Permeabilized Cells Research Articles

Potentiation of the antibiofilm effect of natural compound analogs in a marine bacterium by the cell permeabilization mechanism

The potentiation of the anti-adhesion and anti-biofilm effects of the three compounds (OB1, AS194, AS162) was evaluated in this study. This potentiation is demonstrated by the combination of each of the three compounds with molecules of permeabilizing types which are Polymyxin B nanopeptide hydrochloride (PMBN), Phenylalanine-Arginine Beta-Naphthylamide (PAßN) and Carbonyl cyanide chlorophenylhydrazone (CCCP). The association of this type of molecule with antibiofilm compounds showed a synergistic effect in TC14. This effect was more pronounced with CCCP (5µM). Thus, the AS162 having the highest effect between the three compounds previously induced a reduction in adhesion of up to 30.80% at only 10µM. In combination with the CCCP and at the same concentration, AS162 reduces TC14's adherence by up to 11.23%, a reduction of more than 63.53%. The AS162 also reduces the biofilm mass of the same bacterium to 10.15% in the presence of CCCP, i.e. a reduction of 89.75%. When taking the case of compounds with relatively low anti-biofilm effect such as OB1 and AS194, the reduction rate under the effect of the combinations is found to be higher than that of AS162. Thus, OB1 with an EC50 of 75.39 µM when used alone, in the presence of the CCCP, experiences a reduction of this EC50 up to 3.6 µM, i.e. 95.22% reduction. In AS194 (with a relatively stronger effect than OB1), the EC50 is reduced by 74.02% and 52.79% for AS162. This method has been shown to be effective in enhancing the anti-biofilm effect of the compounds. It is therefore important to note that the less active the compound, the greater the synergistic effect with the permeabilizers.

Intracellular metal enhanced fluorescence utilizing gold nanoparticles embedded in hydrogel droplets for sensitive protein detection in cells

Cellular protein detection is necessary for monitoring various biological pathways associated with complex diseases such as cancer. Detecting intracellular proteins is more challenging than cell surface protein detection because most antibodies cannot freely cross the cellular membrane. Cellular membrane permeabilization is thus required to facilitate the antibody diffusion and achieve adequate detection when combined with various conventional protein detection methods (e.g., flow cytometry and immunocytochemistry). However, these methods come with sensitivity challenges when a large number of cells are unavailable. Therefore, for early disease diagnostics and prognosis, novel methods are needed to achieve higher sensitivity to track disease-related proteins from small amounts of cells at early stages of disease progression. Here we applied a novel biosensing platform that co-entraps cells and gold nanoparticles in 3D hydrogel droplets to achieve sensitive fluorescence-based detection and quantification of intracellular proteins in intact cells. Importantly, intracellular metal enhanced fluorescence (iMEF) was achieved using gold nanoparticles via cell permeabilization. The intracellular gold-nanoparticles, together with the high cell loading capacity of the hydrogel droplets, enhanced the fluorescence signals and in turn improved assay sensitivity compared to conventional methods. After assay optimization, we demonstrated the detection of Heat Shock Protein 70, a common cancer marker, with as little as 250 cells. This technology was also able to distinguish between cancerous and non-cancerous samples when mimicking a clinical fine needle aspiration procedure. Compared to conventional assays measuring the same protein, the assay sensitivity was improved by two to three orders of magnitude, allowing for highly sensitive intracellular protein detection from minimal amounts of cells.

Assessment of the effect of air humidity and temperature on convective drying of apple with pulsed electric field pretreatment

This research aims at assessing the impact of pulsed electric field (PEF) pretreatment and various properties of drying agent (absolute humidity, temperature) on the course of convective drying (CD) of apples. Moreover, the selected quality indicators of obtained dried materials (dry matter content, water activity, hygroscopicity, rehydration, color, total phenolic content, antioxidant activity) have been evaluated. As a result of increased permeabilization of cells, PEF reduced time of CD of apples even by 20%. However, lower total phenolic content and antioxidant activity in PEF-pretreated dried samples were observed. With the use of PEF, the release of phenolic compounds from the cellular structures of the apple tissue could occur, making them more susceptible to thermal degradation. After introducing the dehumidification system, an increase in the drying time of apples was observed. It could be so due to case hardening, which further deteriorated the reconstitution properties of the obtained dried materials.

