Real-time live imaging of cytosolic hydrogen peroxide and Ca2+ of Marchantia polymorpha gemmalings reveal immediate initial responses of plant cells triggered by nonthermal plasma irradiation.

Cold atmospheric pressure plasma generators capable of generating plasma under normal pressure and temperature conditions have recently been developed, and their biological applications have been extensively studied. Plasma irradiation has been reported to affect plant germination and growth; however, the molecular mechanism underlying these effects and initial cellular responses to plasma irradiation remains poorly understood. To unravel the molecular and cellular mechanisms underlying the effects of plasma irradiation on plants, we have been establishing novel experimental systems using a model liverwort Marchantia polymorpha. We here focused on the initial responses of plant cells to plasma irradiation. To investigate immediate cellular responses following plasma irradiation, we developed a new plasma device that allows irradiation under a microscope. Through integration with live fluorescence imaging, we established an experimental setup to track, the dynamics of intracellular concentration of H2O2 and Ca2+ as representative initial cellular responses. We revealed that plasma irradiation induced a rapid and transient increase in intracellular concentration of H2O2 and Ca2+ in Marchantia gemmalings. Pharmacological analyses suggested that the long-lived reactive species, H2O2, generated by the plasma generator was directly delivered into the plant cells. Competitive inhibitors of Ca2+ channels abolished the Ca2+ rise, suggesting that plasma irradiation immediately activate plasma membrane Ca2+ channel(s) to induce Ca2+ influx. Importantly, this study marks the inaugural demonstration of real-time monitoring of cytosolic H2O2 and Ca2+ dynamics in plants, triggered by plasma irradiation.

Effects of Corm Treatment with Cold Plasma and Electromagnetic Field on Growth and Production of Saffron Metabolites in Crocus sativus.

Crocus sativus L. is a widely cultivated traditional plant for obtaining dried red stigmas known as "saffron," the most expensive spice in the world. The response of C. sativus to pre-sowing processing of corms with cold plasma (CP, 3 and 5 min), vacuum (3 min), and electromagnetic field (EMF, 5 min) was assessed to verify how such treatments affect plant performance and the quality and yield of herbal raw materials. The results show that applied physical stressors did not affect the viability of corms but caused stressor-dependent changes in the kinetics of sprouting, growth parameters, leaf trichome density, and secondary metabolite content in stigmas. The effect of CP treatment on plant growth and metabolite content was negative, but all stressors significantly (by 42-74%) increased the number of leaf trichomes. CP3 treatment significantly decreased the length and dry weight of flowers by 43% and 60%, respectively, while EMF treatment increased the length of flowers by 27%. However, longer CP treatment (5 min) delayed germination. Vacuum treatment improved the uniformity of germination by 28% but caused smaller changes in the content of stigma compounds compared with CP and EMF. Twenty-six compounds were identified in total in Crocus stigma samples by the HPLC-DAD method, including 23 crocins, rutin, picrocrocin, and safranal. Processing of Crocus corms with EMF showed the greatest efficiency in increasing the production of secondary metabolites in saffron. EMF increased the content of marker compounds in stigmas (crocin 4: from 8.95 to 431.17 mg/g; crocin 3: from 6.27 to 164.86 mg/g; picrocrocin: from 0.4 to 1.0 mg/g), although the observed effects on growth were neutral or slightly positive. The obtained findings indicate that treatment of C. sativus corms with EMF has the potential application for increasing the quality of saffron by enhancing the amounts of biologically active compounds.

Subchronic toxicity study of indium-tin oxide nanoparticles following intratracheal administration into the lungs of rats.

