two-dimensional membranes and discuss their potential role in membrane structure formation.

References:
(1) Imry Y., and Ma S.K., Random-field instability of the ordered state of continuous symmetry. Phys. Rev. Lett., 35, 1399 (1975).
(2) Zurek W.H., Cosmological experiments in superfluid helium? Nature, 317, 505 (1985).
(3) Kibble T.W.B., Topology of cosmic domains and strings, J. Phys. A: Math. Gen., 9, 1387 (1976).

Mesarec L.*, Faculty of Electrical Engineering, University of Ljubljana, Slovenia
Kralj-Iglič V., Faculty of Health Sciences, University of Ljubljana, Slovenia
Kralj S., Faculty of Natural Sciences and Mathematics, University of Maribor, Slovenia
Iglič A., Faculty of Electrical Engineering, University of Ljubljana, Slovenia
* luka.mesarec@fe.uni-lj.si

In this contribution, we investigate the nematic orientational ordering of curved, flexible nematic molecules on 2D shells. The examples of such molecules in nature are for example liquid crystal molecules and different proteins in biological membranes. In our modeling, we use a mesoscopic Helfrich–Landau–de Gennes-type approach, in which the curvature of the flexible 2D shell and the nematic ordering field are mutually coupled and simultaneously determined through free energy minimization. When the nematic molecules are distributed across the entire 2D surface of the shell, topological defects are always present on shells with spherical topology. However, if the concentration of curved molecules is reduced, it becomes possible for the molecules to arrange themselves on the surface in a way that avoids topological defects, as they can shift away from the regions where such defects would typically occur. In this study, we consider different concentrations of rod-like molecules on 2D surfaces, leading to new types of equilibrium 2D shell shapes and nematic configurations on these shells.

L. Corne, Université de Lyon, ENS de Lyon, France
A. Vagias, Institut Laue Langevin, France
B. Demé, Institut Laue Langevin, France
P. Gutfreund, Institut Laue Langevin, France
N. Paracini, Institut Laue Langevin, France
P.T. de Souza, Molecular Microbiology and Structural Biochemistry, France
J. Peters, University Grenoble Alpes, France
C.M. Marques, Université de Lyon, ENS de Lyon, France

* jpeters@ill.fr

Plastic pollution is widely recognized for its visible impact on oceans and wildlife, but less attention is given to the molecular byproducts that arise as plastics degrade (1). Among these, styrene oligomers (SOs)—small molecules released from polystyrene—may pose hidden risks to living systems, including under the high pressures of deep ocean environments where plastic degradation products have been detected (2). We investigated how SOs interact with model cell membranes composed of dipalmitoylphosphatidylcholine (DPPC). Using neutron reflectivity, neutron diffraction, and molecular dynamics simulations at both ambient and elevated pressures, we found that SOs disrupt the structural organization of lipid bilayers and weaken the membrane’s fluid-to-gel phase transition. These results highlight how plastic-derived molecules can subtly compromise biomembrane stability and function, even under extreme conditions

References:
(1) Morandi, M.I., Kluzek, M., Wolff, J., Schroder, A., Thalmann, F. and Marques, C.M., 2021. Accumulation of styrene oligomers alters lipid membrane phase order and miscibility. PNAS, 118(4), p.e2016037118.
(2) Worm, B., Lotze, H.K., Jubinville, I., Wilcox, C. and Jambeck, J., 2017. Plastic as a persistent marine pollutant. Annual Review of Environment and Resources, 42, pp.1-26.

Magdalena Przybyło*, Lipid Systems sp. z o.o., Wroclaw University of Science and Technology
Langner M. Wrocław University of Science and Technology, Poland

*magda.przybylo@lipid-systems.pl

Liposomes are used as models of biological membranes and delivery vehicle of bioactives to targeted body compartments for some time now. The main difficulty associated with application of liposomes is the low encapsulation efficiency of hydrophilic substances, especially macromolecules and biosimilars. The introduction of mRNA-based pharmaceuticals necessitated application of supramolecular aggregates to ensure mRNA stability, both in storage and after application. To surround mRNA with lipid protective layer the electrostatic interaction between negatively charged nucleic acid and permanent or ionizable cationic lipids were used. However, the resulting topology resample emulsion where polar nucleic acid is immersed in the hydrophobic core of particulate whereas polar lipids form the interface between hydrophobic core and an aqueous phase. At the same time the new liposome formation method, characterized by the high encapsulation efficiency of hydrophilic substance including macromolecules, have been developed. The method is based on careful control of water activity in nonpolar lipid-containing solution when mixing with aqueous phase containing hydrophilic compounds. As a result of the process the viscous liposome gel is formed. The new formation procedure can be used to produce liposomes containing in their inner aqueous phase large quantities of macromolecules such as nucleic acids. The main advantage of the method is its potential for the upscaling up to tones enabling large scale production of sophisticated food and pharmaceutical products. The other major advantage is the possibility of the construction of lipid capsules filled with intact macromolecules without the need for application strong electrostatic interactions.

Bartosz Różycki*, Institute of Physics, Polish Academy of Sciences, Poland
*rozycki@ifpan.edu.pl

Molecular dynamics (MD) simulations provide methods to study the structure, dynamics and functions of biomolecules. Although MD simulations with all-atom force fields are used most commonly, their practical application often is limited due to issues with insufficient simulation time and sampling. These limitations can be overcome by appropreate coarse-grained methods that capture the essential physics of the system under study. In my talk I will discuss applications of various coarse-grained approaches to study biomembranes, proteins and biomolecular condensates. Recently, we used MD simulations with the Martini coarse-grained force field to study lipid transport proteins in contact with biomembranes [1,2] as well as mixed-folded proteins such as galectin-3 in solution [3]. We employed MD simulations of a structure-based one-bead-per-residue model to investigate how off-rates of T-cell receptor/peptide-MHC complexes are affected by the peptide [4]. By using the maximum entropy method, we combined Monte Carlo (MC) simulations of a one-bead-per-residue protein model with data from biophysical experiments (SAXS, PRE, NMR) to delineate conformational ensembles of mixed-folded proteins such as human ataxin-3 [5] and SARS-CoV-2 nucleocapsid protein [6]. We employed dissipative particle dynamics (DPD) simulations to study lipid droplets [7] as well as biomolecular condensates of intrinsically disordered proteins interacting with lipid membranes [8]. We used MC simulations of a lattice-based mesoscale model for membrane adhesion to explore such processes as condensation of cell membrane receptors [9,10] and indirect membrane-mediated interactions between CD47-SIRPα complexes [11,12]. Our synergetic use of coarse-grained MD and mesoscale kinetic MC simulations allowed us to explore both equilibrium properties and dynamical behavior of adhering membranes on length scales between 1 nm and 1 μm on time scales ranging from 0.1 ns all the way up to about 20 s [12].

References:
[1] A. Ballekova, A. Eisenreichova, B. Różycki, E. Boura, J. Humpolickova. Commun. Biol. 7, 1585 (2024).
[2] A. Eisenreichova, M. Klima, M. M. Anila, A. Koukalova, J. Humpolickova, B. Różycki, E. Boura. Cells 12, 1974 (2023).
[3] M. M. Anila, P. Rogowski, B. Różycki. Molecules 29, 2768 (2024).
[4] J. Pettmann, L. Awada, B. Różycki, et al. EMBO J. 42, e111841 (2023).
[5] A. Sicorello, B. Różycki, P. V. Konarev, D. I. Svergun, A. Pastore. Structure 29, 70-81 (2021).
[6] B. Różycki, E. Boura. Biophys. Chem. 288, 106843 (2022).
[7] F. Kazemisabet, A. Bahrami, R. Ghosh, B. Różycki, A. H. Bahrami. Soft Matter 20, 909-922 (2024).
[8] M. M. Anila, R. Ghosh, B. Różycki. Soft Matter 19, 3723-3732 (2023).
[9] L. Li, J. Hu, B. Różycki, F. Song. Nano Lett. 20, 722-728 (2020).
[10] L. Li, R. Hou, X. Shi, J. Ji, B. Różycki, J. Hu, F. Song. Commun. Phys. 7, 174 (2024).
[11] L. Li, C. Gui, J. Hu, B. Różycki. Membranes 13, 871 (2023).
[12] R. Hou, S. Ren, R. Wang, B. Różycki, J. Hu. J. Chem. Theory Comput. 21, 2030-2042 (2025).

Bar L.1,2, Lavrič M.2, Iglič A.1, P. Losada-Pérez3, Daniel M.4, Cordoyiannis G.1*
1Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia
2Condensed Matter Physics Department, Jožef Stefan Institute, Ljubljana, Slovenia
3Experimental Soft Matter and Thermal Physics (EST) Group, Department of Physics, Université Libre de Bruxelles, Brussels, Belgium
4Faculty of Mechanical Engineering, Czech Technical University in Prague, Prague, Czech Republic
*georgios.kordogiannis@ijs.si

Quartz crystal microbalance with dissipation monitoring (QCM-D) is a label-free, surface-sensitive technique widely used to probe molecular events in real-time. We have employed QCM-D to investigate phase transitions in supported lipid vesicles (SLVs). Changes in the phase transition signatures have, in turn, been used to describe a wide range of phenomena (1). Two illustrative examples will be presented.
In the first case, we report on the impact of solid substrate on the lipid phase behavior. The melting of DMPC and DPPC SLVs exhibits a strong dependence on the type of solid substrate, Au, SiO2, or TiO2, as previously indicated (2) yet not explored in detail. Moreover, the pre-transition (i.e. the transition between the gel and the ripple phases) is observed for the first time by QCM-D in the case of DMPC (3).
In the second case, nanoparticle-lipid membrane interactions are probed through analysis of the phase transitions’ signal. Variable strength of interactions is reflected on the phase transition temperature and cooperativity, as well as the appearance of the ripple phase (4).

References:
(1) Cordoyiannis G., Bar L., Losada-Pérez P., Advances in Biomembranes and Lipid Self-Assembly, 34, 107 (2021).
(2) Bibissidis N., Betlem K., Cordoyiannis G., Prista-von Bonhorst F., Goole J., Raval J., Daniel M., Góźdź W., Iglič A., Losada-Pérez P., Journal of Molecular Liquids, 320, 114492 (2020).
(3) Bar L., Lavrič Marta, Iglič A., Cordoyiannis G., unpublished results.
(4) Daniel M., Mendová K., Bar L., Cordoyiannis G., Iglič A., Kralj-Iglič V., unpublished results.

Čechová Petra*, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Paloncýová Markéta, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Šrejber Martin, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Otyepka Michal, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic; IT4Innovations, VŠB – Technical University of Ostrava, Czech Republic
*petra.cechova@upol.cz

Biological membranes play a crucial role in the ability of organisms to maintain cellular homeostasis in various environments, making use of the great chemical variability of lipids species available (1). However, this incredible diversity in membrane composition poses a great challenge when creating computational membrane models for the use in molecular dynamics (2).

A critical step in any biomolecular simulation study is the selection of an appropriate model. On one hand, the model must sufficiently represent the essential features of the biological system; on the other, it must remain manageable with current computational resources and tools. This modelling decision directly impacts the predictive accuracy and relevance of the simulation, making it a crucial yet often underappreciated aspect of study design. Despite its importance, there is a surprising lack of systematic guidelines or best practices for model selection in the literature. Furthermore, many studies of protein-in-a-membrane systems still use a single-lipid (POPC) bilayer, without addressing the experimental composition of the respective membrane.

To evaluate the effect of the lipid composition and membrane complexity on its properties, we constructed a series of membrane models – three complex mixes mimicking plasma membrane composition and four simple model membranes. To observe the effects of membrane composition on a transmembrane protein we used the transmembrane portion of the toll-like receptor (TLR) protein 2 (3).

The main difference between the systems is related to their internal arrangement i.e. the ordered or disordered phases, rather than fine details of composition of the models. The secondary and tertiary structure of the TLR2 fragment also follow this dependence on the membrane phase. Therefore, we conclude that the presence of cholesterol and subsequent phase behaviour has a major influence on the system behaviour and is absolutely necessary when setting up systems containing plasma membrane proteins, in contrast to a lesser role of precise composition.

References:
(1) Sarmento et al., Cellular Molecular Life Sciences, 80, 237 (2023)
(2) Lorent et al., Nature Chemical Biology, 16, 644–652 (2020)
(3) Kornilov et al., Nature Communications, 14, 1503, (2023)

Shubhadeep Sadhukhan, Weizmann Institute of Science, Israel
Nir S. Gov*, Weizmann Institute of Science, Israel; University of Cambridge, U.K.
*nir.gov@weizmann.ac.il

Phagocytosis is an essential process for maintaining our life, as it allows cells (mostly specialized immune cells) to engulf objects that are harmful to our body, such as bacteria and dead cells. We extended our model of “mini-cell” membrane shape, which is driven by curved active membrane proteins [1], to allow it to interact with another vesicle that represents the passive engulfed object. We find that as the engulfed object is made softer the dynamics of this process changes: a rigid object is fully engulfed, while a softer object is only pushed away. We find that for very soft objects the mini-cell is able to engulf a small portion, which corresponds to “biting” a small piece. These processes are observed in experiments of cells engulfing artificial vesicles of different membrane tension, and when engulfing cancer cells that have a large spread of stiffnesses. Our model offers a physical explanation for this diversity of cellular behaviors, guided solely by the forces and shape dynamics.

References:
[1] Sadhu, R. K., Barger, S. R., Penič, S., Iglič, A., Krendel, M., Gauthier, N. C., & Gov, N. S. (2023). A theoretical model of efficient phagocytosis driven by curved membrane proteins and active cytoskeleton forces. Soft Matter, 19(1), 31-43.

Author Mikuláš Klenor*, IOCB Prague, Czechia

Second-Author Vojtěch Košťál, IOCB Prague
*mikulas.klenor@uochb.cas.cz

To achieve reliable simulations, we develop force fields that enable an improved description of ions in MD simulations¹. To target the well-known issue of full-charge force fields of overestimating interactions of divalent cations, we apply the theory grounded Electronic Continuum Correction (ECC), which incorporates electronic polarization by scaling of charges.

Our results demonstrate that ECC-modified CHARMM36 and Slipids parameters for the POPC lipid, in combination with ECC-consistent ion and water models, effectively mitigate calcium overbinding to the membrane.  The ECC-adjusted force field allows for a more accurate representation of ion–ion interactions without relying on empirical corrections or incurring the high computational cost associated with polarizable force fields.

An excellent use case for ECC force field is the description of interactions of Cell Penetrating Peptides and cell membrane. Cell-Penetrating Peptides are short, highly positively charged peptides. Contrary to chemical intuition, they can passively traverse the hydrophobic core of cell membranes. To harness their potential as cargo carries, advanced simulations are required to understand the mechanism at atomic level.

We perform atomistic MD simulations of nonaarginine (R9) as a prototypical CPP. One of the early steps in R9 translocation involves inducing membrane curvature leading to membrane multilamerality². To explore this process, we use model membrane mixture of DOPE, DOPS and DOPC. We investigate the interplay between the membrane curvature and R9 aggregation.

References:

1. Nencini, R. et al. Effective Inclusion of Electronic Polarization Improves the Description of Electrostatic Interactions: The prosECCo75 Biomolecular Force Field. J. Chem. Theory Comput. 20, 7546–7559 (2024).
2. Allolio, C. et al. Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore. Proc. Natl. Acad. Sci. 115, 11923–11928 (2018).

Author Mgr. Adéla Chadalíková (adela.chadalikova01@upol.cz), CATRIN (Czech Advanced Technology and Research Institution), Czech Republic
Second-Author Mgr. Markéta Paloncýová, PhD., CATRIN (Czech Advanced Technology and Research Institution), Czech Republic
Mgr. Petra Kührová, PhD. CATRIN (Czech Advanced Technology and Research Institution), Czech Republic

One of the most advanced forms of vaccination today is the mRNA vaccine. This type of vaccine is emerging as a promising, safe, and universal vaccine. A crucial role in these vaccines is played by mRNA structures. Messenger RNA contains a pathogen sequence in its structure, which is not toxic to humans [1]. For example, in the case of COVID-19 vaccines (Comirnaty or Spikevax)[2], mRNA molecules encode the Spike protein sequence. The vaccines were designed to safely and effectively deliver mRNA to target cells, where translation converts the mRNA sequence into protein. Thanks to our immune system, we can initiate immunisation against these proteins and develop and primarily remember the preparation of antigens [3].
mRNA vaccines use lipid nanoparticles (LNPs) to protect mRNA and deliver it into target cells. LNPs typically contain ionizable lipids (e.g., ALC-0315, SM-102, MC3)[4], helper phospholipids, cholesterol, and PEG-lipids; together, these components stabilise the nanoparticle, facilitate endosomal escape, and improve circulation time. The precise mechanism behind their endosomal escape remains unclear. Current theories propose that the lipid nanoparticle (LNP) carrying the RNA enters target cells via endocytosis [5]. As the LNP is internalised and the endosomal environment acidifies, the ionizable lipids within the LNP gain a positive charge, allowing them to interact with the membrane lipids of the target cell and trigger the release of the RNA cargo. It is believed that the lipids in the LNP reorganise into an inverted hexagonal (HII) phase during this process, although studying this structure remains experimentally and theoretically challenging.
Our goal is to gain deeper insight into the mechanisms of lipid phase changes in lipid nanoparticles and how these phases affect RNA structure.

REFERENCES:
[1] Gote, V., Bolla, P. K., Kommineni, N., Butreddy, A., Nukala, P. K., Palakurthi, S. S., & Khan, W. (2023). A Comprehensive Review of mRNA Vaccines. International Journal of Molecular Sciences, 24(3), 2700. https://doi.org/10.3390/ijms24032700
[2] S. Kashte, A. Gulbake, S. F. El-Amin III, and A. Gupta, ‘COVID-19 vaccines: rapid development, implications, challenges and future prospects’, Hum. Cell, vol. 34, no. 3, pp. 711–733, May 2021, doi: 10.1007/s13577-021-00512-4.
[3] D. E. Speiser, M. F. Bachmann, D. E. Speiser, and M. F. Bachmann, ‘COVID-19: Mechanisms of Vaccination and Immunity’, Vaccines, vol. 8, no. 3, Jul. 2020, doi: 10.3390/vaccines8030404.
[4] X. Hou, T. Zaks, R. Langer, and Y. Dong, ‘Lipid nanoparticles for mRNA delivery’, Nat. Rev. Mater., vol. 6, no. 12, pp. 1078–1094, Dec. 2021, doi: 10.1038/s41578-021-00358-0.
[5] Y. Eygeris, M. Gupta, J. Kim, and G. Sahay, ‘Chemistry of Lipid Nanoparticles for RNA Delivery’, Acc. Chem. Res., vol. 55, no. 1, pp. 2–12, Jan. 2022, doi: 10.1021/acs.accounts.1c00544.

Alicia Galinsoga¹, Delaram Khamari¹, Xabier Osteikoetxea¹, Tamás Visnovitz¹, Agnes Kittel¹, Anita Schamberger¹, Edit I Buzas^1,2

1. Semmelweis University, Department of Genetics, Cell- and Immunobiology, Budapest, Hungary
2. HUN-REN-SU, Budapest, Hungary

Over the past two decades, extracellular vesicle research has focused predominantly on small EVs, while larger and especially very large extracellular vesicles have remained largely unexplored, often dismissed as cellular ”debris”.

Historically, only large oncosomes and apoptotic bodies were recognized in the extracellular vesicle field.

Analysis of the components within these “debris” fractions has led us to the identification of very large-sized novel extracellular vesicle populations with distinct ultrastructural features and molecular composition. Our laboratory has shown the en bloc release of multivesicular body (MVB)-like small extracellular vesicle clusters and the release of secreted amphisomes (amphiectosomes). More recently, we conducted a detailed characterization of very large extracellular vesicles released during apoptosis and pyroptosis. In addition, we identified large-sized secreted autolysosomes within the secretome of cancer cells.

