Redox regulation of macrophages.

Redox signaling, a mode of signal transduction that involves the transfer of electrons from a nucleophilic to electrophilic molecule, has emerged as an essential regulator of inflammatory macrophages. Redox reactions are driven by reactive oxygen/nitrogen species (ROS and RNS) and redox-sensitive metabolites such as fumarate and itaconate, which can post-translationally modify specific cysteine residues in target proteins. In the past decade our understanding of how ROS, RNS, and redox-sensitive metabolites control macrophage function has expanded dramatically. In this review, we discuss the latest evidence of how ROS, RNS, and metabolites regulate macrophage function and how this is dysregulated with disease. We highlight the key tools to assess redox signaling and important questions that remain.

Oxidative Stress and Cerebral Vascular Tone: The Role of Reactive Oxygen and Nitrogen Species.

The brain's unique characteristics make it exceptionally susceptible to oxidative stress, which arises from an imbalance between reactive oxygen species (ROS) production, reactive nitrogen species (RNS) production, and antioxidant defense mechanisms. This review explores the factors contributing to the brain's vascular tone's vulnerability in the presence of oxidative damage, which can be of clinical interest in critically ill patients or those presenting acute brain injuries. The brain's high metabolic rate and inefficient electron transport chain in mitochondria lead to significant ROS generation. Moreover, non-replicating neuronal cells and low repair capacity increase susceptibility to oxidative insult. ROS can influence cerebral vascular tone and permeability, potentially impacting cerebral autoregulation. Different ROS species, including superoxide and hydrogen peroxide, exhibit vasodilatory or vasoconstrictive effects on cerebral blood vessels. RNS, particularly NO and peroxynitrite, also exert vasoactive effects. This review further investigates the neuroprotective effects of antioxidants, including superoxide dismutase (SOD), vitamin C, vitamin E, and the glutathione redox system. Various studies suggest that these antioxidants could be used as adjunct therapies to protect the cerebral vascular tone under conditions of high oxidative stress. Nevertheless, more extensive research is required to comprehensively grasp the relationship between oxidative stress and cerebrovascular tone, and explore the potential benefits of antioxidants as adjunctive therapies in critical illnesses and acute brain injuries.

Tannic acid and zinc ion coordination of nanase for the treatment of inflammatory bowel disease by promoting mucosal repair and removing reactive oxygen and nitrogen species.

Colon mucosal overexpression of reactive oxygen and nitrogen species (RONS) accelerates the development of inflammatory bowel disease (IBD) and destroys the mucosa and its barrier. IBD can be alleviated by removing RONS from the inflamed colon. The preparation of strong and efficient nanoantioxidants remains a challenge despite the development of numerous nanoantioxidants. In this paper, Zn-TA nanoparticles with fine hollow microstructure (HZn-TA) were successfully prepared and could be effectively used to treat IBD. In the first step, ZIF-8 nanoparticles were synthesized by a one-pot method. On this basis, HZn-TA nanoparticles were etched by TA, and a multifunctional nanase was developed for the treatment of IBD. RONS, including reactive oxygen species (ROS) and nitric oxide (NO), can be eliminated to increase cell survival following Hydrogen peroxide (H2O2) stimulation, including reactive oxygen species (ROS) and nitric oxide (NO with hydrogen peroxide (H2O2). In a model for preventing and delaying acute colitis, clearance of RONS has been shown to reduce intestinal inflammation in mice by reducing colon damage, proinflammatory cytokine levels, the spleen index, and body weight. Intestinal mucosal healing can be promoted by HZn-TA nanoparticles, which can upregulate zonula occludens protein 1 (ZO-1) and claudin-1 expression. Based on the results of this study, HZn-TA nanoparticles were able to effectively treat IBD with minimal adverse effects by being biocompatible, multienzyme active, and capable of scavenging RONS. Therefore, we pioneered the application of HZn-TA nanoparticles for the treatment of IBD, which are capable of clearing RONS without significant adverse effects. STATEMENT OF SIGNIFICANCE: ➢ HZn-TA nanoparticles were successfully prepared and could be effectively used to treat IBD. ➢ Intestinal mucosal healing can be promoted by HZn-TA nanoparticles, which can upregulate ZO-1 and claudin-1 expression. ➢ HZn-TA nanoparticles were able to effectively treat IBD with minimal adverse effects by being biocompatible, multienzyme active, and capable of scavenging RONS.