OBJECTIVES: We aimed to analyze the subchronic toxicity and tissue distribution of indium after the intratracheal administration of indium-tin oxide nanoparticles (ITO NPs) to the lungs of rats. METHODS: Male Wistar rats were administered a single intratracheal dose of 10 or 20 mg In/kg body weight (BW) of ITO NPs. The control rats received only an intratracheal dose of distilled water. A subset of rats was periodically euthanized throughout the study from 1 to 20 weeks after administration. Indium concentrations in the serum, lungs, mediastinal lymph nodes, kidneys, liver, and spleen as well as pathological changes in the lungs and kidneys were determined. Additionally, the distribution of ionic indium and indium NPs in the kidneys was analyzed using laser ablation-inductively coupled plasma mass spectrometry. RESULTS: Indium concentrations in the lungs of the 2 ITO NP groups gradually decreased over the 20-week observation period. Conversely, the indium concentrations in the mediastinal lymph nodes of the 2 ITO groups increased and were several hundred times higher than those in the kidneys, spleen, and liver. Pulmonary and renal toxicities were observed histopathologically in both the ITO groups. Both indium NPs and ionic indium were detected in the kidneys, and their distributions were similar to the strong indium signals detected at the sites of inflammatory cell infiltration and tubular epithelial cells. CONCLUSIONS: Our results demonstrate that intratracheal administration of 10 or 20 mg In/kg BW of ITO NPs in male rats produces pulmonary and renal toxicities.

Growth control of Marchantia polymorpha gemmae using nonthermal plasma irradiation.

Several studies have documented that treatment by cold atmospheric pressure plasma (CAPP) on plants foster seed germination and growth in recent years. However, the molecular processes that underlie the action of CAPP on the seeds and plants remain mostly enigmatic. We here introduce gemmae of Marchantia polymorpha, a basal liverwort, as a novel model plant material suitable for CAPP research. Treating the gemmae with CAPP for a constant time interval at low power resulted in consistent growth enhancement, while growth inhibition at higher power in a dose-dependent manner. These results distinctly demonstrate that CAPP irradiation can positively and negatively regulate plant growth depending on the plasma intensity of irradiation, offering a suitable experimental system for understanding the molecular mechanisms underlying the action of CAPP in plants.

Health assessment of rice cultivated and harvested from plasma-irradiated seeds.

This study provides the health effects assessment of rice cultivated from plasma-irradiated seeds. The rice (Oryza sativa L.) cultivated from seeds with plasma irradiation showed a growth improvement (slope-ratios of with plasma to without plasma were 1.066, 1.042, and 1.255 for tiller, and earing, and ripening periods, respectively) and an 4% increase in yield. The cultivated rice was used for repeated oral administrations to mice for 4-week period. Distilled water and rice cultivated from seeds without plasma irradiation were also used as control. The weights of the lung, kidney, liver, and spleen, with corresponding average values of 0.22 g, 0.72 g, 2.1 g, and 0.17 g for w/ plasma group and 0.22 g, 0.68 g, 2.16 g, and 0.14 g for w/o plasma group, respectively, showing no effect due to the administration of rice cultivated from plasma-irradiated seeds. Nutritional status, liver function, kidney function, and lipid, neutral fat profiles, and glucose metabolism have no significant difference between with and without plasma groups. These results show no obvious subacute effects were observed on rice grains cultivated and harvested from the mother plant that experienced growth improvement by plasma irradiation. This study provides a new finding that there is no apparent adverse health effect on the grains harvested from the plasma-irradiated seeds.

Influence of electric potential-induced by atmospheric pressure plasma on cell response.

Plasma irradiation leads not only active species, but also reactive chemical species, ultraviolet light, electric fields, magnetic fields, and shock waves. To date the effects of reactive chemical species have been mainly discussed. To understand the biological effect caused by an electric potential induced with an atmospheric-pressure plasma, the behavior of cell stimulated by electric potential was investigated using HeLa cell. The cell concentration assay revealed that less than 20% of cells inactivated by potential stimulation and the remained cells proliferate afterward. Fluorescent microscopic observation revealed that potential stimulation is appreciable to transport the molecules through membrane. These results show that potential stimulation induces intracellular and extracellular molecular transport, while the stimulation has a low lethal effect. A possible mechanism for this molecular transport by potential stimulation was also shown using numerical simulation based on an equivalent circuit of the experimental system including adhered HeLa cell. The potential formation caused by plasma generation is decisive in the contribution of plasma science to molecular biology and the elucidation of the mechanism underlying a biological response induction by plasma irradiation.

Unraveling the Transport Properties of RONS across Nitro-Oxidized Membranes.

