Oxidative Effects during Irreversible Electroporation of Melanoma Cells—In Vitro Study

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2–4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2–0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2–0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids’ oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.     

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.     

The temperature effect during pulse application on cell membrane fluidity and permeabilization

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.     

Evidence that Pulsed Electric Field Treatment Enhances the Cell Wall Porosity of Yeast Cells

The application of rectangular electric pulses, with 0.1–2 ms duration and field intensity of 2.5–4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2–8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.     

Probing the interplay between amyloidogenic proteins and membranes using lipid monolayers and bilayers

Many degenerative diseases such as Alzheimer's and Parkinson's involve proteins that have a tendency to misfold and aggregate eventually forming amyloid fibers. This review describes the use of monolayers, bilayers, supported membranes, and vesicles as model systems that have helped elucidate the mechanisms and consequences of the interactions between amyloidogenic proteins and membranes. These are twofold: membranes favor the formation of amyloid structures and these induce damage in those membranes. We describe studies that show how interfaces, especially charged ones, favor amyloidogenic protein aggregation by several means. First, surfaces increase the effective protein concentration reducing a three-dimensional system to a two-dimensional one. Second, charged surfaces allow electrostatic interactions with the protein. Anionic lipids as well as rafts, rich in cholesterol and gangliosides, prove to play an especially important role. Finally, these amphipathic systems also offer a hydrophobic environment favoring conformational changes, oligomerization, and eventual formation of mature fibers. In addition, we examine several models for membrane permeabilization: protein pores, leakage induced by extraction of lipids, chaotic pores, and membrane tension, presenting illustrative examples of experimental evidence in support of these models. The picture that emerges from recent work is one where more than one mechanism is in play. Which mechanism prevails depends on the protein, its aggregation state, and the lipid environment in which the interactions occur.     

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light‐programmable, cell‐permeable reporter deliverable simultaneously to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2‐4,5‐dimethoxy 2‐nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single‐cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.     

Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.     

Micro pulsed radio-frequency electroporation chips

Electroporation (EP) is one of the most important physical methods in biotechnology, which employs electrical pulses to transiently permeabilize cell membranes. In this study, a new micro pulsed radio-frequency electroporation cell (microPREP) chip was fabricated using a lift-off technique and SU-8 photolithography. The biological tests were carried out using three different plant protoplasts (cabbage, spinach and oil rape) on the micro EP chip and a pulsed RF electric field was applied to the microchip. The variations of fluorescent intensity and cell viability as functions of the electric pulse amplitude and duration time during the electroporation process were studied in detail at the single-cell level. Using such chip design and test method, one can easily optimize the efficiency and cell viability. Also, a large amount of statistical data can be quickly obtained. Finally, results of this parametric study were presented in the "phase diagram", from which the critical electric field for inducing single-cell electroporation under different conditions can be clearly determined.     

Application of relaxation periods during electrodialysis of a casein solution: Impact on anion-exchange membrane fouling

Electrodialysis (ED) is a membrane process used on a large scale. However, one of the common problems is fouling of ion-exchange membranes stacked in the cell. The use of pulsed power, consisting in applying a constant current density during a fixed time of application (T_on) followed by a pause duration (T_off), was demonstrated recently as an effective fouling mitigation method for electrodialysis. Up until now, no work has investigated the potential of electrodialysis using pulsed electric field on protein fouling. The aim of the present work was to study the influence of pulsed electric field (PEF) with a low frequency square shaped periodic signal (T_on = 10 s–T_off = 10 s, T_on = 10 s–T_off = 40 s) in comparison with dc current during electrodialysis of a casein solution at different current densities (10, 20 and 30 mA/cm²) on membrane fouling. It appeared from these results that PEF, under certain conditions of pulse, would avoid fouling on anion-exchange membranes. For 10 s–40 s pulsed electric field conditions, no fouling was observed with any density, while for 10 s–10 s PEF conditions, fouling appeared only at current density over 10 mA/cm². dc current, whatever the current density conditions, led to a fouling on the diluate side of the AEM. Furthermore, when fouling occurred, magnitude layer thickness and dry weight increased with the applied current density. The nature of the fouling was identified as 97% protein. The protein fouling would be due to the dissociation of water molecules and/or heat increase at the anion-exchange membrane interface. The relaxation time of the pulse would limit both phenomena on the membrane.     

