Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

Copper Tannic Acid Coordination Nanosheet: A Potent Nanozyme for Scavenging ROS from Cigarette Smoke.

The global tobacco epidemic is still a devastating threat to public health. Toxic reactive oxygen species (ROS) in the cigarette smoke cannot be efficiently eliminated by currently available cigarette filters. The resultant oxidative stress causes severe lung injury and further diseases. To tackle this challenge, herein, a novel copper tannic acid coordination (CuTA) nanozyme is reported as a highly active and thermostable ROS scavenger. The CuTA nanozyme exhibits intrinsic superoxide dismutase-like activity, catalase-like activity, and hydroxyl radical elimination capacity. These synergistic antioxidant abilities make the CuTA nanozyme a promising candidate for the improvement of commercial cigarette filters. Mouse model results show that commercial cigarettes loaded with CuTA nanozyme efficiently scavenge ROS in the cigarette smoke, reduce oxidative stress-induced lung inflammation, and minimize the resultant acute lung injury. The developed CuTA nanozyme offers an efficient ROS scavenger with multiple antioxidant ability and opens up new opportunities for the modification of cigarette filters to reduce the toxic effects of cigarette smoke.

Plasma from Patients with Rheumatoid Arthritis Reduces Nitric Oxide Synthesis and Induces Reactive Oxygen Species in A Cell-Based Biosensor.

Rheumatoid arthritis (RA) has been associated with a higher risk of developing cardiovascular (CV) diseases. It has been proposed that systemic inflammation plays a key role in premature atherosclerosis development, and is therefore crucial to determine whether systemic components from RA patients promotes endothelial cell-oxidative stress by affecting reactive oxygen species (ROS) and nitric-oxide (NO) production. The aim of this study was to evaluate whether plasma from RA patients impair NO synthesis and ROS production by using the cell-line ECV-304 as a biosensor. NO synthesis and ROS production were measured in cells incubated with plasma from 73 RA patients and 52 healthy volunteers by fluorimetry. In addition, traditional CV risk factors, inflammatory molecules and disease activity parameters were measured. Cells incubated with plasma from RA patients exhibited reduced NO synthesis and increased ROS production compared to healthy volunteers. Furthermore, the imbalance between NO synthesis and ROS generation in RA patients was not associated with traditional CV risk factors. Our data suggest that ECV-304 cells can be used as a biosensor of systemic inflammation-induced endothelial cell-oxidative stress. We propose that both NO and ROS production are potential biomarkers aimed at improving the current assessment of CV risk in RA.

Redox Trimetallic Nanozyme with Neutral Environment Preference for Brain Injury.

Metal nanozyme has attracted wide interest for biomedicine, and a highly catalytic material in the physiological environment is highly desired. However, catalytic selectivity of nanozyme is still highly challenging, limiting its wide application. Here, we show a trimetallic (triM) nanozyme with highly catalytic activity and environmental selectivity. Enzyme-mimicked investigations find that the triM system possesses multi-enzyme-mimetic activity for removing reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as 1O2, H2O2, •OH, and •NO. Importantly, triM nanozyme exhibits the significant neutral environment preference for removing the •OH, 1O2, and •NO free radical, indicating its highly catalytic selectivity. The density functional theory (DFT) calculations reveal that triM nanozyme can capture electrons very easily and provides more attraction to reactive oxygen and nitrogen species (RONS) radicals in the neutral environment. In vitro experiments show that triM nanozyme can improve the viability of injured neural cell. In the LPS-induced brain injury model, the superoxide dismutase (SOD) activity and lipid peroxidation can be greatly recovered after triM nanozyme treatment. Moreover, the triM nanozyme treatment can significantly improve the survival rate, neuroinflammation, and reference memory of injured mice. Present work provides a feasible route for improving selectivity of nanozyme in the physiological environment as well as exploring potential applications in brain science.

Impaired proteostasis during skeletal muscle aging.