The bisphosphonates alendronate and zoledronate induce adaptations of aerobic metabolism in permanent human endothelial cells

Nitrogen-containing bisphosphonates (NBPs), compounds that are widely used in the treatment of bone disorders, may cause side effects related to endothelial dysfunction. The aim of our study was to investigate the effects of chronic 6-day exposure to two common bone-preserving drugs, alendronate and zoledronate, on endothelial function and oxidative metabolism of cultured human endothelial cells (EA.hy926). NBPs reduced cell viability, induced oxidative stress and a pro-inflammatory state and downregulated the prenylation-dependent ERK1/2 signaling pathway in endothelial cells. In addition, NBPs induced increased anaerobic respiration and slightly increased oxidative mitochondrial capacity, affecting mitochondrial turnover through reduced mitochondrial fission. Moreover, by blocking the mevalonate pathway, NBPs caused a significant decrease in the level of coenzyme Q10, thereby depriving endothelial cells of an important antioxidant and mitochondrial electron carrier. This resulted in increased formation of reactive oxygen species (ROS), upregulation of antioxidant enzymes, and impairment of mitochondrial respiratory function. A general decrease in mitochondrial respiration occurred with stronger reducing fuels (pyruvate and glutamate) in NBP-treated intact endothelial cells, and significantly reduced phosphorylating respiration was observed during the oxidation of succinate and especially malate in NBP-treated permeabilized endothelial cells. The observed changes in oxidative metabolism caused a decrease in ATP levels and an increase in oxygen levels in NBP-treated cells. Thus, NBPs modulate the energy metabolism of endothelial cells, leading to alterations in the cellular energy state, coenzyme Q10 redox balance, mitochondrial respiratory function, and mitochondrial turnover.

Erratum: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa.

An erratum was issued for:High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Resultsections wereupdated. Step 2.4.12 of the Protocol was updated from: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex II inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. to: Finally, inject 1 µL of 5 mM antimycin A (2.5 µM final concentration). This is a complex III inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. Figure 3 in the Representative Results section was updated from: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure. to: Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure.

An Acoustic Device for Ultra High-Speed Quantification of Cell Strain During Cell-Microbubble Interaction.

Microbubbles utilize high-frequency oscillations under ultrasound stimulation to induce a range of therapeutic effects in cells, often through mechanical stimulation and permeabilization of cells. One of the largest challenges remaining in the field is the characterization of interactions between cells and microbubbles at therapeutically relevant frequencies. Technical limitations, such as employing sufficient frame rates and obtaining sufficient image resolution, restrict the quantification of the cell's mechanical response to oscillating microbubbles. Here, a novel methodology was developed to address many of these limitations and improve the image resolution of cell-microbubble interactions at high frame rates. A compact acoustic device was designed to house cells and microbubbles as well as a therapeutically relevant acoustic field while being compatible with a Shimadzu HPV-X camera. Cell viability tests confirmed the successful culture and proliferation of cells, and the attachment of DSPC- and cationic DSEPC-microbubbles to osteosarcoma cells was quantified. Microbubble oscillation was observed within the device at a frame rate of 5 million FPS, confirming suitable acoustic field generation and ultra high-speed image capture. High spatial resolution in these images revealed observable deformation in cells following microbubble oscillation and supported the first use of digital image correlation for strain quantification in a single cell. The novel acoustic device provided a simple, effective method for improving the spatial resolution of cell-microbubble interaction images, presenting the opportunity to develop an understanding of the mechanisms driving the therapeutic effects of oscillating microbubbles upon ultrasound exposure.

Coumarin-based C-2 cycloalkylated histidine derivatives: Design, synthesis, biological evaluation, molecular docking and MD simulation studies as potential antimicrobial agents

