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Compared with other reactive oxygen species, peroxynitrite (ONOO−) has diversified reactions and transformations in organisms, and its specific action mechanism is not very clear. The study of reactive oxygen species is of great significance in the field of physiology and pathology. Recently an effective on/off fluorescent probe HCA-OH was designed by Liu et al. through tethering p-aminophenol to 1,8-naphthalimide directly. The probe HCA-OH could release the fluorophore HCA-NH2 with good photostability and high fluorescence quantum yield under oxidation of ONOO− via dearylation process. In this work, the sensing mechanism and spectrum character of probe HCA-OH were studied in detail under quantum chemistry calculation. The electronic structures, reaction sites and fluorescent properties of the probe were theoretically analyzed to benefit us for in-depth understanding the principle of detection on reactive oxygen species (ONOO−) with the fluorescent probe HCA-OH. These theoretical results could inspire the medical research community to design and synthesize highly efficient fluorescent probe for reactive oxygen species detection.

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (•NO) and superoxide (O2•−) produced by phagocytes contributes to its success as a human pathogen. Recombination of •NO and O2•− generates peroxynitrite (ONOO−), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward •NO and O2•− is well established, how Mtb responds to ONOO− remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both •NO and O2•−, which should combine to produce ONOO−. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO− levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO−. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe–4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe–4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe–S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO− in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO− and targeting Fe–S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).

T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis.

Introduction: Chemodynamic therapy (CDT) holds great promise in achieving cancer therapy through Fenton and Fenton-like reactions, which generate highly toxic reactive species. However, CDT is limited by the lower amount of catalyst ions that can decompose already existing intracellular H2O2 and produce reactive oxygen species (ROS) to attain a therapeutic outcome. Objectives: To overcome these limitations, a tailored approach, which utilizes dual metals cations (Ag+, Fe2+) based silver pentacyanonitrosylferrate or silver nitroprusside (AgNP) were developed for Fenton like reactions that can specifically kill cancer cells by taking advantage of tumor acidic environment without used of any external stimuli. Methods: A simple solution mixing procedure was used to synthesize AgNP as CDT agent. AgNP were structurally and morphologically characterized, and it was observed that a minimal dose of AgNP is required to destroy cancer cells with limited effects on normal cells. Moreover, comprehensive in vitro studies were conducted to evaluate antitumoral mechanism. Results: AgNP have an effective ability to decompose endogenous H2O2 in cells. The decomposed endogenous H2O2 generates several different types of reactive species (•OH, O2•−) including peroxynitrite (ONOO−) species as apoptotic inducers that kill cancer cells, specifically. Cellular internalization data demonstrated that in short time, AgNP enters in lysosomes, avoid degradation and due to the acidic pH of lysosomes significantly generate high ROS levels. These data are further confirmed by the activation of different oxidative genes. Additionally, we demonstrated the biocompatibility of AgNP on mouse liver and ovarian organoids as an ex vivo model while AgNP showed the therapeutic efficacy on patient derived tumor organoids (PDTO). Conclusion: This work demonstrates the therapeutic application of silver nitroprusside as a multiple ROS generator utilizing Fenton like reaction. Thereby, our study exhibits a potential application of CDT against HGSOC (High Grade Serous Ovarian Cancer), a deadly cancer through altering the redox homeostasis.

Gas therapy is emerging as a highly promising therapeutic strategy for cancer treatment. However, there are limitations, including the lack of targeted subcellular organelle accuracy and spatiotemporal release precision, associated with gas therapy. In this study, we developed a series of photoactivatable nitric oxide (NO) donors NRh-R-NO (R = Me, Et, Bn, iPr, and Ph) based on an N-nitrosated upconversion luminescent rhodamine scaffold. Under the irradiation of 808 nm light, only NRh-Ph-NO could effectively release NO and NRh-Ph with a significant turn-on frequency upconversion luminescence (FUCL) signal at 740 nm, ascribed to lower N–N bond dissociation energy. We also investigated the involved multistage near-infrared-controlled cascade release of gas therapy, including the NO released from NRh-Ph-NO along with one NRh-Ph molecule generation, the superoxide anion O2⋅− produced by the photodynamic therapy (PDT) effect of NRh-Ph, and highly toxic peroxynitrite anion (ONOO‒) generated from the co-existence of NO and O2⋅−. After mild nano-modification, the nanogenerator (NRh-Ph-NO NPs) empowered with superior biocompatibility could target mitochondria. Under an 808 nm laser irradiation, NRh-Ph-NO NPs could induce NO/ROS to generate RNS, causing a decrease in the mitochondrial membrane potential and initiating apoptosis by caspase-3 activation, which further induced tumor immunogenic cell death (ICD). In vivo therapeutic results of NRh-Ph-NO NPs showed augmented RNS-potentiated gas therapy, demonstrating excellent biocompatibility and effective tumor inhibition guided by real-time FUCL imaging. Collectively, this versatile strategy defines the targeted RNS-mediated cancer therapy.

The clinical application of nanomaterials for chemodynamic therapy (CDT), which generate multiple reactive oxygen species (ROS), presents significant challenges. These challenges arise due to insufficient levels of endogenous hydrogen peroxide and catalytic ions necessary to initiate Fenton reactions. As a result, sophisticated additional delivery systems are required. In this study, a novel bimetallic copper (II) pentacyanonitrosylferrate (Cu(II)NP, Cu[Fe(CN) 5 NO]) material was developed to address these limitations. This material functions as a multiple ROS generator at tumoral sites by self-inducing hydrogen peroxide and producing peroxynitrite (ONOO-) species. The research findings demonstrate that this material exhibits low toxicity towards normal liver organoids, yet shows potent antitumoral effects on High Grade Serous Ovarian Cancer (HGSOC) organoid patients, regardless of platinum resistance. Significantly, this research introduces a promising therapeutic opportunity by proposing a single system capable of replacing the need for H2O2, additional catalysts, and NO-based delivery systems. This innovative system exhibits remarkable multiple therapeutic mechanisms, paving the way for potential advancements in clinical treatments.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.