侧链磺化聚芳醚质子交换膜的研究进展

燃料电池是以氢气、甲醇等作为燃料的一种新型能量转化装置,其中质子交换膜燃料电池（Proton Exchange Membrane Fuel Cell, PEMFC）凭借其能量功率高、启动速度快和使用寿命长等优点已经在移动电源、潜艇和电动汽车等领域得到了广泛应用。质子交换膜（Proton Exchange Membrane, PEM）对PEMFC的性能影响最大,高效的PEMFC需要PEM具有高的质子电导率、良好的热稳定性和机械性能、低燃料渗透率以及优异的物理化学稳定性等。目前市面上多数使用的均是具有优异质子电导率的Nafion系列膜,但其存在制备困难、成本昂贵、质子电导率严重依赖湿度等缺点,在一定程度上限制了其发展。为了让PEM有更多的选择,科学家一直专注于使用新材料替代Nafion膜。近年来,科学家们模拟Nafion结构,通过合成各种侧链含磺酸基团的聚芳醚结构,使得亲水基团磺酸基和疏水基团之间形成微相分离结构,从而获得了一系列具有优异综合性能的PEM。本文将重点对侧链烷基磺化型、侧链磺化嵌段型、侧链局部密集磺化型、侧链磺化交联型和侧链磺化复合型这几种常见策略的合成方法及性能进行了综述,最后展望了侧链磺化聚芳醚在PEM领域的优势及发展前景。     

Steep pulsed electric fields modulate cell apoptosis through the change of intracellular calcium concentration

A steep electric pulsed field with low intensity (150–250 V/cm) and relative long time (10 min) was applied to adherent liver cancer cell line SMMC-7721 and the liver cell line HL-7702. Results showed that the electric field with intensity of 200 and 250 V/cm could trigger cell apoptosis, whereas the SMMC-7721 cell was more sensitive to the electric stimulation than the HL-7702 cell. Laser Scanning Confocal Microscope (LSCM) was used to measuring the real-time change of cytosolic free Ca²⁺ concentration. When cells were exposed electric pulses with 100 V/cm intensity for 10 min, there was no significant change of intracellular calcium concentration. With the intensity increased to 200 and 250 V/cm, intracellular calcium concentration decreased significantly. Results demonstrated the relationship between the apoptosis and change of intracellular calcium concentration. And the steep electric pulsed field can be used to the cancer therapy.     

Low Cost Compact Nanosecond Pulsed Plasma System for Environmental and Biomedical Applications

Nanosecond pulsed non-thermal atmospheric-pressure plasmas are promising for numerous applications including air and water purification, ozone synthesis, surface sterilization, material processing, and biomedical care. However, the high cost of the nanosecond pulsed power sources has hindered adaptation of the plasma-based technologies for clinical and industrial use. This paper presents a low cost (<100US$) nanosecond pulsed plasma system that consists of a Cockcroft–Walton high voltage charging circuit, a compact nanosecond pulse generator using a spark gap as switch, and a plasma reactor. The nanosecond pulse power source requires only a 12 V DC input, hence is battery operable. Through the optimization of the experimental parameters, pulses with a peak voltage >10 kV, a 3 ns rise time (10 to 90 %), and a 10 ns pulse duration (full width at half maximum) at a pulse repetition rate of up to 500 Hz were achieved in the present study. It has been successfully tested to power three different plasma reactors to form pulsed corona discharges, dielectric barrier discharges, and sliding discharges. The energy efficiency of such a nanosecond pulsed sliding discharge system was assessed in the context of ozone synthesis using air or oxygen as the feed gas, and was found comparable to a previously reported non-thermal plasma system that used commercial high voltage pulsed power sources. This study demonstrated that this low-cost nanosecond pulsed power source can prove to be an energy efficient and simple supply to drive various non-thermal atmospheric-pressure plasma reactors for environmental, medical and other applications.     