Despite numerous attempts to develop new pharmacophores as promising antimicrobials, the coumarin scaffold remains one of the foremost sought moiety in the design of molecules. Herein, we report the new structural class of coumarin-based C-2 cycloalkylated histidine hybrids as potential antimicrobial agents. The most promising compound 11n displayed IC50 of 0.27 µg/mL, 1.1 µg/mL, 4.4 µg/mL and 4.4 µg/mL against Crptococcus neoformans, Candida albicans, Escherichia coli and Staphylococcus aureus, respectively. Interestingly, 11n displays fungicidal activity on growing cryptococcal cells with high selectivity. The presence of 11n lead to the permeabilization of fungal cells as confirmed by propidium iodide uptake study. Moreover, scanning electron microscopy revealed that it causes shrinkage and disruptions in the cell membranes, which is responsible for the cell lysis. Molecular docking analysis revealed that 11n have a distinct H-bonding with TYR409 and TRP94 which are lacking in the co-crystal ligand and are predicted that these are responsible for the potency of the compound. Moreover, the results of the MD simulation analysis showed that the protein-ligand complex exhibits substantial binding interactions with a variety of amino acids and is stable for 100 ns. Additionally, ADMET analysis depicted that these molecules have considerable ADME properties.

Cationic Polymers Enable Internalization of Negatively Charged Chemical Probes into Bacteria.

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate (D) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis. We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells.

Intracellular delivery technologies that are cost-effective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resource-limited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world. These filtroporation devices permeabilize cells by pulling them through the pores of a cell culture insert by the application of vacuum available in biosafety cabinets. In a format that costs less than $10 in materials per experiment, we demonstrate the delivery of fluorescently labeled dextran, expression plasmids, and RNPs for gene knockout to Jurkat cells and human CD34+ hematopoietic stem and progenitor cell populations with delivery efficiencies of up to 40% for RNP knockout and viabilities of >80%. We show that functionalizing the surfaces of the filters with fluorinated silane moieties further enhances the delivery efficiency. These devices are capable of processing 500,000 to 4 million cells per experiment, and when combined with a 3D-printed vacuum application chamber, this throughput can be straightforwardly increased 6-12-fold in parallel experiments.

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH

BackgroundThough multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity.The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).ResultsThe mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.ConclusionA simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.

Design and Fabrication of a Test Rig to Determine Helium Permeability through Fabrics

The fabric of a Lighter-Than-Air (LTA) system experiences permeation of Helium over a period of time. Due to the small size of the molecules of Helium, it permeates through the intermolecular spaces of the fabric. This leakage increases with time due to the degradation of the fabric. LTA systems need to be deployed for long durations. The loss of Helium due to permeation reduces the relative lift of the LTA system. The unknown permeation rates thus lead to uncertainties in the deployment time of the system. This paper describes the design and fabrication of a test rig to determine the permeability of Helium through different fabrics. This method involves a permeation cell divided into upper and lower chambers arranged to hold a specimen fabric between them, with the provision of a continuous supply of Helium in the lower chamber. A Phoenix L300i Helium Leak Detector (HLD) is connected to the upper chamber to detect Helium and measure its concentration. The further objective of this research is to study the variation of helium permeability through the fabrics at constant temperature and input pressure with time.

Antibacterial mechanism of action and in silico molecular docking studies of Cupressus funebris essential oil against drug resistant bacterial strains

The primary objective of this research work was to study the antibacterial effects of Cupressus funebris essential oil (EO) against various drug resistant bacterial pathogens along with studying the molecular docking interactions of the major components of the EO with the key bacterial proteins/enzymes. Gas chromatography-mass spectrometry was used to analyse the chemical composition of the Cupressus funebris EO. The initial antibacterial screening was performed by using disc diffusion and microdilution methods. Scanning electron microscopy was also performed in order to study effects of the EO on bacterial cell morphology. Further, molecular docking studies were performed using Autodock Vina and results were visualised by BIOVIA Discovery Studio. The chemical composition of the EO showed the presence of 15 components with citronellal, terpinene-4-ol, α-phellandrene and 1,8-cineole as the major components of the EO. Results indicated that the EO of Cupressus funebris exhibited dose-dependent as well as time dependent antibacterial effects. The scanning electron microscopy indicated that the Cupressus funebris EO led to membrane rupture and permeabilization of the bacterial cells. Molecular docking studies indicated that the major compounds of the EO (citronellal and terpinene-4ol) showed strong interactions with the active site of the bacterial DNA gyrase enzyme explaining the antibacterial mode of action of the EO. Ciprofloxacin was also used for docking which showed stronger interactions with the target protein than citronellal or terpinene-4-ol. In conclusion, the major findings of the current study were that the EO of Cupressus funebris causes bacterial membrane rupture and permeabilization, shows time-dependent and dose-dependent antibacterial action, along with interacting with crucial bacterial enzyme viz., DNA gyrase as indicated by molecular docking studies.