Diabetes mellitus currently affects ∼10% of the population worldwide, with Type 2 predominating, and this incidence is increasing steadily. Both Type 1 and 2 are complex diseases, involving β-cell death and chronic inflammation, but the pathways involved are unresolved. Chronic inflammation is characterized by increased oxidant formation, with this inducing protein modification, altered function and immunogenicity. Amylin, a peptide hormone co-secreted with insulin by β-cells, has attracted considerable interest for its amyloidogenic properties, however, the effects that oxidants have on amylin aggregation and function are poorly understood. Amylin was exposed in vitro to hypochlorous acid, hydrogen peroxide and peroxynitrous acid/peroxynitrite to investigate the formation of post-translational oxidative modifications (oxPTMs, via mass spectrometry) and fibril formation (via transmission electron microscopy). Amylin free acid (AFA) was also examined to investigate the role of the C-terminal amide in amylin. Oxidant exposure led to changes in aggregate morphology and abundance of oxPTMs in a concentration-dependent manner. The toxicity and immunogenic potential of oxidant-modified amylin or AFA on pancreatic islet cells (INS-1E), human monocyte cell line (THP-1) and monocyte-derived dendritic cells (moDCs) were examined using metabolic activity and cytokine assays, and flow cytometry. No significant changes in vitality or viability were detected, but exposure to oxidant-modified amylin or AFA resulted in altered immunogenicity when compared to the native proteins. THP-1 and moDCs show altered expression of activation markers and changes in cytokine secretion. Furthermore, oxidant-treated amylin and AFA promoted maturation of THP-1 and pre-mature moDCs, as determined by changes in size, and maturation markers.

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO−) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO−, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO−, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO− (50 μM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO− concentrations (500 μM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO−/ONOOH. Both reagent ONOO−/ONOOH and SIN-1 (a thermal source of ONOO−/ONOOH) modified Tyr, Trp and Met residues. ONOO−/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO−/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO−/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

There is a lack of literature on peroxynitrite formation due to sonolysis of aerated water. In this work, the impact of sonication parameters, frequency and power, on ultrasonic peroxynitrite production in aerated alkaline water was investigated. Peroxynitrite formation was clearly established with undeniable evidence at all the tested frequencies in the range of 516–1140 kHz with a typical G-value (energy-specific yield) of 0.777 × 10−10, 0.627 × 10−10, 0.425 × 10−10 and 0.194 × 10−10 mol/J at 516, 558, 860 and 1140 kHz, respectively. The ultrasonication frequency has a direct impact on the sonochemical peroxynitrite production. Increasing the ultrasonication frequency in the interval 321–1140 kHz reduces peroxynitrite formation. The most practical sonochemistry dosimetries, including hydrogen peroxide production, triiodide dosimetry, Fricke dosimetry, and 4-nitrocatechol formation, were compared with the sonochemical efficiency of the reactors used to produce peroxynitrite. The G-value, energy specific yield, for the tested dosimetries was higher than that for peroxynitrite formation, regardless of frequency. For all chemical dosimetries investigated, the same trend of frequency dependence was found as for peroxynitrite generation. The influence of ultrasonication power on peroxynitrite formation by sonication at diverse frequencies in the interval 585–1140 kHz was studied. No peroxynitrite was formed at lower acoustic power levels, regardless of frequency. As the frequency increases, more power is required for peroxynitrite formation. The production of peroxynitrite increased as the acoustic power increased, despite the frequency of ultrasonic waves. Ultrasonic power is a key factor in the production of peroxynitrite by sonolysis. Since peroxynitrite is uniformly distributed in the bulk solution, peroxynitrite-sensitive solutes can be transformed both in the bulk of the solution and in the surfacial region (shell) of the cavitation bubble. The formation of peroxynitrite should be taken into account in sonochemistry, especially at higher pH values. Ultrasonic peroxynitrite formation in alkaline solution (pH 12) can be considered as a kind of chemical dosimetry in sonochemistry.

Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with replication stress-induced senescence termed senescence-associated redox state (SA-redox state). SA-redox state is characterized by its reactivity with superoxide-sensing fluorescent probes such as dihydroethidine, lucigenin and mitosox and peroxynitrite or hydroxyl radical sensing probe hydroxyphenyl fluorescein (HPF) but not the hydrogen peroxide (H2O2) reactive fluorescent probe CM-H2DCFDA. Measurement of GSH and GSSH also reveals that SA-redox state mitigates the level of total GSH rather than oxidizes GSH to GSSG. Moreover, supporting the role of superoxide (O2.-) in the SA-redox state, we show that incubation of senescent RPE1-hTERT cells with the O2.- scavenger, Tiron, decreases the reactivity of SA-redox state with the oxidants’ reactive probes lucigenin and HPF while the H2O2 antioxidant N-acetyl cysteine has no effect. SA-redox state does not participate in the loss of proliferative capacity, G2/M cell cycle arrest or the increase in SA-β-Gal activity. However, SA-redox state is associated with the activation of NF-κB, dictates the profile of the Senescence Associated Secretory Phenotype, increases TFEB protein level, promotes geroconversion evidenced by increased phosphorylation of S6K and S6 proteins, and influences senescent cells response to senolysis. Furthermore, we provide evidence for crosstalk between SA redox state, p53 and p21. While p53 mitigates the establishment of SA-redox state, p21 is critical for the sustained reinforcement of the SA-redox state involved in geroconversion and resistance to senolysis.

Objective: To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. Methods: The in vitro primary cultured cochlear hair cells were subjected to l00 μM peroxynitrite and l00 μM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. Results: The number of surviving hair cells was significantly reduced in the 100 μM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Conclusion: These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. Level of evidence: Level 2.

Lactoferrin (LF) is a multifunctional antimicrobial, anti-inflammatory, and antioxidant protein that occurs naturally in mammals, most notably in exocrine gland tissues and fluids, such as in the eye. Nitrosative stress can promote changes to tyrosine and other amino acid residues of the protein, which also reduces the activity of LF. l-ergothioneine (ET) is a potent anti-inflammatory antioxidant present in the eye and other tissues through nutrition or supplementation and that may play a role in the prevention or treatment of a variety of diseases. Here we investigated the ability of ET to reduce 3-nitrotyrosine (NTyr) formation using two separate substrates, with the goal of determining whether ET can protect the antibacterial function of LF and other proteins when exposed separately to peroxynitrite and tetranitromethane as nitrating reagents. Native human LF was used as a simple protein substrate, and lamb corneal lysate was chosen as one example of mammalian tissue with a more complex mixture of proteins and other biomolecules. Nitration was monitored by absorbance and fluorescence spectroscopy as well as sandwich (nitrated LF) and direct NTyr (corneal lysate) enzyme-linked immunosorbent assays (ELISAs). We found that pretreatment with ET reduced chemical modification of both native LF and corneal lysate samples and loss of antibacterial LF function due to exposure to the nitrating reagents. These initial results suggest that ET, raised to sufficiently elevated levels, could be tailored as a therapeutic agent to reduce effects of nitrosative stress on LF and in turn sustain the protein activity.

