Proteomic profiling of antibiotic-resistant Escherichia coli GW-AmxH19 isolated from hospital wastewater treated with physical plasma.

Microorganisms which are resistant to antibiotics are a global threat to the health of humans and animals. Wastewater treatment plants are known hotspots for the dissemination of antibiotic resistances. Therefore, novel methods for the inactivation of pathogens, and in particular antibiotic-resistant microorganisms (ARM), are of increasing interest. An especially promising method could be a water treatment by physical plasma which provides charged particles, electric fields, UV-radiation, and reactive species. The latter are foremost responsible for the antimicrobial properties of plasma. Thus, with plasma it might be possible to reduce the amount of ARM and to establish this technology as additional treatment stage for wastewater remediation. However, the impact of plasma on microorganisms beyond a mere inactivation was analyzed in more detail by a proteomic approach. Therefore, Escherichia coli GW-AmxH19, isolated from hospital wastewater in Germany, was used. The bacterial solution was treated by a plasma discharge ignited between each of four pins and the liquid surface. The growth of E. coli and the pH-value decreased during plasma treatment in comparison with the untreated control. Proteome and antibiotic resistance profile were analyzed. Concentrations of nitrite and nitrate were determined as long-lived indicative products of a transient chemistry associated with reactive nitrogen species (RNS). Conversely, hydrogen peroxide served as indicator for reactive oxygen species (ROS). Proteome analyses revealed an oxidative stress response as a result of plasma-generated RNS and ROS as well as a pH-balancing reaction as key responses to plasma treatment. Both, the generation of reactive species and a decreased pH-value is characteristic for plasma-treated solutions. The plasma-mediated changes of the proteome are discussed also in comparison with the Gram-positive bacterium Bacillus subtilis. Furthermore, no effect of the plasma treatment, on the antibiotic resistance of E. coli, was determined under the chosen conditions. The knowledge about the physiological changes of ARM in response to plasma is of fundamental interest to understand the molecular basis for the inactivation. This will be important for the further development and implementation of plasma in wastewater remediation.

Hydrogen Peroxide Production of Individual Nanosecond Pulsed Discharges Submerged in Water of Elevated Conductivity.

The production of hydrogen peroxide (H2 O2 ) is a key parameter for the performance of pulsed discharges submerged in water utilized as advanced oxidation process. So far, any related assessment of the underlying mechanism was conducted for the application of several hundred discharges, which did not allow for a correlation with physical processes. Moreover, the production was rarely investigated depending on water conductivity as one of the most important parameters for the development of submerged discharges. Accordingly, hydrogen peroxide generation was investigated here for individual single discharge events instigated with 100 ns high-voltage pulses in water with three different conductivities and was associated with the discharge development, i. e. spatial expansion and dissipated electrical energy. The approach necessitated the improvement of an electrochemical flow injection analysis based on the reaction of Prussian blue with H2 O2 . Hydrogen peroxide concentrations were quadratically increasing with propagation time and stable for different water conductivities. H2 O2 production per unit volume of a discharge was constant over time with an estimated rate constant of 3.2 mol ⋅ m-1 s-1 , averaged over the crosssectional area of all discharge filaments. However, the individually dissipated energy increased with conductivity, hence, the production efficiency decreased from 6.1 g ⋅ kWh-1 to 1.4 g ⋅ kWh-1 , which was explained by increased resistive losses within the bulk liquid.

Uncertainty Quantification and Sensitivity Analysis for the Electrical Impedance Spectroscopy of Changes to Intercellular Junctions Induced by Cold Atmospheric Plasma.