Taken together, our findings accumulated over the years challenge the long-standing “debris” dogma and offer new insights into the biology and significance of very large extracellular vesicles.

Extracellular vesicles (EVs)—including exosomes, microvesicles, and apoptotic bodies—are membrane-bound particles released by cells that play a crucial role in intercellular communication by transferring a wide range of biological signals. The vascular endothelium is a major contributor to the circulating vesicle population and influences key signaling pathways that affect blood cells and modulate endothelial cell plasticity and adaptation through paracrine interactions. The molecular and functional diversity of endothelial cells across different vascular regions underlines their heterogeneity and drives ongoing research into the specific physiological and pathological roles of EVs derived from blood and lymphatic endothelial cells. Endothelial EVs have been implicated in the onset and progression of various vascular disorders and are being explored for their potential as biomarkers and therapeutic targets. Yet, our knowledge on EVs derived from the lymphatic vasculature is still scarce. This keynote lecture will give an overview of the status quo in endothelial EV biology, extending from the blood to the lymphatic vasculature.

Cavitation in water under tension is typically triggered at nanoscale hydrophobic defects that stabilize preexisting nanobubbles. Using atomistic molecular dynamics simulations combined with classical nucleation theory, we show that polar lipids can adsorb onto nanoscale hydrophobic crevices (1), which are the strongest cavitation nuclei. Once coated, these defects no longer stabilize nanobubbles, shifting the cavitation-limiting step from defect-driven bubble growth to the rupture mechanics of the lipid bilayer itself (2). This membrane-mediated suppression of cavitation offers a molecular explanation for the surprising stability of water under tension in biological systems and suggests new strategies for designing cavitation-resistant membranes and coatings.

References:
(1) M. Šako, F. Staniscia, E. Schneck, R.R. Netz, M. Kanduč, “Conditions for the stable adsorption of lipid monolayers to solid surfaces,” PNAS Nexus, pgad190 (2023)
(2) M Šako, S Jansen, HJ Schenk, RR Netz, E Schneck, M Kanduč, “How Lipids Suppress Cavitation in Biological Fluids”, J. Colloid Interface Sci. 703, 139286 (2026)

Ana Kolenc*, Slovenian Institute for Transfusion Medicine, 1000 Ljubljana, Slovenia
Zala Lužnik Marzidovšek, Eye Hospital, University Medical Centre Ljubljana, 1000 Ljubljana, Slovenia
Elvira Maličev, Slovenian Institute for Transfusion Medicine, 1000 Ljubljana, Slovenia
*ana.kolenc@ztm.si

Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells capable of differentiating into multiple cell lineages and exerting immunomodulatory and regenerative effects, making them highly attractive for therapeutic applications (1, 2). MSC-derived extracellular vesicles (EVs) offer possible advantages over direct MSC administration, however, their clinical translation remains limited by critical challenges including biological and methodological factors (3, 4). To address this, we compared the effects of passage number (passage 3-5) and serum-free conditioning duration (24h and 48h) on yield and size of EVs released from three different MSC sources (umbilical cord, adipose tissue, bone marrow). Prior to EV collection, all MSCs were characterised by immunophenotyping (CD73, CD90, CD105, CD45, and HLA-DR) and determination of adipogenic and osteogenic differentiation potential. Using MSC-EV samples obtained under the conditions that produced the highest yield, we assessed their effects on epithelial cells using an in vitro scratch assay.
EV yields were substantially higher in samples collected after 48 hours of culturing in serum-free medium and particle concentration gradually increased with higher passage number. These were trends consistent across all three MSC sources, as confirmed by imaging flow cytometry (CD9, CD63, CD81, CD73 and CD90) and nanoparticle tracking analysis. Umbilical cord-derived MSCs produced the highest EV yield, suggesting a potential advantage in terms of vesicle production. In scratch assay on corneal epithelial cells, all EVs samples improved wound closure compared with negative control, however no statistically significant differences between EVs from different MSC cell sources were found. Continued research on MSC-EVs production and cargo analysis will be essential to generate safe, stable, and reproducible EV preparations, ultimately making the successful clinical translation of MSC-derived EV therapies possible.

References:
(1) Pittenger M.F., Discher D.E., Péault B.M. et al. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regenerative Medicine 4, 22 (2019). https://doi.org/10.1038/s41536-019-0083-6
(2) Maličev E., Jazbec K. An overview of mesenchymal stem cell heterogeneity and concentration. Pharmaceuticals 17, 350 (2024). https://doi.org/10.3390/ph17030350
(3) Gowen A., Shahjin F., Chand S. et al. Mesenchymal stem cell-derived extracellular vesicles: challenges in clinical applications. Frontiers in Cell and Developmental Biology 8, 149 (2020). https://doi.org/10.3389/fcell.2020.00149
(4) Kolenc A., Maličev E. Current methods for analysing mesenchymal stem cell-derived extracellular vesicles. International Journal of Molecular Sciences 25(6), 3439 (2024). https://doi.org/10.3390/ijms25063439

Sofija Glamočlija Jekić¹, Anna Schmid², Nataša Radulović³, Alisa Gruden-Movsesijan¹, Ljiljana Sabljić¹, Sergej Tomić¹, Jelena Đokić⁴, Irma Schabussova², Maja Kosanović^1*

¹Institute for the Application of Nuclear Energy-INEP, University of Belgrade, Belgrade, Serbia; ²Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria; ³Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia; ⁴Institute of molecular genetics and genetical engineering, IMGGI, University of Belgrade, Belgrade, Serbia

* maja@inep.ac.rs

Extracellular vesicles (EVs) releassed by parasitic organisms represent a fascinating frontier in interspecies communication, serving as nanoscale couriers that deliver regulatory cargo to modulate host immune responses and ensure parasite survival. Across diverse phyla, from protozoans deploying EVs to evade innate immunity, to trematodes using them to dampen inflammation, EVs exploit conserved uptake pathways in mammalian cells, reprogramming signaling in recipient cells which can lead to tolerance and alleviate pathology.

Among helminth parasites, nematodes such as Trichinella spiralis employ EVs derived from the excretory-secretory products of their muscle larvae (TsEVs), which encapsulate immunomodulatory glycoproteins, miRNAs, and lipids. These vesicles not only shield the parasite from host immune responces but also mitigate concomitant hypersensitive immune responses in the host, including allergic disorders.

Building on our in vitro findings that TsEVs induce a stable tolerogenic phenotype in human monocyte-derived dendritic cells, which release anti-inflammatory cytokines and stimulate regulatory T cell expansion we translated this cross-kingdom dialogue to an in vivo model of ovalbumin (OVA)-induced allergic airway inflammation in BALB/c mice. Intranasal TsEVs administration lead to reductions in bronchoalveolar lavage eosinophils, serum OVA-specific IgE, and lung infiltrates of macrophages and NK cells. Treated mice had increased presence of CD103⁺ tolerogenic dendritic cells, CD4+Foxp3+ regulatory T cells and decreased CD11b⁺Ly6C⁺ inflammatory monocytes in the lungs,. Ex vivo analyses from lung and spleen isolates confirmed suppressed Th2 cytokine production (IL-4, IL-5, IL-13) and elevated IL-10, highlighting a shift from a Th2-dominated response to a more regulatory profile.

Collectively, these insights position parasitic EVs as evolutionary blueprints for future bioengineered therapeutics, harnessing interspecies EVs signaling to mitigate respiratory allergies and provide precision immunomodulation.

Pietro Parisse*, CNR – IOM, Trieste, Italy
Ana Svetic, University of Trieste, Italy
Elena Babici, University of Trieste, Italy
Elena Ferraguzzi, University of Trieste, Italy
Luca Puricelli, Area Science Park, Trieste, Italy
Loredana Casalis, Elettra Sincrotrone Trieste, Italy

*pietro.parisse@cnr.it

Cell plasma membranes (PM) represent the pivotal step of interaction with extracellular systems, such as pathogens, drugs and extracellular vesicles (EVs). Among the great biochemical heterogeneity of PM, cholesterol is currently acknowledged as one of the fundamental players regulating membrane stability, dynamics and mechanical properties. Here we will present our investigation based on AFM nano-topographic and mechanical measurements on supported lipid bilayers as in-vitro biomimetic models of PM. We chose different mixture of lipids with variable concentration of cholesterol and we focused on the role played by cholesterol, in ruling morphological, topographical, and mechanical properties of the diverse coexistent lipidic phases and in affecting the interaction with Extracellular Vesicles in the biological contexts of triple negative breast cancer and Covid-19 infection.
Altogether, our results aim to get a more detailed insight into the key components and biophysical properties ruling EVs interaction with cellular PM, as a first step for potential applications in the theranostic field, such as novel drug-delivery strategies, antiviral approaches or cancer therapies targeting the membrane lipidic composition.

References:
(1) Paba C. et al., Journal of Colloid and Interface Science 652, 1937-1943 (2023)
(2) Perissinotto F. et al. Nanoscale, 13 (2021)
(3) Helmy S. et al., Journal of Colloid and Interface Science, 690, 137333 (2025)

Radeghieri A.*, Università degli Studi di Brescia, Italy
*annalisa.radeghieri@unibs.it

The extracellular vesicle (EV) interface is a dynamic boundary where the vesicle membrane interacts with the surrounding biological environment through the formation of a biomolecular corona (BC). Originally described for synthetic nanoparticles, the BC is now recognized as a defining feature of extracellular nanoparticles such as EVs, emerging from the selective adsorption of proteins and other biomolecules present in extracellular fluids. BC can substantially reshape EV surface properties, thereby influencing vesicle identity, biological function, cellular interactions, and biodistribution.
Despite growing interest, the mechanisms, specificity, and dynamics governing biomolecular recruitment to the EV interface remain poorly understood, in part due to the experimental challenges of studying these interactions under physiologically relevant conditions. Over the past years, our laboratory has focused on developing new experimental approaches to probe the EV biomolecular interface in situ and in near physiological environments. By combining single-particle and ensemble-level measurements, we investigate how BC form, evolve, and differ across EVs of distinct cellular origin (1), as well as how EVs interact with other circulating nanoparticles such as lipoproteins (2).
In this lecture, I will present our recent findings on EV BC dynamics, highlighting how interfacial interactions shape EV behavior and how novel non-destructive methods enable their study in complex biological fluids. Finally, I will discuss the implications of these insights for EV-based diagnostics (3) and therapeutic applications (4).

References:
(1) Musicò A., Zendrini A., Reyes S.G., Mangolini V., Paolini L., Romano M., Papait A., Silini A.R., Di Gianvincenzo P., Neva A., Cretich M., Parolini O., Almici C., Moya S.E., Radeghieri A., Bergese ., Nanoscale Horiz. 10(1):104-112, (2024)
(2) Musicò A., Frigerio R., Normak K., Scolari S., Gori A., Arosio P., Radeghieri A., Paolini L., Llarena I., Moya S., Zendrini A., Bergese P., ChemRxiv. doi:10.26434/chemrxiv-2025-x10p3. (2025)
(3) Tassoni, S., Bergese, P., Radeghieri, A., Nanomedicine, 20(16), 2013–2021 (2025).
(4) Musicò A., Zenatelli R., Romano M., Zendrini A., Alacqua S., Tassoni S., Paolini L., Urbinati C., Rusnati M., Bergese P., Pomarico G., Radeghieri A., Nanoscale Adv. 5(18):4703-4717(2023)

Bárkai T., Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Lenzinger D., Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Bugyik E., Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Dudás I., Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Mórotz G.M., Semmelweis Univ., Dep. Pharmacology and Pharmacotherapy, Hungary
Sághi M. Semmelweis Univ., Dep. Pathology and Experimental Cancer Research, Hungary
Fintha A., Semmelweis Univ., Dep. Pathology and Experimental Cancer Research, Hungary
Buzás EI., Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Visnovitz T.*, Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
*visnovitz.tamas@semmelweis.hu

Our recent findings provide evidence that amphiectosome release via the “torn bag mechanism” represents a novel small extracellular vesicle (sEV) secretion pathway. It is present in all tested cell lines and mouse tissues (1,2). Recently, we demonstrated that in HEK293 cells under stress conditions, such as calcium ionophore-induced membrane stress or metabolic stress caused by serum starvation, multivesicular endosome (MVE) exocytosis is activated, whereas this process is absent under steady-state conditions. Importantly, we modulated these two mechanisms. The amphiectosome release was dependent on autophagy, while MVE exocytosis was autophagy-independent but RAB27a-dependent.

Here, we aimed to investigate whether a switch in sEV secretion mechanisms occurs in different types of heart failure. Transmission electron microscopy was performed on patient-derived samples as well as on rat and mouse models. Following heart transplantation of the patients, portions of the explanted hearts were fixed, embedded and sectioned for ultrastructural analysis. Our observations suggest that in slowly progressing pathologies such as cardiomyopathy, the predominant sEV secretion mechanism in capillary endothelial cells is the release of large multivesicular extracellular vesicles (MV-lEVs) via the “torn bag mechanism”, while in acute heart failure conditions such as ischemic heart disease, endothelial cells release sEVs through MVE exocytosis. These findings were further validated using animal models.

Previously, we also provided evidence that not just endothelial cells but cardiomyocytes are capable of releasing MV-lEVs (1-3), most likely amphiectosomes. The intraluminal vesicle composition of amphiectosomes appears to differ from that of exosomes secreted via MVE exocytosis (1-3), therefore, this compositional difference in circulating EVs may serve as a potential diagnostic marker for acute pathological changes in the heart.

Funding: NKFIH grants: NKKP ADVANCED_25 152112, NVKP_16-1-2016-0004, VEKOP-2.3.2-16-2016-00002, VEKOP-2.3.3-15-2017-00016, Higher Education Excellence Program (FIKP), Therapeutic Thematic Programme (TKP2021-EGA-23), RRF-2.3.1-21-2022-00003, 2019-2.1.7-ERA-NET-2021-00015; the European Union’s Horizon 2020 (No. 739593); and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences G.M.M and T.V.).

References:
(1) Lenzinger D. et al., bioRxiv, p. 2025.10.29.685290 (2025).
(2) Visnovitz T., Lenziger D., Koncz A. et al, eLife, 13, p. RP95828 (2025).
(3) Koncz A. et al., Membranes, 13, 431 (2025).

Tünde Juhász,1 Kamal et Battioui,1 Sohini Chakraborty,1 Imola Szigyártó,1 Kamilla Ujvári,1 Benjámin Kovács,1 Tasvilla Sonallya,1 Vignesh U. Nagaray,1 Tamás Beke-Somfai,1*

1HUN-REN Research Center for Natural Sciences, Budapest, Hungary
*beke-somfai.tamas@ttk.hu

new antimicrobial solutions have been extensively investigated in the backdrop of the growing resistance towards conventional antibiotic treatments. Recently it has become clear that bacteria have an affinity to release various extracellular vesicles as part of their regular function in colonies. However, should this occur at an increased pace, that could rapidly lead to destroy individual cells. We have recently designed a beta-peptide (named 3K) (1), which here we shot that it can be triggered into extensive nanonet formation by extracellular ATP, a molecular signal of hostile microbial attacks. 3K and ATP co-assembles into highly entangled 3D architectures of infinite fibrils, with high antibacterial activity against E. coli. The mechanism of antibacterial action was tracked by cryo-EM which has revealed that these fibrils not only entrap microbes by entangling them, but these fibrils also enforce various extracellular vesicles to be released from the bacteria, leading eventually to membrane disruption. These mechanistic insights could in turn provide better understanding of the natural molecular processes underlying e.g. the toxic behaviour of natural peptide nanonets, but these also suggest that enforcing increased amount of bacterial extracellular vesicles could be a viable strategy towards the development of conceptually new antimicrobial therapies.

References:
(1) K. El Battioui, S. Chakraborty, A. Wacha, D. Molnár, M. Quemé-Peña, I. Cs. Szigyártó, Cs. L. Szabó, A. Bodor, K. Horváti, G. Gyulai, Sz. Bősze, J. Mihály, B. Jezsó, L. Románszki, J. Tóth, Z. Varga, I. Mándity, T. Juhász, T. Beke-Somfai: In situ captured antibacterial action of membrane incising peptide lamellae, Nat. Commun, 2024, 15, 3424

Fabrizio Cillo, fabrizio.cillo@cnr.it, National Research Council (CNR), Institute for Sustainable Plant Protection, Bari, Italy.
Valeria Daniello, CNR, Institute for Sustainable Plant Protection, Bari, Italy
Wilma Sabetta, CNR, Institute for Biosciences and Bioresources, Bari, Italy

Tetraspanins are conserved four-pass transmembrane proteins involved in membrane organization, protein trafficking, and cellular communication. In plants, they participate in development, stress responses, and pathogen interactions, but remain less studied than their animal counterparts (1).

We identified and characterized 17 tomato (Solanum lycopersicum) tetraspanins (SlTETs) through genome-wide and phylogenetic analyses. Several SlTETs clustered into distinct clades with their Arabidopsis thaliana orthologs, suggesting conserved functions (2). Structural features, including the plant-specific GCCK/RP motif and conserved cysteines in EC2, support their structural conservation and membrane anchoring capacity (3). Structural 3D modeling indicates a cone-shaped transmembrane configuration, consistent with what observed in other species (4).

Expression profiling revealed tissue-specific patterns and changes upon pathogen infection. Transcriptional changes due to virus infections possibly correlate with virus-induced massive modifications of cellular membrane structures (5).

We generated SlTET interaction networks with other proteins. The resulting interactome suggests that individual SlTETs are involved in vesicle trafficking, membrane remodeling, ion transport, and signaling. Some interactor proteins are shared among multiple SlTETs.

Immuno-electron microscopy and immunoblotting using anti-TET8 antibodies confirmed SlTET8 association with nanovesicle membranes, as in Arabidopsis (6).

In conclusion, the characterization of tomato tetraspanins highlights their potential involvement in membrane organization, vesicular trafficking and extracellular transport.

References:

(1)  Reimann, R., Kost, B., & Dettmer, J. (2017). TETRASPANINs in plants. Front. Plant Sci., 8, 545.

(2)  Wang F. et al. (2015). Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development. Plant Physiol., 169(3), 2200–2214.

(3)  Zhu T. et al. (2022). Arabidopsis Tetraspanins Facilitate Virus Infection via Membrane-Recognition GCCK/RP Motif and Cysteine Residues. Front. Plant Sci., 13, 805633.

(4)  Rubinstein E. et al. (2025). Tetraspanins affect membrane structures and the trafficking of molecular partners: what impact on extracellular vesicles? Biochem. Soc. Trans., 53(2), 371–382.

(5)  He, R., et al. (2023). Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants. Viruses, 15(3), 744.

(6)  Liu N. et al. (2024). Arabidopsis TETRASPANIN8 mediates exosome secretion and glycosyl inositol phosphoceramide sorting and trafficking. The Plant Cell, 36(3), 626–641.

Veronika Kralj-Iglič⁵, Aleš Iglič⁵

¹University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia
²University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia

Theoretical and experimental studies of membrane-enclosed entities have been a subject of study for the last 70 years. The most studied systems were erythrocytes and phospholipid vesicles, which are large enough to observe live under the optical microscope. The observed shapes that exhibited heterogeneity depended on the properties of the membrane as well as on the properties of the outer and inner compartment solution. Methods have been developed to theoretically explain the observed shapes of these systems, based on the minimization of the free energy of the membrane at given geometrical constraints. Early models considered the membrane as an elastic continuum. Extracellular vesicles are too small to observe by the optical microscope, but scanning electron micrographs reveal that they may have the same shapes as erythrocytes and phospholipid vesicles indicating that the observed shapes are subject to the same physical laws. However, due to small size of extracellular vesicles, which exposes the membrane constituent dimensions, the theoretical assumptions should be revisited. Models based on the methods of statistical physics, were proposed to allow for in-plane orientational distribution of constituents and explain stability of anisotropic membranous nanostructures, (e.g. nanotubes and narrow necks). Experimental evidences and theoretical considerations of extracellular vesicles within the curvature patch model, will be presented.