Background: After an acute stroke, highly reactive oxygen and nitrogen species (ROS/RNS) lead to microvessel injury and inhibit endogenous mediators to prevent the repair process of endothelial dysfunction and tissue injury. Among them, the peroxynitrite (ONOO−) generation as a result of nitric oxide (NO) and superoxide anion (O2·−) reaction contributes to ischemic brain injury. Purpose: To validate the targeted inhibition of ischemic injury's markers with the delivery of potent antioxidants such as β-carotene (BC) in lipid nanoparticles (LNPs) directly acting with ONOO− compound to prevent the breakdown of blood-brain barrier (BBB). Study design: In the current work, BC in polyethylene glycol (PEG) lipid nanoparticles (PEG-LNPs) with or without attached anti-nitrotyrosine antibody (3-NT) inhibits ONOO− generation in the transient middle cerebral artery occlusion (tMCAO) model. Transmission electron microscopy (TEM) was used to examine structural change in LNPs with or without 3-NT antibody attachment. Fluorescence imaging was employed to study drug encapsulated PEG-LNPs accumulation in mice brain following MCAO and spectrophotometry for the in-vitro drug release profile. Immunoblotting using antibodies specific for spectrin and calcineurin, occludin and ZO-1 and other targets was utilized. The NP3 fluorescent probe was used for the detection of ONOO−generation. Evans Blue was injected 24 h after tMCAO in vehicle and BC PEG-LNPs with 3-NT antibody treated animal groups to observe the tight junction breakdown. Results: The fluorescence's data reveals that attachment of 3-NT antibody has directed the delivery of BC to the ischemic ipsilateral region. BC, as well, is found effective to improve infarct volume and neurological deficit by neuronal protection by inhibiting spectrin and calcineurin (CaN) breakdown. Furthermore, inhibition of ONOO−generation is observed in NP3 probe incubation of brain slices and protects BBB from breakdown in Evans blue extravasation. Conclusion: The current work suggests that inhibiting ONOO−at ischemic site protects the brain microvessels from injury. 3-NT/BC/LNPs carrier system is potentially efficient in delivering BC at the site of ONOO−generation, and possibly suitable for ischemic region-targeted therapy.

Acute lung injury (ALI) is a serious pulmonary inflammatory disease resulting from excessive reactive oxygen species (ROS) which could cause the damage of the alveolar epithelial cells and capillary endothelial cells. Peroxynitrite, as one of short-lived reactive oxygen species, is closely related to the process of ALI. Thus, it is important to monitor the fluctuation of peroxynitrite in living system for understanding the process of ALI. Herein, the novel mitochondria-targeted fluorescent probe BHMT was designed to respond to peroxynitrite and pH with distinct fluorescence properties respectively. The absorption spectrum of the probe BHMT exhibited a notable red shift as the pH value declined from 8.8 to 2.6. Upon reaction with peroxynitrite, BHMT had a significant increase of fluorescence intensity (63-fold) with maintaining a detection limit of only 43.7 nM. Furthermore, BHMT could detect the levels of endogenous peroxynitrite and image the intracellular pH in ratiometric channels utilizing cell imaging. In addition, BHMT was successfully applied to revealing the relationship between the peroxynitrite and the extent of ALI. Thus, these results indicated the probe BHMT could be a potential tool for diagnosing the early stage of ALI and revealed the peroxynitrite was likely to be a crucial therapeutic target in ALI treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ischemic stroke is a major cause of death and disability. The activation of neuronal nitric oxide synthase (nNOS) and the resulting production of nitric oxide (NO) via NMDA receptor-mediated calcium influx play an exacerbating role in cerebral ischemia reperfusion injury. The NO rapidly reacts with superoxide (O 2- ) to form peroxynitrite (ONOO - ), a toxic molecule may modify proteins through tyrosine residue nitration, ultimately worsening neuronal damage. SIRT6 has been proven to be crucial in regulating cell proliferation, death, and aging in various pathological settings. We have previous reported that human SIRT6 tyrosine nitration decreased its intrinsic catalytic activity in vitro. However, the exact role of SIRT6 function in the process of cerebral ischemia reperfusion injury is not yet fully elucidated. Herein, we demonstrated that an increase in the nitration of SIRT6 led to reduce its enzymatic activity and aggravated hippocampal neuronal damage in a rat model of four-artery cerebral ischemia reperfusion. In addition, reducing SIRT6 nitration resulted in increase the activity of SIRT6, alleviating hippocampal neuronal damage. Moreover, SIRT6 nitration affected its downstream molecule activity such as PARP1 and GCN5, promoting the process of neuronal ischemic injury in rat hippocampus. Additionally, treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7-NI, and resveratrol (an antioxidant) diminished SIRT6 nitration and the catalytic activity of downstream molecules like PARP1 and GCN5, thereby reducing neuronal damage. Finally, in the biochemical regulation of SIRT6 activity, tyrosine 257 was essential for its activity and susceptibility to nitration. Replacing tyrosine 257 with phenylalanine in rat SIRT6 attenuated the death of SH-SY5Y neurocytes under oxygen-glucose deprivation (OGD) conditions. These results may offer further understanding of SIRT6 function in the pathogenesis of cerebral ischemic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Peroxynitrite is one of the important reactive oxygen species in the human body and is closely related to the physiological and pathological processes of many diseases. Therefore, the development of probes to detect peroxynitrite is important for diagnostic and pathologic studies of many diseases. In this work, a ratiometric probe was designed using benzopyran as the recognition site, and the sensitivity and selectivity of the probe were tuned by modification of substituents on benzopyran. Upon reaction with peroxynitrite, the color of the solution changes to the naked eye (from blue to yellow), and the fluorescence changes from red to blue. The probe SJ has the advantages of large Stokes shift (237 nm), fast response (≤10 s), wide linear range, good selectivity, low detection line (21.3 nm), and low cytotoxicity. Probe SJ has been successfully used for bioimaging of endogenous and exogenous peroxynitrite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Breakpoint chlorination, an important chemical process relevant to chlorine-based advanced oxidation processes for potable reuse and to traditional water treatment, was investigated for its oxidative capacity, generation of reactive species, and potential impacts on organic contaminant degradation. This work describes a newly recognized HO• radical generation pathway during breakpoint chlorination that may play an important role in water treatment and examines the behavior of the HO• radical and other related reactive species and their potential formation pathways. Experimental data showed that the removal of 1,4-dioxane (1,4-D) positively correlated with chlorine-to-ammonia molar ratio until a molar ratio of ~1.5–2.0 was reached, above which removal efficiency rapidly decreased. Peroxynitrite (ONOO–) and peroxynitrous acid (ONOOH) are proposed as important radical sources that lead to the formation of HO• in the breakpoint process. This is supported by application of tert-butanol as a selective HO• scavenger and the observation that the amendment of reaction solutions with carbonate species suppressed oxidative capacity to a much greater extent than expected based solely on scavenging of HO• by H2CO3 * /CO2 and HCO3– (apparently due to selective scavenging of ONOOH/ONOO– by dissolved CO2). These experiments also provided evidence that reactive species other than HO• contributed to 1,4-D oxidation. The results of this study suggest that breakpoint chlorination can lead to significant degradation of organic contaminants via ONOOH/ONOO–-mediated formation of HO• and other reactive species and may potentially be optimized for enhanced removal of recalcitrant organic contaminants in the context of water reuse, though with due caution to the potential for enhancement of nitrogenous and other disinfection byproduct formation under such conditions.

