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Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects., Competing Interests: The authors have declared that no competing interests exist.

Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, β-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the β-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of β-sheet and β-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of β-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The safety and quality of cereal grain supplies are adversely impacted by microbiological contamination, with novel interventions required to maximise whole grains safety and stability. The microbiological contaminants of wheat grains and the efficacy of Atmospheric Cold Plasma (ACP) for potential to control these risks were investigated. The evaluations were performed using a contained reactor dielectric barrier discharge (DBD) system; samples were treated for 0-20 min using direct and indirect plasma exposure. Amplicon-based metagenomic analysis using bacterial 16S rRNA gene and fungal 18S rRNA gene with internal transcribed spacer (ITS) region was performed to characterize the change in microbial community composition in response to ACP treatment. The antimicrobial efficacy of ACP against a range of bacterial and fungal contaminants of wheat, was assessed to include individual isolates from grains as challenge pathogens. ACP influenced wheat microbiome composition, with a higher microbial diversity as well as abundance found on the untreated control grain samples. Culture and genomic approaches revealed different trends for mycoflora detection and control. A challenge study demonstrated that using direct mode of plasma exposure with 20 min of treatment significantly reduced the concentration of all pathogens. Overall, reduction levels for B. atrophaeus vegetative cells were higher than for all fungal species tested, whereas B. atrophaeus spores were the most resistant to ACP among all microorganisms tested. Of note, repeating sub-lethal plasma treatment did not induce resistance to ACP in either B. atrophaeus or A. flavus spores. ACP process control could be tailored to address diverse microbiological risks for grain stability and safety., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Atmospheric cold plasma (ACP) treatment is an emerging food technology for product safety and quality retention, shelf-life extension, and sustainable processing. The activated chemical species of ACP can act rapidly against microorganisms without leaving chemical residues on food surfaces. The main objectives of this study were to investigate the efficiency and mechanisms of inactivation of fungal spores and biofilms by ACP and to understand the effects of the gas-mediated and liquid-mediated modes of application against important fungal contaminants. Aspergillus flavus was selected as the model microorganism. A. flavus spores were exposed to either gas plasma (GP) or plasma-activated water (PAW), whereas gas plasma alone was used to treat A. flavus biofilms. This study demonstrated that both GP and PAW treatments independently resulted in significant decreases of A. flavus metabolic activity and spore counts, with maximal reductions of 2.2 and 0.6 log 10 units for GP and PAW, respectively. The characterization of the reactive oxygen and nitrogen species in PAW and spore suspensions indicated that the concentration of secondary reactive species was an important factor influencing the antimicrobial activity of the treatment. The biofilm study showed that GP had detrimental effects on biofilm structure; however, the initial inoculum concentration prior to biofilm formation can be a crucial factor influencing the fungicidal effects of ACP. IMPORTANCE The production of mycotoxin-free food remains a challenge in both human and animal food chains. A. flavus , a mycotoxin-producing contaminant of economically important crops, was selected as the model microorganism to investigate the efficiency and mechanisms of ACP technology against fungal contaminants of food. Our study directly compares the antifungal properties of gas plasma (GP) and plasma-activated water (PAW) against fungi as well as reporting the effects of ACP treatment on biofilms produced by A. flavus ., (Copyright © 2020 American Society for Microbiology.)

Antimicrobial coating of medical devices has emerged as a potentially effective tool to prevent or ameliorate device-related infections. In this study the plasma deposition process for direct deposition of pharmaceutical drugs on to a range of surfaces and the retention of structure function relationship and antimicrobial efficacy against mono-species biofilms were investigated. Two selected sample antibiotics-ampicillin and gentamicin, were deposited onto two types of surfaces-polystyrene microtiter plates and stainless steel coupons. The antimicrobial efficacy of the antibiotic-coated surfaces was tested against challenge populations of both planktonic and sessile Escherichia coli and Pseudomonas aeruginosa , with responses monitored for up to 14 days. The plasma deposition process bonded the antibiotic to the surfaces, with localized retention of antibiotic activity. The antibiotics deposited on the test surfaces retained a good efficacy against planktonic cells, and importantly prevented biofilm formation of attached cells for up to 96 h. The antibiotic rapidly eluted from the surface of antibiotic-coated surfaces to the surrounding medium, with retention of effect in this surrounding milieu for up to 2 weeks. Control experiments established that there was no independent antimicrobial or growth promoting effect of the plasma deposition process, where there was no antibiotic in the helium plasma assisted delivery stream. Apart from the flexibility offered through deposition on material surfaces, there was no additive or destructive effect associated with the helium assisted plasma deposition process on the antibiotic. The plasma assisted process was a viable mean of coating clinically relevant materials and developing innovative functional materials with retention of antibiotic activity, without employing a linker or plasma modified polymer, thus minimizing bio-compatibility issues for medical device materials. This offers potential to prevent or control instrumented or non-permanent device associated infection localized to the surgical or implant site., (Copyright © 2019 Los, Ziuzina, Boehm, Han, O'Sullivan, O'Neill and Bourke.)

