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.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

Flucytosine-based prodrug activation by cold physical plasma.

Reactive oxygen species (ROS) are known to trigger drug release from arylboronate-containing ROS-responsive prodrugs. In cancer cells, elevated levels of ROS can be exploited for the selective activation of prodrugs via Baeyer-Villiger type oxidation rearrangement sequences. Here, we report a proof of concept to demonstrate that these cascades can as well be initiated by cold physical plasma (CPP). An analog of a recently reported fluorouracil prodrug based on the less toxic drug 5-fluorocytosine (5-FC) was synthesized with a view to laboratory safety reasons and used as a model compound to prove our hypothesis that CPP is suitable as a trigger for the prodrug activation. Although the envisioned oxidation and rearrangement with successive loss of boronic acid species could be achieved by plasma treatment, the anticipated spontaneous liberation of 5-FC was inefficient in the model case. However, the obtained results suggest that custom-tailored CPP-responsive prodrugs might become an evolving research field.

Efficiency of cold atmospheric plasma, cleaning powders and their combination for biofilm removal on two different titanium implant surfaces.

OBJECTIVES: Biofilm removal is the decisive factor for the control of peri-implantitis. Cold atmospheric pressure plasma (CAP) can become an effective aid due to its ability to destroy and to inactivate bacterial biofilm residues. This study evaluated the cleaning efficiency of CAP, and air-polishing with glycine (APG) or erythritol (APE) containing powders alone or in combination with CAP (APG + CAP, APE + CAP) on sandblasted/acid etched, and anodised titanium implant surface. MATERIALS AND METHODS: On respective titanium discs, a 7-day ex vivo human biofilm was grown. Afterwards, the samples were treated with CAP, APG, APE, APG + CAP, and APE + CAP. Sterile and untreated biofilm discs were used for verification. Directly after treatment and after 5 days of incubation in medium at 37 °C, samples were prepared for examination by fluorescence microscopy. The relative biofilm fluorescence was measured for quantitative analyses. RESULTS: Air-polishing with or without CAP removed biofilms effectively. The combination of air-polishing with CAP showed the best cleaning results compared to single treatments, even on day 5. Immediately after treatment, APE + CAP showed insignificant higher cleansing efficiency than APG + CAP. CONCLUSIONS: CAP supports mechanical cleansing and disinfection to remove and inactivate microbial biofilm on implant surfaces significantly. Here, the type of the powder was not important. The highest cleansing results were obtained on sandblasted/etched surfaces. CLINICAL RELEVANCE: Microbial residuals impede wound healing and re-osseointegration after peri-implantitis treatment. Air-polishing treatment removes biofilms very effectively, but not completely. In combination with CAP, microbial free surfaces can be achieved. The tested treatment regime offers an advantage during treatment of peri-implantitis.

Insight into the Impact of Oxidative Stress on the Barrier Properties of Lipid Bilayer Models.

As a new field of oxidative stress-based therapy, cold physical plasma is a promising tool for several biomedical applications due to its potential to create a broad diversity of reactive oxygen and nitrogen species (RONS). Although proposed, the impact of plasma-derived RONS on the cell membrane lipids and properties is not fully understood. For this purpose, the changes in the lipid bilayer functionality under oxidative stress generated by an argon plasma jet (kINPen) were investigated by electrochemical techniques. In addition, liquid chromatography-tandem mass spectrometry was employed to analyze the plasma-induced modifications on the model lipids. Various asymmetric bilayers mimicking the structure and properties of the erythrocyte cell membrane were transferred onto a gold electrode surface by Langmuir-Blodgett/Langmuir-Schaefer deposition techniques. A strong impact of cholesterol on membrane permeabilization by plasma-derived species was revealed. Moreover, the maintenance of the barrier properties is influenced by the chemical composition of the head group. Mainly the head group size and its hydrogen bonding capacities are relevant, and phosphatidylcholines are significantly more susceptible than phosphatidylserines and other lipid classes, underlining the high relevance of this lipid class in membrane dynamics and cell physiology.

