Exploring the Efficacy of Using Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus, Clay Minerals, and Walnut Nutshells for Mycotoxin Remediation.

The aim of this study was to evaluate the effectiveness of nine different biological compounds to reduce mycotoxins concentrations. The hypothesis of this study was that a static in vitro gastrointestinal tract model, as an initial screening tool, can be used to simulate the efficacy of Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus yeast cell walls and their polysaccharides, red and white clay minerals, and walnuts nutshells claiming to detoxify AFB1, ZEA, DON, and T-2 toxin mycotoxins. Mycotoxin concentrations were analyzed using high-performance liquid chromatography (HPLC) with fluorescent (FLD) and ultraviolet detectors (UV). The greatest effects on reducing mycotoxin concentrations were determined as follows: for AFB1, inserted G. fermentans cell wall polysaccharides and walnut nutshells; for ZEA, inserted R. rubra and G. fermentans cell walls and red clay minerals; for DON, R. rubra cell wall polysaccharides and red clay minerals; and for T-2 toxin, R. rubra cell walls, K. marxianus, and G. fermentans cell wall polysaccharides and walnut nutshells. The present study indicated that selected mycotoxin-detoxifying biological compounds can be used to decrease mycotoxin concentrations.

Antimicrobial Properties of Newly Developed Silver-Enriched Red Onion-Polymer Composites.

Simple low-cost, nontoxic, environmentally friendly plant-extract-based polymer films play an important role in their application in medicine, the food industry, and agriculture. The addition of silver nanoparticles to the composition of these films enhances their antimicrobial capabilities and makes them suitable for the treatment and prevention of infections. In this study, polymer-based gels and films (AgRonPVA) containing silver nanoparticles (AgNPs) were produced at room temperature from fresh red onion peel extract ("Ron"), silver nitrate, and polyvinyl alcohol (PVA). Silver nanoparticles were synthesized directly in a polymer matrix, which was irradiated by UV light. The presence of nanoparticles was approved by analyzing characteristic local surface plasmon resonance peaks occurring in UV-Vis absorbance spectra of irradiated experimental samples. The proof of evidence was supported by the results of XRD and EDX measurements. The diffusion-based method was applied to investigate the antimicrobial activity of several types of microbes located in the environment of the produced samples. Bacteria Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC BAA 747, and Pseudomonas aeruginosa ATCC 15442; yeasts Candida parapsilosis CBS 8836 and Candida albicans ATCC 90028; and microscopic fungi assays Aspergillus flavus BTL G-33 and Aspergillus fumigatus BTL G-38 were used in this investigation. The greatest effect was observed on Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa bacteria, defining these films as potential candidates for antimicrobial applications. The antimicrobial features of the films were less effective against fungi and the weakest against yeasts.

Application of Red Onion Peel Extract for Green Synthesis of Silver Nanoparticles in Hydrogels Exhibiting Antimicrobial Properties.

UV-initiated green synthesis of metal nanoparticles by using plant extracts as photoreducing agents is of particular interest since it is an environmentally friendly, easy-to-maintain, and cost-effective method. Plant molecules that act as reducing agents are assembled in a highly controlled way which makes them suitable for metal nanoparticle synthesis. Depending on the plant species, their application for green synthesis of metal nanoparticles for diverse applications may contribute to the mediation/reduction in organic waste amounts, thus enabling the implementation of the circular economy concept. In this work, UV-initiated green synthesis of Ag nanoparticles in hydrogels and hydrogel's thin films containing gelatin (matrix), red onion peel extract of different concentrations, water, and a small amount of 1 M AgNO3 have been investigated and characterized using UV-Vis spectroscopy, SEM and EDS analysis, XRD technique, performing swelling experiments and antimicrobial tests using bacteria (Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa), yeasts (Candida parapsilosis, Candida albicans) and microscopic fungi (Aspergillus flavus, Aspergillus fumigatus). It was found that the antimicrobial effectiveness of the silver-enriched red onion peel extract-gelatin films was higher at lower AgNO3 concentrations as compared to those usually used in the commercially available antimicrobial products. The enhancement of the antimicrobial effectiveness was analyzed and discussed, assuming the synergy between photoreducing agent (red onion peel extract) and silver nitrate (AgNO3) in the initial gel solutions leading to the intensification of Ag nanoparticles production.

Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance.

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL-1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0-1.5 log10 cfu mL-1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.

Antimicrobial particles based on Cu2ZnSnS4 monograins.