James Parry*, Unchained Labs, USA
Iva Kušec, Altium International, Croatia
*james.parry@unchainedlabs.com, iva.kusec@altium.net

Getting the full picture of your rare extracellular vesicles (EVs) in cell culture or biofluid samples is a challenge – sometimes even if samples are purified. To most techniques, interference from lipoproteins, cell debris and protein aggregates can get in the way or make it hard to be confident that you’re counting the right stuff. The task gets even harder when sample volumes are limited and the EV subpopulations you care about are rare. Leprechaun skips past troublesome background matrix effects by capturing EVs on its Luni consumable to analyze particle size, concentration, and phenotype for exactly the EV particles you care about. With sensitivity down to 5×10^5 particles/mL, analysis down to 35 nm, single particle phenotypic analysis and now the ability to quantify the proportion of an EV subpopulation out of the total EV concentration, Leprechaun is ready to help you paint the whole picture of your EV sample no matter how complex.

Gabriella Pocsfalvi*, Institute of Biosciences and BioResources, National Research Council of Italy
Ani Barbulova, Institute of Biosciences and BioResources, National Research Council of Italy
Ramila Mammadov, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
Feby Wijaya Pratiwi, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
Seppo Vainio, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland
Kim Kwang Pyo Kim, College of Applied Science, Global Campus, Kyung Hee University, Republic of Korea
Jae Won Oh, College of Applied Science, Global Campus, Kyung Hee University, Republic of Korea

*gabriellakatalin.pocsfalvi@cnr.it

Plant extracellular vesicles (EVs) are membrane‑bound nanostructures enclosed by a biomembrane that play natural biofunctional roles in plants, including intercellular communication and stress responses. In contrast, plant‑derived nanovesicles (PDNVs), typically obtained from plant homogenates, do not serve intrinsic functions for the plant but are emerging as innovative biotechnological tools with applications in agriculture and health. To overcome limitations of PDNVs such as variability, pathogen contamination, and heterogeneity, we developed an EV farming approach using in vitro plant cell suspension cultures (CSCs), providing a controlled and reproducible platform for vesicle production.

Within the European FarmEVs project, cell suspension cultures (CSCs) of tomato (Solanum lycopersicum) and bean (Phaseolus vulgaris) were established to produce extracellular vesicles (EVs). Vesicles harvested from conditioned media showed typical morphology and size (100–200 nm) consistent with plant EVs. Proteomic analysis of tomato EVs revealed distinct cargo signatures, including membrane ATPases, transporters, stress‑related proteins, and enzymes involved in lipid metabolism. To explore translational potential, drug‑loading experiments with tolvaptan were performed, demonstrating successful encapsulation of this hydrophobic therapeutic and confirming the suitability of plant EVs as carriers for bioactive molecules.(1)

In summary, CSC‑derived EVs provide a reproducible and scalable platform that overcomes limitations of PDNVs and enables mechanistic studies of vesicle biogenesis and function. Their capacity to incorporate drugs such as tolvaptan highlights promising applications in targeted molecular delivery, contributing to the One Health vision that connects plant biotechnology with human and environmental health.

Funding: This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Staff Exchange project “FarmEVs,” grant agreement No. 101131175, and the Italian Ministry of Foreign Affairs and International Cooperation EV-C@p grant number PGR02032.

References:

• Mammadova et al., Int J Nanomedicine 17;20, 6253–6269 (2025)

Olga Janoušková1, Oksana Batkivska1, Michaela Kocholatá1 and Jan Malý1

1 Centre of Nanomaterials and Biotechnology, Faculty of Science, Jan Evangelista Purkyně University in Ústí nad Labem, Czech Republic

Introduction: Extracellular vesicles (EVs), particularly exosomes (EXs) isolated from various animal and plant sources, can serve as biomarkers and as therapeutic tools. Their properties and performance can be strongly affected by the isolation method and by the detection methodologies used. These methodologies are increasingly essential for in vitro and in vivo testing, including evaluation of labeling efficiency, cellular and tissue uptake, cell-to-cell communication, and downstream transcriptomic, lipidomic, or proteomic analyses. Here, we compared plasma membrane, protein, and RNA labeling strategies for plant- and animal-derived exosomes isolated using several methods, and we assessed cellular uptake, cargo-detection efficiency, and loading capacity.

Methods: EXs from mesenchymal stem cells and EVs derived from different types of plant tissue cultures were isolated by ultracentrifugation or by tangential flow filtration (TFF) followed by size exclusion chromatography (SEC). The vesicles were characterized using BCA, NTA, cryo-TEM, and Western blotting (WB). Exosomes were labeled with two classes of fluorescent dyes targeting either the vesicle lumen or the membrane. Laser scanning confocal microscopy (LSCM) and NanoFCM were used to evaluate labeling efficiency, uptake by plant and human cell lines, and loading capacity.

Results: The isolation method influences not only the yield of exosomes, but also the amount of co-isolated (non-vesicular) proteins and RNA. Depending on the source and isolation approach, exosomes are labeled differently by various fluorescent dyes, which can affect cellular uptake and, consequently, their biological behavior and cargo transport efficiency.

Conclusion: These findings provide important experimental data on the in vitro behavior of EXs, which may influence their downstream applications, as well as their transport, biodistribution, and behavior in vivo.

Funding information: This work was supported by the Ministry of Education, by the project EXREGMET CZ.02.01.01/00/22_008/0004562, funded by OP JAK and project projects UJEP-SGS-2022-53-008-2

Seppo J. Vainio2, Markus Lampimäki1,7, Feby W. Pratiwi2,7, Zoé Brasseur1, Ulla Saarela2, Ramila Mammadova2, Erfan Khamespanah2, Keerthanaa B. Shanthi,2 Genevieve Bart2, Elisabeth Garcia Ruiz2 , Anatoliy Samoylenko2, Marko Mutanen3, Marko Suokas3, Artem Zhyvolozhnyi2, Katrianne Lehtipalo1,4, Soile Jokipii-Lukkari3, Seppo Rytkönen3, Jonathan Duplissy1, Lauri Ahonen1, Juha Kangasluoma1, Juha Röning5, Caglar Elbuken2, Henrikki Liimatainen6, Markku Kulmala1 and Tuukka Petäjä1.

1Institute for Atmospheric and Earth System Research/Physics, Faculty of Science, University of Helsinki, 00014, Helsinki, Finland

2Kvantum Institute, Disease Networks Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90014, Oulu, Finland

3Ecology and Genetics Research Unit, University of Oulu, 90014, Oulu, Finland

4Finnish Meteorological Institute, 00560, Helsinki, Finland

5Biomimetics and Intelligent Systems Group, Faculty of Information Technology and Electrical Engineering, University of Oulu, 90014, Oulu, Finland

6Fibre and Particle Engineering

Various biological particles—including pollen, fungal spores, bacteria, viruses, and biogenic particles originating from forests or marine aerosols—can act as ice-nucleating particles (INPs), influencing ice crystal formation and contributing to biosphere–climate feedback mechanisms. Extracellular vesicles (EVs) constitute a complex and widely active signaling system that facilitates molecular communication across diverse biomes. EVs have been identified in terrestrial and aquatic environments; however, their presence, fate, and functional roles in atmospheric processes during the bioaerosol (BA) phase remain largely unexplored. Notably, EVs may also function as INPs.

Bioaerosols can rapidly alter micro-biogeography upon deposition, acting as critical agents of biodiversity change, ecological dynamics, and potential health effects through the long-distance dispersal of biological material. Here, we demonstrate that plant-derived nanovesicles (NVs) from Norway spruce (Picea abies L. Karst.) and bilberry (Vaccinium myrtillus L.) function as bioaerosols and possess the potential to act as INPs. These NVs can transition into the bioaerosol phase and be recaptured on impactor filters. During this transition, NV size decreases from a liquid-phase range of approximately 50–250 nm to around 30 nm, corresponding to Aitken-mode aerosols—a key size fraction in biogenic atmospheric ice nucleation.

Both spruce- and bilberry-derived NVs exhibited heterogeneous ice nucleation activity at temperatures above −15 °C. Furthermore, atmospheric aerosol samples collected from a boreal forest at the SMEAR II (Station for Measuring Ecosystem–Atmosphere Relations) station contained spruce-like NV particles, along with associated non-coding nucleotides from local insect species, including those linked to spruce ecosystems. In vitro, spruce NVs were internalized by human lung and skin cells and exhibited antioxidant properties, suggesting potential implications for human wellness.

Together, these findings highlight the multifaceted roles of plant-derived NVs, demonstrating their capacity to remain airborne, modulate ice nucleation, and potentially influence cloud formation and biosphere–climate feedback mechanisms.

Kelsey Fletcher, Semmelweis Univ., Inst. of Genetics, Cell- and Immunobiology, Hungary
Tamás Visnovitz, Semmelweis Univ., Inst. of Genetics, Cell- and Immunobiology, Hungary; ELTE, Dep. of Plant Physiology and Molecular Plant Biology, Hungary
Péter Lőrincz, ELTE, Dep. of Anatomy, Cell and Developmental Biology, Hungary
Dorina Lenzinger, Semmelweis Univ., Inst. Genetics, Cell- and Immunobiology, Hungary
Edit I. Buzás, Semmelweis University, Inst. of Genetics, Cell- and Immunobiology, Hungary; HUN-REN-SU Translational Extracellular Vesicle Research Group, Hungary; HCEMM-SU Extracellular Vesicle Research Group, Hungary,
Krisztina V. Vukman*, Semmelweis Univ., Inst. of Genetics, Cell- and Immunobiology, Hungary
*Vukman.krisztina@semmelweis.hu

Mast cells (MC) are well known for their role in allergic reactions, where allergen-induced cross-linking of FcεRI-bound IgE leads to the release of granules containing bioactive mediators, such as histamine and tryptase (1). Interestingly, MC-derived extracellular vesicles (EVs) (2) were also among the first described to carry RNA molecules suggesting a broader immunoregulatory potential beyond classical degranulation (3). However, the relationship between extracellular granules (EGs) and EVs remains poorly defined.

In this study, we aimed to clearly differentiate and characterize the diverse extracellular particle (EP) subtypes released by MCs. Primary murine MCs were stimulated with IgE-antigen complexes (IgE-DNP), lipopolysaccharide (LPS), and calcium ionophore A23187. EPs were isolated using differential centrifugation and density gradient separation. Their identity and properties were assessed by high-resolution flow cytometry, confocal and electron microscopy. Real-time degranulation and release of EPs were also monitored using a CytoFLEX flow cytometer and a Fluoview FV4000 confocal laser scanning microscope.

Our results reveal stimulus-specific EP release patterns: LPS, mimicking type 1 inflammation, primarily induced EV secretion, whereas IgE-mediated (type 2) degranulation mainly produced EGs. Stimulation with Ca²⁺ ionophore led to a mixed release of EGs and EVs. These findings call for a careful re-evaluation of prior interpretations in MC and EV-related research employing A23187. Importantly, we identified a molecular marker, histamine, that allow the distinction between EG and EV populations. In conclusion, our study provides new insights into the differential release and characterization of MC-derived EPs. These findings highlight the distinct and potentially complementary roles of EGs and EVs in immune modulation and point toward refined approaches in MC and EV research.

Funding: NKFIH grants: NKKP STARTING_25 152115, NVKP_16-1-2016-0004, VEKOP-2.3.2-16-2016-00002, VEKOP-2.3.3-15-2017-00016, Higher Education Excellence Program (FIKP), Therapeutic Thematic Programme (TKP2021-EGA-23), RRF-2.3.1-21-2022-00003, 2019-2.1.7-ERA-NET-2021-00015; the European Union’s Horizon 2020 (No. 739593); and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

References:
(1) Vukman K.V. et al, Semin Cells Dev Biol, 67:45-73 (2017).
(2) Vukman K. V. et al, J Extracell Vesicle 10(1):e12023 (2020).
(3) Valadi H. et al, Nat Cell Biol, 9(6):654-9 (2007)Seppo Vainio

Christopher Aisenbrey*, Jasmin Schlauch, Burkhard Bechinger
Institut de Chimie (UMR-7177), CNRS/Université de Strasbourg, 67000 Strasbourg

* aisenbrey@unistra.fr

During the last few years we have developed a fluorescence self-quenching method to investigate the proximity of peptides within the lipid membrane (1,2). The short intrinsic timescale of the fluorescence measurements allows the characterization of very dynamic systems on the membrane surface. The results indicate the presence of certain degree of alignment and local structure. However, those structures seem to feature a large flexibility and allow fast molecular exchange over time. This leads to the idea that those peptides in a membrane rather form a mesophase (3) instead of classical supramolecular structures.
In a classical liquid crystal (or mesophase), the molecules are close enough to establish an attractive intermolecular potential which is the cause of a mesophase organization described for example by the Maier-Saupe mean field theory (4) or the distortion free energy density (5). The cationic peptides within a mesophase on the surface of a lipid membrane are separated by lipid molecules which prevent direct interactions of residues. In addition, they experience a repulsive Coulomb interaction in case of cationic peptides. However cationic peptides disturb the order of the surrounding lipids (6) which leads to an energetic penalty. This energetic penalty is dependent on the special distribution of the peptides, which leads to an effective potential (7–9). The liquid crystalline character of the lipid membrane is transferred towards the incorporated peptides.

(1) Aisenbrey C, Amaro M, Pospíšil P, Hof M, Bechinger B. Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids. Sci Rep. 2020 July 15;10(1):11652.
(2) Aisenbrey C, Bechinger B. Molecular Packing of Amphipathic Peptides on the Surface of Lipid Membranes. Langmuir. 2014 Sept 2;30(34):10374–83.
(3) Friedel G. Les états mésomorphes de la matière. Ann Phys. 1922;9(18):273–474.
(4) Maier W, Saupe A. Eine einfache molekulare Theorie des nematischen kristallinflüssigen Zustandes. Zeitschrift für Naturforschung A. 1958 July 1;13(7):564–6.
(5) Frank FC. I. Liquid crystals. On the theory of liquid crystals. Discuss Faraday Soc. 1958 Jan 1;25(0):19–28.
(6) Harmouche N, Bechinger B. Lipid-Mediated Interactions between the Antimicrobial Peptides Magainin 2 and PGLa in Bilayers. Biophysical Journal. 2018 Sept 18;115(6):1033–44.
(7) Marčelja S. Lipid-mediated protein interaction in membranes. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1976 Nov 11;455(1):1–7.
(8) Pearson LT, Edelman J, Chan SI. Statistical mechanics of lipid membranes. Protein correlation functions and lipid ordering. Biophysical Journal. 1984 May 1;45(5):863–71.
(9) Schröder H. Aggregation of proteins in membranes. An example of fluctuation‐induced interactions in liquid crystals. J Chem Phys. 1977 Aug 15;67(4):1617–9.

Crnković, Ana*, National Institute of Chemistry, Slovenia
Šolinc, Gašper, National Institute of Chemistry, Slovenia
Srnko Marija, National Institute of Chemistry, Slovenia
Spruk, Gregor, National Institute of Chemistry, Slovenia
Podobnik, Marjetka, National Institute of Chemistry, Slovenia
Anderluh, Gregor, National Institute of Chemistry, Slovenia
*ana.crnkovic@ki.si

Nanopore technology has gained significant momentum over the past couple of decades, with DNA sequencing being its most widely recognized application. The canonical detection principle relies on a single protein pore inserted into a lipid bilayer that separates two electrolyte-filled chambers. When a voltage bias is applied, an analyte in one chamber translocates through the pore, generating a characteristic current blockade defined by its amplitude and dwell time. In principle, these event features are expected to be specific to a given analyte, thereby enabling its identification and, in some cases, its quantification.

However, both the protein pore and the analytes can exhibit undesirable behavior. The pore may display elevated noise levels or gating at the voltages required for analysis. Larger analytes, such as proteins, can adopt multiple conformations, collide with or clog the pore, or enter from either the N- or C-terminus, resulting in a wide range of current signatures that complicate event analysis and hinder accurate identification. Conversely, small analytes—including ions and small organic molecules—may translocate too rapidly to be reliably detected.

Here, we demonstrate how directed evolution can be applied to improve protein pore performance in single-channel experiments, and how noncanonical amino acids (ncAAs) can be used to tailor pore selectivity toward specific analytes in complex mixtures. The ncAAs are incorporated site-specifically into the pore using genetic code expansion. Through functional assays, biophysical measurements, and structural characterization, we identify the optimal positions for ncAA incorporation to achieve specific detection of the analyte. Overall, this work highlights the potential of ncAAs to advance nanopore biosensing through their unique specificity and bioorthogonality.

Czogalla A.*, Department of Cytobiochemistry, University of Wrocław, Poland
Hinc P., Department of Cytobiochemistry, University of Wrocław, Poland
Drabik D., Department of Cytobiochemistry, University of Wrocław, Poland & Department of Biomedical Engineering, Wrocław University of Science and Technology, Poland
Pabisz J., Department of Cytobiochemistry, University of Wrocław, Poland
*aleksander.czogalla@uwr.edu.pl

Lipids are essential structural components of all biological membranes. In addition, they play key roles in wide range of cellular functions, including signal transduction and propagation. Until recently, recognition of membrane lipids by peripheral proteins was understood by analogy to ligand-receptor events, based largely on the selectivity of proteins for lipid hydrophilic heads. Each signaling lipid (e.g. phosphatidic acid, PA or ceramide-1-phosphate, C1P) may be entangled in a broad variety of cellular pathways, which implies that their biological activity must be tightly regulated. While the spatiotemporally controlled activity of numerous specific lipid-metabolizing enzymes defines the levels and turnover of signaling lipids, additional molecular mechanisms are indispensable to fine-tune protein-lipid recognition (1). General lipid composition, bilayer organization and geometry, and the presence of ions may lead to altered conformation of lipid head groups, their exposure at the water-bilayer interface and/or domain formation. These and several other features underlie the modulated specificity of lipid recognition by peripheral membrane proteins – a concept we have termed lipid presentation (2).
In our studies, we employ several model membrane systems, including lipid monolayers and vesicles of adjustable size to investigate how individual species of signaling lipid molecules behave in membranes of different composition and under varying conditions, and how this influences their interactions with peripheral membrane proteins. Using state-of-the-art biophysical approaches alongside molecular dynamics simulations, we have discovered previously unknown molecular mechanisms underlying these phenomena. This led us to find that the structure of acyl chains strongly influences the collective behavior of PA and C1P (3-5), which is reflected in altered recognition of these lipids by peripheral proteins (5-6). Additionally, cholesterol proved to be a potent modulator of lipid presentation in the membrane context, although the consequences for protein membrane recruitment and/or activation are strongly correlated with structural features of membrane-binding domains. This suggests that several subspecies of a particular signaling lipid may play different roles within a single cell.
Our results have enabled us to elucidate the mechanisms underlying selective recognition of signaling lipids by effector proteins. This is fundamental to understanding cellular signaling pathways and appreciating additional, so far poorly recognized aspects of their regulation and interdependence.

References:
(1) Zegarlińska J., Piaścik M., Sikorski A.F., Czogalla A. Acta Biochim Pol, 65,163 (2018).
(2) Czogalla A., Grzybek M., Jones W., Coskun U. Biochim Biophys Acta, 1841, 1049 (2014).
(3) Drabik D., Czogalla A. Int J Mol Sci, 22, 11523 (2021).
(4) Drabik D, Drab M, Penič S, Iglič A, Czogalla A. Sci Rep, 13, 18570 (2023).
(5) Drabik D., Hinc P., Cierluk K., Czogalla A. bioRxiv (2025).
(6) Żelasko J., Czogalla A. Cells, 11, 119 (2022).