Effect of reactive oxygen, nitrogen, and sulfur species on signaling pathways in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease in which fats, lipids, cholesterol, calcium, proliferating smooth muscle cells, and immune cells accumulate in the intima of the large arteries, forming atherosclerotic plaques. A complex interplay of various vascular and immune cells takes place during the initiation and progression of atherosclerosis. Multiple reports indicate that tight control of reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) production is critical for maintaining vascular health. Unrestricted ROS and RNS generation may lead to activation of various inflammatory signaling pathways, facilitating atherosclerosis. Given these deleterious consequences, it is important to understand how ROS and RNS affect the signaling processes involved in atherogenesis. Conversely, RSS appears to exhibit an atheroprotective potential and can alleviate the deleterious effects of ROS and RNS. Herein, we review the literature describing the effects of ROS, RNS, and RSS on vascular smooth muscle cells, endothelial cells, and macrophages and focus on how changes in their production affect the initiation and progression of atherosclerosis. This review also discusses the contribution of ROS, RNS, and RSS in mediating various post-translational modifications, such as oxidation, nitrosylation, and sulfation, of the molecules involved in inflammatory signaling.

Biomarkers of Oxidative and Radical Stress.

Reactive oxygen and nitrogen species (ROS/RNS) are generated as a result of normal intracellular metabolism [...].

Enhanced Chemoradiotherapy for MRSA-Infected Osteomyelitis Using Immunomodulatory Polymer-Reinforced Nanotherapeutics.

The eradication of osteomyelitis caused by methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge due to its development of biofilm-induced antibiotic resistance and impaired innate immunity, which often leads to frequent surgical failure. Here, the design, synthesis, and performance of X-ray-activated polymer-reinforced nanotherapeutics that modulate the immunological properties of infectious microenvironments to enhance chemoradiotherapy against multidrug-resistant bacterial deep-tissue infections are reported. Upon X-ray radiation, the proposed polymer-reinforced nanotherapeutic generates reactive oxygen species and reactive nitrogen species. To robustly eradicate MRSA biofilms at deep infection sites, these species can specifically bind to MRSA and penetrate biofilms for enhanced chemoradiotherapy treatment. X-ray-activated nanotherapeutics modulate the innate immunity of macrophages to prevent the recurrence of osteomyelitis. The remarkable anti-infection effects of these nanotherapeutics are validated using a rat osteomyelitis model. This study demonstrates the significant potential of a synergistic chemoradiotherapy and immunotherapy method for treating MRSA biofilm-infected osteomyelitis.

Antioxidant Hydrogels: Antioxidant Mechanisms, Design Strategies, and Applications in the Treatment of Oxidative Stress-Related Diseases.

Oxidative stress is a biochemical process that disrupts the redox balance due to an excess of oxidized substances within the cell. Oxidative stress is closely associated with a multitude of diseases and health issues, including cancer, diabetes, cardiovascular diseases, neurodegenerative disorders, inflammatory conditions, and aging. Therefore, the developing of antioxidant treatment strategies has emerged as a pivotal area of medical research. Hydrogels have garnered considerable attention due to their exceptional biocompatibility, adjustable physicochemical properties, and capabilities for drug delivery. Numerous antioxidant hydrogels have been developed and proven effective in alleviating oxidative stress. In the pursuit of more effective treatments for oxidative stress-related diseases, there is an urgent need for advanced strategies for the fabrication of multifunctional antioxidant hydrogels. Consequently, the authors' focus will be on hydrogels that possess exceptional reactive oxygen species and reactive nitrogen species scavenging capabilities, and their role in oxidative stress therapy will be evaluated. Herein, the antioxidant mechanisms and the design strategies of antioxidant hydrogels and their applications in oxidative stress-related diseases are discussed systematically in order to provide critical insights for further advancements in the field.

