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Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

Dairy and plant-based proteins are widely utilized in various food applications. Several techniques have been employed to improve the techno-functional properties of these proteins. Among them, pulsed electric field (PEF) technology has recently attracted considerable attention as a green technology to enhance the functional properties of food proteins. In this review, we briefly explain the fundamentals of PEF devices, their components, and pulse generation and discuss the impacts of PEF treatment on the structure of dairy and plant proteins. In addition, we cover the PEF-induced changes in the techno-functional properties of proteins (including solubility, gelling, emulsifying, and foaming properties). In this work, we also discuss the main challenges and the possible future trends of PEF applications in the food proteins industry. PEF treatments at high strengths could change the structure of proteins. The PEF treatment conditions markedly affect the treatment results with respect to proteins’ structure and techno-functional properties. Moreover, increasing the electric field strength could enhance the emulsifying properties of proteins and protein-polysaccharide complexes. However, more research and academia-industry collaboration are recommended to build highly effective PEF devices with controlled processing conditions. © 2022 by the authors. Licensee MDPI, Basel, Switzerland., Institute of Solid State Physics, University of Latvia as the Center of Excellence acknowledges funding from the European Union’s Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 under grant agreement No. 739508, project CAMART2.

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.

The design and development of a compact square-wave pulse generator for the electroporation of biological cells is presented. This electroporator can generate square-wave pulses with durations from 3 μs up to 10 ms, voltage amplitudes up to 3500 V, and currents up to 250 A. The quantity of the accumulated energy is optimized by means of a variable capacitor bank. The pulse forming unit design uses a crowbar circuit, which gives better control of the pulse form and its duration, independent of the load impedance. In such cases, the square-wave pulse form ensures better control of electroporation efficiency by choosing parameters determined in advance. The device has an integrated graphic LCD screen and measurement modules for the visualization of the current pulse, allowing for express control of the electroporation quality and does not require an external oscilloscope for current pulse recording. This electroporator was tested on suspensions of Saccharomyces cerevisiae yeast cells, during which, it was demonstrated that the application of such square-wave pulses ensured better control of the electroporation efficiency and cell viability after treatment using the pulsed electric field (PEF)., This article belongs to the Special Issue Information Technologies and Electronics, This work was partly funded by “Geozondas” Ltd. Implementing an EU structural investment project 01.2.1-MITA-T-851-02-0004 under measure “Inocekiai.”

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.

In this research we have evaluated the binding kinetics between an immobilized receptor and several genetically engineered ligands, differing by molecular mass or by the number of binding sites available for the binding to the receptor. Genetically engineered protein (GCSF-Receptor), which contains some antibody parts (Fc domain) and at some extent is similar to antibody because also has two binding sites that selectively bind another protein – glycoprotein granulocyte colony stimulating factor (GCSF), which was immobilized on a thin gold layer in order to design an immunosensor sensitive to GCSF. Three structurally different GCSF-based proteins were genetically-engineered and evaluated as ligands, which selectively bind to immobilized GCSF-Receptor: (i) GCSF monomer (mGCSF), (ii) GCSF-homodimer consisting of two via polypeptide Lα-based linker ‘fused’ GCSF molecules ((GCSF)2Lα) and (iii) GCSF-heterodimer (SCF-Lα-GCSF), which is based on a native GCSF molecule ‘fused’ via Lα-based linker with another protein – a soluble part of stem cell factor (SCF). SCF, unlike GCSF, does not contain any site suitable for GCSF-Receptor binding. The ligands differ by: (i) molecular mass – (GCSF)2Lα and SCF-Lα-GCSF F are two times heavier than mGCS, (ii) number of binding sites – mGCSF and SCF-Lα-GCSF have one binding site, while (GCSF)2Lα has two. The binding kinetics of mGCSF, (GCSF)2Lα, and SCF-Lα-GCSF with immobilized GCSF-Receptor was investigated using total internal reflection ellipsometry. The interaction kinetics of the mGCSF and SCF-Lα-GCSF are both well described using a standard Langmuir kinetics model. However, receptor-ligand association and dissociation rates in the case of SCF-Lα-GCSF ligand are about 10 times lower than that of mGCSF. The association rate of (GCSF)2Lα is about half of that of the mGCSF, which can be explained by the smaller diffusion coefficient of the larger molecule. Moreover, unlike SCF-Lα-GCSF, the (GCSF)2Lα adsorption kinetics cannot be adequately described by the standard Langmuir kinetics model and surface regeneration (induced by ‘washing’) experiments illustrate that (GCSF)2Lα, unlike the mGCSF and SCF-Lα-GCSF, is irreversibly bound to the surface modified by immobilized GCSF-Receptors. Therefore, to describe binding kinetics in the case of (GCSF)2Lα we have applied advanced kinetic model based on three protein association stages (three-stage kinetics model) in which (GCSF)2Lα forms several different intermediate complexes with GCSF-Receptor. This model precisely describes the time-varying surface concentration of (GCSF)2Lα bound to surface modified by immobilized GCSF-Receptors. In addition to the bioanalytical-aspects possible improvement of GCSF-based drugs is discussed.