MECHANISM OF ORIENTATION AND LINEAR DICHROISM OF PHOTOSYNTHETIC PARTICLES IN ELECTRIC FIELDS: CHLOROPLASTS AND CHLOROPHYLL-PROTEIN COMPLEXES

Abstract— The mechanisms of orientation in pulsed and alternating electric fields of thylakoids (derived from the sonication of spinach chloroplasts) and of light-harvesting chlorophyll a/b-protein complexes (CPII) were investigated by utilizing linear dichroism techniques. Comparisons of the linear dichroism spectra of thylakoids and CPII particles suggest that the latter are oriented with their directions of largest electronic polarizabilities (and thus probably their largest dimensions) within the thylakoid membrane planes. At low electric field strengths (< 12 V cm^?1), and at low frequencies of alternating electric fields (< 0.25 Hz), thylakoid membranes tend to align with their normals parallel to the direction of the applied electric field; the mechanism of orientation involves a permanent dipole moment of the thylakoids which is oriented perpendicular to the planes of the membranes. However, at high field strengths and high frequencies of the applied alternating electric fields, the thylakoids tend to orient with their planes parallel to the applied field, thus exhibiting an inversion of the sign of the linear dichroism as the electric field strength is increased. At the higher frequencies and at higher field strengths, the orientation mechanisms of the thylakoids involve induced dipole moments related to anisotropies in the electronic polarizabilities. The polarizability is higher within the plane than along a normal to the plane, thus accounting for the inversion of the dichroism as the electric field strength is increased. The CPII particles align with their largest dimension parallel to the applied field at all field strength, indicating that the induced dipole moment dominates the orientation mechanisms in pulsed electric fields. The magnitude of the absolute linear dichroism of CPII suspensions increases with increasing dilution, indicating that aggregates of lower symmetry are formed at higher concentrations of the CPII complexes.     

Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating Peptide example

A novel solid-state NMR technique for identifying the asymmetric insertion depths of membrane proteins in lipid bilayers is introduced. By applying Mn (2+) ions on the outer but not the inner leaflet of lipid bilayers, the sidedness of protein residues in the lipid bilayer can be determined through paramagnetic relaxation enhancement (PRE) effects. Protein-free lipid membranes with one-side Mn (2+)-bound surfaces exhibit significant residual (31)P and lipid headgroup (13)C intensities, in contrast to two-side Mn (2+)-bound membranes, where lipid headgroup signals are mostly suppressed. Applying this method to a cell-penetrating peptide, penetratin, we found that at low peptide concentrations, penetratin is distributed in both leaflets of the bilayer, in contrast to the prediction of the electroporation model, which predicts that penetratin binds to only the outer lipid leaflet at low peptide concentrations to cause an electric field that drives subsequent peptide translocation. The invalidation of the electroporation model suggests an alternative mechanism for intracellular import of penetratin, which may involve guanidinium-phosphate complexation between the peptide and the lipids.     

电子传感技术在中药材及农产品分析领域的应用研究进展

电子传感技术以其信号易于获得、信息丰富、能够从整体上表征样品性质等优势, 近年来在中药材及农产品分析领域得到日益广泛的应用.对常用的电子传感技术电子眼、电子鼻、电子舌等进行介绍, 对上述技术在中药、烟叶、食品、饮料等领域的应用报道进行综述, 并对其相关的多变量数据分析技术、多源数据融合技术、品牌保护技术及未来仪器研发进行展望, 以期为电子传感技术在中药材及农产品领域的推广应用提供借鉴.     

Conformational and Intermolecular Interaction Dynamics of Photolyase/Cryptochrome Proteins Monitored by the Time‐Resolved Diffusion Technique

Cryptochrome (CRY), a blue light sensor protein, possesses a similar domain structure to photolyase (PHR) that, upon absorption of light, repairs DNA damage. In this review, we compare the reaction dynamics of these systems by monitoring the reaction kinetics of conformational change and intermolecular interaction change based on time‐dependent diffusion coefficient measurements obtained by using the pulsed laser‐induced transient grating technique. Using this method, time‐dependent biomolecular interactions, such as transient dissociation reactions in solution, have been successfully detected in real time. Conformational change in (6‐4) PHR has not been detected after the photoexcitation by monitoring the diffusion coefficient. However, the repaired DNA dissociates from PHR with a time constant of 50 μs, which must relate to a minor conformational change. However, CRY exhibits a considerable diffusion change with a time constant of 400 ms, which indicates that the protein–solvent interaction is changed by the conformational change. The C‐terminal domain of CRY is shown to be responsible for this change.     