Protein tyrosine residue (Y) nitration, a post-translational chemical-modification mode, has been associated with changes in protein activity and function; hence the accumulation of specific nitrated proteins in tissues may be used to monitor the onset and progression of pathological disorders. To verify the possible impact of nitration on postnatal muscle growth and regeneration, a pilot study was designed to examine the nitration/dysfunction of hepatocyte growth factor (HGF), a key ligand that is released from the extracellular tethering and activates myogenic stem satellite cells to enter the cell cycle upon muscle stretch and injury. Exposure of recombinant HGF (a hetero-dimer of α- and β-chains) to peroxynitrite induces Y nitration in HGF α-chain under physiological conditions. Physiological significance of this finding was emphasized by Western blotting that showed the NK1 segment of HGF (including a K1 domain critical for signaling-receptor c-met binding) undergoes nitration with a primary target of Y198. Peroxynitrite treatment abolished HGF-agonistic activity of the NK1 segment, as revealed by in vitro c-met binding and bromodeoxyuridine-incorporation assays. Importantly, direct-immunofluorescence microscopy of rat lower hind-limb muscles from two aged-groups (2-month-old “young” and 12-month-old “retired/adult”) provided in vivo evidence for age-related nitration of extracellular HGF (Y198). Overall, findings provide the insight that HGF/NK1 nitration/dysfunction perturbs myogenic stem cell dynamics and homeostasis; hence NK1 nitration may stimulate progression of muscular disorders and diseases including sarcopenia.

Objective: To assess the preventive role of metformin on rat ovarian ischemia reperfusion injury. Materials and methods: Forty rats were divided equally into five groups; Group 1: sham, Group 2: surgical control with 3-hr torsion and detorsion, Group 3: 50 mg/kg p.o. metformin 30 min before 3-hr torsion, Group 4; metformin just after detorsion, Group 5; metformin 30 min before torsion and just after detorsion. Bilateral ovaries and blood sample were obtained seven days after detorsion for biochemical and histopathological evaluation. Results: Ovarian tissue total anti-oxidant status (TAS) levels were significantly increased in group 4 when compared to group 1, 2 and 3 (all p

Alzheimer's disease (AD), characterized by its incurable nature and prevalence among the elderly, has remained a focal point in medical research. Increasing evidence suggests that peroxynitrite (ONOO - ) serves as a crucial biomarker for the diagnosis of AD. In this study, we present a novel, easily available, high-yield, and cost-effective near-infrared (NIR) fluorescent probe, CDCI-ONOO. This probe utilizes a coumarin-dicyanoisophorone conjugate as the fluorophore and diphenylphosphinic chloride as the recognition site, enabling the detection of ONOO - both in vitro and in vivo. Upon interaction with ONOO - , CDCI-ONOO exhibits a distinct maximum emission peak at 715 nm with a substantial Stokes shift of 184 nm. The probe demonstrates excellent selectivity and sensitivity (LOD = 144 nM), along with noticeable colorimetric and fluorescence changes after the reaction. Comprehensive analyses using high-performance liquid chromatography (HPLC), high-resolution mass spectrometry (HRMS), and density functional theory (DFT) calculations confirm that the reaction with ONOO - restores the initially quenched Intramolecular Charge Transfer (ICT), resulting in the formation of CDCI-OH, a product that emitting fluorescence in the near-infrared region. Furthermore, we demonstrated the successful application of CDCI-ONOO for ONOO - detection in neuronal cells and imaging of ONOO - in the brains of mice. These findings underscore the potential of CDCI-ONOO as a near-infrared fluorescent probe for in vivo ONOO - detection, offering a significant avenue for advancing our understanding of AD pathology and diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acute kidney injury (AKI) can result in a sudden decline in kidney function and, if not promptly diagnosed and treated, can lead to a high mortality rate. Therefore, there is a critical need for the development of a non-invasive and dependable early diagnostic method for AKI to prevent its progression and deterioration. To address the risk of misdiagnosis or overlooked diagnosis due to reliance on a single biomarker, we developed a novel molecular fluorescent probe (HX-GP) to simultaneously detect and image two biomarkers, γ-Glutamyl transpeptidase (γ-GGT) and Peroxynitrite (ONOO - ), in the AKI process. HX-GP can specifically detect γ-GGT in the red fluorescence channel (λ em  = 613 nm) and ONOO - in the green fluorescence channel (λ em  = 518 nm). HX-GP demonstrated high sensitivity, selectivity, and rapid response, showing excellent biocompatibility and detection performance. In addition, HX-GP was successful in imaging experiments in a cell model of cisplatin-induced AKI, a result that highlights its potential application value in early diagnosis of AKI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nonalcoholic fatty liver disease (NAFLD) poses a significant global health concern, necessitating precise diagnostic tools and effective treatment strategies. Peroxynitrite (ONOO - ), a reactive oxygen species, plays a pivotal role in NAFLD pathogenesis, highlighting its potential as a biomarker for disease diagnosis and therapeutic evaluation. This study reports on the development of a near-infrared (NIR) fluorescent probe, designated DRP-O, for the selective detection of ONOO - with high sensitivity and photostability. DRP-O exhibits rapid response kinetics (within 2 min) and an impressive detection limit of 2.3 nM, enabling real-time monitoring of ONOO - dynamics in living cells. Notably, DRP-O demonstrates excellent photostability under continuous laser irradiation, ensuring reliable long-term monitoring in complex biological systems. We apply DRP-O to visualize endogenous ONOO - in living cells, demonstrating its potential for diagnosing and monitoring NAFLD-related oxidative stress. Furthermore, DRP-O effectively evaluates the efficacy of therapeutic drugs in NAFLD cell models, underscoring its potential utility in drug screening studies. Moreover, we confirm DRP-O to enable selective identification of fatty liver tissues in a mouse model of NAFLD, indicating its potential for the early diagnosis of NAFLD. Collectively, DRP-O represents a valuable tool for studying ONOO - dynamics, evaluating drug efficacy, and diagnosing NAFLD, offering insights into novel therapeutic strategies for this prevalent liver disorder., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The diffusion-limited reaction of nitric oxide (NO) and superoxide (O2−) produces peroxynitrite (ONOO−), a biological oxidant that has been implicated in a number of pathological conditions, including neurodegenerative disorders. We previously reported that incubation of PC12 cells with peroxynitrite triggers apoptosis by simultaneously inhibiting the PI3K/Akt survival pathway, and activating the p38 and JNK MAP kinase pathways. We also reported that peroxynitrite-treated Heat Shock Protein 90 (Hsp90) stimulates PC12 cell death. Here, we show that nitrated Hsp90 mediates peroxynitrite-induced apoptosis by regulating specific signaling pathways triggered by activation of the purine receptor P2X7 (P2X7R) and downstream activation of PTEN. Intracellular delivery of peroxynitrite-treated Hsp90 was sufficient to stimulate PC12 cell death. In contrast, intracellular delivery of peroxynitrite-treated Hsp90 in which the five tyrosine (Tyr) residues susceptible to nitration were replaced by nitration-resistant phenylalanine had no effect on PC12 cell survival. Further, only nitration of Hsp90 at Tyr 56 was necessary and sufficient to stimulate PC12 cell apoptosis, and incubation of PC12 cells with peroxynitrite resulted in Hsp90 nitration at Tyr 56. Inhibition of P2X7R or downstream inhibition of PTEN prevented PC12 cell death stimulated by both incubation with peroxynitrite and nitrated Hsp90 (Hsp90NY). Peroxynitrite, Hsp90NY, and P2X7R activation all increased p38 and JNK MAP kinases activity, while inhibiting the Akt survival pathway. These results suggest that, in undifferentiated PC12 cells, peroxynitrite triggers apoptosis via nitration of Hsp90 at Tyr 56, which in turn activates P2X7R and PTEN. These results contrast with observations in motor neurons where the nitration of either Tyr 33 or Tyr 56 in Hsp90 stimulates apoptosis, suggesting that the targets of peroxynitrite may be different in different cell types. However, uncovering the pathways through which peroxynitrite triggers cell death in neurodegenerative conditions will provide new potential targets for therapeutic treatment.