Atmospheric cold plasma (ACP) is an effective method for microbiological decontamination. This study evaluated an alternative water-based decontamination approach for inactivation of bacterial population from fresh produce and in the wash water generated from fresh produce washing. The study characterised ACP inactivation of attached Listeria innocua and Pseudomonas fluorescens inoculated on lettuce in comparison to chlorine treatment. P. fluorescens was sensitive to ACP treatment and was reduced below detection limit within 3 min of treatment. L. innocua population was reduced by ∼2.4 Log 10  CFU/g after 5 min of treatment; showing similar inactivation efficacy to chlorine treatment. The microbial load in wash water was continuously decreased and was below detection limits after 10 min of ACP treatment. Micro-bubbling along with agitation assisted the bacterial detachment and distribution of reactive species, thus increasing bacterial inactivation efficacy from fresh produce and wash water. A shift in pH of plasma functionalised water was observed along with high concentration of nitrate and ozone with a relative amount of nitrites which increased with plasma exposure time. Further, L. innocua treated at different independent pH conditions showed minimal or no effect of pH on ACP bacterial inactivation efficacy. Aqueous ACP treatment poses a promising alternative for decontamination of fresh produce and the associated wash-waters which could be applied in the food industry to replace continuous chlorine dosing of process waters., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Antibiotics, such as ofloxacin (OFX) and ciprofloxacin (CFX), are often detected in considerable concentrations in both wastewater effluents and surface water. This poses a risk to non-target organisms and to human health. The aim of this work was to study atmospheric cold plasma (ACP) degradation of antibiotics in water and meat effluent and to explore any residual antimicrobial activity of samples submitted to the plasma process. The results revealed that ACP successfully degraded the studied antibiotics and that the reaction mechanism is principally related to attack by hydroxyl radicals and ozone. According to the disk diffusion assay, the activity of both antibiotics was considerably reduced by the plasma treatment. However, a microdilution method demonstrated that CFX exhibited higher antimicrobial activity after ACP treatment than the corresponding control revealing a potentially new platform for future research to improve the efficiency of conventional antibiotic treatments. Importantly, short-term exposures to sub-lethal concentrations of the antibiotic equally reduced bacterial susceptibility to both ACP treated and untreated CFX. As a remediation process, ACP removal of antibiotics in complex wastewater effluents is possible. However, it is recommended that plasma encompass degradant structure activity relationships to ensure that biological activity is eliminated against non-target organisms and that life cycle safety of antibiotic compounds is achieved.

Cold atmospheric plasma is a versatile new tool in the biomedical field with applications ranging from disinfection, wound healing and tissue regeneration to blood coagulation, and cancer treatment. Along with improved insights into the underlying physical, chemical and biological principles, plasma medicine has also made important advances in the introduction into the clinic. However, in the absence of a standard plasma 'dose' definition, the diversity of the field poses certain difficulties in terms of comparability of plasma devices, treatment parameters and resulting biological effects, particularly with regards to the question of what constitutes a safe plasma application. Data from various in vitro cytotoxic and genotoxic studies along with in vivo findings from animal and human trials are reviewed to provide an overview of the current state of knowledge on the safety of plasma for biological applications. Treatment parameters employed in clinical studies were well tolerated but intense treatment conditions can also induce tissue damage or genotoxicity. There is a need identified to establish both guidelines and safety limits that ensure an absence of (long-term) side effects and to define treatments as safe for applications, where cell stimulation is desired, e.g. in wound healing, or those aimed at inducing cell death in the treatment of cancer.

Bacterial infection and antibiotic resistance are major threats to human health and very few solutions are available to combat this eventuality. A growing number of studies indicate that cold (non-thermal) plasma treatment can be used to prevent or eliminate infection from bacteria, bacterial biofilms, fungi and viruses. Mechanistically, a cold plasma discharge is composed of high-energy electrons that generate short-lived reactive oxygen and nitrogen species which further react to form more stable compounds (NO2, H2O2, NH2Cl and others) depending on the gas mixture and plasma parameters. Cold plasma devices are being developed for medical applications including infection, cancer, plastic surgery applications and more. Thus, in this review we explore the potential utility of cold plasma as a non-antibiotic approach for treating post-surgical orthopedic infections.