Optimizing Lectin Staining Methodology to Assess Glycocalyx Composition of Legionella-Infected Cells

Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. Legionella utilizes effector proteins to hijack ER-Golgi vesicle trafficking to sustain proliferation in its intracellular niche. Legionella has a considerable influence on O-glycosylation but not N-glycosylation events in the Golgi of infected cells. This research aims to optimize the use of fluorescent lectins, which are proteins that bind carbohydrates, to effectively label host-cell glycocalyx during Legionella infection. Methods: Epifluorescence imaging or flow cytometry were used to optimize the lectin staining methodology. We noted that the most effective conditions for lectin-labeling were when live HeLa cells were incubated with lectins diluted in Hank’s balanced salt solution (HBSS) with 3% Bovine serum albumin (BSA) for 10-30 minutes at 4 °C. Results: Incubating suspended cells with lectins necessitated smaller lectin concentrations, whereas lectin labeling of adherent cells required considerably larger concentrations. Wheat germ agglutinin (WGA) lectin mean fluorescence intensity (MFI) was concentration-dependent, but Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did not alter substantially with increasing lectin concentrations. Discussion: The optimal lectin concentration required was lectin-specific and based on whether the lectin fluorescence was assessed using flow cytometry or epifluorescence. Furthermore, the use of phosphate-buffered saline (PBS) for lectin dilution, cell permeabilization for intracellular labelling, and incubation of lectins in fixed cells reduced productive labelling of lectins on cell surfaces because it inhibited the lectin's ability to effectively bind the associated carbohydrate structure. Conclusion: Further research using diverse lectins on U937 macrophages is necessary to reach a definitive conclusion on the effect of Legionella on the overall host-cell glycocalyx composition during infection of these relevant immune cells.

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

First person – Kevin Zhang

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Kevin Zhang is first author on ‘ Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells’, published in DMM. Kevin is an MD/PhD student in the lab of Dr Joshua Dunaief at the Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA, investigating the cell biology and metabolism of the eye.

Investigation of osmotic shock effect on pulsed electric field treated S. cerevisiae yeast cells

Pulsed electric field (PEF) treatment is known to cause plasma membrane permeabilization of microorganisms, an effect known as electroporation. PEF treatment is very attractive since it can achieve permeabilization with or without lethal damage in accordance with desired results. This study aimed to expand the accomplishment of electroporation outcomes by applying sudden post-PEF osmotic composition change of the media. Changes in yeast cells’ viability, size and plasma membrane regeneration rate were evaluated. However, we still have questions about the intracellular biochemical processes responsible for plasma membrane recovery after electroporation. Our suggested candidate is the high osmolarity glycerol (HOG) kinase pathway. The HOG pathway in Saccharomyces cerevisiae yeasts is responsible for volume recovery after dangerous shape modifications and intracellular water disbalance caused by environmental osmotic pressure changes. Thus, we evaluated the HOG pathway inactivation effect on S. cerevisiae’s reaction to PEF treatment. Results showed that Hog1 deficient S. cerevisiae cells were considerably more sensitive to electric field treatment, confirming a link between the HOG pathway and S. cerevisiae recovery process after electroporation. By suddenly changing the osmolarity of the media after PEF we influenced the cells’ plasma membrane recovery rate, severity of permeabilization and survivability of yeast cells. Studies of electroporation in combination with various treatments might improve electric field application range, efficiency, and optimization of the process.

Cell disruption and permeabilization methods for obtaining yeast bioproducts

Yeasts are used to produce several bioproducts, including functional bio-molecules, enzymes, biofuels, lipids, pigments, vitamins, organic acids, and other value-added bioproducts. When the production of the bioproduct occurs intracellularly, methods of disruption are traditionally used (mechanical and non-mechanical), which promote the release of bioproducts, but also the total degradation of the cell wall with consequent loss of yeast viability. As an alternative, cell permeabilization methods can be used through the use of external agents (chemical or physical), which form pores that increase the transfer of the product through the membrane, facilitating the separation of material, increasing the production of metabolites and also acting as an effective way of maintaining cellular viability - at least partially. In this review, we summarize the advances in yeast cell wall permeabilization compared to traditional methods of cell rupture. We also present the methods available for evaluating cell disruption and yeast permeabilization for yeast.