Semeraro E.F.*, University of Graz, Field of Excellence BioHealth, Graz, Austria
Piller P., University of Graz, Field of Excellence BioHealth, Graz, Austria
Bartoš L., CEITEC & NCBR, Masaryk University, Brno, Czech Republic
Deb R., CEITEC & NCBR, Masaryk University, Brno, Czech Republic
Keller S., University of Graz, Field of Excellence BioHealth, Graz, Austria
Vácha R., CEITEC & NCBR, Masaryk University, Brno, Czech Republic
Pabst G., University of Graz, Field of Excellence BioHealth, Graz, Austria
*enrico.semeraro@uni-graz.at

Understanding the dynamic interplay between lipid bilayers and integral membrane proteins (IMPs) is essential for grasping the physicochemical properties of cell membranes. Unlike classic model membranes, which reflect the bulk properties of bilayers, structural stress in more complex membrane systems can affect the arrangement and function of IMPs (1,2,3). When the hydrophobic portion of an IMP differs from the thickness of the acyl-chain region of surrounding lipids—a phenomenon known as hydrophobic mismatching—the insertion of such IMPs leads to a free energy penalty that is alleviated by, e.g., membrane deformations. Currently, experimental measurements of lipid thickness near membrane proteins are limited and, to the best of our knowledge, have only recently been explored through NMR (4, 5).

In this study we present a comprehensive experimental and computational investigation of the outer membrane protein phospholipase A (OmpLA). OmpLA, an integral enzyme that hydrolyzes phospholipids upon dimerization, is reconstituted in large unilamellar vesicles—referred to as proteoliposomes—composed of either 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). We selected POPC and DLPC for their differing hydrophobic thicknesses, which respectively exceed or fall short of the OmpLA hydrophobic patch. We propose a multi-scale model for small-angle X-ray scattering (SAXS) that focuses on simultaneously probing the membrane trans-bilayer structure and the accurate copy number of OmpLA per proteoliposome. Additionally, we combined SAXS modeling with results from all-atom molecular dynamics (MD) simulations.

Combining MD results and SAXS data modeling gave us a comprehensive overview of the fine structure in proteoliposomes. This includes characterizing (i) the OmpLA monomeric/dimeric state, (ii) the extension and profiles of membrane deformations at the protein-membrane interfaces, (iii) as well as the detecting the absence of bulk lipids as a function of OmpLA concentration within proteoliposomes.

References:
(1) Piller P., Semeraro E.F., Rechberger G. N., Keller S., Pabst G., PNAS Nexus, 2(5) (2023).
(2) Piller P., Reiterer P., Semeraro E. F., Pabst G., RSC Applied Interfaces, 2(1), 69-73 (2025).
(3) Huster D., Biophysical Journal, 124(16), 2569-2570 (2025).
(4) Engberg O., Ulbricht D., Döbel V., Siebert V., Frie C., Penk A., Lemberg M.K., Huster D., Science Advances, 8(38), 8303 (2022).
(5) Soubias O., Sodt A.J., Teague W.E., Hines K.G., Gawrisch K., Biophysical Journal, 122(6), 973-983 (2023).

David Šťasný, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
Priti Sengupta, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
Zuzana Johanovská, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
Barbora Svobodová, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
Erdinc Sezgin, Science for Life Laboratory, Department of Applied Physics, Sweden
Martin Hof, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
Radek Šachl, J. Heyrovský Institute of Physical Chemistry, Czech Rep.
*radek.sachl@jh-inst.cas.cz

Gangliosides are a distinct class of glycosphingolipids (GSLs) enriched in neuronal plasma membranes, notable for their ability to self-organize into nanoscopic domains and to mediate interactions with extracellular molecules. Disruption of this interaction platform has been linked to various diseases and proposed to contribute to downstream signaling; however, the precise role of ganglioside nanodomains in ligand binding and their physicochemical properties remain poorly understood. To address this, we investigated the nanoscale organization and dynamics of gangliosides using a complementary set of advanced biophysical approaches, including FLIM-FRET (Fluorescence Lifetime Imaging of Förster Resonance Energy Transfer), which provides nanometer-scale insights into nanodomain architecture, and STED-FCS (Stimulated Emission Depletion–Fluorescence Correlation Spectroscopy), which resolves molecular dynamics within and outside nanoscopic domains. Our results demonstrate that ganglioside nanodomains are highly dynamic membrane entities that not only regulate the binding of incoming protein ligands but are also remodeled by ligand engagement. Furthermore, their physical characteristics critically depend on the overall lipid environment, lipid membrane asymmetry and local curvature as well as the cross-linking capacity of interacting ligands.

References:
(1) B. Svobodová, D. Šťastný, H. Blom, I. Mikhalyov, N. Gretskaya, A. Balleková, E. Sezgin, M. Hof, R. Šachl, Revised Diffusion Law Permits Quantitative Nanoscale Characterization of Membrane Organization, Analytical Chemsitry, 97, 11478-11485 (2025).

(2) D. Davidović, M. Kukulka, MJ. Sarmento, I. Mikhalyov, N. Gretskaya, B. Chmelová, JC Ricardo, M. Hof, L. Cwiklik, R. Šachl Which Moiety Drives Gangliosides to Form Nanodomains ? J. Phys. Chem. Lett., 14, 5791–5797, (2023).

Mario Vazdar*, University of Chemistry and Technology, Prague, Czechia
Zuzana Johanovská, J. Heyrovský Institute of Physical Chemistry, Prague, Czechia Kamila Yesmurzayeva, University of Chemistry and Technology, Prague, Czechia
Katarina Baxova, Institute of Organic Chemistry and Biochemistry, Prague, Czechia
Denys Biriukov, CEITEC, Masaryk University, Brno, Czechia
Barbara Pem, Rudjer Boskovic Institute, Zagreb, Croatia
Radek Šachl, J. Heyrovský Institute of Physical Chemistry, Prague, Czechia

*mario.vazdar@vscht.cz

Ethylenediaminetetraacetic acid (EDTA) is widely used in lipid bilayer experiments to chelate ions such as Ca²⁺ from membranes and bulk solution. We previously showed using molecular dynamics (MD) simulations and Langmuir monolayers that, beyond Ca²⁺ removal, EDTA anions unexpectedly bind to phosphatidylcholine (PC) monolayers and may affect the binding of positively charged species. (1)

Here, using fluorescence microscopy and MD simulations, we demonstrate that EDTA strongly alters the translocation of arginine-rich cell-penetrating peptides (CPPs). (2,3) Addition of 0.1 mM EDTA significantly reduces Arg₉ translocation across PC giant unilamellar vesicles (GUVs) to levels comparable with Lys₉ and a fluorescent probe. MD simulations reveal that EDTA, present in large excess, binds Arg₉ in solution, preventing its membrane adsorption and subsequent translocation. These findings challenge a common experimental assumption and caution against the use of EDTA in studies involving positively charged species such as CPPs.

(1) K. Vazdar, C. Tempra, A. Olżyńska, D. Biriukov, L. Cwiklik, M. Vazdar, J. Phys. Chem. B 127, 5462 (2023)

(2) M. Vazdar, J. Heyda, P. E. Mason, G. Tesei, C. Allolio, M. Lund, P. Jungwirth, Acc. Chem. Res 51, 1455 (2018)

(3) Ü. Langel, Cell Penetrating Peptides, Springer, Singapore (2019).

Bar L* 1, Lavrič M 2, Iglič A 1, Cordoyiannis G 2
1Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Slovenia
2Condensed Matter Physics Department, Jožef Stefan Institute, Ljubljana, Slovenia
*Laure.Bar@fe.uni-lj.si

The complex and dynamic architecture of lipid membranes makes their interactions with nanoparticles (NPs) challenging to understand. Despite numerous studies carried out over the last years, deep insights into the factors governing these interactions are still necessary in order to evaluate the efficiency or cytotoxicity of the NPs in biomedical applications.
The lipid membrane-NP interactions can lead to diverse outcomes, ranging from simple superficial NP adhesion to membrane wrapping and vesicle disruption, depending on the NPs and lipid membrane properties such as size or charge (1-2). In this work, we explore the interaction between gold NPs and lipid assemblies: supported lipid vesicles and bilayers. Experiments were conducted using quartz crystal microbalance with dissipation monitoring (QCM-D), a surface-sensitive technique that enables real-time tracking of lipid membrane-NP interactions. To this end, NPs are injected over supported lipid bilayers or supported vesicle layers via a fluidic flow (the supports being quartz crystal sensors). QCM-D is highly efficient for probing lipid membrane-NP interactions (3-4).
Several parameters that impact the interactions have been studied: the charge (role of electrostatic attraction), the membrane curvature (comparison between planar bilayers and vesicles), and the lipid phase (contribution of the lipid order to the NPs’ binding capability). Interestingly, on curved systems, a strong influence of the lipid phase state is observed, most notably, a phenomenon of vesicle rupture when the vesicles are in the gel phase. The experimental results, supported by theoretical considerations, will be discussed (5-6).

References:
(1) Contini C., Hindley J.W., Macdonald T.J., Barritt J.D., Ces O., Quirke N., Commun. Chem., 3, 130 (2020)
(2) Lunnoo T., Assawakhajornsak J., Puangmali T., J. Phys. Chem. C, 123, 3801–3810 (2019)
(3) Chen Q., Xu S., Liu Q., Masliyah J., Xu Z., Adv. Colloid Interface Sci., 233, 94–114 (2016)
(4) Yousefi N., Tufenkji N., Front. Chem. 4 (2016).
(5) Van der Pol R.W.I., Brinkmann B.W., Sevink G.J.A., J. Chem. Theory Comput., 20, 2888–2900 (2024).
(6) Bar L.*, Lavrič M., Caf M., Kralj S., Kumar Sadhu R., Losada-Pérez P., Iglič A., Cordoyiannis G., unpublished results.

Taoufiq Bourakadi*, INSERM U1248 Pharmacology & Transplantation, University of Limoges, France
Mehdi Benmameri, InSiliBio, France
Patrick Trouillas, INSERM U1248 Pharmacology & Transplantation, University of Limoges, France ; InSiliBio, France ; CATRIN – RCPTM, Palacký University Olomouc, Czech Republic
Gabin Fabre, INSERM U1248 Pharmacology & Transplantation, University of Limoges, France
* ahmed_taoufiq.bourakadi@unilim.fr

Passive permeation of xenobiotics, such as drugs, across biological membranes is a fundamental process that influences their disposition within the body. Rationalizing the underlying molecular mechanisms allows to optimize drug design and delivery. Molecular dynamics simulations provide a powerful computational approach to predict permeation coefficients by characterizing the interactions of xenobiotics with the lipid bilayer environment, thereby offering insights into their dynamic behavior.

Our in-house model, MemCross(1), provides a framework to investigate passive permeation at an all-atom resolution. Previously, we have been exclusively focusing on single-molecule passive permeation using a simple POPC lipid bilayer. To broaden the scope of our model, we aim to extend it towards new permeation mechanisms that reflect with greater accuracy the complexity of physiological conditions.

Firstly, we examine the impact of lipid composition on passive permeation. Biological membranes consist of a variety of lipid species, differing across numerous tissues and organs. By simulating xenobiotic diffusion across various lipid mixtures membrane models, we can determine to which extent different lipid environments modulate permeation barriers and through which physicochemical mechanisms such results are brought.

Secondly, we explore the interactions between xenobiotics and the surface of transmembrane protein domains. Such interactions could introduce preferential pathways that alter the energy landscape of transmembrane crossing. We select proteins with specific features to define the contribution of each to the free energy landscape.

Through these advancements, we try to provide a more comprehensive understanding of the process of passive permeation in regards to more complex experimental models. These insights would help to guide the design of pharmaceuticals with improved membrane permeability.

(1) Benmameri, M.; Chantemargue, B.; Humeau, A.; Trouillas, P.; Fabre, G. MemCross: Accelerated Weight Histogram Method to Assess Membrane Permeability. Biochimica et Biophysica Acta (BBA) – Biomembranes 2023, 1865 (3), 184120. https://doi.org/10.1016/j.bbamem.2023.184120.

Dlouhý O.*, University of Ostrava, Czech Republic
Böde K., University of Ostrava, Czech Republic
Jurečka, P., University of Ostrava, Czech Republic
Zgarbová M., University of Ostrava, Czech Republic
Fehér B., Semmelweis University, HUN-REN, Hungary
Karlický V., University of Ostrava, Czech Republic
Špunda V., University of Ostrava, Czech Republic
Bicout, D., Institut Laue-Langevin, France
Peters, J., Univ. Grenoble Alpes, LiPhy, ILL, Grenoble, France
Garab G., University of Ostrava, Czech Republic; HUN-REN BRC, Szeged, Hungary
Demé B., Institut Laue-Langevin, France

*ondrej.dlouhy@osu.cz

Bilayer lipid membranes are essential for maintaining the structure and function of cells. The membrane dynamics are described by the dynamic Matryoshka model (dMm), based on quasi-elastic neutron scattering (QENS) experiments and theoretical approach using nested hierarchical convolution of the motional processes (1). However, dMm does not consider the presence of non-bilayer lipids and lipid phases, which have been shown to play very significant roles in the two main energy-converting membranes of the biosphere, the inner-mitochondrial membranes (IMMs) and plant thylakoid membranes (TMs) (2).

The overall goal of this project is to extend dMm to membranes containing non-bilayer lipids and displaying polymorphic lipid phase behavior. To this end, we have carried QENS (IN5@ILL) and membrane diffraction experiments (D16@ILL) on model membranes mimicking the composition of IMMs, using mixtures of a bilayer lipid DOPC (dioleoyl-phosphatidylcholine) and non-bilayer propensities, DOPE (dioleoyl-phosphatidyl-ethanolamine), respectively, at different temperatures and hydrations.

QENS experiments – performed on DOPC:DOPE mixtures (100:0, 50:50, 20:80 mol%) under controlled hydrations (10 and 30 mol% water contents) and temperatures (5 and 50 °C) – revealed at least three different kinds of motions which were clearly different in the samples containing 80% DOPE compared to membranes with lower DOPE contents. Membrane diffraction experiments – carried out at 5, 15, 25, 35 and 50 °C, and relative humidities (RH, 50% and fine-tuned between 80 and 100%) – have revealed an H_II phase clearly emerging already in 50:50 mol% DOPC:DOPE at 5 °C at 80% RH, and at lower RHs at all temperatures; and in 20:80 mol% DOPC:DOPE at ≥25 °C even at 100% RH. In DOPE containing assemblies both the lamellar and H_II phase d-spacings increased at higher RHs and decreased at higher temperatures – reflecting in the lamellar phase an increase of the repeat distance arising from bilayer thickness and interlamellar water, while in the H_II phase the changes originated from swelling of the lipid monolayer geometry and the associated water-filled tubular cores.

These data are being complemented with model calculations using coarse-grain molecular dynamics simulations and time-resolved fluorescence spectroscopy using lipophilic dyes – under conditions comparable to the QENS and membrane diffraction experiments.

Our data show that the structure of DOPC:DOPE model membranes, and their lipid phases, in particular, dynamically respond to changes in their physico-chemical environment.

References:

• Garab G. et al. Physiologia Plantarum. 2025, 177(2). ISSN 0031-9317.
• Bicout D. J. et al. BBA – Biomembranes. 2022, 1864(9). ISSN 0005-2736

Lavrič M.*, Jožef Stefan Institute, Ljubljana, Slovenia
Bar L., Faculty of Electrical Engineering, Ljubljana, Slovenia
Cordoyiannis G., Jožef Stefan Institute, Ljubljana, Slovenia
*marta.lavric@ijs.si

Low-complexity biomimetic membranes such as supported lipid bilayers serve as platforms to study processes connected by membrane organization, lipid transfer and interactions with nanoparticles or biomolecules.

Quartz Crystal Microbalance with Dissipation monitoring QCM-D is a highly sensitive and versatile experimental technique, using the acoustic waves generated by the oscillation of a piezoelectric crystal quartz sensor to monitor mass variations, down to only a few ng/cm2, at the sensor-sample interface (1). Supported lipid bilayers (SLBs) used as biomimetic membranes in studies with QCM-D should be of good quality.

We investigated the influence of several parameters on the quality of SLBs formed on Au- and SiO2-coated sensors. The influence of the aqueous medium (i.e., buffer type) and the adsorption temperature, above and below the lipid melting point, was explored for SLBs of DMPC and DPPC formed by a solvent exchange (2).

References:
(1) Reviakine, I., Johannsmann, D., Richter, R.P., Anal. Chem., 83, 8838–8848, (2011).
(2) Lavrič M., Bar L., Villanueva M.E., Losada-Pérez P., Iglič A., Novak N., Cordoyiannis G., Sensors, 24, 6093 (2024).

Paloncýová Markéta,* The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Kührová Petra, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Šrejber Martin, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Čechová Petra, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic
Otyepka Michal, The Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Czech Republic; IT4Innovations, VŠB – Technical University of Ostrava, Czech Republic

*marketa.paloncyova@upol.cz

Lipid nanoparticles (LNPs) have emerged as key vehicles for nucleic acid (NA) delivery, promising novel tool for vaccination, cancer and rare diseases therapy. LNPs are complex structures, composed of ionizable lipids (ILs), helper lipids, PEGylated lipids and cargos. ILs are responsible for efficient encapsulation of the NA cargo and for NA release during endosome maturation. The dynamic nature of LNP pH dependent behavior inside a target cell is a major challenge in experimental research of LNP behavior, therefore a full understanding of LNP structure and processes related to the cargo release are still missing.

Molecular dynamics (MD) simulations with multiscale resolution offer a powerful approach to explore LNP organization and function in various environments (1). All-atom simulations can describe the interactions of individual lipid functional groups with NAs and their mutual effect on their structure and stability, but are limited to either small models or a short simulation of a prebuilt LNP. On the other hand, coarse-grained (CG) simulations can be used to simulate the formation of a whole LNP in tens of nanometers and microseconds scale, predicting the internal LNP organization, distinguishing between lipid inverse hexagonal and lamellar phase (2). The lower computational costs allow CG simulations to study LNP in a systematic way, manipulating the composition and ratio of lipid species or in a desired bioenvironment in its path through the body, getting us closer to the description of the mechanism of the endosomal escape process.

The potentials and limitations of both the resolutions can be efficiently combined to a valuable workflow, advancing our understanding of LNP structure, stability and behavior. The provided insight can lead to a targeted in-silico design of next-generation delivery platforms, increasing the cargo delivery efficiency and decreasing the costs. Their integration into formulation workflows represents a promising direction for predictive, mechanism-informed design of therapeutic systems.

References:
(1) Paloncýová, M. et al., Mol. Pharm, 22, 1110–1141 (2025).
(2) Kjølbye, L. R. et al., J. Chem. Theory Comput. https://doi.org/10.1021/acs.jctc.5c01207. (2025)

Žiga Pandur*, Faculty of Mechanical Engineering, University of Ljubljana, Aškerčeva cesta 6, 1000 Ljubljana, Slovenia
Mitja Drab, Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, 1000 Ljubljana, Slovenia
Aleš Iglič, Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, 1000 Ljubljana, Slovenia
David Stopar, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
*e-mail address: ziga.pandur@fs.uni-lj.si

Cavitation, rapid vaporization and bubble collapse due to a local change in pressure, is a widely used method in industry and research for cleaning, disinfection, dispersion, cell disruption, isolation of cell components, drug delivery, etc. (1). Furthermore, it is recognized as effective and chemical free novel advanced water and wastewater treatment process (1-3). The macroscopic effects of cavitation on bacteria are the results of implosions of a large number of bubbles (1,4). However, the effects of the large bubble clusters do not reveal the inherent nature of cavitation and a plethora of possible cavitation modes of action on bacteria (mechanical, physical, chemical). As stated by Prosperetti, thousands of papers have been devoted to the subject of bubbles, yet the exact mode of action of the bubble has not been elucidated (5). To evaluate the effect of cavitation on bacteria at a fundamental level, one needs to downscale the cavitation process to a single cavitation bubble which is similar in size to a bacterial cell.