Cold plasma science and technology is increasingly investigated for translation to a plethora of issues in the agriculture and food sectors. The diversity of the mechanisms of action of cold plasma, and the flexibility as a standalone technology or one that can integrate with other technologies, provide a rich resource for driving innovative solutions. The emerging understanding of the longer-term role of cold plasma reactive species and follow-on effects across a range of systems will suggest how cold plasma may be optimally applied to biological systems in the agricultural and food sectors. Here we present the current status, emerging issues, regulatory context, and opportunities of cold plasma with respect to the broad stages of primary and secondary food production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Contamination of cereal grains as a key global food resource with insects or microorganisms is a persistent concern for the grain industry due to irreversible damage to quality and safety characteristics and economic losses. Atmospheric cold plasma presents an alternative to conventional grain decontamination methods owing to the high antimicrobial potential of reactive species generated during the treatment, but effects against product specific microflora are required to understand how to optimally develop this approach for grains. This work investigated the influence of ACP processing parameters for both cereal grain decontamination and grain quality as important criteria for grain or seed use. A high voltage (HV) (80 kV) dielectric barrier discharge (DBD) closed system was used to assess the potential for control of native microflora and pathogenic bacterial and fungal challenge microorganisms, in tandem with effects on grain functional properties. Response surface modelling of experimental data probed the key factors in relation to microbial control and seed germination promotion. The maximal reductions of barley background microbiota were 2.4 and 2.1 log 10  CFU/g and of wheat - 1.5 and 2.5 log 10  CFU/g for bacteria and fungi, respectively, which required direct treatment for 20 min followed by a 24 h sealed post-treatment retention time. In the case of challenge organisms inoculated on barley grains, the highest resistance was observed for Bacillus atrophaeus endospores, which, regardless of retention time, were maximally reduced by 2.4 log 10  CFU/g after 20 min of direct treatment. The efficacy of the plasma treatment against selected microorganisms decreased in the following order: E. coli > P. verrucosum (spores) > B. atrophaeus (vegetative cells) > B. atrophaeus (endospores). The challenge microorganisms were more susceptible to ACP treatment than naturally present background microbiota. No major effect of short term plasma treatment on the retention of quality parameters was observed. Germination percentage measured after 7 days cultivation was similar for samples treated for up to 5 min, but this was decreased after 20 min of direct treatment. Overall, ACP proved effective for cereal grain decontamination, but it is noted that the diverse native micro-flora may pose greater resistance to the closed, surface decontamination approach than the individual fungal or bacterial challenges, which warrants investigation of grain microbiome responses to ACP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The use of plasma-activated liquids such as PBS, medium or simply plasma-activated water (PAW) has been receiving increasing attention for applications in cancer treatments. Amongst the reactive species contained in these solutions, hydrogen peroxide appears to play a pivotal role in causing cytotoxic effects. H 2 O 2 concentrations can be correlated with reduced cell viability and growth and used as an indicator of the potential efficacy of a plasma-activated liquid., Objective: To investigate the cytotoxic mediators generated in water specific to high-voltage DBD-ACP., Method: Using a high-voltage dielectric barrier atmospheric cold plasma (DBD-ACP) system, we examined PAW-mediated cytotoxic effects on different mammalian cell lines employing a set-up where short-lived reactive species can be discounted and activated liquids with long-term stability are generated., Results: The PAW potency could be modulated using voltage level, treatment time and post-treatment storage time and target-related characteristics such as surface to volume ratio. All of these parameters effected cell viability in a hydrogen peroxide concentration correlated manner. The susceptibility of two cancer cell lines to PAW was similar to that observed for two non-cancer cell lines and the toxicity of plasma-activated water exceeded that of the corresponding hydrogen peroxide concentrations., Conclusion: In cytotoxic plasma activated water an essential role for H 2 O 2 has been demonstrated multi-fold, yet further contributing factors are apparent and remain to be identified., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Brochothrix thermosphacta is the predominant spoilage microorganism in meat and its control in processing environments is important to maintain meat product quality. Atmospheric cold plasma is of interest for control of pathogenic and spoilage microorganisms in foods. This study ascertained the potential of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) for control of B. thermosphacta, taking microbial and food environment factors into consideration, and investigated the shelf-life of lamb chop after in-package plasma treatment in modified atmosphere. Community profiling was used to assess the treatment effects on the lamb microflora. ACP treatment (80 kV) for 30s inactivated B. thermosphacta populations below detection levels in PBS, while 5 min treatment achieved a 2 Log cycle reduction using a complex meat model medium and attached cells. The antimicrobial efficacy of plasma was reduced but still apparent on lamb chop surface-inoculated with high concentrations of B. thermosphacta. Lamb chop treated under modified atmosphere exhibited reduced microbial growth over the product shelf-life and community profiling showed no evident changes to the microbial populations after the treatment. The overall results indicated potential of ACP to enhance microbial control leading to meat storage life extension through adjusting the modality of treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims: This study investigated the effect of atmospheric cold plasma (ACP) exposure-induced stress on microbial inactivation patterns and the regulation of genes involved in the microbial stress response in conjunction with key processing parameters of exposure time and post-treatment storage time., Methods and Results: Cell suspensions of Escherichia coli BW 25113 and its isogenic knock-out mutants in rpoS, soxR, soxS, oxyR and dnaK genes were treated with high-voltage ACP in a sealed package for 1, 3 and 5 min followed by 0-, 1- and 24-h post-treatment storage. Reactive oxygen species (ROS) densities and colony formation were determined. ΔrpoS strain showed higher microbial reduction and greater cell permeability than other mutants, while ΔoxyR only showed this effect after 5 min of treatment. With increased post-treatment storage time, ΔsoxS and ΔsoxR had increased sensitivity and resistance respectively. ΔdnaK cell suspensions had much higher ROS than other strains and showed increased sensitivity with 24 h post-treatment storage., Conclusions: RpoS and oxyR genes have both short-term and long-term regulatory effects under plasma stress. However, knocking out dnaK gene had an immediate response on ROS scavenging and long-term repairing mechanisms. ΔsoxR and ΔsoxS had different responses to ACP treatment with the increase in post-treatment time in relation to clearance of reactive species implying the different characteristics and functions as subunits., Significance and Impact of the Study: By comparing the response of mutants under ACP exposure to key processing parameters, the mechanism of microbial inactivation was partly revealed with respect to cellular regulation and repairing genes., (© 2016 The Society for Applied Microbiology.)