The potential of cold atmospheric plasma (CAP) in biomedical applications has received significant interest, due to its ability to generate reactive oxygen and nitrogen species (RONS). Upon exposure to living cells, CAP triggers alterations in various cellular components, such as the cell membrane. However, the permeation of RONS across nitrated and oxidized membranes remains understudied. To address this gap, we conducted molecular dynamics simulations, to investigate the permeation capabilities of RONS across modified cell membranes. This computational study investigated the translocation processes of less hydrophilic and hydrophilic RONS across the phospholipid bilayer (PLB), with various degrees of oxidation and nitration, and elucidated the impact of RONS on PLB permeability. The simulation results showed that less hydrophilic species, i.e., NO, NO2, N2O4, and O3, have a higher penetration ability through nitro-oxidized PLB compared to hydrophilic RONS, i.e., HNO3, s-cis-HONO, s-trans-HONO, H2O2, HO2, and OH. In particular, nitro-oxidation of PLB, induced by, e.g., cold atmospheric plasma, has minimal impact on the penetration of free energy barriers of less hydrophilic species, while it lowers these barriers for hydrophilic RONS, thereby enhancing their translocation across nitro-oxidized PLB. This research contributes to a better understanding of the translocation abilities of RONS in the field of plasma biomedical applications and highlights the need for further analysis of their role in intracellular signaling pathways.

Cold Plasma-Induced Changes in Stevia rebaudiana Morphometric and Biochemical Parameter Correlations.

Stevia rebaudiana Bertoni is an economically important source of natural low-calorie sweeteners, steviol glycosides (SGs), with stevioside (Stev) and rebaudioside A (RebA) being the most abundant. Pre-sowing seed treatment with cold plasma (CP) was shown to stimulate SGs biosynthesis/accumulation up to several fold. This study aimed to evaluate the possibility to predict CP-induced biochemical changes in plants from morphometric parameters. Principle component analysis (PCA) was applied to two different sets of data: morphometric parameters versus SGs concentrations and ratio, and morphometric parameters versus other secondary metabolites (total phenolic content (TPC), total flavonoid content (TFC)) and antioxidant activity (AA). Seeds were treated for 2, 5 and 7 min with CP (CP2, CP5 and CP7 groups) before sowing. CP treatment stimulated SGs production. CP5 induced the highest increase of RebA, Stev and RebA+Stev concentrations (2.5-, 1.6-, and 1.8-fold, respectively). CP did not affect TPC, TFC or AA and had a duration-dependent tendency to decrease leaf dry mass and plant height. The correlation analysis of individual plant traits revealed that at least one morphometric parameter negatively correlates with Stev orRebA+Stev concentration after CP treatment.

Effects of plasma-activated Ringer's lactate solution on cancer cells: evaluation of genotoxicity.

BACKGROUND: Non-thermal atmospheric pressure plasma technologies form the core of many scientific advances, including in the electronic, industrial, and biotechnological fields. The use of plasma as a cancer therapy has recently attracted significant attention due to its cancer cell killing activity. Plasma-activated Ringer's lactate solution (PAL) exhibits such activity. In addition to ROS, PAL contains active compounds or species that cause cancer cell death, but the potential mutagenic risks of PAL have not been studied. RESULTS: PAL has a low pH value and a high concentration of H2O2. H2O2 was removed from PAL using catalase and catalase-treated PAL with a pH of 5.9 retained a killing effect on HeLa cells whereas this effect was not observed if the PAL was adjusted to pH 7.2. Catalase-treated PAL at pH 5.9 had no significant effect on mutation frequency, the expression of γH2AX, or G2 arrest in HeLa cells. CONCLUSION: PAL contains one or more active compounds or species in addition to H2O2 that have a killing effect on HeLa cells. The compound(s) is active at lower pH conditions and apparently exhibits no genotoxicity. This study suggested that identification of the active compound(s) in PAL could lead to the development of novel anticancer drugs for future cancer therapy.

Changes in Content of Bioactive Compounds and Antioxidant Activity Induced in Needles of Different Half-Sib Families of Norway Spruce (Picea abies (L.) H. Karst) by Seed Treatment with Cold Plasma.