The amino acid metabolism is essential for evading physical plasma-induced tumour cell death.

BACKGROUND: Recent studies have emphasised the important role of amino acids in cancer metabolism. Cold physical plasma is an evolving technology employed to target tumour cells by introducing reactive oxygen species (ROS). However, limited understanding is available on the role of metabolic reprogramming in tumour cells fostering or reducing plasma-induced cancer cell death. METHODS: The utilisation and impact of major metabolic substrates of fatty acid, amino acid and TCA pathways were investigated in several tumour cell lines following plasma exposure by qPCR, immunoblotting and cell death analysis. RESULTS: Metabolic substrates were utilised in Panc-1 and HeLa but not in OVCAR3 and SK-MEL-28 cells following plasma treatment. Among the key genes governing these pathways, ASCT2 and SLC3A2 were consistently upregulated in Panc-1, Miapaca2GR, HeLa and MeWo cells. siRNA-mediated knockdown of ASCT2, glutamine depletion and pharmacological inhibition with V9302 sensitised HeLa cells to the plasma-induced cell death. Exogenous supplementation of glutamine, valine or tyrosine led to improved metabolism and viability of tumour cells following plasma treatment. CONCLUSION: These data suggest the amino acid influx driving metabolic reprogramming in tumour cells exposed to physical plasma, governing the extent of cell death. This pathway could be targeted in combination with existing anti-tumour agents.

Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation.

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2 ) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma-treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.

Argon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells.

Cold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1α, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment.

Combination of Gas Plasma and Radiotherapy Has Immunostimulatory Potential and Additive Toxicity in Murine Melanoma Cells in Vitro.

Despite continuous advances in therapy, malignant melanoma is still among the deadliest types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants. Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and radiotherapy, and translational tumor models are needed to develop this concept further.

Fluorescence measurements of peroxynitrite/peroxynitrous acid in cold air plasma treated aqueous solutions.

Qualitative detection of peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) as one of the key bactericidal agents produced in cold air plasma activated aqueous solutions is presented. We examined the use of the 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye to detect ONOO-/ONOOH in plasma activated non-buffered water (PAW) or buffered solution (PAPB) generated by DC-driven self-pulsed transient spark discharge at atmospheric pressure in ambient air. The diagnostic selectivity of H2DCFDA to reactive oxygen and nitrogen species (RONS) typical of plasma activated aqueous solutions was examined by using various scavengers of RONS. This cross-reactivity study showed the highest sensitivity of the H2DCFDA dye to ONOO-/ONOOH. However, besides ONOO-/ONOOH, H2DCFDA also exhibited sensitivity to hypochlorite anions/hypochlorous acid (OCl-/HOCl), showing that for a selective study it is important to have an idea about the possible constituents in the studied solutions. The sensitivity of H2DCFDA to other RONS even in much higher concentrations was negligible. The presence of nitrites (NO2-) and hydrogen peroxide (H2O2) in PAW led predominantly to the production of peroxynitrous acid with a strong fluorescence response of H2DCFDA in PAW. Plasma treatment of buffered solutions led to the weak response of H2DCFDA. The fluorescence induced in PAW decreased after scavenging individual reactants, namely NO2- and H2O2, as well as by scavenging the product of the peroxynitrite forming reaction, proving that the fluorescence response of H2DCFDA is primarily due to the formation of ONOO-/ONOOH. A chemical kinetics analysis of post-discharge processes and the pseudo-second order reaction between H2O2 and NO2- confirms formation of peroxynitrous acid in PAW with a rate in the order of tens of nM per second. The post-discharge evolution of the ONOOH formation rate was clearly correlated with the parallel detection of ONOO-/ONOOH by fluorescence spectroscopy using the H2DCFDA dye.

Stimulation of melanin synthesis in melanoma cells by cold plasma.