Aims: Preferential expression of receptors for TNF-family related apoptosis inducing ligand (TRAIL), DR4 and DR5 makes TRAIL an attractive anti-cancer therapeutic. However, the efficacy of targeting death receptors has not been extensively studied in nasopharyngeal cancer (NPC). Here we investigated TRAIL sensitivity and its underlying mechanism in NPC cell lines, and assessed the potential of TRAIL as a therapeutic option against NPC. Results: Using two established NPC cell lines, we report the expression of DR4 and DR5, which respond to TRAIL ligation by triggering efficient Type II apoptosis. Mechanistically, early activation of caspase-3 and its membrane recruitment is identified in NPC cell lines, which is associated with, hitherto unreported, interaction with transmembrane and tetratricopeptide repeat containing 2 (TMTC2) in the lipid raft domains. TMTC2 expression is induced upon exposure to TRAIL and involves intracellular increase in peroxynitrite (ONOO−) production. While ONOO− increase is downstream of caspase-8 activation, it is involved in the upregulation of TMTC2, gene knockdown of which abrogated TRAIL-induced apoptotic execution. Bioinformatics analyses also provide evidence for a strong correlation between TMTC2 and DR4 or caspase-3 as well as a significantly better disease-free survival in patients with high TMTC2 expression. Innovation and conclusion: Collectively, redox-dependent execution of NPC cells upon ligation of TRAIL receptors reintroduces the possible therapeutic use of TRAIL in NPC as well as underscores the potential of using TMTC2 as a biomarker of TRAIL sensitivity.