Aging is a complex phenomenon that has detrimental effects on tissue homeostasis. The skeletal muscle is one of the earliest tissues to be affected and to manifest age-related changes such as functional impairment and the loss of mass. Common to these alterations and to most of tissues during aging is the disruption of the proteostasis network by detrimental changes in the ubiquitin-proteasomal system (UPS) and the autophagy-lysosomal system (ALS). In fact, during aging the accumulation of protein aggregates, a process mainly driven by increased levels of oxidative stress, has been observed, clearly demonstrating UPS and ALS dysregulation. Since the UPS and ALS are the two most important pathways for the removal of misfolded and aggregated proteins and also of damaged organelles, we provide here an overview on the current knowledge regarding the connection between the loss of proteostasis and skeletal muscle functional impairment and also how redox regulation can play a role during aging. Therefore, this review serves for a better understanding of skeletal muscle aging in regard to the loss of proteostasis and how redox regulation can impact its function and maintenance.

Reactive oxygen species-induced alterations in H19-Igf2 methylation patterns, seminal plasma metabolites, and semen quality.

PURPOSE: This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction. METHODS: Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out. RESULT(S): The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = - 0.20, P < 0.05), tryptophan (r = - 0.45, P < 0.05), sperm motility (r = - 0.20, P < 0.05), sperm viability (r = - 0.23, P < 0.01), and sperm concentration (r = - 0.30, P < 0.001). CONCLUSION(S): Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART).

3,2-Hydroxypyridinone-Grafted Chitosan Oligosaccharide Nanoparticles as Efficient Decorporation Agents for Simultaneous Removal of Uranium and Radiation-Induced Reactive Oxygen Species in Vivo.

Most of the key radionuclides in the nuclear fuel cycle, such as actinides, possess a combination of heavy metal chemotoxicity and radiotoxicity and therefore represent a severe threat to the ecological environment and public safety. The radiotoxicity originates from direct radiation-induced organ damage and indirect damage, mostly through radiation-induced reactive oxygen species (ROS). Although effective chelating agents that can accelerate the excretion of actinides, such as uranium, have been developed in the past several decades, very few of them can reduce radiation-induced damage from internal contamination. In fact, the strategy of simultaneous removal of actinides and their induced-ROS in vivo has scarcely been considered. Here, we report a 3,2-hydroxypyridinone-grafted chitosan oligosaccharide nanoparticle (COS-HOPO) as a new type of decorporation agent that is effective for the removal of both uranium and ROS in vivo. The cytotoxicity and decorporation assays indicate that the marriage of chitosan oligosaccharide (COS) and hydroxypyridinone (HOPO) gives rise to a remarkable decrease in toxicity and promotion of the uranium removal capability from both kidneys and femurs. The decorporation efficacy can reach up to 43% in rat proximal tubular epithelial cells (NRK-52E), 44% in kidneys, and 32% in femurs. Moreover, the ROS levels of the cells treated with COS-HOPO are significantly lower than those of the control group, implying a promising radiation protection effect. The detoxification mechanism of COS-HOPO is closely related to both chelating U(VI)- and scavenging U(VI)-induced intracellular ROS.

Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions.

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.

Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants.

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networks.

Behavior of Neutrophil Granulocytes during Toxoplasma gondii Infection in the Central Nervous System.

Cerebral toxoplasmosis is characterized by activation of brain resident cells and recruitment of specific immune cell subsets from the periphery to the central nervous system (CNS). Our studies revealed that the rapidly invaded Ly6G+ neutrophil granulocytes are an early non-lymphoid source of interferon-gamma (IFN-γ), the cytokine known to be the major mediator of host resistance to Toxoplasma gondii (T. gondii). Upon selective depletion of Ly6G+ neutrophils, we detected reduced IFN-γ production and increased parasite burden in the CNS. Ablation of Ly6G+ cells resulted in diminished recruitment of Ly6Chi monocytes into the CNS, indicating a pronounced interplay. Additionally, we identified infiltrated Ly6G+ neutrophils to be a heterogeneous population. The Ly6G+CD62-LhiCXCR4+ subset released cathelicidin-related antimicrobial peptide (CRAMP), which can promote monocyte dynamics. On the other hand, the Ly6G+CD62-LloCXCR4+ subset produced IFN-γ to establish early inflammatory response. Collectively, our findings revealed that the recruited Ly6G+CXCR4+ neutrophil granulocytes display a heterogeneity in the CNS with a repertoire of effector functions crucial in parasite control and immune regulation upon experimental cerebral toxoplasmosis.