In this study, the metabolism of yeast cells (Saccharomyces cerevisiae) was utilized for the synthesis of the conducting polymer - polypyrrole (Ppy).Yeast cells were modified in situ by synthesized Ppy. The Ppy was formed in the cell wall by redox-cycling of [Fe(CN)6]3-/4-, performed by the yeast cells. Fluorescence microscopy, enzymatic digestions, atomic force microscopy and isotope ratio mass spectroscopy were applied to determine both the polymerization reaction itself and the polymer location in yeast cells. Ppy formation resulted in enhanced resistance to lytic enzymes, significant increase of elasticity and alteration of other mechanical cell wall properties evaluated by atomic force microscopy (AFM). The suggested method of polymer synthesis allows the introduction of polypyrrole structures within the cell wall, which is build up from polymers consisting of carbohydrates. This cell wall modification strategy could increase the usefulness of yeast as an alternative energy source in biofuel cells, and in cell based biosensors.

Saccharomyces cerevisiae yeast cells were used as a model organism to investigate the effects of various pulsed electric fields on the programed death of such cells. These were exposed to electric field pulses with field strengths (E) of up to 220 kV/cm. The effects of square shaped pulses having different durations (τ = 10–90 ns) and different pulse numbers (pn = 1–5) were then analysed. The obtained results show that nanosecond pulses can induce the death of such cells, which in turn is dependent on the electric field pulse parameters and increase with the rise in E, τ and pn. The decrease of the cells' viability was accompanied by an increase in the active form of intracellular yeast metacaspases. It was thus shown that nanosecond electric field pulses induced the caspase-dependent yeast cell death.

Prolonged storage of frozen dough worsens the structure of thawed dough. The main reason is the inhibition of yeast activity. In this study we investigated applicability of pulsed electric field treatment for introduction of cryoprotectant into yeast cells. We showed that pre-treatment of cells suspended in a trehalose solution improves freezing tolerance and results in higher viability after thawing. Viability increased with rise in electric field strength (from 3 to 4.5 kV/cm) and incubation time (from 0 to 60 min) after exposure. Pretreatment resulted in lower decrease in the viability of thawed cells, viability of untreated cells dropped to 10%, while pre-treatment with PEF and trehalose tripled the viability.

The present paper shows a green method for the formation of polypyrrole by two different MIUG 12p and MIUG 4.88 strains of Streptomyces spp. bacteria. According to our knowledge, such method has never been proposed before for pyrrole oxidation. For this experiment pyrrole monomer was added into bacterial medium after 6 days of incubation and after 4 days polypyrrole (PPy) based 10–25 μm microspheres has been observed in the medium around living cells, The microsphere diameter dependence on the bacterial strains was observed. The morphological characterization of the PPy microspheres was subsequently performed by optical microscopy (OM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy. The characterization of formed hollow microspheres confirmed that they consist of polypyrrole.

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)6](3-)ions, which were generated from K4[Fe(CN)6], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)6](4-). After 24h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated.

Prospects of pulsed electric field technology application on acid whey concentrate pretreatment were analyzed. Stationary and flow pre-treatment systems were combined with different treatment parameters: electric field strength (E = 39 kV/cm, 95 kV/cm, 92 kV/cm), pulse duration (τ = 60 ns, 90 ns, 1000 ns) and pulse number (pn = up to 100 pulses). Isolates of Saccharomyces sp. and Lactobacillus sp. were predominant in concentrate. Significant non-thermal inactivation effect was achieved after PEF treatment. Exposure to short pulses selectively inactivated yeast cells, as a result PEF technology can be applied for low-energy acid whey processing.

Spectroscopic total internal reflection ellipsometry (TIRE) in its dynamic data acquisition mode has been applied for the in situ study of the interactions of monomeric (mGCSF) and dimeric (dGCSF) granulocyte colony stimulating factor and its receptor (GCSF-R) which was immobilized on a thin gold layer. Regression results have shown that the account for signal non-linearity due to analysis of spectroscopic dynamic TIRE data has allowed us to detect small differences in association and dissociation times through the precise determination of the refractive index dispersion. Different binding models were applied in the study of mGCSF and dGCSF interaction with a monolayer of its receptor GCSF-R due to their distinctive changes of refractive index over time.

