Eglė Lastauskienė

Formic Acid and Acetic Acid Induce a Programmed Cell Death in Pathogenic Candida Species

Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds—acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid—were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2–3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100–250 μs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

Measurement of Transient Permeability of Sp2/0 Myeloma Cells: Flow Cytometric Study

Abstract Electroporation is an electric field induced phenomenon occurring when the permeability of the cell membrane is increased due to the excess of critical transmembrane potential. Fluorescent dye assays are frequently used for evaluation of the permeabilization rate, however, the protocols vary, which negatively affects the repeatability of the results. In this work we have designed experiments to investigate the protocols and threshold concentrations of the Propidium Iodide (PI) and YO-PRO-1 (YP) fluorescent dyes for evaluation of mammalian cell permeabilization induced by electroporation. The Sp2/0 mouse myeloma cells were used and the bursts of 100 μs × 8 electrical pulses of 0.8-2 kV/cm were applied. It has been shown that the dye concentration has an influence on the detectable permeabilization, and the concentrations below 30 μM for PI and 1 μM for YP should be avoided for measurement of electropermeabilization efficacy due to unreliable fluorescence signals. Further, based on the experimental data, the permeabilization curve for the Sp2/0 myeloma cells in the 0.8-2 kV/cm range has been presented.

Growth Inhibition and Membrane Permeabilization of Candida lusitaniae Using Varied Pulse Shape Electroporation

Candida lusitaniae is an opportunistic yeast pathogen, which can readily develop resistance to antifungal compounds and result in a complex long-term treatment. The efficient treatment is difficult since structure and metabolic properties of the fungal cells are similar to those of eukaryotic host. One of the potential methods to improve the inhibition rate or the cell permeability to inhibitors is the application of electroporation. In this work we investigated the dynamics of the growth inhibition and membrane permeabilization of C. lusitaniae by utilizing the various pulse shape and duration electric field pulses. Our results indicated that single electroporation procedure using 8 kV/cm electric field may result in up to 51 ± 5% inhibition rate. Also it has been experimentally shown that the electroporation pulse shape may influence the inhibitory effect; however, the amplitude of the electric field and the pulse energy remain the most important parameters for definition of the treatment outcome. The dynamics of the cell membrane permeabilization in the 2–8 kV/cm electric field were overviewed.

Ras/PKA signal transduction pathway participates in the regulation of Saccharomyces cerevisiae cell apoptosis in an acidic environment

The acidification of the medium is observed during yeast cell growth. This process contributes to the emission of organic acids, mainly acetic acid. Acetic acid is known as the inducer of apoptosis in the yeast Saccharomyces cerevisiae. In this study, we showed that hydrochloric acid can also induce apoptosis in yeast cells, and the apoptotic phenotype triggered by treating yeast cells with hydrochloric acid is modulated by the Ras/PKA pathway. The Ras/PKA pathway is highly conserved between all eukaryotic organisms, as well as cell processes that are related to apoptosis and aging. In this research, we demonstrated that the activation of the Ras/PKA pathway by insertion of Ras2Val19 allele or deletion of PDE2 gene increases cell death, displaying the markers of apoptosis in an acidic environment. Downregulation of the pathway by deletion of RAS2, RAS1, PDE1, and insertion of the Ha‐ras gene increases the cell viability and diminishes cell death with the apoptotic phenotypes. The deletion of PDE1 gene and double deletion of both phosphodiesterase genes prevent the induction of apoptosis in the cells. Modulations in the Ras/PKA pathway affect cell viability and apoptosis during natural gradual acidification of the medium as well as in acid stress conditions.

Activation of Tryptophan and Phenylalanine Catabolism in the Remission Phase of Allergic Contact Dermatitis: A Pilot Study

Background: Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The severity and duration of this disease are associated with many different factors. Some of these factors may represent markers for monitoring disease activity and the individual response to an intervention. Methods: We used a targeted metabolomics approach to find such factors in the serum of individuals with ACD. Metabolomics profiles were examined and compared in the acute phase of the disease and also in the absence of disease activity. Results: Our study identified a significant remission phase of ACD-associated systemic biochemical shifts in 2 metabolic pathways: tryptophan-kynurenine and phenylalanine-tyrosine. Conclusions: Although the responsible mechanisms are unclear, these results suggest that the remission phase of ACD is linked to tryptophan metabolism via kynurenine and phenylalanine-tyrosine pathways. However, further replication studies with a larger number of subjects and their subgroups are necessary to validate our results. These studies may provide a new perspective with which to understand the mechanism of and find potential biomarkers of ACD, as well as a new reference for personalized treatment.