In this research, Cu2ZnSnS4 (CZTS) particles were successfully fabricated via the molten salt approach from the copper, zinc and tin sulphides as raw precursors. SEM analysis revealed that CZTS particles are tetragonal-shaped with sharp edges, smooth flat plane morphology, and crystal size varying from 10.8 to 28.7 µm. The phase and crystalline structure of synthesized powders were investigated using XRD analysis, which confirms the presence of a tetragonal crystal structure kesterite phase. The chemical composition of CZTS particles was evaluated by EDX spectroscopy, which identified the nearly stoichiometric composition with an averaged formula of Cu1.88Zn1.04SnS3.97. The TG/DTA-MS and ICP-OES analysis showed the possible decomposition pathways and predicted their degradation rate in aqueous solutions. The CZTS particles possessed highly effective concentration and time-dependent antimicrobial properties against medically relevant bacteria and yeast strains. The CZTS particles (1 g L-1) exhibited over 95.7 ± 1.9% killing efficiency towards M. luteus. In contrast, higher dosages (3.5 and 5 g L-1) led to its complete inactivation and reduced the P. aeruginosa cell viability to 43.2 ± 3.2% and 4.1 ± 1.1%, respectively. Moreover, the CZTS particles (0.5 g L-1) are responsible for causing 54.8 ± 1.8% of C. krusei and 89.7 ± 2.1% of C. parapsilosis yeasts death within the 24 h of exposure, which expanded to almost 100% when yeasts were treated with two times higher CZTS concentration (1.0 g L-1). The mechanism of action has been proposed and evidenced by monitoring the 2',7'-dichlorofluorescein (DCF) fluorescence, which revealed that the overproduction of reactive oxygen species (ROS) is responsible for microorganism death.

Microbial Fuel Cell Based on Nitrogen-Fixing Rhizobium anhuiense Bacteria.

In this study, the nitrogen-fixing, Gram-negative soil bacteria Rhizobium anhuiense was successfully utilized as the main biocatalyst in a bacteria-based microbial fuel cell (MFC) device. This research investigates the double-chambered, H-type R. anhuiense-based MFC that was operated in modified Norris medium (pH = 7) under ambient conditions using potassium ferricyanide as an electron acceptor in the cathodic compartment. The designed MFC exhibited an open-circuit voltage (OCV) of 635 mV and a power output of 1.07 mW m-2 with its maximum power registered at 245 mV. These values were further enhanced by re-feeding the anode bath with 25 mM glucose, which has been utilized herein as the main carbon source. This substrate addition led to better performance of the constructed MFC with a power output of 2.59 mW m-2 estimated at an operating voltage of 281 mV. The R. anhuiense-based MFC was further developed by improving the charge transfer through the bacterial cell membrane by applying 2-methyl-1,4-naphthoquinone (menadione, MD) as a soluble redox mediator. The MD-mediated MFC device showed better performance, resulting in a slightly higher OCV value of 683 mV and an almost five-fold increase in power density to 4.93 mW cm-2. The influence of different concentrations of MD on the viability of R. anhuiense bacteria was investigated by estimating the optical density at 600 nm (OD600) and comparing the obtained results with the control aliquot. The results show that lower concentrations of MD, ranging from 1 to 10 µM, can be successfully used in an anode compartment in which R. anhuiense bacteria cells remain viable and act as a main biocatalyst for MFC applications.

Impact of Soil Chemical Properties on the Growth Promotion Ability of Trichoderma ghanense, T. tomentosum and Their Complex on Rye in Different Land-Use Systems.

Microbial-based biostimulants that increase plant performance and ensure sustainable restoration of degraded soils are of great importance. The aim of the present study was to evaluate the growth promotion ability of indigenous Trichoderma ghanense, T. tomentosum and their complex on early rye seedlings in sustained grassland and arable soil. The impact of soil chemical properties on the ability of selected Trichoderma strains and their complex to promote plant growth was determined by the evaluation of the rye (Secale cereale L.) early seedling growth-measuring the length of shoots and roots as well as their dry weight. Trichoderma species were tested for their ability to produce extracellular degradative enzymes on solid media. Furthermore, the soil properties and CM-cellulase activity of soil were estimated. The indigenous Trichoderma strains possess the capacity to produce enzymes such as peroxidase, laccase, tyrosinase, and endoglucanase. The results indicated a significant (p < 0.05) increase in plant growth and the improvement of some soil chemical properties (total N, mobile humic and fulvic acids, exchangeable K2O, soil CM-cellulase activity) in inoculated soils when compared to the control. The growth of the roots of rye seedlings in sustained grassland was enhanced when T. tomentosum was applied (p = 0.005). There was an increase in total weight and shoot weight of rye seedlings when T. ghanense was used in the arable soil (p = 0.014 and p = 0.024). The expected beneficial effect of Trichoderma spp. complex on rye growth promotion was not observed in any tested soil. The results could find application in the development of new and efficient biostimulants, since not only do physiological characteristics of fungi play an important role but also the quality of the soil has an impact.