In order to ensure sufficient food resources for a constantly growing human population, new technologies (e.g., cold plasma technologies) are being developed for increasing the germination and seedling growth without negative effects on the environment. Pinaceae species are considered a natural source of antioxidant compounds and are valued for their pharmaceutical and nutraceutical properties. In this study, the seeds of seven different Norway spruce half-sib families were processed for one or two minutes with cold plasma (CP) using dielectric barrier discharge (DBD) plasma equipment. At the end of the second vegetation season, the total flavonoid content (TFC), DPPH (2,2- diphenyl-1-picryl-hydrazyl-hydrate), and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) antioxidant activity, and the amounts of six organic acids (folic, malic, citric, oxalic, succinic, and ascorbic) were determined in the needles of different half-sib families of Norway spruce seedlings. The results show that the TFC, antioxidant activity, and amounts of organic acids in the seedling needles depended on both the treatment duration and the genetic family. The strongest positive effect on the TFC was determined in the seedlings of the 477, 599, and 541 half-sib families after seed treatment with CP for 1 min (CP1). The TFC in these families increased from 118.06 mg g-1 to 312.6 mg g-1 compared to the control. Moreover, seed treatment with CP1 resulted in the strongest increase in the antioxidant activity of the needles of the 541 half-sib family seedlings; the antioxidant activity, determined by DPPH and ABTS tests, increased by 30 and 23%, respectively, compared to the control. The obtained results indicate that the CP effect on the amount of organic acids in the needles was dependent on the half-sib family. It was determined that treatment with CP1 increased the amount of five organic acids in the needles of the 541 half-sib family seedlings. The presented results show future possibilities for using cold plasma seed treatment in the food and pharmacy industries.

Detection of NO3- introduced in plasma-irradiated dry lettuce seeds using liquid chromatography-electrospray ionization quantum mass spectrometry (LC-ESI QMS).

Discharge plasma irradiates seeds with reactive oxygen and nitrogen species (RONS). However, RONS introduced in seeds by plasma irradiation have not been successfully detected thus far. This study provides experimental evidence that nitrate ion NO3- is introduced in lettuce seeds as RONS upon irradiation with atmospheric-pressure air dielectric barrier discharge plasma. Plasma irradiation for 5 min promotes seed germination. The components of the plasma-irradiated seeds were examined using electrospray ionization quantum mass spectrometry (ESI QMS), which revealed that the plasma irradiation introduced an ion with a mass of 62 m/z in detectable amounts. This ion was identified as NO3- by liquid chromatography (LC), multiple wavelength detector (MWD), and LC-ESI QMS. A one-dimensional simulation at electron temperature Te = 1 eV, electron density Ne = 1013/m3, and gas temperature Tg = 300 K indicated the introduction of NO3-, involving nitric oxide NO. NO3- is one of the most important ions that trigger signal transduction for germination when introduced in seeds. The scanning electron microscopy (SEM) images revealed that there was no change on the surface of the seeds after plasma irradiation. Plasma irradiation is an effective method of introducing NO3- in seeds in a dry process without causing damage.

Mechanistic Insight into Permeation of Plasma-Generated Species from Vacuum into Water Bulk.

Due to their potential benefits, cold atmospheric plasmas (CAPs), as biotechnological tools, have been used for various purposes, especially in medical and agricultural applications. The main effect of CAP is associated with reactive oxygen and nitrogen species (RONS). In order to deliver these RONS to the target, direct or indirect treatment approaches have been employed. The indirect method is put into practice via plasma-activated water (PAW). Despite many studies being available in the field, the permeation mechanisms of RONS into water at the molecular level still remain elusive. Here, we performed molecular dynamics simulations to study the permeation of RONS from vacuum into the water interface and bulk. The calculated free energy profiles unravel the most favourable accumulation positions of RONS. Our results, therefore, provide fundamental insights into PAW and RONS chemistry to increase the efficiency of PAW in biological applications.

Nanostructured Ge and GeSn films by high-pressure He plasma sputtering for high-capacity Li ion battery anodes.