Serum Biomarkers of Allergic Contact Dermatitis: A Pilot Study

Background: Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by repeated skin exposure to contact allergens. The goal of this pilot study was to identify inflammatory proteins which can serve as biomarkers for ACD. Methods: We measured levels of 102 cytokines, chemokines, and growth factors in the sera of 16 ACD patients during acute and remission phases, and 16 healthy volunteers. Results: Serum levels of adiponectin, chemokine (C-C motif) ligand 5 (CCL5), C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), complement factor D (CFD), endoglin, lipocalin-2, osteopontin, retinol-binding protein 4 (RBP4), and platelet factor 4 (PF4) were significantly higher, whereas levels of trefoil factor 3 (TFF3) were significantly lower, in ACD patients than in healthy controls. In ACD patients, serum levels of CCL5 were elevated, whereas levels of TFF3, soluble intercellular adhesion molecule-1 (sICAM-1), and platelet-derived growth factor (PDGF)-AB/BB were found to be lower during the remission phase of the disease. Conclusions: Serum levels of adiponectin, CCL5, CRP, CHI3L1, CFD, endoglin, lipocalin-2, osteopontin, RBP4, PF4, and TFF3 might be exploited as biomarkers for ACD, whereas levels of CCL5, TFF3, sICAM-1, and PDGF-AB/BB might be exploited for evaluation of disease progression and efficacy of ACD treatment.

Non-invasive nanosecond electroporation for biocontrol of surface infections: an in vivo study

Invasive infections caused by drug-resistant bacteria are frequently responsible for fatal sepsis, morbidity and mortality rates. In this work, we propose a new methodology based on nanosecond high frequency electric field bursts, which enables successful eradication of bacteria in vivo. High frequency (15 kHz) 15–25 kV/cm 300–900 ns pulsing bursts were used separately and in combination with acetic acid (0.1–1%) to treat Pseudomonas aeruginosa in a murine model. Acetic acid 1% alone was effective resulting in almost 10-fold reduction of bacteria viability, however combination of nanosecond electric field and acetic acid 1% treatment was the most successful showing almost full eradication (0.01% survival compared to control) of the bacteria in the contaminated area. The short duration of the pulses (sub-microsecond) and high frequency (kHz range) of the burst enabled reduction of the muscle contractions to barely detectable level while the proposed applicators ensured predominantly topical treatment, without electroporation of deeper tissues. The results of our study have direct application for treatment of wounds and ulcers when chemical treatment is no longer effective.

Induction of Different Sensitization Patterns of MRSA to Antibiotics Using Electroporation

Treatment of bacteria-associated infections is complicated and antibiotic treatment alone is often inadequate to overcome biofilm infections. Physical methods allow overcoming this problem and propose solutions that are non-dependent on drug resistance. In this work, we investigated the feasibility of pulsed electric fields for sensitization of MRSA to common antibiotics. We analyzed the efficacy of inactivation of methicillin-resistant Staphylococcus aureus in 5–20 kV/cm electric field separately and in combination with gentamicin, doxycycline, ciprofloxacin, sulfamethoxazole, and vancomycin. Combined treatment allowed using up to 1000-fold smaller concentrations of antibiotics to induce the same inactivation of S. aureus.

Different permeabilization patterns of splenocytes and thymocytes to combination of pulsed electric and magnetic field treatments

Genetic manipulation of T cells is frequently inefficient, however, when combined with physical methods (i.e. electroporation) a promising alliance with immunotherapy can be formed. This study presents new data on permeabilization of murine thymocytes and splenocytes as a T cell model using pulsed electric (PEF) and electromagnetic field (EMF). The 300ns, 500ns, 2μs and 100μs pulse bursts in a broad range of PEF 0-8kV/cm were applied separately and in combination with 3.3T, 0.2kV/cm EMF pulses. The permeabilization efficiency was evaluated using fluorescent dye (YO-PRO-1) and flow cytometry. It was shown that a >14% increase in thymocytes permeabilization is achieved when electroporation is applied in combination with EMF, however splenocytes responded in a different manner - a statistically significant (P<0.05) reduction in permeabilization was observed. The cytokine secretion patterns were mainly unaltered independently on the applied treatment parameters determined by secretion of IFNγ, IL-4 and IL-17 - the main cytokines of Th1, Th2 and Th17 cells. The results of this study are useful for development of pulsed power protocols for effective genetic modification of T cells.