Peroxynitrite (ONOO - ) and cell plasma membrane (CPM) are two key factors in cell pyroptosis during the progression of abdominal aortic aneurysm (AAA). However, their combined temporal and spatial roles in initiating AAA pathogenesis remain unclear. Herein, we developed a two-photon fluorescence probe, BH-Vis, enabling real-time dynamic detection of CPM and ONOO - changes, and revealing their interplay in AAA. BH-Vis precisely targets CPM with reduced red fluorescence intensity correlating with diminished CPM tension. Concurrently, a blue shift of the fluorescence signal of BH-Vis occurs in response to ONOO - offering a reliable ratiometric detection mode with enhanced accuracy by minimizing external testing variables. More importantly, two photon confocal imaging with palmitic acid (PA) and ganglioside (GM1) manipulation, which modulating cell pyroptosis, showcases reliable fluorescence fluctuations. This groundbreaking application of BH-Vis in a mouse AAA model demonstrates its significant potential for accurately identifying cell pyroptosis levels during AAA development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Over-consumption of drugs can result in drug-induced liver damage (DILI), which can worsen liver failure. Numerous studies have shown the significant role ferroptosis plays in the pathophysiology of DILI, which is typified by a marked imbalance between the generation and breakdown of lipid reactive oxygen species (ROS). The content of peroxynitrite (ONOO - ) rapidly increased during this process and was thought to be a significant marker of early liver injury. Therefore, the construction of fluorescence probe for the detection and imaging of ONOO - holds immense importance in the early diagnosis and treatment of ferroptosis-mediated DILI., Results: We designed a probe DILI-ONOO based on the ICT mechanism for the purpose of measuring and visualizing ONOO - in ferroptosis-mediated DILI processes and associated studies. This probe exhibited significant fluorescence changes with good sensitivity, selectivity, and can image exogenous and endogenous ONOO - in cells with low cytotoxicity. Using this probe, we were able to show changes in ONOO - content in ferroptosis-mediated DILI cells and mice models induced by the intervention of acetaminophen (APAP) and isoniazid (INH). By measuring the concentration of ferroptosis-related indicators in mice liver tissue, we were able to validate the role of ferroptosis in DILI. It is worth mentioning that compared to existing alanine transaminase (ALT) and aspartate aminotransferase (AST) detection methods, this probe can achieve early identification of DILI prior to serious liver injury., Significance: This work has significant reference value in researching the relationship between ferroptosis and DILI and visualizing research. The results indicate a strong correlation between the progression of DILI and ferroptosis. Additionally, the use of DILI-ONOO shows promise in investigating the DILI process and assessing the effectiveness of medications in treating DILI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Acute kidney injury (AKI) poses a severe risk to public health, mostly manifested by damage and death of renal tubular epithelial cells. However, routine blood examination, a conventional approach for clinical detection of AKI, is not available for identifying early-stage AKI. Plenty of reported methods were lack of early biomarkers and real time evaluation tools, which resulted in a vital challenge for early diagnosis of AKI. Therefore, developing novel probes for early detection and assessment of AKI is exceedingly crucial., Results: Based on ESIPT mechanism, a new fluorescent probe (MEO-NO) with 2-(2'-hydroxyphenyl) benzothiazole (HBT) derivatives as fluorophore has been synthesized for dynamic imaging peroxynitrite (ONOO - ) levels in ferroptosis-mediated AKI. Upon the addition of ONOO - , MEO-NO exhibited obvious fluorescence changes, a significant Stokes shift (130 nm) and rapid response (approximately 45 s), and featured exceptional sensitivity (LOD = 7.28 nM) as well as high selectivity from the competitive species at physiological pH. In addition, MEO-NO was conducive to the biological depth imaging ONOO - in cells, zebrafish, and mice. Importantly, MEO-NO could monitor ONOO - levels during sorafenib-induced ferroptosis and CP-induced AKI. With the assistance of MEO-NO, we successfully visualized and tracked ONOO - variations for early detection and assessment of ferroptosis-mediated AKI in cells, zebrafish and mice models., Significance and Novelty: Benefiting from the superior performance of MEO-NO, experimental results further demonstrated that the levels of ONOO - was overexpressed during ferroptosis-mediated AKI in cells, zebrafish, and mice models. The developed novel probe MEO-NO provided a strong visualization tool for imagining ONOO - , which might be a potential method for the prevention, diagnosis, and treatment of ferroptosis-mediated AKI., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Excessive peroxynitrite (ONOO - ) is closely related to the occurrence and progression of inflammation. Therefore, the development of an efficacious ONOO - activatable probe holds great potential for the early diagnosis of pathological inflammation, and the direct evaluation of the therapeutic efficacy of active protectants. In this work, a new ONOO - -activated fluorescent probe (SZP) which greatly improved the specificity and sensitivity (LOD = 8.03 nM) with large Stokes shift (150 nm) through introducing two reaction triggers (diphenyl phosphinate moiety, CC unsaturated bond) was rationally designed for rapid detecting ONOO - (within 2 min). The excellent properties of probe SZP enable it to realize the fluorescence-guided diagnosis of inflammation. More importantly, probe SZP has also been utilized to assess the anti-inflammatory efficacy of traditional Chinese medicines (TCMs) active ingredients for the remediation of inflammation by monitoring ONOO - fluctuation for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Peroxynitrite, a short-lived and reactive oxidant, emerges from the diffusion-controlled reaction between the superoxide radical and nitric oxide. Evidence shows that peroxynitrite is a critical mediator in physiological and pathological processes such as the immune response, inflammation, cancer, neurodegeneration, vascular dysfunction, and aging. The biochemistry of peroxynitrite is multifaceted, involving one- or two-electron oxidations and nitration reactions. This minireview highlights recent findings of peroxynitrite acting as a metabolic mediator in processes ranging from oxidative killing to redox signaling. Selected examples of nitrated proteins (i.e., 3-nitrotyrosine) are surveyed to underscore the role of this post-translational modification on cell homeostasis. While accumulated evidence shows that large amounts of peroxynitrite participates of broad oxidation and nitration events in invading pathogens and host tissues, a closer look supports that low to moderate levels selectively trigger signal transduction cascades. Peroxynitrite probes and redox-based pharmacology are instrumental to further understand the biological actions of this reactive metabolite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Severe anorexia limits the clinical application of cisplatin, and even leads to the discontinuation of treatment. However, the mechanisms underlying cisplatin-induced anorexia are unknown. Herein, we demonstrated that cisplatin could affect neuronal gamma oscillations and induce abnormal neuronal theta-gamma phase-amplitude coupling in the arcuate nucleus (Arc) of the hypothalamus, and these findings were associated with significantly decreased food intake and weight loss in mice. Chemogenetic activation of AgRP neurons in the Arc reversed the cisplatin-induced food intake reduction in mice. We further demonstrated that endothelial peroxynitrite (ONOO−) formation in the Arc induced nitrosative stress following cisplatin treatment via a previously uncharacterized pathway involving neuronal caspase-1 activation. Strikingly, treatment with the ONOO− scavenger uric acid (UA) reversed the reduced action potential (AP) frequency of AgRP neurons and increased the AP frequency of POMC neurons induced by SIN1, a donor of ONOO−, in the Arc, as determined by whole-cell patch-clamp electrophysiological recording. Consistent with these findings, UA treatment effectively alleviated cisplatin-induced dysfunction of neuronal oscillations and neuronal theta-gamma phase-amplitude coupling in the Arc of mice. Taken together, these results suggest, for the first time, that targeting the overproduction of endothelial ONOO− can regulate cisplatin-induced neurotoxicity through neuronal caspase-1, and thereby serve as a potential therapeutic approach to alleviate chemotherapy-induced anorexia and weight loss.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by ‘oxidant-mediated thiol-disulfide reactions’ via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

Peroxynitrite (ONOO - ) is a highly reactive oxygen species that plays a critical role in many physiological and pathological processes of cell function. This study aimed to propose a ratiometric fluorescent probe BDHCA derived from coumarin for determining the ONOO - level. ONOO - could specifically induce oxidative cleavage of the conjugated C = C double bond in probe BDHCA, providing a fluorescent ratiometric output. The response of probe BDHCA to ONOO - was selective, fast, and highly sensitive, with a detection limit of 50.3 nM. Biological imaging experiments suggested that probe BDHCA could be used to image ONOO - in living RAW264.7 cells and zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