The study examines how a single cavitation event on a microscale affects attached bacterial cells. The method enables accurate spatial and temporal manipulation of generation of cavitation microbubble with optical microscopy to follow the response up of individual bacterial cells. The results show that permeabilization of E. coli cells across occurs on a microsecond-to-minute timescales and micrometer-scale distances. Fluorescence imaging of propidium iodide uptake showed that cells nearer to the cavitation center experienced faster and more extensive membrane disruption. A modified Goldman equation successfully modeled first-order uptake kinetics and matched the observed fluorescence dynamics. The model revealed that permeability decays exponentially over minutes and decreases with distance as 1/r, indicating active pore-resealing. Overall, the findings quantify spatial and temporal patterns of cavitation-induced cell damage and align with sonoporation studies and offer novel insights for optimizing cavitation-assisted drug delivery and biofilm disruption strategies.

References:
(1) Zupanc, M. et al. Effects of cavitation on different microorganisms: The current understanding of the mechanisms taking place behind the phenomenon. A review and proposals for further research. Ultrasonics Sonochemistry 57, 147–165 (2019).
(2) Šarc, A., Oder, M. & Dular, M. Can rapid pressure decrease induced by supercavitation efficiently eradicate Legionella pneumophila bacteria? Desalination and Water Treatment 57, 2184–2194 (2016).
(3) Zupanc, M. et al. Shear-induced hydrodynamic cavitation as a tool for pharmaceutical micropollutants removal from urban wastewater. Ultrasonics Sonochemistry 21, 1213–1221 (2014).
(4) Gogate, P. R. & Pandit, A. B. A review and assessment of hydrodynamic cavitation as a technology for the future. Ultrasonics Sonochemistry 12, 21–27 (2005).
(5) Prosperetti, A. Bubbles. Physics of Fluids 16, 1852–1865 (2004).

Sifre van Teeffelen*¹, Eulalie Lafarge², André P. Schroder³, Pierre Muller², Fabrice Thalmann², Yann Bretonnière¹, Léo Corne¹, and Carlos M. Marques¹
¹ University of Lyon, ENS Lyon, CNRS UMR 5182, Chemistry Laboratory, Lyon, 69342, France
² Charles Sadron Institute, CNRS UPR22 & University of Strasbourg, Strasbourg, 67000, France
³ CNRS, INSA Lyon, LaMCoS, UMR5259, Villeurbanne, 69621, France
*sifre.van_teeffelen@ens-lyon.fr

Lipid peroxidation is a defining feature of oxidative stress and a major factor in membrane dysfunction during aging, inflammation, and disease. While oxidation-induced changes in bilayer structure are well recognized (1), their impact on the main phase transition of unsaturated phospholipids remains unexplored (2). Here, we investigate how hydroperoxidation of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) influences its phase behavior.

Mixtures of SOPC and its oxidized form (SOPC–OOH) were analyzed using differential scanning calorimetry (DSC) and steady-state fluorescence spectroscopy with two environment-sensitive probes: Laurdan (3) and a new solvatochromic dye A10 (4). The incorporation of hydroperoxidized acyl chains perturbed membrane thermodynamics, revealing a complex phase-transition landscape governed by the interplay between acyl-chain ordering and the spatial distribution of OOH groups.

These results highlight the sensitivity of lipid phase behavior to oxidative modifications at the molecular level and provide new insights into how lipid peroxidation modulates membrane organization in cellular and biomimetic systems.

References:
(1) Páez Pérez M., Vyšniauskas A., López Duarte I., Lafarge E.J., López Ríos De Castro R., Marques C.M., Schröder A.P., Müller P., Lorenz C.D., Brooks N.J., and Kuimova M.K., Commun. Chem., 6, 15 (2023).
(2) Wang G., Lin H.N., Li S., and Huang C.H., J. Biol. Chem., 270, 22738 (1995).
(3) Parasassi T., De Stasio G., Ravagnan G., Rusch R.M., and Gratton E., Biophys. J., 60, 179 (1991).
(4) Zheng Z., Caraguel F., Liao Y.-Y., Andraud C., van der Sanden B., and Bretonnière Y., RSC Adv., 6, 94200 (2016).

Villanueva M. E.*, Experimental Soft Matter and Thermal Physics (EST) group, Université libre de Bruxelles, Belgium.
Ruysschaert J. M., Structure and Functions of Biological Membranes, Université libre de Bruxelles, Belgium.
Bouchet A. M., Lifesome Therapeutics S. L., Madrid, Spain.
Losada-Pérez P. M., Experimental Soft Matter and Thermal Physics (EST) group, Université libre de Bruxelles, Belgium.
*martin.villanueva@ulb.be

The paradigm of asymmetry in lipid membranes lies in the differing physicochemical properties of the inner and outer bilayer leaflets, but it also occurs intramolecularly within lipid moieties (1). Though less explored, this intramolecular asymmetry critically defines molecular shape and dictates self-assembly in aqueous environments.

In this context, we have been studying a particular glycolipid called Ohmline (OHM), characterized by a significant asymmetry between its hydrophobic chain lengths. This lipid can spontaneously self-assemble into helical tubes (~ 15 nm thickness and tens of μm length) upon its deposition onto Au surfaces below its main transition temperature Tm (2). Beyond its biophysical interest, this lipid has also shown promise as an antimetastatic drug at low, non-toxic concentrations (3).

Here, we explore how mixing OHM with classical phospholipids modifies both nanotube formation and the equilibrium between competing self-assembled species. Using a combination of thermophysical characterization and atomic force microscopy, we mapped phase diagrams, assessed mixing behavior, and correlated lipid composition with the morphology of the resulting LNTs. We demonstrate that compositional control provides a straightforward and versatile means to tune the formation temperature and structural properties of LNTs. This opens new possibilities for the controlled design of next-generation nano(bio)structures with potential applications in different fields such as drug delivery, biosensing, and antifouling. Finally, we present preliminary yeast cell adhesion assays that highlight the influence of OHM-based assemblies and illustrate the complex interplay of factors governing cell–lipid interactions in different scenarios.

References:
(1) Feigerson G. W., et. al., J. Am. Chem. Soc. 145, 21717-21722 (2023).
(2) Villanueva, M. E., et. al., J. Colloid Interface Sci. 671, 410-422 (2024).
(3) Herrera, F. E., et al. ACS Omega 2, 6361–6370 (2017).

Iglič A. *, University of Ljubljana, Faculty of Electrical Engineering,  Ljubljana, Slovenia
Gongadze E., University of Ljubljana, Faculty of Electrical Engineering,  Ljubljana, Slovenia
Shubhadeep S., Weizmann Institute of Science, Rehovot, Israel
Sadhu R.K., Indian Institute of Technology, Kharagpur, India
Gov N., Weizmann Institute of Science, Rehovot, Israel and University of  Cambridge, Cambridge, UK
Kralj-Iglič V., University of Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia

*ales.iglic@fe.uni-lj.si

The membrane lipid bilayer composed of   zwiterionic  or  negatively charged  lipids  in contact with aqueous solution of monovalent salt ions was studied theoretically by using  mean-field theoretical approach and  simulations. Charge  distribution of   lipid head groups in lipid bilayer  is theoretically described  by  the  negatively charged planar surface which accounts for  negatively charged phosphate groups,  while other  positively and negatively charged  groups  are assumed to be fixed on the rod-like structures with rotational degree of freedom.   The spatial variation of relative permittivity  within the lipid head group region and outside in  electrolyte solution   is  derived within strict statistical mechanical approach considering also the orientational ordering of water molecules. It is shown that in the saturation regime close to the charged lipid surface, water dipole ordering and depletion of water molecules may result in a substantial  local decrease of  relative permittivity. An analytical expression for the osmotic pressure of the electrolyte solution between the zwitterionic lipid surface and a charged particle (macroion) also is derived. In addition, the theoretical description of the possible origin of the experimentally observed attractive interactions between like-charged membrane surfaces mediated by charged macroions with distinctive internal charge distribution is given. At the end the engulfment of  rigid or soft objects/particles  by  giant lipid vesicle or cell membrane is briefly  described by using   theoretical models and simulations, where the  electrostatic forces are also taken into account.

Kucharski M.*, Wrocław University of Science and Technology, Poland
Piątek F., Wrocław University of Science and Technology, Poland
Langner M, Wrocław University of Science and Technology, Poland
Magdalena Przybyło, Wrocław University of Science and Technology, Poland

*278733@student.pwr.edu.pl

Ascorbate is critical for many critical metabolic processes necessitating its sufficient intracellular level, which depends on the cell metabolic activity and location. The intracellular ascorbate concentration varies from 0.05 mM in erythrocytes to about 20 mM in corneal endothelium. In humans, these cells are imbedded in body fluids with ascorbate levels dependent on the temporal metabolic activity and the rate of exchange with surrounding, which is facilitated by epithelial cells (1).

On the cellular level ascorbate homeostasis is affected by the cell metabolic activity and transmembrane transport, both active and passive. The active transport relies on two ascorbate specific sodium coupled transporters (SVCT1 and SVCT2) (2), (3). We selected three distinct, from the ascorbate homeostasis point if views, cell types: two of them (cancer and differentiated cells) immerse in body fluids and enterocyte located on the border between intestine and serum. In the first two cell types only SVCT2 is present whereas epithelial cell has both SVCT1 and SVCT2 transporters, located on the apical and basolateral membranes, respectively (4). All three cell types are differing, among other things, in membrane potential values. Cancer cells have in general membrane potential lower than 50 mV, whereas membrane potential for differentiated cells is higher than 50 mV (5). Epithelial cell represented by enterocyte has two different membrane potentials, one on the apical and the other on the basolateral sides (6). The theoretical model of the cellular ascorbate homeostasis was constructed and used to evaluate its intracellular concentration depending on the cell location and membrane electrical potential difference across the plasma membrane. Simulations show that in cancer cells intracellular concentration equilibrates on the higher level than in differentiated cells and that the ascorbate may flow across enterocyte in both directions but at different rates. The ascorbate intake being more efficient than its release to the intestine.

References:

(1) M. Dosedel, E. Jirkovsky, K. Macakova, L. K. Krcmova, L. Javorska, J. Pourova, L. Mercolini, F. Remiao, L. Novakova, P. Mladenka and O. On Behalf Of The, Nutrients, 2021, 13.
(2) I. Savini, A. Rossi, C. Pierro, L. Avigliano and M. V. Catani, Amino Acids, 2008, 34, 347-355.
(3) H. Takanaga, B. Mackenzie and M. A. Hediger, Pflug Arch Eur J Phy, 2004, 447, 677-682.
(4) J. C. Boyer, C. E. Campbell, W. J. Sigurdson and S. M. Kuo, Biochem Bioph Res Co, 2005, 334, 150-156.
(5) E. Di Gregorio, S. Israel, M. Staelens, G. Tankel, K. Shankar and J. A. Tuszynski, Phys Life Rev, 2022, 43, 139-188.
(6) K. Thorsen, T. Drengstig and P. Ruoff, Am J Physiol Cell Physiol, 2014, 307, C320-337.

Primožič, Urša, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
Potočnik, Tjaša, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
Ladurantie, Caroline, Institute of Pharmacology and Structural Biology, Toulouse, France
Kolosnjaj Tabi, Jelena, Institute of Pharmacology and Structural Biology, Toulouse, France
Rols, Marie-Pierre, Institute of Pharmacology and Structural Biology, Toulouse, France
Maček Lebar, Alenka*, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
*alenka.macek.lebar@fe.uni-lj.si

Electroporation is a technique in which short electric pulses increase cell membrane permeability. It is used in medical applications like electrochemotherapy (ECT), pulsed field ablation (PFA), and gene electrotransfer (GET). Depending on pulse parameters, electroporation can be reversible or irreversible, induces mechanical stress, activates membrane resealing, and can lead to cell death. These processes may also stimulate extracellular vesicle (EV) release.

We compared the concentration and size of EVs released from Chinese hamster ovary (CHO) cells 2 and 4 hours after electroporation using three protocols commonly applied in ECT (eight 100 µs pulses, 1 Hz), GET (eight 5 ms pulses, 1 Hz), and PFA (50 bursts of 50 bipolar 2 µs pulses). EV size distribution and concentration were measured by nanoparticle tracking analysis (NanoSight LM10).

All electroporation protocols increased EV release compared to control cells (5.74 × 10⁶ particles/ml). The highest EV concentration was observed after PFA pulse protocol (86.56 × 10⁶ particles/ml), significantly exceeding that after ECT (21.54 × 10⁶ particles/ml) and GET (59.33 × 10⁶ particles/ml) pulse protocols. EVs released after GET pulse protocol were significantly larger (119 nm) than those released after ECT (107 nm) and PFA (105.5 nm) pulse protocols and control cells (103 nm). The concentration of released EVs using PEF protocol was significantly lower after 4 h of incubation than after 2 h.

Author: Matan Mussel, Department of Physics, University of Haifa, Israel
E-mail: mmussel@univ.haifa.ac.il

Abstract: We present a theoretical framework that captures the electrical response of lipid membranes to thermodynamic perturbations, revealing two key phenomena observed experimentally in nonliving systems. First, we show that nonlinear compression waves propagating within the membrane are accompanied by co-propagating voltage signals, resulting from variations in the membrane’s charge density. Second, by incorporating phase-dependent changes in membrane permeability, we identify a regime of spontaneous, repetitive voltage oscillations across the membrane. The resulting voltage and time scales align with experimental observations. Interestingly, many of these features resemble fundamental properties of nerve action potentials. We discuss these findings in the context of the ongoing debate about the physical nature of the action potential, proposing new predictions and highlighting unresolved questions.

References:
(1) M Mussel, and MF Schneider (2019), It sounds like an action potential: unification of electrical, chemical and mechanical aspects of acoustic pulses in lipids, Journal of the Royal Society Interface, 16(151): 20180743.
(2) S Das and M Mussel (2023), Characterizing oscillatory and excitability regimes in a protein-free lipid membrane, Langmuir, 39(16): 5752–5760.
(3) M Mussel (2023), Information propagated by longitudinal pulses near a van der Waals phase transition, Physical Review E, 108: 034209.

Rems L.*, University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia
Čagalj J., University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia
*lea.rems@fe.uni-lj.si

Electroporation enhances membrane permeability for therapeutic applications [1], but understanding where pores preferentially form in cellular membranes remains challenging. While molecular dynamics (MD) simulations have revealed pore formation in both lipid domains and voltage-gated ion channels (VGICs) [2], the relative likelihood of these events in actual plasma membranes is unclear due to complex transmembrane voltage dynamics during cellular electroporation.

We systematically compared poration rates using atomistic MD simulations of POPC bilayers, plasma membrane lipid domains with complex composition [3], and membranes containing clinically relevant VGICs. Our results demonstrate that pore formation time decreases exponentially with increasing transmembrane voltage across all systems. Notably, some VGICs exhibited enhanced susceptibility to poration, with “complex pores” forming within voltage-sensor domains and becoming stabilized by both lipid head-groups and amino-acid residues.

These findings suggest that electroporation treatments may have specific effects on excitable tissues beyond simple membrane permeabilization, particularly relevant for cardiac ablation and skeletal muscle gene delivery. The structural perturbation of voltage sensors aligns with experimentally observed decreases in voltage-dependent ionic currents following pulse treatment. Future work incorporating free energy calculations could enable quantitative predictions for optimizing electroporation protocols while minimizing unintended ion channel dysfunction.

References:
(1) Campelo S.N., Huang P.-H., Buie C.R., Davalos R.V, Annu. Rev. Biomed. Eng., 25, 77-100 (2023).
(2) Rems L., Kasimova M.A., Testa I., Delemotte L., Biophys. J., 119, 190-205 (2020).
(3) Rems L., Tang X., Zhao F., Perez-Conesa S., Testa I., Delemotte L., eLife, 11, e74773 (2022).

Aneta Stefanovska*, Physics Department, Lancaster University, Lancaster, UK

*aneta@lancaster.ac.uk

The resting membrane potential is commonly treated as a static equilibrium quantity set by ionic gradients and membrane conductances. Yet, high-resolution electrophysiological recordings reveal persistent fluctuations even under steady resting conditions, raising fundamental questions about their physical origin and physiological relevance. From a biophysical perspective, it remains unclear to what extent these fluctuations reflect thermal noise, stochastic channel dynamics, or active, energy-dependent processes.

In this talk, I will present patch-clamp measurements of resting membrane potential fluctuations obtained under controlled variations of extracellular sodium, potassium, and chloride concentrations. By systematically modifying ionic driving forces, we probe how individual ion species shape both the mean membrane potential and its temporal variability. Time-resolved analysis of the recorded signals reveals structured fluctuations that cannot be fully explained by equilibrium noise models.

These results support a view of the resting membrane potential as a dynamically maintained, non-equilibrium state, rather than a passive baseline. Interpreting membrane potential fluctuations as a biophysical signature of ongoing ionic and channel activity provides new insight into cellular stability, excitability, and the limits of classical resting-state descriptions.

Brocklehurst, J.* School of Chemical and Process Engineering, University of Leeds, UK
Connell, S. School Physics and Astronomy, Molecular and Nanoscale Physics, University of Leeds, UK
Rappolt, M. School of Food Science and Nutrition, University of Leeds, UK
*ll16j2b@leeds.ac.uk

The binary phase diagram of dimyristoylphosphatidylcholine (DMPC)/cholesterol has been widely studied for a better understanding of cholesterol’s role in regulating the fluidity of plasma cell membranes. In this study, we focus on the interplay between the lipids and the confined water at the bilayer/water interface. Applying the recently published Three-Water Layer (TWL) model (1), DMPC/cholesterol dispersions spanning 0-35 mol% cholesterol were analysed, using small-angle X-ray scattering over a 15-60 °C temperature range. The data were analysed using a global fitting method, wherein the electron density profile was refined with a three Gaussian model, mimicking the lipid headgroup, the methyl trough region, and the cholesterol insertion within the lipid leaflets. Dilatometry was performed over a 26-60 °C temperature range, to obtain volumetric data.

Key structural parameters of the bilayer concern the Gibbs dividing surface, the headgroup-to-headgroup distance, the headgroup extension, hydrocarbon chain length, and area per lipid. Additionally, three water layers are discerned, relating to bound water at the headgroup, perturbed water, and unperturbed water. Generally, the thickness of the perturbed water layer increases with temperature and membrane fluidity, whereas the thickness of the free water core exhibits the opposite trend. The relative position of the cholesterol within the bilayer, depends on both the hydration and structural properties of the membrane, displaying the deepest insertion in the miscibility gap of the liquid disordered, Ld, and liquid ordered, Lo, phases. In sum, the extracted structural parameters and membrane hydration data from the TWL model gave us a greater insight of the hydration behaviour of the Ld and Lo phases.

Reference:
(1) Vancuylenberg, G., Sadeghpour, A., Tyler, A.I.I., Rappolt, M. Soft Matter, 19, 5179 (2023)

Nasim Hosseinlar, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Marija Srnko, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Franci Merzel, National Institute of Chemistry, Theory Department, Ljubljana, Slovenia
Gregor Anderluh, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
* Nasim.hosseinlar@ki.si

Pore-forming proteins (PFPs) are a structurally and functionally diverse class of membrane-associated proteins with critical biological roles and emerging biotechnological applications. Actinoporins, a subclass of α-pore-forming toxins from marine organisms, form oligomeric pores in sphingomyelin- and cholesterol-rich membranes1. The actinoporin-like protein Fav, derived from the coral Orbicella faveolata, has attracted interest for nanopore sensing due to its lipid specificity and pore-forming capability2.
To enhance Fav’s structural stability, we engineered cysteine substitutions at selected sites to promote disulfide bond formation. Molecular dynamics simulations revealed that these mutations lead to a narrower pore radius and reduced backbone fluctuations, as indicated by lower RMSD and RMSF values compared to the wild type. This suggests a more rigid and stable pore conformation, potentially improving Fav’s robustness for biosensing applications.
Complementary experimental work is underway to validate the computational findings. Preliminary purification results indicate that most engineered variants are soluble after affinity column chromatography, suggesting that the introduced disulfide bonds do not impair protein folding. These initial observations support the feasibility of stabilizing Fav through targeted cysteine engineering for future structural and functional studies.