The progression of inorganic nanoparticles and natural products for inflammatory bowel disease.

There is a growing body of evidence indicating a close association between inflammatory bowel disease (IBD) and disrupted intestinal homeostasis. Excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), along with an increase in M1 proinflammatory macrophage infiltration during the activation of intestinal inflammation, plays a pivotal role in disrupting intestinal homeostasis in IBD. The overabundance of ROS/RNS can cause intestinal tissue damage and the disruption of crucial gut proteins, which ultimately compromises the integrity of the intestinal barrier. The proliferation of M1 macrophages contributes to an exaggerated immune response, further compromising the intestinal immune barrier. Currently, intestinal nanomaterials have gained widespread attention in the context of IBD due to their notable characteristics, including the ability to specifically target regions of interest, clear excess ROS/RNS, and mimic biological enzymes. In this review, we initially elucidated the gut microenvironment in IBD. Subsequently, we delineate therapeutic strategies involving two distinct types of nanomedicine, namely inorganic nanoparticles and natural product nanomaterials. Finally, we present a comprehensive overview of the promising prospects associated with the application of nanomedicine in future clinical settings for the treatment of IBD (graphic abstract). Different classes of nanomedicine are used to treat IBD. This review primarily elucidates the current etiology of inflammatory bowel disease and explores two prominent nanomaterial-based therapeutic approaches. First, it aims to eliminate excessive reactive oxygen species and reactive nitrogen species. Second, they focus on modulating the polarization of inflammatory macrophages and reducing the proportion of pro-inflammatory macrophages. Additionally, this article delves into the treatment of inflammatory bowel disease using inorganic metal nanomaterials and natural product nanomaterials.

Plasma-Activated Media Produced by a Microwave-Excited Atmospheric Pressure Plasma Jet Is Effective against Cisplatin-Resistant Human Bladder Cancer Cells In Vitro.

Media exposed to atmospheric pressure plasma (APP) produce reactive oxygen and nitrogen species (RONS), with hydrogen peroxide (H2O2), nitrite (NO2-), and nitrate (NO3-) being among the most detected species due to their relatively long lifetime. In this study, a standardized microwave-excited (ME) APP jet (APPJ) source was employed to produce gaseous RONS to treat liquid samples. The source was a commercially available plasma jet, which generated argon plasma utilizing a coaxial transmission line resonator at the operating frequency of 2.45 GHz. An ultraviolet-visible spectrophotometer was used to measure the concentrations of H2O2 and NO3- in plasma-activated media (PAM). Three different types of media (deionized water, Hank's balanced salt solution, and cell culture solution Dulbecco's modified eagles medium [DMEM]) were utilized as liquid samples. Among these media, the plasma-treated DMEM was observed to have the highest levels of H2O2 and NO3-. Subsequently, the feasibility of using argon ME-APPJ-activated DMEM (PAM) as an adjuvant to enhance the therapeutic effects of cisplatin on human bladder cancer cells (T-24) was investigated. Various cancer cell lines, including T-24 cells, treated with PAM were observed in vitro for changes in cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. A viability reduction was detected in the various cancer cells after incubation in PAM. Furthermore, the study's results revealed that PAM was effective against cisplatin-resistant T-24 cells in vitro. In addition, a possible connection between HER expression and cell viability was sketched.

The molecular paradigm of reactive oxygen species (ROS) and reactive nitrogen species (RNS) with different phytohormone signaling pathways during drought stress in plants.