Skin color is derived from epidermal melanocytes that contain specialized organelles in which melanin is formed. The formation of melanin is a well-orchestrated process, and reactive oxygen species (ROS) play a role in numerous enzymatic conversions, such as the reactions catalyzed by tyrosinase and tyrosine hydroxylase. Currently, there is ample evidence that cold plasma exerts biological effects on cells through the impact of ROS and reactive nitrogen species (RNS). Modulation of melanin biosynthesis by cold plasma has not yet been investigated. This study investigated melanin biosynthesis of human melanoma cell lines with different endogenous melanin contents (SK-Mel 28, G-361, FM-55-P and MNT-1) in response to cold plasma-derived reactive species. Initially, the distribution of melanosomes, via immunofluorescence, and the influence of microphthalmia-associated transcription factor (MiTF), as a key transcription factor, was investigated. In our experimental setup, all of the tested cell lines had an elevated melanin content after exposure to cold plasma. These promising results suggest a novel potential application of cold plasma for the regulation of melanogenesis and may be a useful tool for influencing skin color in the future.

Chemistry and biochemistry of cold physical plasma derived reactive species in liquids.

Reactive oxygen and nitrogen species deposited by cold physical plasma are proposed as predominant effectors in the interaction between discharge and biomedical application. Most reactive species found in plasma sources are known in biology for inter- and intracellular communication (redox signaling) and mammalian cells are equipped to interpret the plasma derived redox signal. As such, considerable effort has been put into the investigation of potential clinical applications and the underlying mechanism, with a special emphasis on conditions orchestrated significantly via redox signaling. Among these, immune system control in wound healing and cancer control stands out with promising in vitro and in vivo effects. From the fundamental point of view, further insight in the interaction of the plasma-derived species with biological systems is desired to (a) optimize treatment conditions, (b) identify new fields of application, (c) to improve plasma source design, and (d) to identify the trajectories of reactive species. Knowledge on the biochemical reactivity of non-thermal plasmas is compiled and discussed. While there is considerable knowledge on proteins, lipids and carbohydrates have not received the attention deserved. Nucleic acids have been profoundly investigated yet focusing on molecule functionality rather than chemistry. The data collected underline the efforts taken to understand the fundamentals of plasma medicine but also indicate 'no man's lands' waiting to be discovered.

Biological and medical applications of plasma-activated media, water and solutions.

Non-thermal atmospheric pressure plasma has been proposed as a new tool for various biological and medical applications. Plasma in close proximity to cell culture media or water creates reactive oxygen and nitrogen species containing solutions known as plasma-activated media (PAM) or plasma-activated water (PAW) - the latter even displays acidification. These plasma-treated solutions remain stable for several days with respect to the storage temperature. Recently, PAM and PAW have been widely studied for many biomedical applications. Here, we reviewed promising reports demonstrating plasma-liquid interaction chemistry and the application of PAM or PAW as an anti-cancer, anti-metastatic, antimicrobial, regenerative medicine for blood coagulation and even as a dental treatment agent. We also discuss the role of PAM on cancer initiation cells (spheroids or cancer stem cells), on the epithelial mesenchymal transition (EMT), and when used for metastasis inhibition considering its anticancer effects. The roles of PAW in controlling plant disease, seed decontamination, seed germination and plant growth are also considered in this review. Finally, we emphasize the future prospects of PAM, PAW or plasma-activated solutions in biomedical applications with a discussion of the mechanisms and the stability and safety issues in relation to humans.

Non-touching plasma-liquid interaction - where is aqueous nitric oxide generated?

Nitric oxide is a relatively stable free radical and an important signal molecule in plants, animals, and humans with high relevance for biological processes involving inflammatory processes, e.g. wound healing or cancer. The molecule can be detected in the gas phase of non-thermal plasma jets making it a valuable tool for clinical intervention, but transport efficiency from the gas phase into the liquid phase or tissue remains to be clarified. To elucidate this fact, the nitric oxide concentration in buffered solutions is determined using electron paramagnetic resonance spectroscopy. The origin of the nitric oxide in the liquid could be excluded, therefore, potential precursors such as hydroxyl radicals, superoxide anions, atomic hydrogen and stable species (nitrite, nitrate and hydrogen peroxide) were detected and the potential formation pathway as well as ways of enhancing the production of nitric oxide by alteration of the feed gas and the surrounding gas composition during plasma treatment of the liquid have been pointed out.