The influence of pertinent parameters of a Cole-Cole model in the impedimetric assessment of cell-monolayers was investigated with respect to the significance of their individual contribution. The analysis enables conclusions on characteristics, such as intercellular junctions. Especially cold atmospheric plasma (CAP) has been proven to influence intercellular junctions which may become a key factor in CAP-related biological effects. Therefore, the response of rat liver epithelial cells (WB-F344) and their malignant counterpart (WB-ras) was studied by electrical impedance spectroscopy (EIS). Cell monolayers before and after CAP treatment were analyzed. An uncertainty quantification (UQ) of Cole parameters revealed the frequency cut-off point between low and high frequency resistances. A sensitivity analysis (SA) showed that the Cole parameters, R0 and α were the most sensitive, while Rinf and τ were the least sensitive. The temporal development of major Cole parameters indicates that CAP induced reversible changes in intercellular junctions, but not significant changes in membrane permeability. Sustained changes of τ suggested that long-lived ROS, such as H2O2, might play an important role. The proposed analysis confirms that an inherent advantage of EIS is the real time observation for CAP-induced changes on intercellular junctions, with a label-free and in situ method manner.

Degradation of glyphosate in water by the application of surface corona discharges.

Glyphosate (GLP) is one of the most widely applied herbicides, and is found ubiquitously in the environment. The removal of glyphosate from waste water and soil is challenging and can be achieved with chemical or biological methods, which, nevertheless, suffer from different disadvantages. The application of a physical plasma for the removal of GLP in water was examined by the application of surface corona discharges in a wire-to-cylinder setup filled with argon. The plasma was ignited at the liquid surface without any additives. By applying a photometric method, GLP was detected after derivatisation with fluorenyl methoxycarbonyl chloride, whereas phosphate was determined with ammonium molybdate. A GLP degradation rate of 90.8% could be achieved within a treatment time of 30 minutes with an estimated energy efficiency of 0.32 g/kWh.

Combined In Vitro Toxicity and Immunogenicity of Cold Plasma and Pulsed Electric Fields.

In modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.

Analysis of microstructural parameters of trabecular bone based on electrical impedance spectroscopy and deep neural networks.

The potential of electrical impedance spectroscopy (EIS) was demonstrated for the investigation of microstructural properties of osseous tissue. Therefore, a deep neural network (DNN) was implemented for a sensitive assessment of different structural features that were derived on the basis of dielectric parameters, especially relative permittivities, recorded over a frequency range from 40 Hz to 5 MHz. The advantages of the developed method over conventional approaches, including equivalent circuit models (ECMs), linear regression and effective medium approximation (EMA), is the comprehensive quantification of bone morphologies by several microstructural parameters simultaneously, such as bone volume fraction (BV/TV), bone surface-volume-ratio (BS/BV), structure model index (SMI), trabecular number (Tb.N) and trabecular thickness (Tb.Th). The comparison of predictions of the DNN with an analysis of µCT-images confirmed a high accuracy for different microstructural parameters, which was indicated by corresponding Pearson correlation coefficients, especially for Tb.Th (r = 0.89) and BS/BV (r = 0.80). Concurrently, the approach was able to unambiguously discriminate anatomically similar bone regions (femoral head, greater trochanter and femoral neck) and therefore was capable to determine the morphological status of osseous tissue in detail. The classification was more discriminative than one based on classical linear discriminant analysis (LDA), due to the distinguishing features extracted by the DNN model. Accordingly, the method and model can serve as a potential tool for evaluating bone quality and bone status.

Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields.

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4-1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

Assessment of Phycocyanin Extraction from Cyanidium caldarium by Spark Discharges, Compared to Freeze-Thaw Cycles, Sonication, and Pulsed Electric Fields.

Phycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields.

Changes in [Ca(2+)](i) response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca(2+)](i) was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca(2+)](i) response was due to the release of Ca(2+) from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca(2+)](i) was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca(2+)](i) exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca(2+)](i) response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca(2+) release may be due to nanopore formation in the endoplasmic reticulum.

Enhanced resolution impedimetric analysis of cell responses from the distribution of relaxation times.