Glycine-functionalized copper(ii) hydroxide nanoparticles with high intrinsic superoxide dismutase activity.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O2-) radicals into molecular oxygen (O2) and H2O2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH)2 NPs) are functional SOD mimics, whereas bulk Cu(OH)2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH)2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH)2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >1016 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH)2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.

Switching on a transient endogenous ROS production in mammalian cells and tissues.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Quantification of hydrogen peroxide in plant tissues using Amplex Red.

Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H2O2), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H2O2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H2O2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H2O2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg-1 FW range. Commonly used additives for H2O2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue.

In situ production of ROS in the skin by photodynamic therapy as a powerful tool in clinical dermatology.

Photodynamic therapy (PDT) is a clinical modality of photochemotherapy based on the accumulation of a photosensitizer in target cells and subsequent irradiation of the tissue with light of adequate wavelength promoting reactive oxygen species (ROS) formation and cell death. PDT is used in several medical specialties as an organ-specific therapy for different entities. In this review we focus on the current dermatological procedure of PDT. In the most widely used PDT protocol in dermatology, ROS production occurs by accumulation of the endogenous photosensitizer protoporphyrin IX after treatment with the metabolic precursors 5-methylaminolevulinic acid (MAL) or 5-aminolevulinic acid (ALA). To date, current approved dermatological indications of PDT include actinic keratoses (AK), basal cell carcinoma (BCC) and in situ squamous cell carcinoma (SCC) also known as Bowen disease (BD). With regards to AKs, PDT can also treat the cancerization field carrying an oncogenic risk. In addition, an increasing number of pathologies, such as other skin cancers, infectious, inflammatory or pilosebaceous diseases are being considered as potentially treatable entities with PDT. Besides the known therapeutic properties of PDT, there is a modality used for skin rejuvenation and aesthetic purposes defined as photodynamic photorejuvenation. This technique enables the remodelling of collagen, which in turn prevents and treats photoaging stygmata. Finally we explore a new potential treatment field for PDT determined by the activation of follicular bulge stem cells caused by in situ ROS formation.

A microdialysis method to measure in vivo hydrogen peroxide and superoxide in various rodent tissues.

Reactive oxygen species (ROS) play a critical role in cell signaling and disease pathogenesis. Despite their biological importance, assessment of ROS often involves measurement of indirect byproducts or measurement of ROS from excised tissue. Herein, we describe a microdialysis technique that utilizes the Amplex Ultrared assay to directly measure hydrogen peroxide (H2O2) and superoxide in tissue of living, anesthetized rats and mice. We demonstrate the application of this methodology in the penis, adipose tissue, skeletal muscle, kidney, and liver. We provide data demonstrating the impact of important methodological considerations such as membrane length, perfusion rate, and time-dependence upon probe insertion. In this report, we provide a complete list of equipment, troubleshooting tips, and suggestions for implementing this technique in a new system. The data herein demonstrate the feasibility of measuring both in vivo H2O2 and superoxide in the extracellular environment of various rodent tissues, providing a technique with potential application to a vast array of disease states which are subject to oxidative stress.

Exerting better control and specificity with singlet oxygen experiments in live mammalian cells.

Singlet molecular oxygen, O2(a1Δg), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O2(a1Δg) in this regard is acknowledged, an understanding of the targets and mechanisms of O2(a1Δg) action is inadequate. Thus, methods that better facilitate studies of O2(a1Δg) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O2(a1Δg) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O2(a1Δg) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O2(a1Δg) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O2(a1Δg) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O2(a1Δg) at the expense of other ROS. We also comment on our recent efforts to monitor O2(a1Δg) in cells and to monitor the cell's response to O2(a1Δg).

Automated image analysis for quantification of reactive oxygen species in plant leaves.