A cold plasma jet accelerates wound healing in a murine model of full-thickness skin wounds.

Cold plasma has been successfully applied in several fields of medicine that require, for example, pathogen inactivation, implant functionalization or alteration of cellular activity. Previous studies have provided evidence that plasma supports the healing of wounds owing to its beneficial mixtures of reactive species and modulation of inflammation in cells and tissues. To investigate the wound healing activity of an atmospheric pressure plasma jet in vivo, we examined the cold plasma's efficacy on dermal regeneration in a murine model of dermal full-thickness ear wound. Over 14 days, female mice received daily plasma treatment. Quantitative analysis by transmitted light microscopy demonstrated a significantly accelerated wound re-epithelialization at days 3-9 in comparison with untreated controls. In vitro, cold plasma altered keratinocyte and fibroblast migration, while both cell types showed significant stimulation resulting in accelerated closure of gaps in scratch assays. This plasma effect correlated with the downregulation of the gap junctional protein connexin 43 which is thought to be important in the regulation of wound healing. In addition, plasma induced profound changes in adherence junctions and cytoskeletal dynamics as shown by downregulation of E-cadherin and several integrins as well as actin reorganization. Our results theorize cold plasma to be a beneficial treatment option supplementing existing wound therapies.

Osteoblast growth, after cleaning of biofilm-covered titanium discs with air-polishing and cold plasma.

AIM: To investigate the effects of a combined biofilm removal with an optimized air polishing and a cold plasma device on cells in vitro. MATERIALS AND METHODS: A 7-day-old biofilm was removed from rough titanium discs with an air-polishing device with erythritol powder (AP) or with a cold atmospheric pressure argon plasma (CAP) device or in combination of both (AP + CAP). The removal efficacy was evaluated by subsequent cell seeding of osteoblast-like cells (MG-63). The cell spreading was analysed after 5 days of incubation by scanning electron microscopy. Separately, the surface hydrophilicity was analysed by measuring the water contact angle (WCA) of the disc for each treatment method. RESULTS: The mechanical plaque removal with AP rendered specimen conducive for cell growth, 85% of the surface was covered with cells. An advantage of the combination of AP + CAP was not detectable compared to AP (cell coverage ranged from 57% up to 75%). After sole CAP treatment, microorganisms re-grew and destroyed all cells. The WCA was reduced by all treatment methods. CONCLUSION: An AP treatment has the potential to remove biofilm from rough implant surfaces completely. In contrast to our hypothesis, the combination of plasma and AP treatment did not enhance osteoblast spreading.

Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.

In vitro treatment of Candida albicans biofilms on denture base material with volume dielectric barrier discharge plasma (VDBD) compared with common chemical antiseptics.

OBJECTIVES: To prevent oral candidiasis, it is crucial to inactivate Candida-based biofilms on dentures. Common denture cleansing solutions cannot sufficiently inactivate Candida albicans. Therefore, we investigated the anticandidal efficacy of a physical plasma against C. albicans biofilms in vitro. MATERIALS AND METHODS: Argon or argon plasma with 1 % oxygen admixture was applied on C. albicans biofilms grown for 2, 7, or 16 days on polymethylmethacrylate discs; 0.1 % chlorhexidine digluconate (CHX) and 0.6 % sodium hypochlorite (NaOCl) solutions served as positive treatment controls. In addition, these two solutions were applied in combination with plasma for 30 min to assess potential synergistic effects. The anticandidal efficacy was determined by the number of colony forming units (CFU) in log(10) and expressed as reduction factor (RF, the difference between control and treated specimen). RESULTS: On 2-day-biofilms, plasma treatment alone or combined with 30 min CHX treatment led to significant differences of means of CFU (RF = 4.2 and RF = 4.3), clearly superior to CHX treatment alone (RF = 0.6). Plasma treatment of 7-day-or 16-day-old biofilms revealed no significant CFU reduction. The treatment of 7-day-old (RF = 1.7) and 16-day-old (RF = 1.3) biofilms was slightly more effective with NaOCl alone than with the combined treatment of NaOCl and plasma (RF = 1.6/RF = 1.9). The combination of CHX and plasma increased the RF immaterially. CONCLUSION: The use of plasma alone and in combination with antiseptics is promising anticandidal regimens for daily use on dentures when biofilms are not older than 2 days. CLINICAL RELEVANCE: Plasma could help to reduce denture-associated candidiasis.

Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology.

Modern non-thermal atmospheric pressure plasma sources enable controllable interaction with biological systems. Their future applications - e.g. wound management - are based on their unique mixture of reactive components sparking both stimulatory as well as inhibitory processes. To gain detailed understanding of plasma-cell interaction and with respect to risk awareness, key mechanisms need to be identified. This study focuses on the impact of an argon non-thermal atmospheric pressure plasma jet (kINPen 09) on human HaCaT keratinocytes. With increasing duration, cell viability decreased. In accordance, cells accumulated in G2/M phase within the following 24 h. DNA single-strand breaks were detected immediately after treatment and receded in the aftermath, returning to control levels after 24 h. No directly plasma-related DNA double-strand breaks were detected over the same time. Concurrently, DNA synthesis decreased. Coincident with treatment time, an increase in intracellular 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) conversion increased reactive oxygen species (ROS) levels. The radical scavenging activity of culture medium crucially influenced these effects. Thus, ROS changed DNA integrity, and the effectiveness of cellular defence mechanisms characterises the interaction of non-thermal plasma and eukaryotic cells. Effects were time-dependent, indicating an active response of the eukaryotic cells. Hence, a stimulation of eukaryotic cells using short-term non-thermal plasma treatment seems possible, eg in the context of chronic wound care. Long-term plasma treatments stopped in cell proliferation and apoptosis, which might be relevant in controlling neoplastic conditions.

YAP/TAZ, beta-catenin, and TGFb pathway activation in medical plasma-induced wound healing in diabetic mice.

INTRODUCTION: Hippo is a signaling pathway that is evolutionarily conserved and plays critical roles in wound healing and tissue regeneration. Disruption of the transcriptional activity of both Hippo-associated factors, the yes-associated protein (YAP), and the transcriptional co-activator with PDZ binding motif (TAZ) has been associated with cardiovascular diseases, fibrosis, and cancer. This makes the Hippo pathway an appealing target for therapeutic interventions. OBJECTIVES: Prior research has indicated that medical gas plasma promotes wound healing by delivering a combination of reactive species directly to the affected areas. However, the involvement of YAP/TAZ and other signaling pathways in diabetic wound healing remains unexplored. METHODS: To this extent, ear wounds were generated and treated with gas plasma in streptozotocin (STZ)-induced diabetic mice. Transcriptome profiling at two wound healing stages (days 9 and 20 post-wounding) was performed in female and male mice. Additionally, we employed gene and protein expression analyses, utilizing immunohistological and -chemical staining of various targets as well as quantitative PCR and Western blot analysis. RESULTS: Gas plasma treatment accelerated healing by increasing re-epithelialization and modifying extracellular matrix components. Transcriptomic profiling charting the major alterations in gene expression following plasma treatment was followed by a validation of several targets using transcriptional and translational quantification as well as localization analyses. CONCLUSION: Our study evaluated the cellular regulation of essential targets of the Hippo and related pathways such as YAP/TAZ, ß-catenin, tumor growth factor ß, and oxidative stress signaling after plasma treatment. The activation of genes, pathways, and their regulators is an attractive therapeutic aim for a therapeutic intervention in dermal skin repair in diabetic diseases using medical gas plasmas.