A universal strategy for the sensitive investigation of cell responses to external stimuli, in particular nanosecond pulsed electric fields (nsPEFs), was developed based on electrical impedance spectroscopy (EIS) in combination with a multi-peak analysis for the distribution of relaxation times (DRT). The DRT method provides high resolution for the identification of different polarization processes without a priori assumptions, as they are needed by more conventional approaches, such as an evaluation by equivalent circuit models. Accordingly, the physical properties of cells and their changes due to external stimuli can be uncovered and visualized and dispersion mechanisms introduced by Schwan et al. clearly identified. These are in particular relaxation processes at about 100 kHz that are associated with cell membrane characteristics and dominating respective changes of the distribution function for epithelial cell monolayers after exposure. A relatively moderate evolution at about 10 kHz may represent the polarization of extracellular matrices. Relaxation processes at around 1 MHz were suggested to be associated with intracellular changes. Conversely, the distribution of relaxation times can aid the optimization of the experimental design with respect to intended responses by an external stimulus.

Enhancing the Extraction Yield from Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes), by Pulsed Electric Fields Treatment.

Medicinal mushrooms contain highly valuable substances with proven positive effects on human health. To extract these components, different methods are available. Most of them suffer from individual disadvantages, therefore making them economically unviable. Pulsed electric fields (PEFs) could provide an opportunity to improve these processes. PEFs cause pore formation of cell membranes, facilitating substance transport out of cells. Thus, the influence of this technique on the extraction yield of medicinal mushrooms was studied for the first time. Lentinus edodes was used as model case and PEF treatment was compared with standard Soxhlet extraction alone. A square pulse generator (Electro Square Porator™ ECM 830) with a voltage of 3 kV and pulse length of 100 µs was used for PEF treatment. Extraction was studied for fresh and dried fruiting bodies, and dichloromethane and hot water extracts were analyzed. Extracts were quantified gravimetrically, and carbohydrate yields were also determined qualitatively with GC-MS and quantitatively with anthrone method. PEFs could increase in particular the yield of water-soluble compounds of fresh mushroom material. However, the lipid fraction was not affected by PEF in neither fresh nor dried material.

Cylindrospermopsin is effectively degraded in water by pulsed corona-like and dielectric barrier discharges.

Cylindrospermopsin (CYN) is an important cyanobacterial toxin posing a major threat to surface waters during cyanobacterial blooms. Hence, methods for cyanotoxin removal are required to confront seasonal or local incidences to sustain the safety of potable water reservoirs. Non-thermal plasmas provide the possibility for an environmentally benign treatment which can be adapted to specific concentrations and environmental conditions without the need of additional chemicals. We therefore investigated the potential of two different non-thermal plasma approaches for CYN degradation, operated either in a water mist, i.e. in air, or submerged in water. A degradation efficacy of 0.03 ± 0.00 g kWh-1 L-1 was found for a dielectric barrier discharge (DBD) operated in air, while a submerged pulsed corona-like discharge resulted in an efficacy of 0.24 ± 0.02 g kWh-1 L-1. CYN degradation followed a pseudo zeroth order or pseudo first order reaction kinetic, respectively. Treatment efficacy of the corona-like discharge submerged in water increased with pH values of the initial solution changing from 5.0 to 7.5. Notably, a pH-depending residual oxidative effect was observed for the submerged discharge, resulting in ongoing CYN degradation, even without further plasma treatment. In this case hydroxyl radicals were identified as the dominant oxidants of CYN at acidic pH values. In comparison, degradation by the DBD could be related primarily to the generation of ozone.

Combination Treatment with Cold Physical Plasma and Pulsed Electric Fields Augments ROS Production and Cytotoxicity in Lymphoma.