Fluid flow influences ultrasound-assisted endothelial membrane permeabilization and calcium flux.

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1MHz ultrasound with 20-cycle bursts, 1ms PRI, and 2s duration at a peak negative pressure of 305kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t=1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.

NANOSECOND PULSED ELECTRIC FIELD MODULATES IMMUNOPHENOTYPE OF LYMPHOCYTES

Introduction: Nanosecond pulsed electric fields (nsPEFs) have been shown to have anticancer effects, but little is known about the mechanisms modulating the immune landscape. According to the applied parameters (voltage, pulse duration, pulse delivery frequency, and a number of pulses), it is possible to achieve a specific effect modulating the immunophenotype of cancer cells. This study aims to identify changes in the expression profile of antigens defining lymphocyte maturity and function. Materials and Methods: We treated the Jurkat cell line with PEF. We tested different parameters of electroporation (2.5, 5, 7.5, 10, and 12.5 kV/cm, 100, 200 and 100 ns pulses, 25, 100, 250, and 500 pulses, 1, 10 and Hz). For the detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was analyzed after 24h by MTT Assay. ATP levels in culture were measured using Cell-Titer-Glo® Assay after 24 hours. The expression of cell markers was determined using flow cytometry. Results: The obtained results confirmed that electroporation permeabilizes the cell membrane and modulates the immunophenotype of Jurkat cells. Moreover, we observed that the ATP release increases when higher electric fields are applied. Application of 10kHz-100Hz and 25 pulses of nsPEF induces an increase in the expression of CD154 costimulatory molecule, which regulates the immune response by priming T cells and activating immune cells. Simultaneously, we increase the expression of the early activation marker CD69, indicating the ability to activate lymphocytes. The application of the 10kHz parameter upregulated the expression of antigens involved in the recruitment of inflammatory cells (CD183), controlling homeostasis of peripheral T lymphocytes (CD127) and regulating platelet adhesion (CD61). Importantly, we thus noted there was no alteration in the expression of CD95, which can mediate the induction of apoptosis in cancer cells. On the contrary, significant CD7 subexpression was observed when 10Hz nsPEF was applied. In general, the cells have not undergone cell death with lower electric field parameters but have modified their immunophenotype. Based on our studies, we propose a mechanism in which the cells: (1) permeabilize the cell membrane, (2) increase the expression of lymphocyte-activation antigens, and (3) downregulate the expression of CD7. Conclusions: Concluding, we can state that nsPEF can be a promising approach in immunotherapy via enhancing the presentation of lymphocyte-activation antigens. Also, we proved that application of electric field treatment could modulate the expression profile of lymphocytes while avoiding cell death. Encore Abstract - previously submitted to regional or national meetings (up to < 1’000 attendees) The research was funded by: This research was founded by the Polish National Centre of Science of DAINA 2 (2020/38/L/NZ7/00342; PI: J. Kulbacka), and the Research Council of Lithuania grant (Nr. S-LL-21-4, PI: V. Novickij). Keywords: Diagnostic and Prognostic Biomarkers, Basic and Translational Science - Other No conflicts of interests pertinent to the abstract.

Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair.

Exosomes are an extracellular vesicle (EV) subtype that is secreted upon the fusion of multivesicular bodies (MVBs) with the plasma membrane. Exosomes may participate in intercellular communication and have utility as disease biomarkers; however, little is known regarding the physiological stimuli that induce their secretion. Ca2+ influx promotes exosome secretion, raising the possibility that exosomes are secreted during the Ca2+-dependent plasma membrane repair of tissues damaged by mechanical stress in vivo. To determine whether exosomes are secreted upon plasma membrane damage, we developed sensitive assays to measure exosome secretion in intact and permeabilized cells. Our results suggest that exosome secretion is coupled to Ca2+-dependent plasma membrane repair. We find that annexin A6 (ANXA6), a well-known plasma membrane repair protein, is recruited to MVBs in the presence of Ca2+ and required for Ca2+-dependent exosome secretion, both in intact and in permeabilized cells. ANXA6 depletion stalls MVBs at the cell periphery, and ANXA6 truncations localize to different membranes, suggesting that ANXA6 may serve to tether MVBs to the plasma membrane. We find that cells secrete exosomes and other EVs upon plasma membrane damage and propose that repair-induced secretion may contribute to the pool of EVs present within biological fluids.