1. Rojko, N., Dalla Serra, M., MačEk, P. & Anderluh, G. Pore formation by actinoporins, cytolysins from sea anemones. Biochimica et Biophysica Acta (BBA) – Biomembranes 1858, 446–456 (2016).
2. Šolinc, G. et al. Cryo-EM structures of a protein pore reveal a cluster of cholesterol molecules and diverse roles of membrane lipids. Nature Communications 2025 16:1 16, 1–11 (2025).

The EV Unit at the Life Sciences Core Facilities of the Weizmann Institute of Science offers a comprehensive, multidisciplinary platform for advanced extracellular vesicle (EV) research, supporting projects from the early stage of the research to translational applications. Our mission is to empower academic and industry collaborators with cutting-edge, reproducible, and scalable solutions across EV characterization, functional studies, cargo analysis and therapeutic development.
We provide integrated, end-to-end workflows encompassing EV production, isolation, and in-deep characterization, leveraging advanced biophysical analyses (NTA, DLS, zeta potential), high-resolution imaging (cryo-TEM, super-resolution microscopy, IncuCyte), and small-particle flow cytometry. These capabilities are further enhanced by multi-omics profiling, including next-generation sequencing (NGS), high-resolution proteomics, and targeted metabolomics.
Our unit collaborates closely with other state-of-the-art Core Facilities at Weizmann such as electron microscopy, image stream flow cytometry, and omics units to deliver fully customized and multidimensional EV analysis pipelines. This synergistic approach enables us to support diverse research goals, from fundamental mechanistic studies in basic science to preclinical pipelines in drug development, diagnostics, and delivery systems.

Matija Ruparčič*, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Gregor Anderluh, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
*matija.ruparcic@ki.si

Cone snails are marine gastropods that successfully hunt worms, fish, or other mollusks by utilizing their complex venom [1]. The main components of the venom are so called conotoxins – short bioactive peptides that target membrane receptors. The venom is also comprised of larger proteins, which are believed to be involved either in conotoxin maturation or envenomation [2]. Among the latter are actinoporin-like proteins – conoporins – which are highly expressed in the venom glands of several species, suggesting a key role in envenomation. Since only one conoporin has been functionally characterized to date, the biological roles of these pore-forming toxins remain unclear [3]. Based on known mechanisms of other pore-forming toxins, we propose four potential roles for conoporins: (i) permeabilization of epithelial barriers, (ii) facilitation of conotoxin translocation through the pore, (iii) antimicrobial activity, and (iv) involvement in digestion [4].

Through bioinformatic analysis, we identified 95 unique conoporin sequences from 27 species. Of these, 22 are vermivorous, 5 are piscivorous, and interestingly, no conoporin sequences were found in molluscivorous snails. Compared to actinoporins, conoporins contain extensions at the N- and C-termini, while retaining a conserved β-sandwich core, similar to other molluscan actinoporin-like proteins [3,5]. When plotted on a phylogenetic tree with actinoporins and other actinoporin-like proteins, conoporins cluster together and form at least three distinct clades, with the main driver for this appearing to be sequence variation in the N- and C-termini.

References:
1. Tucker, J.K.; Tenorio, M.J. Illustrated Catalog of the Living Cone Shells; MdM Publishing: Wellington, FL, USA, 2013; ISBN 978-0-9847140-2-5.
2. Robinson, S.D.; Norton, R.S. Conotoxin Gene Superfamilies. Mar. Drugs 2014, 12, 6058–6101, doi:10.3390/md12126058.
3. Koritnik, N.; Gerdol, M.; Šolinc, G.; Švigelj, T.; Caserman, S.; Merzel, F.; Holden, E.; Benesch, J.L.P.; Trenti, F.; Guella, G.; et al. Expansion and Neofunctionalization of Actinoporin-like Genes in Mediterranean Mussel (Mytilus Galloprovincialis). Genome Biol. Evol. 2022, 14, doi:10.1093/gbe/evac151.
4. Ruparčič, M.; Šolinc, G.; Caserman, S.; Galindo, J.C.G.; Tenorio, M.J.; Anderluh, G. The Biological Role of Conoporins, Actinoporin-like Pore-Forming Toxins from Cone Snails. Toxins 2025, 17, 291, doi:10.3390/toxins17060291.
5. Gorbushin, A.; Ruparčič, M.; Anderluh, G. Littoporins: Novel Actinoporin-like Proteins in Caenogastropod Genus Littorina. Fish Shellfish Immunol. 2024, 151, 109698, doi:10.1016/j.fsi.2024.109698.

Fabrizio Cillo, fabrizio.cillo@cnr.it, National Research Council (CNR), Institute for Sustainable Plant Protection, Bari, Italy.
Valeria Daniello, CNR, Institute for Sustainable Plant Protection, Bari, Italy
Wilma Sabetta, CNR, Institute for Biosciences and Bioresources, Bari, Italy

Tetraspanins are conserved four-pass transmembrane proteins involved in membrane organization, protein trafficking, and cellular communication. In plants, they participate in development, stress responses, and pathogen interactions, but remain less studied than their animal counterparts [1].
We identified and characterized 17 tomato (Solanum lycopersicum) tetraspanins (SlTETs) through genome-wide and phylogenetic analyses. Several SlTETs clustered into distinct clades with their Arabidopsis thaliana orthologs, suggesting conserved functions [2]. Structural features, including the plant-specific GCCK/RP motif and conserved cysteines in EC2, support their structural conservation and membrane anchoring capacity [3]. Structural 3D modeling indicates a cone-shaped transmembrane configuration, consistent with what observed in other species [4].
Expression profiling revealed tissue-specific patterns and changes upon pathogen infection. Transcriptional changes due to virus infections possibly correlate with virus-induced massive modifications of cellular membrane structures [5].
We generated SlTET interaction networks with other proteins. The resulting interactome suggests that individual SlTETs are involved in vesicle trafficking, membrane remodeling, ion transport, and signaling. Some interactor proteins are shared among multiple SlTETs.
Immuno-electron microscopy and immunoblotting using anti-TET8 antibodies confirmed SlTET8 association with nanovesicle membranes, as in Arabidopsis [6].
In conclusion, the characterization of tomato tetraspanins highlights their potential involvement in membrane organization, vesicular trafficking and extracellular transport.

Cited Bibliography:

1. Reimann, R., Kost, B., & Dettmer, J. (2017). TETRASPANINs in plants. Front. Plant Sci., 8, 545.
2. Wang F. et al. (2015). Functional Analysis of the Arabidopsis TETRASPANIN Gene Family in Plant Growth and Development. Plant Physiol., 169(3), 2200–2214.
3. Zhu T. et al. (2022). Arabidopsis Tetraspanins Facilitate Virus Infection via Membrane-Recognition GCCK/RP Motif and Cysteine Residues. Front. Plant Sci., 13, 805633.
4. Rubinstein E. et al. (2025). Tetraspanins affect membrane structures and the trafficking of molecular partners: what impact on extracellular vesicles? Biochem. Soc. Trans., 53(2), 371–382.
5. He, R., et al. (2023). Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants. Viruses, 15(3), 744.
6. Liu N. et al. (2024). Arabidopsis TETRASPANIN8 mediates exosome secretion and glycosyl inositol phosphoceramide sorting and trafficking. The Plant Cell, 36(3), 626–641.

Spruk G., National Institute of Chemistry, Slovenia
Šolinc G., National Institute of Chemistry, Slovenia
Anderluh G., National Institute of Chemistry, Slovenia
Crnković A., National Institute of Chemistry, Slovenia
gregor.spruk@ki.si

Nanopore sensing is a versatile method that can be adapted for detection of a wide variety of analytes. Since its successful application in nucleic acid sequencing, attention in this field has shifted to protein sequencing. However, translocation of protein analytes through a nanopore necessitates their denaturation with chaotropic agents, which can also destabilize the protein nanopores most commonly used in these experiments. To minimize the effects of chaotropic agents on the pore, we used genetic code expansion to engineer stabilized nanopore variants of the pore-forming protein bryoporin (Bry) (1), incorporating non-canonical amino acids (ncAAs).

To produce stabilized Bry pores, we introduced either m-chloro-tyrosine (ClY) or p-pentafluorosulfanyl phenylalanine (SF5) at various positions on the interfaces between Bry pore protomers. We tested the efficiency of ncAA incorporation and the solubility of the produced variants using SDS polyacrylamide gel electrophoresis (PAGE). The oligomerization ability of the soluble variants with successfully incorporated ncAAs was then tested using native PAGE, and the stability of the formed oligomers in guanidinium chloride (GdmCl) was assessed by nano-differential scanning fluorimetry (nanoDSF). Finally, pores of the most stable variants were purified using ion exchange chromatography for characterization by cryo-electron microscopy and experiments on planar lipid bilayers.

While most variants containing SF5 showed promising results in nanoDSF experiments, they had very poor solubility and their pores tended to aggregate, preventing their use in further experiments. In contrast, ClY-containing variants were much more soluble, but the ncAA positions that stabilized the pores were less common. For the most stable ClY-containing variant, we observed pore insertions into lipid bilayers in up to 3 M GdmCl, whereas Bry pores without ncAAs did not insert if the GdmCl concentration exceeded 1.75 M. These results are promising for achieving our next goal of translocating and identifying full-length model protein analytes.

References:
(1) Šolinc G., Švigelj T., Omersa N., Snoj T., Pirc K., Žnidaršič N., Yamaji-Hasegawa A., Kobayashi T., Anderluh G., Podobnik M., Journal of Biological Chemistry, 298(10), 15 (2022)

Piątek F.*, Wrocław University of Science and Technology, Poland
Langner M., Lipid Systems sp. z o.o., Wrocław, Poland
Borowik T. Lipid Systems sp. z o.o., Wrocław, Poland

*278786@student.pwr.edu.pl

Abstract

Phosphatidylcholine-based liposomes are widely recognized as effective drug delivery systems, synthesized through the hydration of a lipid-rich organic phase by an aqueous phase. The critical step in defining their final physicochemical properties is extrusion—a mechanical process forcing the lipid suspension through polycarbonate membranes. While the impact of lipid concentration is known, the complex interplay between the solvent composition, ionic environment, and the mechanical energy required for processing remains underexplored. This study aims to elucidate how these variables affect both the liposome characteristics and the energy requirement of the manufacturing process. To investigate the impact of the ionic environment, the aqueous phase was modified with chaotropic and kosmotropic salts, as well as surface active compound – ascorbate. The formation of liposomes was evaluated by self designed, force – controlled extrusion system and the energy consumption during extrusion was quantified by processing force-time profiles at constant velocity, using trapezoidal integration scaled by the piston speed. Liposomes homogeneity was characterized by dynamic light scattering technique and zeta potential. Our study indicates that solvent composition acts as a primary modulator of vesicle formation characteristics, and that the distinct chaotropic and kosmotropic properties of the additives influence the membrane formation process. The quantitative analysis revealed a direct correlation between the formulation’s rheological behavior and the mechanical energy input required for extrusion.

The study confirms that balancing the interplay between solvent ratios and ionic additives is crucial for optimizing phospholipid liposomes formation process. By carefully selecting the aqueous phase composition, it is possible to tune the Critical Quality Attributes of the nanocarriers while monitoring and potentially optimizing the energy load of the extrusion process to achieve the high-quality liposome suspension.

References:

1. Hunter, D. G., & Frisken, B. J. (1998). Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles. Biophysical Journal, 74(6), 2996-3002
2. Doskocz, J., Dałek, P., Foryś, A., Trzebicka, B., Przybyło, M., Mesarec, L., Iglic, A., & Langner, M. (2020). The effect of lipid phase on liposome stability upon exposure to the mechanical stress. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1862(10), 183361
3. Lo Nostro, P., & Ninham, B. W. (2012). Hofmeister phenomena: an update. Chemical Reviews, 112(4), 2286-2322

Jelena Danilović Luković¹, Anna Romolo²,  Matej Hočevar³, Darja Feizpour³, Barbara Šetina Batič³, Tina Sever³, Isidora Santrač¹, Marija Tanović¹, Valentina Ćurić⁴, Ana Obaha⁵, Jan Dolinar⁶, Andrej Race⁷, Aleš Iglič⁸, Vankata Subba Rao Jampani⁹, Marko Novinec⁵, Ivan Jerman⁷, Veronika Kralj-Iglič²

¹Institute of Multidisciplinary Research, University of Belgrade, Belgrade, Serbia, ²University of Ljubljana, Faculty of Health Sciences,Ljubljana, Slovenia, ³Institute of Metals and Technology, Ljubljana, Slovenia, ⁴University of Belgrade, Faculty of Biology, Belgrade, Serbia, ⁵University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia, ⁶University of Ljubljana, Faculty of Physics, Ljubljana, Slovenia, ⁷ National Institute of Chemistry, Ljubljana, Slovenia, ⁸ University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia, ⁹J. Stefan Institute, Ljubljana, Slovenia

Microalgae are of great importance for ecology and industry; however, the mechanisms of ecosystem resiliency and production of economically relevant compounds should be better understood. Here, we focus on adaptive response of microalgae to heavy metals, specifically, manganese (Mn) and copper (Cu). In line with the observation on human neuroblastoma cells in vitro, which expel the metals outside the cell using extracellular particles (EPs) to resist toxicity, we investigated if this could be applied to microalgae. Chlorella sorokiniana cultures were treated with 1mM MnCl₂ and CuCl₂ at the early stationary phase (20 days old). During 4 days after treatment, supernatants and pellets (obtained by centrifugation) were observed using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) with Energy Dispersive Spectroscopy (EDS). We assessed number density n and hydrodynamic diameter D_h of EPs by Interferometric Light Microscopy (ILM). We performed mass photometry and Raman spectroscopy. SEM revealed numerous microalgae surrounded by amorphous mucilage. In supernatant, we observed fractal structures, which were particularly abundant in metal-treated samples. Controls composed of water-added phosphates and copper in the same concentrations as in the culture showed the presence of similarly shaped, albeit larger globular structures. In the pellet of control and Cu-treated samples, SEM and TEM revealed small amount of globular particles. Raman spectroscopy revealed signal of beta carotene in all samples. Mass photometry showed small amount of molecules between 100 and 200 kDa. Isolates was poor in content. Our results indicate that the extracellular particles observed in microalgae samples were formed by phosphates contained in the mucilage shed by microalgae. ILM showed the presence of numerous particles (in the range of 10⁹ per mL in the culture and up to 10 times larger) in the supernatant while the detected number density in the culture was under the detection limit. Measurements of the pellets were not feasible due to the presence of the mucilage. TEM EDS of Cu-treated samples exhibited in the globular particles an increased content of metals with respect to the surroundings. Our results support the assumption of clearance of copper by EPs composed by extracellular material shed by microalgae.

Zuzana Janáčková*, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic

Semen Yesylevskyy, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic

Pavel Jungwirth, Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech Republic

*email: zuzana.janackova@uochb.cas.cz

Cell-penetrating peptides (CPPs) hold great promise as novel drug delivery vehicles, yet the molecular mechanisms underlying their passive membrane translocation remain poorly understood. Among them, oligoarginine peptides are particularly potent CPPs and serve as a minimal model system to investigate translocation mechanisms. (1, 2) Here, we explore the role of membrane curvature and multilamellarity in peptide–membrane interactions using a combination of atomistic molecular dynamics simulations, enhanced sampling methods, and cryo-electron microscopy (cryo-EM). Cryo-EM data reveal that oligoarginine peptides markedly remodel membrane vesicles and cellular membranes, inducing invaginations and multilamellar structures. Complementary simulations show that oligoarginines exhibit distinct binding affinities to flat versus curved membranes. In double-membrane systems, R9 peptides effectively bridge adjacent bilayers, driving their spontaneous “zipping,” in agreement with the multilamellar morphologies observed experimentally. Together, these findings provide direct atomistic insight into peptide-induced membrane remodeling and identify curvature and multilamellarity as key factors contributing to the passive translocation of oligoarginine peptides.

(1) Bechara, C.; Sagan, S. FEBS Letters, 587, 1693–1702 (2013).

(2) Allolio, C.; Magarkar, A.; Jurkiewicz, P.; Baxová, K.; Javanainen, M.; Mason, P. E.; Šachl, R.; Cebecauer, M.; Hof, M.; Horinek, D.; Heinz, V.; Rachel, R.; Ziegler, C. M.; Schröfel, A.; Jungwirth, P. Proceedings of the National Academy of Sciences, 115, 11923–11928 (2018).

Sani Muhammad Ismail*, National Institute of chemistry Ljubljana, Slovenia
Andreja Prešern., National Institute of chemistry Ljubljana, Slovenia
Katerina Naumoska, National Institute of chemistry Ljubljana, Slovenia
Alen Albreht, National Institute of chemistry Ljubljana, Slovenia
Gregor Anderluh, National Institute of chemistry Ljubljana, Slovenia
*sanimuhammad.ismail@ki.si

Glycosylinositol phosphorylceramides (GIPCs) are essential plant sphingolipids that play critical structural roles in plant cells. Their interaction with specific pathogen-derived proteins, such as necrosis- and ethylene-inducing peptide 1–like proteins (NLPs), can trigger plant cell necrosis, highlighting their importance in plant-pathogen interactions and defense mechanisms. Despite their significance, current methods for GIPC extraction are labor-intensive, time-consuming, and require large volumes of solvents, limiting detailed biochemical and functional studies. In this study, we aimed to develop a more efficient and reproducible approach for the extraction and purification of GIPCs from leek (Allium porrum). We systematically evaluated a range of solvents and solvent mixtures to identify conditions that maximize GIPC yield while preserving lipid integrity. The most promising extraction procedure was subsequently upscaled and further optimized to improve efficiency and reproducibility, while minimizing solvent consumption. In the final purification step, flash chromatography was applied to isolate GIPC series B, and the identity of the fraction was confirmed by LC-MSn. This workflow was developed as a high-throughput alternative to our recently reported HPTLC-based GIPC isolation, providing high-quality material suitable for downstream biochemical analyses, including structural characterization and functional assays. The approach presented here offers a practical solution for researchers studying plant sphingolipids and their roles in cellular processes and plant defense.

Renzo A. Condori Tolentino*, LBMC and Chem. Lab., Univ. Lyon, ENS- Lyon, 69342 Lyon, France
Paulo C. T. Souza, LBMC, Univ. Lyon, ENS- Lyon, 69342 Lyon, France
Carlos M. Marques, Chem. Lab., Univ. Lyon, ENS- Lyon, 69342 Lyon, France
*renzo.condori_tolentino@ens-lyon.fr

Plastic-derived pollution has reached most corners of our planet, including those where the physical and physico-chemical parameters of the environment deviate significantly from their average on Earth. Microplastics and their degradation products reach even extreme environments such as those with high salinities like in the Dead Sea or Lake Tyrrell. Understanding how such pollutants affect cellular function thus requires studying their impact across the full range of parameters relevant to extremophile life. Among polystyrene degradation products, SOs are small, hydrophobic molecules that partition into lipid membranes and disrupt their thermodynamic balance (1). Although trace amounts can alter the physicochemical equilibrium underlying biological function (1), their behaviour under extreme salinity remains poorly understood—particularly for halophilic and other extremophilic organisms, for which both SOs and ions serve as key stressors.

To address this gap, we employ coarse-grained molecular dynamics simulations using the Martini 3 force field (2) to examine how monovalent and divalent salts (NaCl, CaCl₂, MgCl₂) modulate lipid bilayer structure and dynamics. Structural metrics such as area per lipid, thickness, lateral diffusion and order parameters quantify salinity-dependent perturbations, while varying SO concentration reveals how pollutant accumulation reorganizes membranes across ionic conditions. Because bilayer physical state governs packing and permeability (3, 4), we compare gel and liquid phases to identify phase-dependent mechanisms of destabilization. Overall, this work provides molecular-level insight into how salinity and nanopollutants jointly modulate membrane integrity under environmental extremes.