Drought is undoubtedly a major environmental constraint that negatively affects agricultural yield and productivity throughout the globe. Plants are extremely vulnerable to drought which imposes several physiological, biochemical and molecular perturbations. Increased generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in different plant organs is one of the inevitable consequences of drought. ROS and RNS are toxic byproducts of metabolic reactions and poise oxidative stress and nitrosative stress that are detrimental for plants. In spite of toxic effects, these potentially active radicals also play a beneficial role in mediating several signal transduction events that lead to plant acclimation and enhanced survival under harsh environmental conditions. The precise understanding of ROS and RNS signaling and their molecular paradigm with different phytohormones, such as auxin, gibberellin, cytokinin, abscisic acid, ethylene, brassinosteroids, strigolactones, jasmonic acid, salicylic acid and melatonin play a pivotal role for maintaining plant fitness and resilience to counteract drought toxicity. Therefore, the present review provides an overview of integrated systemic signaling between ROS, RNS and phytohormones during drought stress based on past and recent advancements and their influential role in conferring protection against drought-induced damages in different plant species. Indeed, it would not be presumptuous to hope that the detailed knowledge provided in this review will be helpful for designing drought-tolerant crop cultivars in the forthcoming times.

Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism.

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.

Reactive oxygen species- and nitric oxide-dependent regulation of ion and metal homeostasis in plants.

Deterioration and impoverishment of soil, caused by environmental pollution and climate change, result in reduced crop productivity. To adapt to hostile soils, plants have developed a complex network of factors involved in stress sensing, signal transduction, and adaptive responses. The chemical properties of reactive oxygen species (ROS) and reactive nitrogen species (RNS) allow them to participate in integrating the perception of external signals by fine-tuning protein redox regulation and signal transduction, triggering specific gene expression. Here, we update and summarize progress in understanding the mechanistic basis of ROS and RNS production at the subcellular level in plants and their role in the regulation of ion channels/transporters at both transcriptional and post-translational levels. We have also carried out an in silico analysis of different redox-dependent modifications of ion channels/transporters and identified cysteine and tyrosine targets of nitric oxide in metal transporters. Further, we summarize possible ROS- and RNS-dependent sensors involved in metal stress sensing, such as kinases and phosphatases, as well as some ROS/RNS-regulated transcription factors that could be involved in metal homeostasis. Understanding ROS- and RNS-dependent signaling events is crucial to designing new strategies to fortify crops and improve plant tolerance of nutritional imbalance and metal toxicity.

Reactive nitrogen species as therapeutic targets for autophagy/mitophagy modulation to relieve neurodegeneration in multiple sclerosis: Potential application for drug discovery.

Multiple sclerosis (MS) is a neuroinflammatory disease with limited therapeutic effects, eventually developing into handicap. Seeking novel therapeutic strategies for MS is timely important. Active autophagy/mitophagy could mediate neurodegeneration, while its roles in MS remain controversial. To elucidate the exact roles of autophagy/mitophagy and reveal its in-depth regulatory mechanisms, we conduct a systematic literature study and analyze the factors that might be responsible for divergent results obtained. The dynamic change levels of autophagy/mitophagy appear to be a determining factor for final neuron fate during MS pathology. Excessive neuronal autophagy/mitophagy contributes to neurodegeneration after disease onset at the active MS phase. Reactive nitrogen species (RNS) serve as key regulators for redox-related modifications and participate in autophagy/mitophagy modulation in MS. Nitric oxide (•NO) and peroxynitrite (ONOO-), two representative RNS, could nitrate or nitrosate Drp1/parkin/PINK1 pathway, activating excessive mitophagy and aggravating neuronal injury. Targeting RNS-mediated excessive autophagy/mitophagy could be a promising strategy for developing novel anti-MS drugs. In this review, we highlight the important roles of RNS-mediated autophagy/mitophagy in neuronal injury and review the potential therapeutic compounds with the bioactivities of inhibiting RNS-mediated autophagy/mitophagy activation and attenuating MS progression. Overall, we conclude that reactive nitrogen species could be promising therapeutic targets to regulate autophagy/mitophagy for multiple sclerosis treatment.

Medical gas plasma technology: Roadmap on cancer treatment and immunotherapy.