The paper presents an image processing method for the quantitative assessment of ROS accumulation areas in leaves stained with DAB or NBT for H2O2 and O2- detection, respectively. Three types of images determined by the combination of staining method and background color are considered. The method is based on the principle of supervised machine learning with manually labeled image patterns used for training. The method's algorithm is developed as a JavaScript macro in the public domain Fiji (ImageJ) environment. It allows to select the stained regions of ROS-mediated histochemical reactions, subsequently fractionated according to the weak, medium and intense staining intensity and thus ROS accumulation. It also evaluates total leaf blade area. The precision of ROS accumulation area detection is validated by the Dice Similarity Coefficient in the case of manual patterns. The proposed framework reduces the computation complexity, once prepared, requires less image processing expertise than the competitive methods and represents a routine quantitative imaging assay for a general histochemical image classification.

Approaches to unravel pathways of reactive oxygen species in the photoinactivation of bacteria induced by a dicationic fulleropyrrolidinium derivative.

The photodynamic mechanism sensitized by N,N-dimethyl-2-[4-(3-N,N,N-trimethylammoniopropoxy)phenyl]fulleropyrrolidinium (DPC602+) was investigated in Staphylococcus aureus cells. Different experimental conditions were used to detect reactive oxygen species (ROS) in S. aureus cell suspensions. First, a photoinactivation of 4 log decrease of S. aureus viability was chosen using 0.5µM DPC602+ and 15min irradiation. An anoxic atmosphere indicated that oxygen was required for an effective photoinactivation. Also, photoprotection was found in the presence of sodium azide, whereas the photocytotoxicity induced by DPC602+ increased in D2O. The addition of diazabicyclo[2.2.2]octane or d-mannitol produced a reduction in the S. aureus photokilling. Moreover, singlet molecular oxygen, O2(1Δg), was detected by the reaction with 9,10-dimethylanthracene into the S. aureus cells. A decrease in the photoinactivation of S. aureus was observed in the presence of ß-nicotinamide adenine dinucleotide reduced form, which was dependent on the NADH concentration. Therefore, under aerobic condition the photocytotoxicity activity induced by DPC602+ was mediated by mainly a contribution of type II process. Moreover, photoinactivation of S. aureus was possible with DPC602+ in the presence of azide anions under anoxic condition. However, these conditions were not effective to photoinactivate Escherichia coli. On the other hand, the addition of potassium iodide produced an increase in the photokilling of bacteria, depending on the KI concentration and irradiation times. The formation of reactive iodine species may be contributing to inactivate S. aureus cells photoinduced by DPC602+.

Quantifying ROS levels using CM-H2DCFDA and HyPer.

At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).

In Vivo Detection of Reactive Oxygen Species and Redox Status in Caenorhabditis elegans.

SIGNIFICANCE: Due to its large families of redox-active enzymes, genetic amenability, and complete transparency, the nematode Caenorhabditis elegans has the potential to become an important model for the in vivo study of redox biology. RECENT ADVANCES: The recent development of several genetically encoded ratiometric reactive oxygen species (ROS) and redox sensors has revolutionized the quantification and precise localization of ROS and redox signals in living organisms. Only few exploratory studies have applied these sensors in C. elegans and undoubtedly much remains to be discovered in this model. As a follow-up to our recent findings that the C. elegans somatic gonad uses superoxide and hydrogen peroxide (H2O2) signals to communicate with the germline, we here analyze the patterns of H2O2 inside the C. elegans germline. CRITICAL ISSUES: Despite the advantages of genetically encoded ROS and redox sensors over classic chemical sensors, still several general as well as C. elegans-specific issues need to be addressed. The major concerns for the application of these sensors in C. elegans are (i) decreased vitality of some reporter strains, (ii) interference of autofluorescent compartments with the sensor signal, and (iii) the use of immobilization methods that do not influence the worm's redox physiology. FUTURE DIRECTIONS: We propose that several of the current issues may be solved by designing reporter strains carrying single copies of codon-optimized sensors. Preferably, these sensors should have their emission wavelengths in the red region, where autofluorescence is absent. Worm analysis could be optimized using four-dimensional ratiometric fluorescence microscopy of worms immobilized in microfluidic chips. Antioxid. Redox Signal. 25, 577-592.