References:
(1) Morandi, M. I., Kluzek, M., Wolff, J., Schroder, A., Thalmann, F., and Marques, C. M. Proc. Natl. Acad. Sci. USA 118, e2016037118 (2021).
(2) Souza, P. C. T., Alessandri, R., Barnoud, J., Thallmair, S., Faustino, I., Grünewald, F., Patmanidis, I., Abdizadeh, H., Bruininks, B. M. H., Wassenaar, T. A., Kroon, P. C., Melcr, J., Nieto, V., Corradi, V., Khan, H. M., Domański, J., Javanainen, M., Martinez-Seara, H., Reuter, N., Best, R. B., Vattulainen, I., Monticelli, L., Periole, X., Tieleman, D. P., de Vries, A. H., and Marrink, S. J. Nat. Methods 18, 382–388 (2021).
(3) Dimova, R., and Marques, C. M., The Giant Vesicle Book, CRC Press, Boca Raton (2019).(4) Pabst, G., Hodzic, A., Strancar, J., Danner, S., Rappolt, M., Laggner, P. Biophysical Journal, 93, 2688–2696 (2007).

Primožič, Urša *, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
Potočnik, Tjaša, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
Ladurantie, Caroline, Institute of Pharmacology and Structural Biology, Toulouse, France
Kolosnjaj Tabi, Jelena, Institute of Pharmacology and Structural Biology, Toulouse, France
Novickij, Vitalij, Vilnius Gediminas Technical University, Faculty of Electronics, Institute of High Magnetic Fields, Vilnius, Lithuania
Rols, Marie-Pierre, Institute of Pharmacology and Structural Biology, Toulouse, France
Maček Lebar, Alenka, University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Ljubljana, Slovenia
*ursa.primozic@fe.uni-lj.si

Extracellular vesicles (EVs) are phospholipid bilayer membrane vesicles that nearly all cell types secrete into the extracellular space. Modulation of cell culture conditions and parameters, through the application of chemical, physical, or environmental stresses, increases the secretion of EVs. EVs are used as disease biomarkers and as therapeutic agents. One commonly used method for increasing the production of EVs is nutrient starvation, which deprives cells of essential nutrients. Electroporation, a process in which cells are exposed to short pulses of an electric field that increase membrane permeability, is widely used for loading EVs with therapeutic molecules. However, because electroporation also imposes significant physical stress on cells, it has the potential to serve as a mechanism for stimulating EV production.

We compared the concentration and size of released EVs from Chinese hamster ovary (CHO) cells after starving the cells, exposing them to 350 20-second bursts of a High Intensity Pulsed Electromagnetic Field (HI-PEMF) with repetition frequency of 1 Hz and amplitude of 6.7 T, and electroporation protocols with eight 100 µs long pulses with repetition frequency of 1 Hz at different electric field strengths.

The results show that cells after electroporation release a higher (21.54*10^6 particles/ml) concentration of EVs compared to HI-PEMF (6.72*10^6 particles/ml), starvation (11.5*10^6 particles/ml), and control (5.74*10^6 particles/ml). HI-PEMF (124 nm) and starvation (119.5 nm) produce slightly larger EVs, compared to control (103 nm). When comparing the concentration and size of EVs at different electric field strengths, the concentration of released EVs increases with electric field strength.

Mitja Drab*, Faculty of Electrical Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia
Yoav Ravid, Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 7610001, Israel
Luka Mesarec, Faculty of Electrical Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia
Nir S. Gov, Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 7610001, Israel
Veronika Kralj-Iglič, Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, 1000 Ljubljana, Slovenia
Aleš Iglič, Faculty of Electrical Engineering, University of Ljubljana, 1000 Ljubljana, Slovenia

*mitja.drab@fe.uni-lj.si

Eukaryotic cells continuously change their shape by altering their membrane composition and restructuring their underlying cytoskeleton. We present further studies and extensions of a minimal physical model, describing a closed vesicle with mobile curved membrane components (CMCs) that have an anisotropic shape. The cytoskeletal forces include a protrusive force due to actin polymerization, which is recruited to the membrane by the curved protein complexes. We characterize the phase diagrams of this model as a function of the magnitude of the active forces, nearest-neighbor protein interactions, and the proteins’ spontaneous curvature, which can be either arc- or saddle-shaped. We distinguish between the direction of the active forces, which can be normal to the membrane or bundled in an interaction that aligns the forces exerted on the membrane by each CMC bound in a cluster.

Kristina Elersic Filipic*, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Apolonija Bedina Zavec*, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Marjetka Podobnik, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia
Gregor Anderluh, National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia

*kristina.elersic.filipic@ki.si
*polona.bedina@ki.si

Biophysical approaches provide essential insights into toxin–membrane interactions and allow us to explore the key physicochemical factors governing toxin binding to membranes. Understanding the interplay between membrane composition, phase behavior, and toxin-induced structural rearrangements is crucial for elucidating mechanisms of selective cytotoxicity. This research has direct implications for drug discovery, particularly in the design of lipid-targeting drugs. Additionally, venom-derived compounds hold promise as novel drug scaffolds, highlighting the translational potential of this work across biophysics, pharmacology, and structural biology. In our Department of Molecular Biology and Nanobiotechnology at the National Institute of Chemistry, we employ a range of advanced techniques to study toxin–membrane interactions, including Surface Plasmon Resonance (SPR) for real-time binding, Isothermal Titration Calorimetry (ITC) for thermodynamic profiling, Microscale Thermophoresis (MST) for precise affinity measurements, Prometheus Panta for assessing liposome stability in the presence of toxin, Synergy microplate reader for Calcein release assays to measure membrane permeability in the presence of toxin, and Cryo Transmission Electron Microscopy (Cryo-TEM) for visualizing differences in liposomes. We also use advanced techniques to characterize liposomes or extracellular vesicles as membrane models before employing them in toxin interaction experiments. Their size, heterogeneity, and temperature stability are assessed by Prometheus Panta using four technologies: nano Differential Scanning Fluorimetry (nanoDSF), backreflection, Dynamic Light Scattering (DLS), and Static Light Scattering (SLS). In biology, there are different types of membranes. We present the differences in toxin interactions with model membranes of eukaryotes, Gram-positive bacteria, and Gram-negative bacteria. Such experiments allow us to observe the mechanisms that play an important role in the binding of toxins to different cell types. By combining multiple biophysical techniques, we aim to clarify our understanding of toxin–membrane interactions, contributing to the advancement of biophysical research and the development of innovative therapeutic strategies

T. V. Plavec 1,2,*, K. Žagar Soderžnik 1, G. Della Pelle 1, Š. Zupančič 2, R. Vidmar 1, and A. Berlec 1,2

1 Jožef Stefan Institute, Ljubljana, Slovenia
2 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia

*tina.plavec@ijs.si

Lactococcus cremoris is a Gram-positive bacterium widely recognized as a model system for recombinant protein expression. Recently, extracellular vesicles (EVs) produced by Gram-positive bacteria have attracted increasing interest due to their potential physiological and therapeutic roles. Harnessing bacteria engineered to produce recombinant proteins offers a powerful approach for packaging bioactive proteins into EVs. In this study, we investigated how the expression of various recombinant proteins in L. cremoris influences EV formation and alters their protein composition.
EVs were isolated following a standard protocol using an ultracentrifuge at 130.000 × g for 2 hours, followed by removal of the supernatant and collection of the EVs. EVs were characterised with transmission electron microscopy (TEM), flow cytometry, polydispersity index measurement and proteomic analysis.
Characterization by TEM and flow cytometry revealed differences in both quantity and heterogeneity of the secreted EVs, depending on the recombinant protein expressed in L. cremoris. The size of the isolated EVs was within the expected nano-scale range. The presence and quantity of individual recombinant proteins inside the EV was confirmed by proteomic analysis, e.g. western blotting and mass spectrometry.
In summary, we have shown that recombinant L. cremoris can serve as a platform to produce EVs enriched with recombinant therapeutic proteins. These results support the potential of Gram-positive bacterial EVs as novel bioengineered delivery systems.

FUNDING
ARIS J3-60060 Extracellular vesicles from recombinant Lactococcus lactis as novel protein delivery vectors for treatment of intestinal inflammation and prevention of colorectal cancer
ARIS N3-0184 Small protein blockers of IL-23/IL-17 axis as intestinal inflammation inhibitors secreted by probiotic bacteria
ARIS P4-0127 Pharmaceutical biotechnology: Science for health

Amina Sajjad*, Department of Applied Chemistry, Institute of Natural Science, Kyung Hee University, Yongin 17104, Republic of Korea
Jaewon Oh*, Department of Applied Chemistry, Institute of Natural Science, Kyung Hee University, Yongin 17104, Republic of Korea
Jeon Yujin, Department of Applied Chemistry, Institute of Natural Science, Kyung Hee University, Yongin 17104, Republic of Korea
Gabriella Pocsfalvi, Institute of Biosciences and BioResources, National Reseach Council of Italy
Kwang Pyo Kim, Department of Applied Chemistry, Institute of Natural Science, Kyung Hee University, Yongin 17104, Republic of Korea

E-mail: kpkim@khu.ac.kr

Abstract
Plant-derived nanovesicles (PDNVs) are emerging bioactive carriers whose molecular composition may be shaped by agricultural practices. To examine how cultivation conditions influence PDNV cargo, the proteomes of vesicles from conventionally grown (CONV) and organically grown (ORG) tomatoes are compared. A total of 3,947 protein groups are identified, with 225 proteins showing higher abundance in ORG and 158 in CONV samples.

Gene ontology (GO) enrichment analysis indicates that CONV PDNVs are associated with extracellular and cell-wall remodeling functions, carbohydrate metabolism, detoxification pathways, and oxidative-stress responses, whereas ORG PDNVs show enrichment for chloroplast and thylakoid functions, redox regulation, oxidoreductase activity, and vesicle-trafficking processes.

High-abundance protein families reveal additional differences: both PDNV types contain chaperones, electron-transport proteins, TCA-cycle enzymes, and redox-detoxification components, but ORG samples contribute slightly more ribosomal, TCA-cycle, and transport-associated proteins, while CONV samples display a modest shift toward glycolytic proteins.

Proteins previously described in the literature as having antioxidant or anti-inflammatory potential such as superoxide dismutase, catalase, heat-shock proteins, 14-3-3 proteins, thioredoxin-domain proteins, and several glutathione S-transferases are also identified; however, their functional activity within PDNVs remains uncertain and would require dedicated validation. Overall, cultivation environment exerts a clear influence on PDNV composition, directing vesicles toward detoxification-oriented profiles in conventional growth and toward adaptive, redox-regulatory profiles in organic growth.

Klara Bulc Rozman*, Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, 1000 Ljubljana, Slovenia
Lea Rems, Faculty of Electrical Engineering, University of Ljubljana, Tržaška cesta 25, 1000 Ljubljana, Slovenia
* Klara.BulcRozman@fe.uni-lj.si

Electroporation is a technique that uses short electric pulses to transiently increase cell membrane permeability, leading to reversible or irreversible alterations in cellular homeostasis1. Electroporation is widely utilized for intracellular delivery of biomolecules; however, its impact on the structural integrity of cardiomyocytes remains poorly understood. In this study, we investigate the relationship between electric field strength and cytoskeletal disruption in cardiomyocytes, focusing on tubulin and actin filament integrity. Using adult and neonatal primary cardiomyocytes, as well as the AC16 human cardiomyocyte cell line, we investigate how eight 100 μs pulses of different amplitudes induce microtubule and actin fiber breaks. High-resolution imaging and quantitative analysis of cytoskeletal architecture post-electroporation reveal distinct susceptibility profiles among the three cell types, suggesting developmental and phenotypic differences in structural resilience. These findings provide critical insights into cytoskeletal damage relevant for optimizing electroporation protocols for cardiac research and therapeutic applications2,3.

References:
(1) Kotnik, T., Rems, L., Tarek, M. & Miklavčič, D. Membrane Electroporation and Electropermeabilization: Mechanisms and Models. Annu. Rev. Biophys. 48, 63–91 (2019).
(2) O’Donnell, C., Mikhailov, A., Yoo, S., Ghosh, A. & Arora, R. Gene Therapies in Atrial Fibrillation. J. Cardiovasc. Transl. Res. 18, 1503–1510 (2025).
(3) Miklavčič, D., Rems, L., Jan, M. & Kos, B. Pulsed Field Ablation: disrupting technology in cardiac electrophysiology. Heart Rhythm S1547527125031601 (2025)

Teja Lavrin1, Marija Holcar1, Samuel Žvanut1, Valentina Levak2,3, Magda Tušek Žnidarič2, Metka Lenassi1

1Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. 2 Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana Slovenia. 3Jozef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia.

*teja.lavrin@mf.uni-lj.si

Introduction: More than 95% of HIV-1 proviruses persisting in brain microglia are defective, yet they can still express viral proteins, including Nef. Nef has been strongly implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND), however, its extracellular trafficking and biological roles remain poorly understood. In this study, we established an inducible human microglia model to enable comprehensive characterization of extracellular vesicles (EVs) released in response to Nef expression.

Methods: Human microglia (h-microglia) were stably transduced with lentiviral vectors encoding doxycycline-inducible Nef.GFP (LV-NefSF2-EGFP) or GFP gene alone (LV-EGFP). Cell viability, morphology, and transduction efficiency were assessed by flow cytometry and (live-cell) fluorescence microscopy. Small EVs were isolated from conditioned media by sequential centrifugation followed by iodixanol density gradient separation. EV yield, size distribution, protein composition, and Nef.GFP content were analyzed using NTA, nano-flow cytometry, immunoblotting, an in-house Nef ELISA, and (immunogold) TEM.

Results: Doxycycline treatment induced rapid and robust Nef.GFP expression in LV-Nef.GFP h-microglia, with 96.6 ± 5.0% of cells fluorescent at 48 h while maintaining high viability (95.4 ± 4.1%). EVs isolated from Nef.GFP-expressing microglia exhibited a 2.3-fold increase in particle number compared to GFP controls, with comparable size distributions (157.2 ± 6.3 nm vs. 144.9 ± 6.1 nm). Nano-flow cytometry revealed a 5.4-fold increase in fluorescence-positive EVs in the Nef.GFP group, with 45.5 ± 15.8% of EVs positive for Nef.GFP. Quantitative Nef ELISA detected 2.14 ± 2.40 ng of Nef.GFP per 10⁹ EVs. EV preparations were enriched in typical EV-associated markers, including Alix, GAPDH, CD81, and CD9. Immunogold TEM demonstrated that Nef.GFP was predominantly localized within the EV lumen, as antibody labeling was observed only following membrane permeabilization.

Conclusion: Inducible expression of HIV-1 Nef in human microglia promotes the release of small EVs carrying intraluminal Nef cargo. This study provides a well-characterized EV platform for investigating Nef-mediated intercellular communication in the central nervous system and its potential contribution to HAND pathogenesis.

Funding information: The Slovenian Research Agency grant P1-0170 and young researcher scholarship.

Samuel Žvanut1*, Teja Lavrin1, Marija Holcar1, Nina Mavec1, Aleksandra Usenik2,3, Sara Pintar2,3, Dušan Turk2,3, Metka Lenassi1

1Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 2Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia; 3Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia

*samuel.zvanut@mf.uni-lj.si

Introduction: HIV-1–associated neurocognitive disorders remain a challenge despite effective antiretroviral therapy. HIV-1 reservoirs persist in the central nervous system, where ongoing viral protein expression drives neuropathology. The viral protein Nef, released within extracellular vesicles (EVs), is implicated in HIV-1 pathogenesis, yet reliable methods to measure its release are lacking. We introduce a novel nanobody-based ELISA (nanoNef ELISA) for detecting EV-associated Nef and validate it using a model of translationally active microglial HIV-1 reservoirs.

Methods: Microglial HIV-1 reservoirs were modeled in vitro by co-culturing human microglia expressing inducible Nef-EGFP with Nef-negative microglia at defined ratios (0-100%). After 48 hours of induction, Nef-EGFP–positive cells were quantified by flow cytometry, and Nef in conditioned media was measured using nanoNef ELISA. EV-enriched fractions were isolated by ultracentrifugation and density gradient separation, then analyzed by nanoNef ELISA, immunoblotting, and nano–flow cytometry. To assess translational potential of the assay, concentrated conditioned media was spiked into cerebrospinal fluid (CSF) or PBS to evaluate signal recovery and matrix effects.

Results: Nef concentrations in conditioned media correlated strongly with the proportion of Nef-EGFP–expressing microglia (r = 0.9149). The 100,000 × g pellets from conditioned media of Nef-EGFP–expressing cells contained Nef and EV markers as shown by immunoblotting, and nano–flow cytometry revealed that 61.2% of EVs were Nef-EGFP–positive. Nef concentrations in EV-enriched pellets reached 51.11 ± 5.59 ng per million microglia. Density gradient separation demonstrated that Nef localized to discrete iodixanol fractions enriched for EV biomarkers. The assay reliably detected vesicle-associated Nef released by Nef-expressing microglia. Regression analysis of spiked conditioned media revealed a proportional decrease in signal with dilution (R² = 0.92 in PBS and 0.88 in CSF), with parallel slopes indicating minimal matrix interference.

Conclusions: nanoNef ELISA enables sensitive and quantitative measurement of EV-associated Nef released from translationally competent HIV-1 microglial reservoirs in vitro. This assay provides a robust platform for studying Nef secretion dynamics and EV-mediated mechanisms of HIV-1 neuropathogenesis, supporting future applications in translational and biofluid-based studies

Funding information: The study was supported by research grant P1-0170 funded by Slovenian Research and Innovation Agency.

Vodušek Maja*1,2, Lenassi Metka2, Arnol Miha1,3
1Department of Nephrology, University Medical Centre Ljubljana, Ljubljana, Slovenia
2Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
3Department of Internal Medicine, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
*maja.vodusek@kclj.si

Early, non-invasive detection of kidney allograft injury remains a significant challenge, because conventional biomarkers often fail to detect subclinical injury. Urinary extracellular vesicles (uEVs), which are predominantly kidney-derived, reflect kidney pathophysiology and carry nucleic acids released during injury.

We evaluated whether uEV-bound nucleic acids could serve as biomarkers of kidney allograft injury in 38 kidney transplant recipients assessed at protocol or indication biopsy (median 942 days post-transplantation). Patients were classified as kidney injury (KI; n=28, 74%) or normal histology (NH; n=10, 26%), and KI cases were further stratified by underlying pathology: recurrent glomerulonephritis (rGN; n=10), chronic antibody-mediated rejection (cABMR; n=10), and BK virus nephropathy (BKVN; n=8). uEVs were isolated from 20 mL of urine, stained with the nucleic acid dye SYTO™16 (Thermo Fisher Scientific), and analyzed using nano-flow cytometry (nanoFCM).

KI patients exhibited a higher proportion of SYTO 16–positive uEVs (10.2% vs. 6.5%, p=0.0017) and greater normalized concentration (16 × 10¹¹ vs. 4.6 × 10¹¹ uEV/mmol urinary creatinine, p = 0.0029) compared with NH (Mann–Whitney U test). Across all four groups, both the proportion (p = 0.0222) and normalized concentration (p = 0.0111) of positive uEVs differed significantly. Dunn’s multiple comparisons test revealed that the proportion of positive uEVs was significantly higher in patients with cABMR than in those with NH (10.8% vs. 6.5%, p = 0.0233), while the normalized concentration was significantly higher in rGN compared with NH (19.5 × 10¹¹ vs. 4.6 × 10¹¹ uEV/mmol urinary creatinine, p = 0.0074). ROC curve analysis for KI versus NH showed that the percentage of positive uEVs yielded an AUC of 0.83 (95% CI: 0.67–0.95, p = 0.0039; sensitivity 75%, specificity 90%; cut-off 7.9%), while normalized uEV concentration yielded an AUC of 0.81 (95% CI: 0.69–0.96, p = 0.0026; sensitivity 57%, specificity 100%; cut-off >14 × 10¹¹ uEV/mmol U-creatinine). The ROC curves for individual comparisons between different injury phenotypes are shown in the Figure.