Despite continuous therapeutic progress, cancer remains an often fatal disease. In the early 2010s, first evidence in rodent models suggested promising antitumor action of gas plasma technology. Medical gas plasma is a partially ionized gas depositing multiple physico-chemical effectors onto tissues, especially reactive oxygen and nitrogen species (ROS/RNS). Today, an evergrowing body of experimental evidence suggests multifaceted roles of medical gas plasma-derived therapeutic ROS/RNS in targeting cancer alone or in combination with oncological treatment schemes such as ionizing radiation, chemotherapy, and immunotherapy. Intriguingly, gas plasma technology was recently unraveled to have an immunological dimension by inducing immunogenic cell death, which could ultimately promote existing cancer immunotherapies via in situ or autologous tumor vaccine schemes. Together with first clinical evidence reporting beneficial effects in cancer patients following gas plasma therapy, it is time to summarize the main concepts along with the chances and limitations of medical gas plasma onco-therapy from a biological, immunological, clinical, and technological point of view.

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.

How living cells are affected during the cold atmospheric pressure plasma treatment.

When the electric discharge process is limited by high voltage electrodes shielding, the ionization measure would be controlled to less than one percent and the temperature to less than 37 °C even at atmospheric pressure, so-called cold atmospheric pressure plasma (CAP). CAP has been found to have profound medical applications in association with its reactive oxygen and nitrogen species (ROS/RNS). In this way that during plasma exposure, the subjected medium (e.g. cell cytoplasmic membrane in plasma therapy) interacts with ROS/RNS. Accordingly, a precise study of the mentioned interactions and their consequences on the cells' behavior changes, is necessary. The results lead to the reduction of possible risks and provide the opportunity of optimizing the efficacy of CAP before the development of CAP applications in the field of plasma medicine. In this report molecular dynamic (MD) simulation is used to investigate the mentioned interactions and a proper and compatible comparison with the experimental results is presented. Based on this, the effects of H2O2, NO and O2 on the living cell's membrane are investigated in biological conditions. Our results show that: i) The hydration of phospholipid polar heads would be enhanced associated with the H2O2 presence. ii) A new definition of the surface area assigned to each phospholipid (APL), more reliable and compatible with the physical expectations, is introduced. iii) The long-term behavior of NO and O2 is their penetration into the lipid bilayer and sometimes passing through the membrane into the cell. The latter would be an indication of internal cells' pathways activation leading to modification of cells' function.

The Impact of Serum Levels of Reactive Oxygen and Nitrogen Species on the Disease Severity of COVID-19.

Elucidation of the redox pathways in severe coronavirus disease 2019 (COVID-19) might aid in the treatment and management of the disease. However, the roles of individual reactive oxygen species (ROS) and individual reactive nitrogen species (RNS) in COVID-19 severity have not been studied to date. The main objective of this research was to assess the levels of individual ROS and RNS in the sera of COVID-19 patients. The roles of individual ROS and RNS in COVID-19 severity and their usefulness as potential disease severity biomarkers were also clarified for the first time. The current case-control study enrolled 110 COVID-19-positive patients and 50 healthy controls of both genders. The serum levels of three individual RNS (nitric oxide (NO•), nitrogen dioxide (ONO-), and peroxynitrite (ONOO-)) and four ROS (superoxide anion (O2•-), hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2)) were measured. All subjects underwent thorough clinical and routine laboratory evaluations. The main biochemical markers for disease severity were measured and correlated with the ROS and RNS levels, and they included tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), the neutrophil-to-lymphocyte ratio (NLR), and angiotensin-converting enzyme 2 (ACE2). The results indicated that the serum levels of individual ROS and RNS were significantly higher in COVID-19 patients than in healthy subjects. The correlations between the serum levels of ROS and RNS and the biochemical markers ranged from moderate to very strongly positive. Moreover, significantly elevated serum levels of ROS and RNS were observed in intensive care unit (ICU) patients compared with non-ICU patients. Thus, ROS and RNS concentrations in serum can be used as biomarkers to track the prognosis of COVID-19. This investigation demonstrated that oxidative and nitrative stress play a role in the etiology of COVID-19 and contribute to disease severity; thus, ROS and RNS are probable innovative targets in COVID-19 therapeutics.