As two of important highly reactive species / nitrogen species, hypochloric acid (HClO) and peroxynitrite (ONOO - ) are involved in various pathological and physiological processes, which are important factors that affect and reflect the functional state of lysosome. Nevertheless, many of their roles are still indefinite because of lack of suitable analytical methods for HClO and ONOO - detection in lysosome. Herein, we designed a lysosome-targeted probe to monitor HClO and ONOO - , which was a hydrid of the benzothiazole derivative, methyl thioether (HClO recognition site) and morpholino hydrazone (ONOO - recognition and lysosome target site). The probe exhibited high sensitivity, good selectivity and fast response toward HClO and ONOO - without spectral crosstalk, and can be employed for quantitative monitoring HClO and ONOO - with LOD of 63 and 83 nM, respectively. In addition, the dual-site probe was lysosome targetable and could be used for detection of HClO and ONOO - in living cells. Furthermore, the excellent behavior made it was suitable for imaging of HClO and ONOO - in zebrafish. Thus, the present probe provides a potent tool for distinguishing monitoring HClO and ONOO - and exploring the role of HClO and ONOO - in biological systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As the most common cause of liver diseases, nonalcoholic fatty liver disease (NAFLD) can be classified into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). While NAFL is generally benign, the transition from NAFL to NASH is a cardinal feature of the non-benign liver disease that leads to cirrhosis and cancer, which indicates that tracking the transformation of NAFL to NASH timely is significant for precision management of liver diseases. Therefore, two fluorescent probes (CNFCl and DRNO) have been developed to visualize this pathological event. α-Fluorochloroacetamide and α-ketoamide was employed as the recognition site for carboxylesterase (CE) in CNFCl and peroxynitrite (ONOO - ) in DRNO, respectively. CNFCl (λ em  = 445 nm) and DRNO (λ em  = 560 nm) showed high specificity and sensitivity towards CE and ONOO - respectively. By incubating with CE/ONOO - for 0.5 h respectively, both the emission intensity of CNFCl (linear range: 0-0.2 U/mL) and DRNO (linear range: 0-17.5 μM) displayed significant enhancement. As a result, the detection limit of CNFCl and DRNO for CE and ONOO - was calculated as 4.2 mU/L and 0.05 μM respectively. More importantly, the emission spectra of CNFCl and DRNO in the presence of CE and ONOO - respectively were cross-talk free under the two-photon excitation of 720 nm. This greatly facilitated the simultaneous detection of CE and ONOO - at distinctive channel, thus ensuring the high fidelity of the detection. These two probes were combined to image the fluctuation of CE and ONOO - during the conversion of NAFL to NASH in vitro and in vivo. It was found that while CE displayed a tendency to rise and then reduce during the transition from NAFL to NASH, ONOO - increased continuously, confirming that the combined imaging by CNFCl and DRNO might visualize the transformation of NAFL to NASH. The results provide robust visual tool to decipher the relationship between the stage of NAFLD and the level of CE/ONOO - . We anticipate this study may open new avenues to distinguish NASH from NAFL, which may further promote the study of intracellular biological activities of CE and the development of NAFLD diagnostic methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The liver plays a crucial role in several important processes in the human body, including metabolism, detoxification, and immune function. When the liver experiences acute injury, it can cause significant harm and requires prompt detection. Traditional biomarkers lack specificity and cannot detect changes in real-time, making them unsuitable for monitoring pathological processes. Recent studies have shown that acute liver injury (ALI) is closely related to oxidative stress, with peroxynitrite (ONOO - ) being a vital byproduct of liver metabolism and become a critical biomarker for detecting liver damage. As a result, this research developed an activatable near-infrared fluorescent probe W-3a that can be used to detect endogenous ONOO - in a mouse model of ALI induced by lipopolysaccharides (LPS). The probe has high selectivity and anti-interference ability, with a reaction time <10 min and a detection limit of 85 nM. It was successfully utilized in detecting endogenous ONOO - in cells and live imaging of ALI mice., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Acute myocardial infarction (AMI) patients are at an elevated risk for life-threatening myocardial ischemia/reperfusion injury. Early-stage nonradioactive and noninvasive diagnosis of AMI is imperative for the subsequent disease treatment, yet it presents substantial challenges. After AMI, the myocardium typically exhibits elevated levels of peroxynitrite (ONOO - ), constituting a distinct microenvironmental feature. In this context, the near-infrared imaging probe (BBEB) is employed to precisely delineate the boundaries of AMI lesions with a high level of sensitivity and specificity by monitoring endogenous ONOO - . This probe allows for the early detection of myocardial damage at cellular and animal levels, providing exceptional temporal and spatial resolution. Notably, BBEB enables visualization of ONOO - level alterations during AMI treatment incorporating antioxidant drugs. Overall, BBEB can rapidly and accurately visualize myocardial injury, particularly in the early stages, and can further facilitate antioxidant drug screening., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite.For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs.Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o’-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2–1000 pmol for parent amino acids, 80 fmol–20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o’-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+–αA-crystallin complex (Kdiss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the Kdiss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+–αB-crystallin complexes (K1 and K2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K1 and K2 values are equal to 4.3 and 70 µM, respectively.

In addition to the primary symptoms arising from inflammatory processes in the joints, muscle weakness is commonly reported by patients with rheumatoid arthritis (RA). Muscle weakness not only reduces the quality of life for the affected patients, but also dramatically increases the burden on society since patients' work ability decreases. A 25–70% reduction in muscular strength has been observed in pateints with RA when compared with age-matched healthy controls. The reduction in muscle strength is often larger than what could be explained by the reduction in muscle size in patients with RA, which indicates that intracellular (intrinsic) muscle dysfunction plays an important role in the underlying mechanism of muscle weakness associated with RA. In this review, we highlight the present understanding of RA-associated muscle weakness with special focus on how enhanced Ca2+ release from the ryanodine receptor and free radicals (reactive oxygen/nitrogen species) contributes to muscle weakness, and recent developments of novel therapeutic interventions.

This article contains representative fluorescence excitation spectra of roGFP2-based probes used for ratiometric analysis of redox changes as presented in the article "Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species" [1]. The recombinant probes roGFP2, roGFP2-Orp1, and Grx1-roGFP2 were exposed to various oxidative and nitrosative species, including hydrogen peroxide (H2O2), aldrithiol-2 (AT-2), glutathione disulfide (GSSG), hypochlorous acid (HOCl), S-nitrosoglutathione (GSNO), peroxynitrite (ONOO−), potassium polysulfide (K2Sx), spermine NONOate (SperNO), and diethyl amino NONOate (DeaNO) at different molar ratios. Fluorescence excitation spectra of the probes were recorded in the excitation wavelength range between 350 and 500 nm and for a total of 60 min. Analysis and interpretation of the data is presented in an associated article [1].

Acetaminophen (APAP) hepatotoxicity is characterized by an extensive oxidative stress. However, its source, pathophysiological role and possible therapeutic potential if targeted, have been controversially described. Earlier studies argued for cytochrome P450-generated reactive oxygen species (ROS) during APAP metabolism, which resulted in massive lipid peroxidation and subsequent liver injury. However, subsequent studies convincingly challenged this assumption and the current paradigm suggests that mitochondria are the main source of ROS, which impair mitochondrial function and are responsible for cell signaling resulting in cell death. Although immune cells can be a source of ROS in other models, no reliable evidence exists to support a role for immune cell-derived ROS in APAP hepatotoxicity. Recent studies suggest that mitochondrial targeted antioxidants can be viable therapeutic agents against hepatotoxicity induced by APAP overdose, and re-purposing existing drugs to target oxidative stress and other concurrent signaling events can be a promising strategy to increase its potential application in patients with APAP overdose.