The exposure of aqueous solutions to atmospheric plasmas results in the generation of relatively long-lived secondary products such as hydrogen peroxide which are biologically active and have demonstrated anti-microbial and cytotoxic activity. The use of plasma-activated solutions in applications such as microbial decontamination or anti-cancer treatments requires not only adequate performance on target cells but also a safe operating window regarding the impact on surrounding tissues. Furthermore the generation of plasma-activated fluids needs to be considered as a by-stander effect of subjecting tissue to plasma discharges. Cytotoxicity and mutagenicity assays using mammalian cell lines were used to elucidate the effects of solutions treated with di-electric barrier discharge atmospheric cold plasma. Plasma-treated PBS inhibited cell growth in a treatment time-dependent manner showing a linear correlation to the solutions' peroxide concentration which remained stable over several weeks. Plasma-treated foetal bovine serum (FBS) acting as a model for complex bio-fluids showed not only cytotoxic effects but also exhibited increased mutagenic potential as determined using the mammalian HPRT assay. Further studies are warranted to determine the nature, causes and effects of the cyto- and genotoxic potential of solutions exposed to plasma discharges to ensure long-term safety of novel plasma applications in medicine and healthcare.

Atmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP) against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin, elastase (Las B) and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated 'inpack' using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence for the inhibition of QS and mechanisms by which this may occur.

Plasma treated liquids (PTLs) provide a means to convey a broad range of effects of relevance for food, environmental, or clinical decontamination, plant growth promotion, and therapeutic applications. Devising the reactive species ingredients and controlling the biological response of PTLs are of great interest. We demonstrate an approach by using an open-air hybrid mode discharge (HMD) to control the principal reactive species composition within plasma treated water (PTW), which is then demonstrated to regulate the cytotoxicity of PTW. The cytotoxicity of HMD produced PTW demonstrates a non-monotonic change over the discharge time. Although hydrogen peroxide and nitrite are not the sole effectors for cell death caused by PTW, using them as principal reactive species indicators, cytotoxicity can be removed and/or enhanced by formulating their concentrations and composition through adjusting the discharge mode and time on-line during PTW generation without the addition of additional working gas or chemical scavengers. This work demonstrates that a hybrid mode discharge can be employed to generate a PTW formulation to control a biological response such as cytotoxicity. This provides insights into how plasma treated liquids may be harnessed for biological applications in a specific and controllable manner. Published by [ABSTRACT FROM AUTHOR]