Simultaneous removal of norfloxacin and chloramphenicol using cold atmospheric plasma jet (CAPJ): Enhanced performance, synergistic effect, plasma-activated water (PAW) contribution, mechanism and toxicity evaluation.

The extensive abuse and inadvertent discharge of various antibiotics into the environment has become a serious problem for posing a big threat to human health. In order to deal with this problem, we utilized cold atmospheric plasma jet (CAPJ) to treat two different antibiotics, namely, norfloxacin and chloramphenicol, and investigated the efficiencies and corresponding mechanisms for removing the mixed antibiotics. In the application of the CAPJ technique, we made use of not only the direct plasma processing, but also the indirect plasma-activated water (PAW) treatment. The efficiency for mixed antibiotics treatment was considerably enhanced as compared to the efficiency for treatment of single antibiotics. The contributions from the CAPJ-induced reactive oxygen/nitrogen species (RONS) were examined, showing that ·OH and 1O2 played a major role in the degradation of norfloxacin and chloramphenicol in the direct plasma treatment, while 1O2 played a major role in the PAW treatment. The bio-toxicity evaluation was also provided to verify the ecological safety of the CAPJ treatment. As such, this work has not only showed the effectiveness of CAPJ treatment of mixed antibiotics, but also elucidated the mechanisms for the enhanced treatment efficiency, which may provide a new solution for treatment of antibiotics in the environment.

Effect of Plasma On-Time with a Fixed Duty Ratio on Reactive Species in Plasma-Treated Medium and Its Significance in Biological Applications.

Optimizing the therapeutic range of nonthermal atmospheric pressure plasma (NTAPP) for biomedical applications is an active research topic. For the first time, we examined the effect of plasma on-times in this study while keeping the duty ratio and treatment time fixed. We have evaluated the electrical, optical, and soft jet properties for two different duty ratios of 10% and 36%, using the plasma on-times of 25, 50, 75, and 100 ms. Furthermore, the influence of plasma on-time on reactive oxygen and nitrogen species (ROS/RNS) levels in plasma treated medium (PTM) was also investigated. Following treatment, the characteristics of (DMEM media) and PTM (pH, EC, and ORP) were also examined. While EC and ORP rose by raising plasma on-time, pH remained unchanged. Finally, the PTM was used to observe the cell viability and ATP levels in U87-MG brain cancer cells. We found it interesting that, by increasing the plasma on-time, the levels of ROS/RNS dramatically increased in PTM and significantly affected the viability and ATP levels of the U87-MG cell line. The results of this study provide a significant indication of advancement by introducing the optimization of plasma on-time to increase the efficacy of the soft plasma jet for biomedical applications.

Assay of Reactive Oxygen/Nitrogen Species (ROS/RNS) in Arabidopsis Peroxisomes Through Fluorescent Protein Containing a Type 1 Peroxisomal Targeting Signal (PTS1).

Plant peroxisomes have an active nitro-oxidative metabolism. However, the assay of reactive oxygen and nitrogen species (ROS/RNS) could be a challenge since the purification of peroxisomes is technically a high time-consuming approach that needs to be optimized for each tissue/organ (root, leaf, fruit) and plant species. Arabidopsis thaliana, as a model plant for biochemical and molecular studies, has become a useful tool to study the basic metabolism, including also that of ROS/RNS. The combination of specific fluorescent probes with Arabidopsis plants expressing a fluorescent protein containing a type 1 peroxisomal targeting signal (PTS1) is a powerful tool to address the profile of ROS/RNS in peroxisomes by confocal laser scanning microscope (CLSM). This chapter provides a detailed description to detect the content and distribution of ROS and RNS in Arabidopsis peroxisomes, together with a critical analysis of their potentialities and limitations, since these approaches require appropriate controls to corroborate the obtained data.