Methylglyoxal (MGO); a reducing sugar and a dicarbonyl; attaches to the biomolecules (proteins, lipids, and DNA) leading to glycation and accumulation of oxidative stress in cells and tissues. Superoxide anion formed under such conditions entraps free nitric oxide radical (NO) to form peroxynitrite (PON). Nitro-oxidative stress due to PON is well established. Human fibrinogen plays a key role in haemostasis and is a highly vulnerable target for oxidation. Modifications of fibrinogen can potentially disrupt its structure and function. Earlier evidence suggested that glycation and nitro-oxidation lead to protein aggregation by making it resistant to lysis. This study aims to reveal the structural perturbations on fibrinogen in the presence of MGO and PON synergistically. The in vitro glyco-nitro-oxidation of human fibrinogen by MGO and PON leads to substantial structural alterations, as evident by biophysical and biochemical studies. In-silico results revealed the formation of stable complexes. UV-visible, intrinsic fluorescence, and circular dichroism investigations confirmed the synergistic effect of MGO and PON caused micro-structural modifications leading to secondary structural alterations. AGEs formation in MGO-modified fibrinogen reduced the free lysine and free arginine residues which were quantified by TNBS and phenanthrenequinone assays. Enhanced oxidative status was confirmed by estimating carbonyl content. ANS fluorophore validated exposure of hydrophobic patches in modified protein and thioflavin-T showed maximum binding with synergistically modified fibrinogen, indicated the formation of β-sheet. Confocal and electron microscope results corroborated the formation of aggregates. This study, therefore, evaluated the impact of MGO and PON on the structural integrity, oxidative status and aggregate formation of fibrinogen that can aggravate metabolic complications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Venous thromboembolism is a sudden cardiovascular disease that can lead to death, and its pathologic development is closely related to vascular viscosity and inflammation. However, direct evidence from in vivo is really scarce. The key limitation is that the combined probes cannot detect multiple markers simultaneously, which may lead to unreliable results. Therefore, to develop a single probe that can simultaneously monitor the variations of viscosity in the vascular microenvironment as well as inflammation level during venous thrombosis., Results: A dual-responsive two-photon fluorescent probe, Cou-ONOO, was designed and synthesized. Cou-ONOO provides a visualization tool for monitoring the viscosity of the vascular as well as the inflammatory marker ONOO‾ during thromboembolism via dual-channel simultaneous imaging. As a single probe that can recognize dual targets, Cou-ONOO effectively avoids the problems from unreliable results caused by complex synthesis and differences in intracellular localization, diffusion, and metabolism of different dyes as using combinatorial probes. Using Cou-ONOO, simultaneous imaging the variations of viscosity and ONOO‾at the cellular and tissue levels was successfully performed. In addition, Cou-ONOO also successfully visualized and tracked the viscosity of the vascular microenvironment and ONOO‾ during venous embolism in mice., Significance: Experimental results show that both viscosity and inflammation are abnormally overexpressed in the microenvironment at the thrombus site during venous thrombosis. An intuitive visualization tool to elucidate the variations of viscosity as well as inflammation level in the vascular microenvironment during thrombosis was provided, which will facilitate a better clinical understanding of the pathological process of thrombosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pyroptosis, a recently discovered form of programmed cell death, plays a pivotal role in oncological treatment. Howbeit, the mechanisms underlying pyroptosis in tumor treatment remain unclear. Previous research has demonstrated that the occurrence of pyroptosis generally accompanies a surge of reactive oxygen species (ROS) generation, with ONOO - being one of these ROS and closely linked to numerous diseases. Therefore, it is imperative to investigate the potential association between ONOO - and pyroptosis. Herein, a highly sensitive and rapidly responsive near-infrared (NIR) probe, Rd700-PN, is fabricated for exploring unrevealed relationships between ONOO - and pyroptosis. We successfully harness Rd700-PN to detect ONOO - fluctuation during cellular pyroptosis for the first time. Furthermore, the results demonstrate that Rd700-PN can scout the chemotherapeutic drug's induction ability of tumor pyroptosis in vivo. Notably, this study provides an excellent means to shed light on the correlation between ONOO - and pyroptosis and to screen antitumor drugs activating pyroptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Gas therapy is emerging as a highly promising therapeutic strategy for cancer treatment. However, there are limitations, including the lack of targeted subcellular organelle accuracy and spatiotemporal release precision, associated with gas therapy. In this study, we developed a series of photoactivatable nitric oxide (NO) donors NRh-R-NO (R = Me, Et, Bn, i Pr, and Ph) based on an N -nitrosated upconversion luminescent rhodamine scaffold. Under the irradiation of 808 nm light, only NRh-Ph-NO could effectively release NO and NRh-Ph with a significant turn-on frequency upconversion luminescence (FUCL) signal at 740 nm, ascribed to lower N-N bond dissociation energy. We also investigated the involved multistage near-infrared-controlled cascade release of gas therapy, including the NO released from NRh-Ph-NO along with one NRh-Ph molecule generation, the superoxide anion O 2 produced by the photodynamic therapy (PDT) effect of NRh-Ph, and highly toxic peroxynitrite anion (ONOO ⋅- produced by the photodynamic therapy (PDT) effect of NRh-Ph, and highly toxic peroxynitrite anion (ONOO ‒ ) generated from the co-existence of NO and O 2 ⋅- . After mild nano-modification, the nanogenerator (NRh-Ph-NO NPs) empowered with superior biocompatibility could target mitochondria. Under an 808 nm laser irradiation, NRh-Ph-NO NPs could induce NO/ROS to generate RNS, causing a decrease in the mitochondrial membrane potential and initiating apoptosis by caspase-3 activation, which further induced tumor immunogenic cell death (ICD). In vivo therapeutic results of NRh-Ph-NO NPs showed augmented RNS-potentiated gas therapy, demonstrating excellent biocompatibility and effective tumor inhibition guided by real-time FUCL imaging. Collectively, this versatile strategy defines the targeted RNS-mediated cancer therapy., Competing Interests: The authors declared no conflicts of interest., (© 2024 The Authors.)

Peroxynitrite (ONOO - ), an endogenous reactive nitrogen species, plays an important role in maintaining intracellular homeostasis. Abnormal levels of ONOO - in cells could cause protein oxidation which is confirmed that related with Alzheimer's diseases, so accurate monitoring of ONOO - in cells is crucial. Herein, a novel fluorescent probe (XPC) based on dicyanomethylene-4H-benzothiopyran was developed by regulating its intramolecular charge transfer (ICT) effect to detect ONOO - . Once reaction with ONOO - , the fluorescence of XPC was turned on and the emission wavelength could reach up to 750 nm. Furthermore, XPC exhibited satisfactory performances for ONOO - such as large Stokes shift (200 nm), good sensitivity (Limit of detection = 13 nM), high selectivity to ONOO - over other a reactive nitrogen species (RNS)/reactive oxygen species (ROS). More importantly, XPC was successfully applied for monitoring the fluctuations of ONOO - in living cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)