We fabricated nanostructured Ge and GeSn films using He radio-frequency magnetron plasma sputtering deposition. Monodisperse amorphous Ge and GeSn nanoparticles of 30-40 nm size were arranged without aggregation by off-axis sputtering deposition in the high He-gas-pressure range of 0.1 Torr. The Ge film porosity was over 30%. We tested the charge/discharge cycle performance of Li-ion batteries with nanostructured Ge and GeSn anodes. The Ge anode with a dispersed arrangement of nanoparticles showed a Li-storage capacity of 565 mAh/g after the 60th cycle. The capacity retention was markedly improved by the addition of 3 at% Sn in Ge anode. The GeSn anode (3 at% Sn) achieved a higher capacity of 1128 mAh/g after 60 cycles with 92% capacity retention. Precise control of the nano-morphology and electrical characteristics by a single step procedure using low temperature plasma is effective for stable cycling of high-capacity Ge anodes.

Impact of Reactive Oxygen and Nitrogen Species Produced by Plasma on Mdm2-p53 Complex.

The study of protein-protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)-tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2-p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.

Plasma treatment causes structural modifications in lysozyme, and increases cytotoxicity towards cancer cells.

Bacterial and mammalian proteins, such as lysozyme, are gaining increasing interest as anticancer drugs. This study aims to modify the lysozyme structure using cold atmospheric plasma to boost its cancer cell killing effect. We investigated the structure at acidic and neutral pH using various experimental techniques (circular dichroism, fluorescence, and mass spectrometry) and molecular dynamics simulations. The controlled structural modification of lysozyme at neutral pH enhances its activity, while the activity was lost at acidic pH at the same treatment conditions. Indeed, a larger number of amino acids were oxidized at acidic pH after plasma treatment, which results in a greater distortion of the lysozyme structure, whereas only limited structural changes were observed in lysozyme after plasma treatment at neutral pH. We found that the plasma-treated lysozyme significantly induced apoptosis to the cancer cells. Our results reveal that plasma-treated lysozyme could have potential as a new cancer cell killing drug.

Impact of seed color and storage time on the radish seed germination and sprout growth in plasma agriculture.

The use of low-temperature plasma for the pre-sowing seed treatment is still in the early stage of research; thus, numerous factors affecting germination percentage, seedling growth, and yield remains unknown. This study aimed to estimate how two critical factors, such as harvest year and seed coat color, affect the percentage of germination and seedling growth after plasma treatment. Radish seeds stored for 2 and 1 year after harvesting (harvested in 2017 and 2018) were sorted into two colors (brown and grey) to investigate the plasma effect on harvest year and seed coat color. We analyzed the amounts of seed phytohormones and antioxidant (γ-tocopherol) were analyzed using mass spectrometry, and physical changes were studied using SEM, EDX, and EPR to understand the mechanism of plasma-induced changes in radish seeds. The obtained results revealed that plasma treatment on seeds affects the germination kinetics, and the maximal germination percentage depends on seed color and the time of seed storage after harvest. Through this study, for the first time, we demonstrated that physical and chemical changes in radish seeds after plasma treatment depends upon the seed color and harvest year. Positive effects of plasma treatment on growth are stronger for sprouts from seeds harvested in 2017 than in 2018. The plasma treatment effect on the sprouts germinated from grey seeds effect was stronger than sprouts from brown radish seeds. The amounts of gibberellin A3 and abscisic acid in control seeds strongly depended on the seed color, and plasma induced changes were better in grey seeds harvested in 2017. Therefore, this study reveals that Air scalar-DBD plasma's reactive oxygen and nitrogen species (RONS) can efficiently accelerate germination and growth in older seeds.

Green route for ammonium nitrate synthesis: fertilizer for plant growth enhancement.