α-Terpinyl Acetate: Occurrence in Essential Oils Bearing Thymus pulegioides, Phytotoxicity, and Antimicrobial Effects.

The aim of this study was to evaluate occurrence of T. pulegioides α-terpinyl acetate chemotype, as source of natural origin α-terpinyl acetate, to determine its phytotoxic and antimicrobial features. Were investigated 131 T. pulegioides habitats. Essential oils were isolated by hydrodistillation and analyzed by GC-FID and GC-MS. Phytotoxic effect of essential oil of this chemotype on monocotyledons and dicotyledons through water and air was carried out in laboratory conditions; the broth microdilution method was used to screen essential oil effect against human pathogenic microorganisms. Results showed that α-terpinyl acetate was very rare compound in essential oil of T. pulegioides: it was found only in 35% of investigated T. pulegioides habitats. α-Terpinyl acetate (in essential oil and pure) demonstrated different behavior on investigated plants. Phytotoxic effect of α-terpinyl acetate was stronger on investigated monocotyledons than on dicotyledons. α-Terpinyl acetate essential oil inhibited seeds germination and radicles growth for high economic productivity forage grass monocotyledon Poa pratensis, but stimulated seed germination for high economic productive forage legume dicotyledon Trifolium pretense. α-Terpinyl acetate essential oil showed high antimicrobial effect against fungi and dermatophytes but lower effect against bacteria and Candida yeasts. Therefore, T. pulegioides α-terpinyl acetate chemotype could be a potential compound for developing preventive measures or/and drugs for mycosis.

Features of iron accumulation at high concentration in pulcherrimin-producing Metschnikowia yeast biomass.

In previous studies it was found that the antimicrobial properties of pulcherrimin-producing Metschnikowia species are related to the formation of a red pigment-pulcherrimin and sequestration of free iron from their growth medium. For strains of Metschnikowia pulcherrima, M. sinensis, M. shaxiensis, and M. fructicola, at a high, ≈80 mg/kg, elemental Fe concentration in agar growth media we observed the essentially different (metal luster, non-glossy rust like, and colored) yeast biomass coatings. For the studied strains the optical and scanning electron microscopies showed the increased formation of chlamydospores that accumulate a red pigment-insoluble pulcherrimin rich in iron. The chlamydospore formation and decay depended on the iron concentration. In this study pulcherrimin in biomass of the selected Metschnikowia strains was detected by Mössbauer spectroscopy. At ≈80 mg/kg elemental Fe concentration the Mössbauer spectra of biomass of the studied strains were almost identical to these of purified pulcherrimin. Iron in pulcherrimin reached ≈1% of biomass by weight which is very high in comparison with elemental Fe percentage in growth medium and is not necessary for yeast growth. The pulcherrimin in biomass was also observed by Mössbauer spectroscopy at lower, ≈5 mg/kg, elemental Fe concentration. Through chemical binding of iron pulcherrimin sequestrates the soluble Fe in the growth media. However, at high Fe concentrations, the chemical and biochemical processes lead to the pulcherrimin accumulation in biomass chlamydospores. When soluble iron is sequestrated or removed from the growth media in this way, it becomes inaccessible for other microorganisms.

Highly efficient antimicrobial agents based on sulfur-enriched, hydrophilic molybdenum disulfide nano/microparticles and coatings functionalized with palladium nanoparticles.