Direct observation of protein motion during the photochemical reaction cycle of bacteriorhodopsin

Platinum-coated, conductive atomic force microscope cantilevers were used to deposit electrophoretically purple membranes from Halobacterium salinarum on the bottom part of the cantilevers. By illuminating the bacteriorhodopsin-containing purple membranes, the protein goes through its photochemical reaction cycle, during which a conformational change happens in the protein, changing its shape and size. The size change of the protein acts upon the cantilever by causing its deflection, which can be monitored by the detection system of the atomic force microscope. The shape of the signal, the action spectrum of the deflection amplitude, and the blue light inhibition of the deflection all prove that the origin of the signal is the conformational change arising in the bacteriorhodopsin during the photocycle. From the size of the signal, the magnitude of the protein motion could be estimated. Using polarized light, the orientation of the motion was determined, relative to the transition moment of the retinal.     

Microstructure,texture and oral processing: New ways to reduce sugar and salt in foods

Food oral processing as the bridge between food texture, microstructure and sensory perception has gained enormous interest in the last decade. This review provides an overview of the role of the microstructure of soft- and semi-solid foods in food oral processing and sensory perception. Phase separated mixed protein–polysaccharide gels and emulsion-filled gels are described as suitable model foods to investigate food oral processing systematically. Special attention is given to the sensory perception of texture, taste and interactions thereof. Several approaches to reduce the salt and sugar content of semi- and soft-solid foods without compromising taste are reviewed. These reduction approaches are based on an understanding of food oral processing in relation to the microstructure of the foods and its breakdown.     

核桃蛋白酶解物的制备及抗氧化活性

采用碱性蛋白酶对核桃蛋白进行酶解, 检测了所得酶解物的抗氧化能力, 包括对1,1-二苯基-2-三硝基苯肼(DPPH)和羟基自由基(·OH)的清除能力; 利用Sephadex G-25 凝胶层析柱和反相柱对核桃蛋白酶解物进行分离纯化; 采用液相色谱-质谱(LC-ESI-Q-TOF)联用方法测得抗氧化能力最强的多肽的序列为Ala-Gly-Gly-Ala, 其还原力和还原型谷胱甘肽相当.     

Protein-Polysaccharide Interactions for Stabilization of Food Emulsions

Proteins, polysaccharides and their blends, as examples of natural biopolymers, are surface active materials. Biopolymers may be considered as amphiphilic macromolecules that play an essential role in stabilizing food formulations (foams, emulsions and dispersions). Under specific conditions (such as protein-to-polysaccharide ratio, pH, ionic strength, temperature, mixing processing), it has been stated that proteins and polysaccharides form hybrids (complexes) with enhanced functional properties in comparison to the proteins and polysaccharides alone. Different protein-polysaccharide pairs are reviewed with particular attention to the emulsification capability of their mixtures. In the case of uncomplexed blends of biopolymers, competitive adsorption onto hydrophobic surfaces is generally reported. Conversely, electrostatic complexation between oppositely charged proteins and polysaccharides allows better anchoring of the new-formed macro-molecular amphiphile onto oil-water interfaces. Moreover, improved thermal stability and increased resistance to external treatment (high pressure) involved in food processing are obtained. This review presents basic and applied knowledge on protein-polysaccharide interactions in aqueous medium and at the oil-water interface in food emulsion systems. Electrostatic interactions and thermodynamic incompatibility in mixed biopolymer solutions are correlated to the functional properties (rheology, surface hydrophobiciry, emulsification power) of these interesting blends. Basic and industrial selected systems of different families of hydrocolloids (as gum Arabic, galactomannans, pectins) and protein (caseins, whey, soya, gelatin) mixtures are reviewed.