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.

This nanosecond electric pulse generator is designed for the electroporation of biological cells suspended in a liquid media. It is based on a spark-gap switch, which is optically triggered by a 0.45-ns duration and 1-mJ energy laser pulse (wavelength 1062 nm). This system can also be triggered manually by changing the distance between the spark-gap electrodes. It is able to generate in a 75- Ω impedance transmission line near-perfect square-shaped electric pulses (rise and fall times ) with durations of 10, 40, 60, or 92 ns. The maximal amplitude of such pulses is 12.5 kV. The main advantage of this system is its ability to generate single pulses, the amplitude and duration of which can be precisely set in advance. To treat the cells, a coaxial cuvette with a 0.03-mL active volume and a 1-mm distance between the 28.3- mm2 circular-shaped electrodes was used. The system was tested on human erythrocytes. It was demonstrated that for the 92- and 40-ns duration pulse, the amplitude required to electroporate 50% of the cells was 20 and 65 kV/cm, respectively.

The baker’s yeast cells (Saccharomyces cerevisiae) are widely used in industry for fermentation control of food and drinks, and as a leavening agent in baking. They are widely available and rapidly reproducible. As an object for investigation the yeast cells distinguish ease of handling and manipulation. The baker’s yeast cells have complex internal cell structure and are well suited as a model organism for scientific studies. Most recent topics of yeast application in industry is related to generation of green-electricity1–3 by microbial fuel cells,4 5 and ethanol production in biofuel technology.6 For this reason baker’s yeasts are very popular among the scientific community for versatile applications. To improve the characteristics of living cells foreign components are introduced into the cell through membrane/wall holes opened by transformations, which are induced by: (i) chemical compounds, (ii) strong magnetic field7 or (iii) electrical discharge. Electrical discharge based method is known as electroporation. The electroporation is usually applied for modification of living cells by introducing into the cell foreign components such as genes, proteins, and other macromolecules8 9 or even specialized nanoparticles,10 which are used for modification of natural chemical processes inside the living cell. Nowadays various nanoparticles offers number of interesting options including fluorescence (e.g., Q-dots)11–14 catalytic ability15–18 and magnetic properties19–23 which could have been artificially added to the cells. Due to mentioned properties very interesting topic is how to introduce them into the cell and one of possible way to solve this problem is application of electroporation. The success of the electroporation usually is controlled by observation

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.

High power pulsed generator for non-contact cell electroporation application in pulsed magnetic fields is presented. Finite element method analysis of the magnetic field and the analysis of the Jurkat T lymphoblasts electroporation experimental data are overviewed. The dependence of cell electroporation efficiency on the structure of the microcoil was acquired experimentally. Possible problems of the technique, further developments and result overview are discussed.

The electrical field-induced changes of the yeast Saccharomyces cerevisiae cells permeabilization to tetraphenylphosphonium (TPP+) ions were studied using square-shaped, nanosecond duration high power electrical pulses. It was obtained that pulses having durations ranging from 10 ns to 60 ns, and generating electric field strengths up to 190 kV/cm significantly (up to 65 times) increase the absorption rate of TPP+ ions without any detectible influence on the yeast cell viability. The modelling of the TPP+ absorption process using a second order rate equation demonstrates that depending on the duration of the pulses, yeast cell clusters of different sizes are homogeniously permeabilized. It was concluded, that nanosecond pulse-induced permeabilization can be applied to increase the operational speed of whole cell biosensors.

The distribution of Lutra lutra in Lithuania was studied in 2007-2008 by surveying spraints and other signs of its presence (so-called standard monitoring methods). In total, 745 sites were checked in various water bodies: rivers, lakes, reclamation ditches, fish farms, and artificial reservoirs. Out of them, 584 (78.4%) were found positive. Fish farms were the most intensively used habitat - otters were found in all surveyed farms. Artificial reservoirs, medium-sized rivers, and streams were also frequently used. Within 100 m in the environs of a water body only anthropogenic landscape (towns, villages) was found to be a negative factor for otter presence. Other habitats both within 100 m in the environs and on the bank within 20 m, as well as regulation of a water body, did not influence the presence of otters. It is concluded that the situation of otters in Lithuania is good. The population is widely distributed across the country and inhabits various water bodies.