In this research the molybdenum disulfide (MoS2)-based nano/microparticles and coatings were synthesized through a simple, one-step hydrothermal approach without any other additives. Composition, structure, and morphology of the synthesized MoS2-based materials were investigated using ultraviolet-visible spectroscopy (UV-Vis), inductively coupled plasma optical emission spectrometry (ICP-OES), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy (EDX) techniques. The fabricated materials exhibited relatively small (Δθ = 18.7 ± 2.5°) contact angle and prominent hydrophilic properties, which are attributable to sulfur-enriched MoS2 composite as evidenced by simultaneous thermal analysis (STA) coupled with mass spectrometric (MS) analysis of evolving gaseous species (TG/DTA-MS) analysis. Such nanostructures exhibit a better adhesion of biomolecules, thus facilitating the interaction between them, as confirmed by highly effective antimicrobial action. The present study examines antimicrobial properties of hydrophilic, sulfur-enriched MoS2 nano/microparticles as well as MoS2-based coatings against various humans' pathogenic bacteria such as Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Micrococcus luteus, and two Candida yeast strains (C. parapsilosis, C. krusei). The MoS2-ns (40 µg mL-1) showed over 90% killing efficiency against S. aureus MRSA bacteria and both Candida yeast when exposed for 24 h. Petal-like MoS2 microstructures and heterostructured MoS2/Ti and Pd/MoS2/Ti coatings also possessed high antimicrobial potential and are considered as a promising antimicrobial agent. The MoS2-induced production of intracellular reactive oxygen species (ROS) was evidenced by measuring the standard DCF dye fluorescence.

Initiation of radioecological monitoring of forest soils and plants at the Lithuanian border region before the start of the Belarusian nuclear power plant operation.

Knowledge of the background activity concentrations of anthropogenic radionuclides before the start of operation of the new nuclear power plant in Belarus, BelNPP, is an issue of great importance for neighbouring countries. In this study, we provide the pilot characterisation of the Lithuanian part of the 30-km zone of the BelNPP, emphasising the forest plants, terrestrial mosses, forest organic and mineral topsoil to describe the preoperational radioecological state of the pine forest ecosystem. Key anthropogenic radionuclides (14C, 3H, 137Cs and 239,240Pu) were analysed. The 14C specific activity varied from 97.80 ± 1.30 to 102.40 ± 0.79 pMC. The 3H specific activity in the tissue-free water tritium form varied from 13.2 ± 2.2 TU to 20.8 ± 2.3 TU, which corresponded to the 3H level of precipitation in this region. The activity concentrations of 239,240Pu in soil and moss samples did not exceed 1 Bq/kg and were mainly due to global fallout after nuclear tests. The 137Cs inventory in the pine forest soils of the Lithuanian part of the BelNPP 30-km zone varied from 930 ± 70 to 1650 ± 430 Bq/m2. High variation of the inventory and uneven distribution in the soil profile conditioned a wide range of 137Сs activity in terrestrial plants from 1.0 ± 0.5 to 40.5 ± 1.8 Bq/kg dry weight. The abundance of microorganisms in different seasons and soil depths do not exceed the natural levels. According to PCA loads, the number of microorganisms and variability of 137Cs specific activity is determined by soil abiotic parameters.

Candida Distribution in Onychomycosis and in vitro Susceptibility to Antifungal Agents.

The aim of this study was to determine distribution of the Candida (C.) species in onychomycosis and analyses in vitro susceptibility to fluconazole and itraconazole. In recent years, cases of onychomycosis in Lithuania caused by Candida have increased significantly. In the period between 2009 and 2016, a total of 8149 clinical cases (outpatients and inpatients) were investigated at the Vilnius University Hospital Santaros Clinics (VUH SC). Candida yeasts were identified using VITEK 2 (BioMerieux, France) and IVD Maldi biotyper 2.3 (Bruker Daltonik GmbH, Germany), automated systems for identification of yeasts. The antifungal susceptibility to the Candida species were determined by disc diffusion. Candida spp. were the most frequently isolated pathogens in onychomycosis during the investigation period. The main species in onychomycosis were C. albicans (38.6%), followed by C. krusei (33.7%), C. tropicalis (11.1%), C. parapsilosis (7.9%), and other Candida (8.7%). The different antifungal susceptibility patterns among Candida species confirm the need to perform antifungal susceptibility in vitro testing of yeasts from patients with onychomycosis.

Ultra-small methionine-capped Au0/Au+ nanoparticles as efficient drug against the antibiotic-resistant bacteria.