New approaches in oncotherapy rely on the combination of different treatments to enhance the efficacy of established monotherapies. Pulsed electric fields (PEFs) are an established method (electrochemotherapy) for enhancing cellular drug uptake while cold physical plasma is an emerging and promising anticancer technology. This study aimed to combine both technologies to elucidate their cytotoxic potential as well as the underlying mechanisms of the effects observed. An electric field generator (0.9-1.0 kV/cm and 100-µs pulse duration) and an atmospheric pressure argon plasma jet were employed for the treatment of lymphoma cell lines as a model system. PEF but not plasma treatment induced cell membrane permeabilization. Additive cytotoxicity was observed for the metabolic activity and viability of the cells while the sequence of treatment in the combination played only a minor role. Intriguingly, a parallel combination was more effective compared to a 15-min pause between both treatment regimens. A combination effect was also found for lipid peroxidation; however, none could be observed in the cytosolic and mitochondrial reactive oxygen species (ROS) production. The supplementation with either antioxidant, a pan-caspase-inhibitor or a ferroptosis inhibitor, all partially rescued lymphoma cells from terminal cell death, which contributes to the mechanistic understanding of this combination treatment.

A new pulsed electric field therapy for melanoma disrupts the tumor's blood supply and causes complete remission without recurrence.

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 microsec. The application of 300 high-voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4-month period after the initial melanoma had disappeared. These pulses generate small, long-lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to small molecules and ions, as well as an increase in intracellular Ca(2+), DNA fragmentation, disruption of the tumor's blood supply and the initiation of apoptosis. Apoptosis was indicated by a 3-fold increase in Bad labeling and a 72% decrease in Bcl-2 labeling. In addition, microvessel density within the treated tumors fell by 93%. This new therapy utilizing nanosecond pulsed electric fields has the advantages of highly localized targeting of tumor cells and a total exposure time of only 180 microsec. These pulses penetrate into the interior of every tumor cell and initiate DNA fragmentation and apoptosis while at the same time reducing blood flow to the tumor. This new physical tumor therapy is drug free, highly localized, uses low energy, has no significant side effects and results in very little scarring.

Kilovolt Blumlein pulse generator with variable pulse duration and polarity.

A Blumlein pulse generator which utilizes the superposition of electrical pulses launched from two individually switched pulse forming lines has been designed and tested. By using a power metal-oxide-semiconductor field-effect transistor as a switch on each end of the Blumlein line, we were able to generate pulses with amplitudes of 1 kV across a 100 Omega load. Pulse duration and polarity can be controlled by the temporal delay in the triggering of the two switches. Using this technique, we have demonstrated the generation of pulses with durations between 8 and 60 ns. The lower limit in pulse duration was determined by the switch closing time and the upper limit by the length of the pulse forming line. A further advantage of the concept is that pulse distortions caused by the non-negligible on-resistance of a line with a single switch can be eliminated by using switches with identical characteristics.

Bioimpedance Analysis of Epithelial Monolayers after Exposure to Nanosecond Pulsed Electric Fields.

Exposures to pulsed electric fields (PEFs) are known to affect cell membranes and consequently also cell-cell interactions as well as associated characteristics. Bioimpedance analysis offers direct and non-invasive insights into structural and functional changes of cell membranes and extracellular matrices through a rigorous evaluation of electrical parameters. Accordingly, the multi-frequency impedance of confluent monolayers of rat liver epithelial WB-F344 cells was monitored in situ before and after exposure to nanosecond PEFs (nsPEFs). The results were fitted by two Cole models in series to obtain the Cole parameters for the monolayer. For an interpretation of the results, dielectric parameters, were correlated with changes of the TJ protein zonula occludens (ZO-1) and the paracellular permeability of the monolayer Cole parameters in general change as a function of pulse number and time. The findings demonstrate that impedance analysis is an effective method to monitor changes of TJs cell-cell contacts and paracellular permeability and relate them to exposure parameters.

Degradation and intermediates of diclofenac as instructive example for decomposition of recalcitrant pharmaceuticals by hydroxyl radicals generated with pulsed corona plasma in water.