These findings demonstrate that uEV-bound nucleic acids are elevated in kidney allograft injury and differ by injury phenotype, supporting their potential as a non-invasive biomarker for early detection of allograft injury.

Megi Tinev*, Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia
Luka Kristanc, Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia; Faculty of Health Sciences, University of Novo mesto, Slovenia
Gregor Gomišček, Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia; Faculty of Health Sciences, University of Ljubljana, Slovenia
Bojan Božič, Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia
*megi.tinev@mf.uni-lj.si

The polyene nystatin is one of the key antifungal agents (1,2) and shows potential for the treatment of skin infections caused by intracellular pathogens such as Leishmania (3). Nystatin acts by forming channels in cell membranes, which increases their permeability and thus leads to cell death (2). Channel formation occurs more prominently in ergosterol-containing membranes, making nystatin particularly effective against fungi and protozoan parasites. However, due to its affinity for cholesterol, it can also cause toxic effects in human cells (4).

Although the mechanism of action of nystatin on membranes is well understood, its ability to pass through the phospholipid bilayer remains unclear. A better understanding of nystatin’s membrane permeability could improve its specificity in targeting intracellular forms of pathogens and contribute to reducing side effects. To this end, we studied the behavior of giant unilamellar vesicles (GUVs) and multivesicular vesicles (MVVs) composed of POPC membranes with 15 or 45 mol% ergosterol, after exposure to nystatin solutions with concentrations of 250 and 500 μM. We measured the time from exposure to nystatin solutions to the rupture of GUVs and the outer vesicles (outGUVs) and inner vesicles (inGUVs) in MVVs. The results showed that inGUVs ruptured significantly faster than GUVs of the same size after the rupture of the outGUV of the same MVV, indicating that nystatin can pass through the ergosterol-containing membrane.

Based on our observations, we proposed improved mechanism of action of nystatin, according to which it simultaneously passes through the phospholipid bilayer while forming channels in the membrane.

References:
(1) M.A. Ghannoum, L.B. Rice, Clin. Microbiol. Rev., 12, 501–517 (1999).
(2) J. Bolard, Biochim. Biophys. Acta, 864, 257–304 (1986).
(3) E.S. Yamamoto, idr., Curr. Top. Med. Chem., 18, 2338–2346 (2018).
(4) R.A. Zager, Am. J. Kidney Dis., 36, 238–249 (2000).

Grabovská S.* 1, Wrobel D. 1, Kocholatá M. 1, Budková K. 1, Janoušková O. 1, Malý J. 1
1 Jan Evangelista Purkyně University in Ústí nad Labem, Faculty of Science, Czech Republic
*grabovska.simona@email.cz

Exosome-liposome hybrid vesicles represent promising nanocarriers with high potential for targeted drug delivery. Polyethylene glycol (PEG) is a biocompatible compound widely used as a fusogenic agent for membrane fusion. This method has previously been established as an efficient strategy for enriching mammalian extracellular vesicles (EVs) with exogenous hydrophilic and lipophilic compounds while preserving their intrinsic cargo and biological properties. However, the applicability of this approach to plant-derived EVs has not yet been systematically investigated.

In this study, we evaluated PEG-mediated fusion of liposomes with plant-derived exosomes isolated from Arabidopsis spp. and compared their fusion behavior with mammalian mesenchymal stem cell (MSC)-derived exosomes. Fusion of vesicles was performed using PEG 1000 at varying concentrations (0, 15, 30, and 45%) and temperatures (25, 37, and 40 °C) with an incubation time of 6 h to identify optimal fusion conditions. The fusion process was validated via Förster resonance energy transfer (FRET)-based assay.

Our results revealed distinct differences in the fusion efficiency of plant- and mammalian-derived exosomes with liposomes. While MSC-derived exosomes exhibited comparable fusion efficiency at 30% and 45% PEG 1000, plant-derived exosomes showed maximal fusion efficiency at 45% PEG 1000. Temperature had a limited effect on fusion efficiency in plant exosomes; in contrast, fusion of MSC exosomes was significantly enhanced at 40 °C. Additionally, prolonged incubation times in the presence of PEG positively affected fusion efficiency in plant-derived exosomes.

Overall, our findings demonstrate that PEG-mediated liposome fusion is a viable strategy for the modification of plant-derived exosomes. However, optimal fusion conditions differ substantially from those established for mammalian exosomes, underscoring the necessity of system-specific optimization. This study provides a practical framework for future experimental design involving plant exosome-based nanocarriers and their functional modification.

This work was supported from project No. CZ.02.01.01/00/23_021/0008398 “Materials and technologies for bioapplications and medicine”

Andrea Nedělníková*, Regional Center of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Šlechtitelů 27, 779 00 Olomouc, Czech Republic
Faculty of Electrical Engineering and Computer Science, VSB – Technical University of Ostrava, 17. listopadu 2172/15, 708 00 Ostrava – Poruba, Czech Republic
Markéta Paloncýová, Regional Center of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Šlechtitelů 27, 779 00 Olomouc, Czech Republic
*andrea.nedelnikova@upol.cz

Alzheimer’s and Parkinson’s diseases are progressive neurodegenerative disorders that currently have no cure. Deep brain stimulation (DBS), which delivers electrical signals to specific brain regions via implanted electrodes, is used to alleviate symptoms. However, conventional metal electrodes often trigger inflammatory responses (1), motivating the search for more biocompatible alternatives. Graphene-based materials emerge as promising candidates due to their excellent electrical and mechanical properties, as well as their potential biocompatibility (2).

In this study, we investigate pristine graphene, reduced graphene oxide, and graphene oxide in interaction with lipid membranes representative of microglial cells, neurons, myelin sheaths, and the SH-SY5Y neuroblastoma cell line, which is widely used as an in vitro model for assessing cytotoxicity in brain cells. We present a comprehensive molecular dynamics analysis that reveals distinct interaction mechanisms among the graphene derivatives. Membrane insertion is observed only in a limited number of cases; however, when insertion occurs, the penetration depth depends strongly on the degree of surface oxidation. Overall, surface oxidation plays a critical role in modulating membrane affinity, insertion behavior, and disruption potential. Potential of mean force (PMF) calculations quantify the energetics of membrane penetration and demonstrate that both the chemical nature of the graphene surface and the lipid composition of the target membrane strongly influence interaction outcomes.

These findings provide molecular-level insight into the design of graphene-based neurointerfaces and establish key parameters for optimizing biocompatibility in future DBS electrode applications.

References:
(1) Bernstein, J. E., Kashyap, S., Ray, K. & Ananda, A. Infections in deep brain stimulator surgery. Cureus 11, 5440 (2019).
(2) Xu, B., Pei, J., Feng, L. & Zhang, X.-D. Graphene and graphene-related materials as brain electrodes. J. Mater. Chem. B 9, 9485–9496 (2021).

Veronika Skockova*, Faculty of Biochemistry and Molecular medicine (FBMM), University of Oulu, Finland
Eva Bozo, FBMM, University of Oulu, Finland
Samar Ahmad, FBMM, University of Oulu, Finland
Pradnya Patil, FBMM, University of Oulu, Finland
Caglar Elbuken, FBMM, University of Oulu, Finland
Seppo Vainio, FBMM, University of Oulu, Finland
*veronika.skockova@oulu.fi

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles produced by all types of cells, e.g., animal, plant, and bacterial cells. EVs reflect the original cell, including its membrane composition and the molecules within the lumen. In contrast, lipid nanoparticles (LNPs) are artificially produced particles with a defined composition that can be easily loaded with various cargo.

Fusion of EVs and LNPs results in a new particle: a hybrid EV that combines the advantages of the original particles. The EV provides surface markers that mask the particle’s artificial origin and help to avoid triggering an immune response in the organism. The LNP delivers the defined cargo and can facilitate its escape from the endosome.

We used EVs isolated from milk and LNPs prepared using a microfluidic system. Using a microfluidic chip to prepare LNPs allows precise control of their properties, composition, and cargo loading. We induced fusion by adding polyethylene glycol (PEG) or by subjecting the particles to cycles of freezing and thawing. The efficacy of fusion was determined by flow cytometry. Freeze/thaw cycles produced better results and were used for subsequent drug loading into EVs.

Adrienn Molnár*, MTA-ELTE Lendület (Momentum) Ion Mobility Mass Spectrometry Research group, ELTE Eötvös Loránd University, Budapest, Hungary
Anna Romolo, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia
Boštjan Korenjak, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia
Alenka Svete Nemec, University of Ljubljana, Veterinary Faculty, Small Animal Clinic, Ljubljana, Slovenia
Vladimira Erjavec, University of Ljubljana, Veterinary Faculty, Small Animal Clinic, Ljubljana, Slovenia
Veronika Kralj-Iglič, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia
Gitta Schlosser, MTA-ELTE Lendület (Momentum) Ion Mobility Mass Spectrometry Research group, ELTE Eötvös Loránd University, Budapest, Hungary

*molnaradrienn97@gmail.com

Brachycephalic obstructive airway syndrome (BOAS) is a common and debilitating condition in brachycephalic dog breeds, characterised by chronic upper airway obstruction and broader systematic physiological effects. While surgical intervention is commonly used to alleviate clinical symptoms, the molecular effects of surgery on metabolism are not well understood yet. In this study, we investigated changes in the lipidomic profile of plasma from dogs with BOAS before and after airway correction surgery.

Plasma samples were collected from dogs diagnosed with BOAS before and after surgery. Comprehensive lipidomic analysis was performed using cyclic ion mobility mass spectrometry (cIM-MS), which allowed high-resolution separation and characterisation of lipid species. The results showed significant differences between the pre- and post-surgical lipidomic profiles, which suggested a marked metabolic shift after surgery. The pre-surgical state was characterised by the abundance of plasmalogen lipids such as PE(P-34:1), PE(P-36:2), and PE(P-38:4). Meanwhile, post-surgical samples showed an increase in other phospholipids such as PE(36:4), PC(34:4), PE(38:5), PC(36:5), PE(38:4), and PC(36:4).

Plasmalogen lipids are known to be involved in oxidative stress regulation and have been reported to be altered in hypoxia-associated conditions (1). The observed lipidomic changes, particularly the decrease in plasmalogen lipids, were consistent with the metabolic adaptations associated with chronic intermittent hypoxia previously described in BOAS and its decrease after surgery.

Overall, this study demonstrates the utility of cIM-MS-based lipidomics to elucidate systematic metabolic alterations in BOAS and provides new insights into the biochemical effects of corrective surgery, which may contribute to a better understanding of the pathophysiology of BOAS.

Acknowledgement

This project was supported by the Lendület (Momentum) Program of the Hungarian Academy of Sciences (HAS, MTA). Project no. SNN 148580 has been implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development and Innovation Fund, financed under the SNN24 funding scheme, and by the Slovenian Research and Innovation Agency (ARIS) project J3-60063 and programs P3-0388 and P4-0053.

References:

(1)  Curran, C. S.; Remaley, A. T.; Torabi-Parizi, P. Plasmalogens as biomarkers and therapeutic targets. Journal of Lipid Research, 66 (12), 100925. (2025)

Hee Jung Kwon, Minho Kim
Department of Applied Chemistry, Kyung Hee University, Korea

Oxygen Evolution Reaction (OER) have attracted interest for renewable conversion of electricity based on an electrochemical water splitting method. While OER under an alkaline condition is highly developed over decades, OER under an acidic media has been questioned for low stability of noble metal oxides (RuO₂ and IrO₂) despite its advantage of high proton conductivity. In this study, we suggest a design principle of a highly active and stable Ir-based oxide catalyst based on a nanoparticle model on MnO₂ surface to maintain efficiency and stability under acidic conditions while reducing costs. We compared three categories of nanostructures – rutile IrO₂ as bulk structure, nanocluster IrO₂ as a confined but finite-sized nanostructure, and single-atom Ir catalyst on MnO₂. Density functional theory (DFT) calculations confirmed that the nanocluster IrO₂ on MnO₂ shows the lowest overpotential of OER among them, indicating its highest OER activity. Additionally, the nanocluster IrO₂ showed the highest dissolution potential of an Ir atom, confirming its stability under working potentials. This work can suggest a promising catalyst to conduct efficient OER with high stability and activity in acidic media based on the size-controlled preparation of IrO₂ nanocatalysts with support materials.

Youngha Kweon, Minho Kim
Department of Applied Chemistry, Kyung Hee University, Korea

Alkaline water electrolysis is a promising route for sustainable hydrogen production, but the hydrogen evolution reaction (HER) under alkaline conditions is hindered by sluggish kinetics due to the additional water dissociation step. While NiFe-based materials have shown experimental potential as low-cost electrocatalysts, atomic-scale understanding of their active sites and mechanisms remains limited. Density functional theory (DFT) studies are thus crucial for uncovering the thermodynamic and electronic factors that govern the HER activity in alkaline media.
In this study, we perform DFT calculations to investigate the alkaline HER performance of three NiFe-based catalysts: NiFeP, NiFe₂O₄, and NiFeO_x. Among them, NiFeP shows the most favorable hydrogen adsorption with a ΔG_H* value closest to zero, indicating superior catalytic activity. We also identify a correlation between H₂O adsorption energy and H adsorption energy, emphasizing the role of phosphides during water dissociation in alkaline HER.
Further analysis using d-band center and Bader charge calculations reveals that NiFeP offers an electronic environment well-suited for hydrogen binding. Unlike the oxide-based catalysts, phosphorus in NiFeP acts as a secondary active site for proton adsorption and enables the formation of highly active Fe–Ni bridge sites, which are key to its enhanced HER activity.
These findings offer valuable theoretical insights into the structure–activity relationship of NiFe-based catalysts and provide guidance for designing efficient and cost-effective materials for alkaline water electrolysis.

Harkai Saša*, Department of Biomechanics, Faculty of Mechanical Engineering, Czech Technical University, Czech Republic

Daniel Matej, Department of Biomechanics, Faculty of Mechanical Engineering, Czech Technical University, Czech Republic
*sasa.harkai@cvut.cz

We discuss the typical response of liposomes when indented during an atomic force microscope (AFM) measurement. The typical liposome response is a nonlinear force-deformation curve for small indentation depths, and linear for larger depths. However, in our experimental measurements, we have noticed a purely linear response for small depth indentations, which has not yet been properly explained.

We present a mathematical model explaining both types of responses. We hypothesize that the two different responses come from two different types of liposome adhesion to the substrate, namely weak and strong adhesion, shown in Figure 1 below. Liposomes with weak adhesion to the substrate have a nonlinear initial response to depression due to them still keeping their energy-efficient circular shape, which causes the initial depression with the AFM to require little force. Liposomes with strong adhesion already have a deformed shape, which causes the prestress in liposome membrane, effectively skipping the nonlinear response. We also discuss the properties of the model and its general applicability and applications in cell mechanics.

Wróbel D.^*, J. E. Purkyne University
Kocholata M., J. E. Purkyne University
Maly J., J. E. Purkyne University

*dominika.wrobel@ujep.cz

The NF-κB signal transduction pathway plays a central regulatory role in the healing process by coordinating the early inflammatory response and subsequent tissue repair (1). Upon activation by injury-related signals, NF-κB induces the expression of cytokines, chemokines, and adhesion molecules that recruit immune cells to the wound site, enabling pathogen clearance and initiating inflammation. As healing progresses, NF-κB regulates genes involved in cell proliferation, angiogenesis, extracellular matrix remodeling, and the resolution of inflammation, thereby facilitating the transition from an acute inflammatory state to a tissue-regenerative environment.

The aryl hydrocarbon receptor (AhR) also contributes to wound healing by modulating inflammation, immune cell activity, and keratinocyte differentiation in response to endogenous and environmental ligands (2). AhR activation influences transcriptional programs regulating cytokine production, oxidative stress responses, and barrier-restorative functions. In skin and mucosal tissues, AhR signaling supports re-epithelialization and maintains extracellular matrix organization, underscoring its importance in tissue regeneration.

Calendula (Calendula officinalis L.) and comfrey (Symphytum officinale L.) are medicinal plants known for their regenerative and wound-healing properties (3,4). The present study investigated the therapeutic potential of exosomes isolated from these species. Their effects on NF-κB and AhR activation were evaluated using THP1-Lucia NF-κB and HT29-Lucia AhR cell lines, respectively. The results revealed that both types of exosomes possess the ability to activate the investigated pathways. These findings provide promising evidence for the potential application of herbal exosomes in supporting tissue healing processes.

References:

(1) Park Y.R., Sultan M.T., Park H.J., Lee J.M., Ju H.W., Lee O.J., Lee D.J., Kaplan D.L., Park C.H., Acta Biomater., 67, 183-195 (2008)

(2)  Barouti N., Mainetti C., Fontao L., Sorg O., Dermatology, 230 (4), 332–339 (2015)

(3)  Givol O., Kornhaber R., Visentin D., Cleary M., Haik J., Harats M., Wound Repair Regen., 27(5), 548-561 (2019).

(4) Staiger C., Phytother Res., 26(10), 1441-8 (2012)

The authors acknowledge the assistance provided by the “Excellence in Regenerative Medicine”project, supported by the Ministry of Education, Youth, and Sports of the Czech Republic. Project No. CZ.02.01.01/00/22_008/0004562, co-funded by the European Union.

Mostafa Bakouei, University of Oulu, Finland
Tatiana Avsievich, University of Oulu, Finland
Indraja Sundara Raju, University of Oulu, Finland
Benny Ryplida, University of Oulu, Finland
Caglar Elbuken*, University of Oulu, Finland

*caglar.elbuken@oulu.fi

We present an experimental system to generate microfluidic liposomes and study membrane budding processes. The microscale liposomes were generated using double emulsion templates (1), where the membrane forming unit is dissolved in two types of solvent (2). As the poor solvent is removed from the double emulsion shell, the interfacial tensions vary dynamically. This leads to membrane budding leading to the formation of the bilipid layer. By precisely controlling the solvent removal rate, this process can be controlled to obtain cell-size liposomes.

The formation of double emulsions was achieved using a glass-PDMS hybrid microfluidic device that eliminated the need for surface treatment (3). The volume ratio of core and shell of the double emulsion can be controlled as well as the total size. The aqueous inner phase of the liposomes are composed of 8% polyethylene glycol, whereas outer aqueous phase is composed of 5 wt% glycerol. During double emulsion formation, a 60/40 (v/v) ratio of hexanol and paraffin oil was used as lipid solvents (DOPC 8 mg/ml). The double emulsions are then collected in a chamber where the middle phase solvent removal rate is controlled. The change in interfacial tension during the solvent removal phase is quantified to explain the budding process.

This system provides an experimental platform to study biological membrane processes as well as a robust mechanism to achieve microscale liposomes. Finally, a counterflow mechanism was used to detach the solvent part of the budded droplets. We extend the understanding in interfacial tension driven membrane budding mechanisms and demonstrate that microfluidically generated double emulsions can be engineered to study membrane budding processes.

This work was primarily funded by the Academy of Finland, NanoEngineered Self-Assembling Vesicle Production Line (NESAV) project (no. 342448) and GeneCellNano (GCN) Flagship.

References:

(1) Shum, H. C., Lee, D., Yoon, I., Kodger, T. & Weitz, D. A. Langmuir 24, 7651–7653 (2008)
(2) Deng, N. N., Yelleswarapu, M. & Huck, W. T. S., J Am Chem Soc 138, 7584–7591 (2016)
(3) Bakouei, M., Kalantarifard A., Sundara Raju, I., Avsievich T. Rannste L., Kreivi M., Microsyst Nanoeng 10, 1–11 (2024).