In this study we examined the influence of ultra-small gold and magnetite­gold nanoparticles (NPs) stabilized with d,l-methionine, Fe3O4@Au@Met, on their antibacterial efficacy against three of twelve the worst bacterial family members included in the World Health Organization (WHO) list. In particular, gram-negative Acinetobacter baumannii, Salmonella enterica and gram-positive methicillin-resistant Staphylococcus aureus and Micrococcus luteus were tested. Apart from the synthesis, gold species reduction and NP stabilization, an excess of methionine has been used herein to detach ultra-small gold NPs from the Fe3O4@Au@Met surface, collect them and investigate. The antimicrobial efficiency of the ultra-small (Ø ~ 1.8 nm) Au@Met NPs and Fe3O4@Au@Met NPs was evaluated through the quantitative analysis by comparing with that of naked magnetite NPs, d,l-Met and BSA. It has been determined that compared with the control sample, 70 mg L-1 probe of Au@Met NPs exhibited the killing efficiency of 84.4-58.5% against gram-negative bacteria and 89.1-75.7% against gram-positive bacteria. The composition, structure, and morphology of the synthesized and tested herein NPs were investigated by inductively coupled plasma optical emission spectrometry, magnetic measurements, FTIR, XRD, XPS, AFM and HRTEM.

Low concentrations of acetic and formic acids enhance the inactivation of Staphylococcus aureus and Pseudomonas aeruginosa with pulsed electric fields.

BACKGROUND: Skin infections, particularly caused by drug-resistant pathogens, represent a clinical challenge due to being a frequent cause of morbidity and mortality. The objectives of this study were to examine if low concentrations of acetic and formic acids can increase sensitivity of Staphylococcus aureus and Pseudomonas aeruginosa to pulsed electric field (PEF) and thus, promote a fast and efficient treatment methodology for wound treatment. RESULTS: We have shown that the combination of PEF (10-30 kV/cm) with organic acids (0.1% formic and acetic acids) increased the bactericidal properties of treatment. The effect was apparent for both acids. The proposed methodology allowed to reduce the energy of electrical pulses and the inhibitory concentrations of acids, while still maintain high efficiency of bacteria eradication. CONCLUSIONS: Application of weak organic acids as bactericidal agents has many advantages over antibiotics because they do not trigger development of drug-resistance in bacteria. The combination with PEF can make the treatment effective even against biofilms. The results of this study are particularly useful for the development of new methodologies for the treatment of extreme cases of wound infections when the chemical treatment is no longer effective or hinders wound healing.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

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.

Influence of plant origin natural α-pinene with different enantiomeric composition on bacteria, yeasts and fungi.

Although the nature-identical chemical compounds are cheaper, they not always repeat biological activity of plant origin natural chemical compounds, often react allergies and resistance of microorganisms. The aim of this study was to investigate effects of Juniperus communis origin α-pinene with different enantiomeric composition on bacteria, yeasts and fungi. Results showed that different enantiomeric composition of α-pinene have different activities on microorganisms: essential oil with (1S)-(-) ≈ (1R)-(+) enantiomeric composition of α-pinene influenced on some microorganisms stronger than essential oil with (1S)-(-) < (1R)-(+) enantiomeric composition of α-pinene; the pure natural α-pinene with enantiomeric composition S < R more strongly inhibited growth of investigated bacteria and Candida yeasts, α-pinene with enantiomeric composition S ≈ R - growth of Trichophyton and Aspergillus. (1S)-(-) and (1R)-(+) enantiomeric forms of α-pinene can have also different synergistic effects with other compounds of essential oil. The results of study showed that the same amount of α-pinene with different enantiomeric composition can have diverse antimicrobial potential due different specific interactions with other chemical compounds of essential oil. Therefore, it is very important to determine and present the enantiomeric composition of those plant origin compounds, which are characterized by enantiomerisation, during the course of research of biological activities of natural plant products (essential oils and other) and their isolated compounds.

Membrane Permeabilization of Pathogenic Yeast in Alternating Sub-microsecond Electromagnetic Fields in Combination with Conventional Electroporation.

Recently, a novel contactless treatment method based on high-power pulsed electromagnetic fields (PEMF) was proposed, which results in cell membrane permeabilization effects similar to electroporation. In this work, a new PEMF generator based on multi-stage Marx circuit topology, which is capable of delivering 3.3 T, 0.19 kV/cm sub-microsecond pulses was used to permeabilize pathogenic yeast Candida albicans separately and in combination with conventional square wave electroporation (8-17 kV/cm, 100 µs). Bursts of 10, 25, and 50 PEMF pulses were used. The yeast permeabilization rate was evaluated using flow cytometric analysis and propidium iodide (PI) assay. A statistically significant (P < 0.05) combinatorial effect of electroporation and PEMF treatment was detected. Also the PEMF treatment (3.3 T, 50 pulses) resulted in up to 21% loss of yeast viability, and a dose-dependent additive effect with pulsed electric field was observed. As expected, increase of the dB/dt and subsequently the induced electric field amplitude resulted in a detectable effect solely by PEMF, which was not achievable before for yeasts in vitro.