Seven recalcitrant pharmaceutical residues (diclofenac, 17α-ethinylestradiol, carbamazepine, ibuprofen, trimethoprim, diazepam, diatrizoate) were decomposed by pulsed corona plasma generated directly in water. The detailed degradation pathway was investigated for diclofenac and 21 intermediates could be identified in the degradation cascade. Hydroxyl radicals have been found primarily responsible for decomposition steps. By spin trap enhanced electron paramagnetic resonance spectroscopy (EPR), OH-adducts and superoxide anion radical adducts were detected and could be distinguished applying BMPO as a spin trap. The increase of concentrations of adducts follows qualitatively the increase of hydrogen peroxide concentrations. Hydrogen peroxide is eventually consumed in Fenton-like processes but the concentration is continuously increasing to about 2mM for a plasma treatment of 70min. Degradation of diclofenac is inversely following hydrogen peroxide concentrations. No qualitative differences between byproducts formed during plasma treatment or due to degradation via Fenton-induced processes were observed. Findings on degradation kinetics of diclofenac provide an instructive understanding of decomposition rates for recalcitrant pharmaceuticals with respect to their chemical structure. Accordingly, conclusions can be drawn for further development and a first risk assessment of the method which can also be applied towards other AOPs that rely on the generation of hydroxyl radicals.

Cell stimulation versus cell death induced by sequential treatments with pulsed electric fields and cold atmospheric pressure plasma.

Pulsed electric fields (PEFs) and cold atmospheric pressure plasma (CAP) are currently both investigated for medical applications. The exposure of cells to PEFs can induce the formation of pores in cell membranes and consequently facilitate the uptake of molecules. In contrast, CAP mainly acts through reactive species that are generated in the liquid environment. The objective of this study was to determine, if PEFs combined with plasma-treated cell culture medium can mutually reinforce effects on viability of mammalian cells. Experiments were conducted with rat liver epithelial WB-F344 cells and their tumorigenic counterpart WB-ras for a direct comparison of non-tumorigenic and tumorigenic cells from the same origin. Viability after treatments strongly depended on cell type and applied field strength. Notably, tumorigenic WB-ras cells responded more sensitive to the respective treatments than non-tumorigenic WB-F344 cells. More cells were killed when plasma-treated medium was applied first in combination with treatments with 100-µs PEFs. For the reversed treatment order, i.e. application of PEFs first, the combination with 100-ns PEFs resulted in a stimulating effect for non-tumorigenic but not for tumorigenic cells. The results suggest that other mechanisms, besides simple pore formation, contributed to the mutually reinforcing effects of the two methods.

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation.

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10ns, 150kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by approximately 2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.

Long-lasting plasma membrane permeabilization in mammalian cells by nanosecond pulsed electric field (nsPEF).

The barrier function of plasma membrane in nsPEF-exposed mammalian cells was examined using whole-cell patch-clamp techniques. A specialized setup for nsPEF exposure of individual cells in culture was developed and characterized for artifact-free compatibility with the patch-clamp method. For the first time, our study provides experimental evidence that even a single 60-ns pulse at 12 kV/cm can cause a profound and long-lasting (minutes) reduction of the cell membrane resistance (R(m)), accompanied by the loss of the membrane potential. R(m) measured in GH3, PC-12, and Jurkat cells (but not in HeLa cells) in 80-120 s after nsPEF exposure was decreased about threefold, and its gradual recovery could take 15 min. Multiple pulses enhanced permeabilization, for example, R(m) in GH3 cells fell about 10-fold after a train of five pulses. Within studied limits, permeabilization did not depend on the presence of Ca(2+), Mg(2+), K(+), Cs(+), Cd(2+), EGTA, tetraethylammonium, or 4-aminopyridine in the pipette or bath solutions. Our results supported theoretical model predictions of plasma membrane poration by nsPEF. However, the extended decrease in R(m), assumed to be related to the life span of the pores, and different nsPEF sensitivity of individual cell lines have yet to be explained. The phenomenon of long-lived membrane permeabilization provides new insights on the nature of nsPEF-opened conductance pores and on molecular mechanisms that underlie nsPEF bioeffects.