Soil fertility management is of great importance for farmers. The use of synthetic nitrogen (N)-fertilizer increased by 20 fold in the last 50 years to feed the increasingly hungry population. This study aims to enrich the plant soil with nitrogen content (NH4NO3 fertilizer in soil) using the low-temperature and low-pressure plasma [without H2 and catalyst]. Subsequently, we used plasma N-enriched soil for plant (radish and tomato) growth. We investigated the germination percentage, seedling growth, seedling weight, phytohormones and antioxidant activity of radish and tomato plants after treatment with plasma N-enriched soil and compared with control soil and soil + commercial N-fertilizer. The plasma N-enriched soil treatment results in significant growth enhancement for both radish and tomato plants. Further, substantial changes in phytohormone and antioxidant levels were observed for the plants grown in plasma N-enriched soil compared to control soil and soil + commercial N-fertilizer. The energy consumption (EC) for total N-fixation was 12 MJ mol-1. EC for ammonia and nitrate fixation was 17 and 41 MJ mol-1, respectively, without H2 gas. Further to understand the plasma chemistry, we performed 1D simulation using COMSOL Multiphysics® software. This study showed that direct N-fixation in the soil by plasma could be used as fertilizer for the plants and open a new window for future decentralized N-fertilizer production at the farm site.

Cold Plasma Treatment of Sunflower Seeds Modulates Plant-Associated Microbiome and Stimulates Root and Lateral Organ Growth.

Cold atmospheric pressure (CP) plasma irradiation of seeds has been shown to promote plant growth, but the molecular basis of this phenomenon is poorly understood. In our study, optimum irradiation of common sunflower seeds using a dielectric barrier discharge CP device stimulated growth of sunflower lateral organs and roots by 9-14% compared to the control. Metagenomic analysis revealed that the structure of plant-associated bacterial assembly was greatly modified upon CP treatment and could be attributed to the antimicrobial effect of CP-generated reactive species. The treatment resulted in the domination of spore forming Mycobacterium sp. in the above-ground tissues of the seedlings. While the overall bacterial diversity in the roots was barely affected, the CP-induced shift in microbial composition is the likely basis for the observed seedling root growth stimulation and the long-term effect on lateral organ growth and could be mediated by increase in water uptake and/or direct root signaling. Low amplitude protein abundance differences were detected in the roots of the emerging seedlings that are characteristic to low intensity stress stimuli response and could be linked to the changes in plant-associated microbiome upon CP treatment.

Structural modification of NADPH oxidase activator (Noxa 1) by oxidative stress: An experimental and computational study.

NADPH oxidases 1 (NOX1) derived reactive oxygen species (ROS) play an important role in the progression of cancer through signaling pathways. Therefore, in this paper, we demonstrate the effect of cold atmospheric plasma (CAP) on the structural changes of Noxa1 SH3 protein, one of the regulatory subunits of NOX1. For this purpose, firstly we purified the Noxa1 SH3 protein and analyzed the structure using X-ray crystallography, and subsequently, we treated the protein with two types of CAP reactors such as pulsed dielectric barrier discharge (DBD) and Soft Jet for different time intervals. The structural deformation of Noxa1 SH3 protein was analyzed by various experimental methods (circular dichroism, fluorescence, and NMR spectroscopy) and by MD simulations. Additionally, we demonstrate the effect of CAP (DBD and Soft Jet) on the viability and expression of NOX1 in A375 cancer cells. Our results are useful to understand the structural modification/oxidation occur in protein due to reactive oxygen and nitrogen (RONS) species generated by CAP.

Characteristics of crystalline sputtered LaFeO3 thin films as photoelectrochemical water splitting photocathodes.

Stable photoelectrochemical (PEC) operation is a critical issue for the commercialization of PEC water-splitting systems. Unfortunately, most semiconductor photocathodes generating hydrogen in these systems are unstable in aqueous solutions. This is a huge limitation for the development of durable PEC water-splitting systems. Lanthanum iron oxide (LaFeO3) is a promising p-type semiconductor to overcome this drawback because of its stability in an aqueous solution and its proper energy level for reducing water. In this study, we fabricated a crystalline LaFeO3 thin film by radio frequency magnetron sputtering deposition and a post-annealing process in air for use as a PEC photocathode. Based on the morphological, compositional, optical and electronic characterizations, we found that it was ideal for a visible light-responsive PEC photocathode and tandem PEC water-splitting system with a small band gap absorber behind it. Furthermore, it showed stable PEC performance in a strong alkaline solution during PEC operation without any protection layers. Therefore, the crystalline sputtered LaFeO3 thin film suggested in this study would be feasible to apply as a PEC photocathode for durable, simple and low-cost PEC water splitting.