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Dichloramine (NHCl2) naturally exists in reverse osmosis (RO) permeate due to its application as an antifouling chemical in membrane-based potable reuse treatment. This study investigated mechanisms of background NHCl2 hydrolysis associated with the generation of oxidative radical species in RO permeate, established a kinetic model to predict the oxidative capacity, and examined its removal efficiency on trace organic contaminants in potable reuse. Results showed that NHCl2 hydrolysis generated transient peroxynitrite (ONOO-) and subsequently dissociated into hydroxyl radical (HO•). The maximal HO• exposure was observed at an RO permeate pH of 8.4, higher than that from typical ultraviolet (UV)-based advanced oxidation processes. The HO• exposure during NHCl2 hydrolysis also peaked at a NH2Cl-to-NHCl2 molar ratio of 1:1. The oxidative capacity rapidly degraded 1,4-dioxane, carbamazepine, atenolol, and sulfamethoxazole in RO permeate. Furthermore, background elevated carbonate in fresh RO permeate can convert HO• to carbonate radical (CO3•-). Aeration of the RO permeate removed total carbonate, significantly increased HO• exposure, and enhanced the degradation kinetics of trace organic contaminants. The kinetic model of NHCl2 hydrolysis predicted well the degradation of contaminants in RO permeate. This study provides new mechanistic insights into NHCl2 hydrolysis that contributes to the oxidative degradation of trace organic contaminants in potable reuse systems.

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ABSTRACT To investigate the biological reactivity of plasma‐activated water (PAW), we prepared PAW using atmospheric air spark discharge over water at 4°C and 25°C, with Escherichia Coli (E. coli) as the microbial model. The biological reactivity of PAW at 25°C decreased rapidly by 3.32 Logs within 20 s of storage. At 4°C, PAW showed high reactivity within 20–30 s of storage but decreased by 3.50 Logs between 30 and 60 s. Our analysis revealed that PAW reactivity is dependent on short‐lived peroxynitrite (ONOOH), formed through H2O2 and HNO2 under acidic conditions. ONOOH actively reacts with amino acid functional groups, as measured by high‐resolution LC‐MS, likely contributing to the rapid inactivation of E. coli. [ABSTRACT FROM AUTHOR]

Living therapeutics is an emerging antitumor modality by living microorganisms capable of selective tropism and effective therapeutics. Nevertheless, primitive microbes could only present limited therapeutic functionalities against tumors. Hybridization of the microbes with multifunctional nanocatalysts is of great significance to achieve enhanced tumor catalytic therapy. In the present work, nitric oxide synthase (NOS)-engineered Escherichia coli strain MG1655 (NOBac) was used to hybridize with the sonopiezocatalytic BaTiO3 nanoparticles (BTO NPs) for efficient tumor-targeted accumulation and antitumor therapy. Under ultrasound irradiation, superoxide anions created by the piezocatalytic reaction of BTO NPs could immediately react with nitric oxide (NO) generated from NOBac to produce highly oxidative peroxynitrite ONOO-species in cascade, resulting in robust tumor piezocatalytic therapeutic efficacy, prompting prominent and sustained antitumoral immunoactivation simultaneously. The present work presents a promising cancer immunotherapy based on the engineered and hybridized microbes for highly selective and sonopiezo-controllable tumor catalytic therapy. [ABSTRACT FROM AUTHOR]

Acanthopanax senticosus belongs to Araliaceae family and is traditionally used as a tonic. The roots and stems are mainly used as treatments for hypodynamia, rheumatism, and hypertension, but their frequent use may lead to extinction. However, comprehensive and simultaneous analysis of the remaining parts were still limited. There is a need to reorganize them for standardization of functional foods. In this study, 50 phenolic compounds and 82 triterpenoid saponins from the shoots, leaves, fruits, and stems of were characterized using UPLC-QTOF-MS and UPLC-QTRAP-MS/MS. Among them, 52 compounds were newly determined as the cis and malonyl-bound phenolic acids and were found to be structural isomers of Acanthopanax flavonoids and saponins. All compounds were absolutely/relatively quantified, and shoots had the highest content. Peroxynitrite and α-glucosidase inhibitory activities were performed, followed by evaluation of structure-activity relationships. Particularly, hederasaponin B and ciwujianoside B showed remarkable efficacy, which were affected by the C-23 hydroxylation, the C-20(29) double bond, and the presence of rhamnose. These detailed profiling can be used as fundamental data for increasing the utilization of A. senticosus and developing them into functional foods. [ABSTRACT FROM AUTHOR]

Graphene quantum dots (GQDs), small graphene domains with lateral dimensions lower than 10 nm, are increasingly used in electronics, composites, and biomedicine. Chemical doping of GQDs allows tuning their optical properties. Immune cells are among the first cells exposed to nanomaterials entering a living body, rapidly triggering a downstream immune response. However, the assessment of the impact of chemically‐doped GQDs on the immune system remains rather limited if not absent. In this context, the effects and the biodegradability of sulfur‐doped and nitrogen‐doped GQDs (S‐GQDs and N‐GQDs) on human monocytes and macrophages are evaluated. The metabolic activity, membrane integrity, apoptosis, and intracellular reactive oxygen species (ROS) generation are studied. In parallel, the degradation of GQDs using human myeloperoxidase and a peroxynitrite‐mediated system is investigated in test tube. Their degradation in macrophages is also pursued. High‐resolution transmission electron microscopy (HRTEM), fluorescence spectroscopy, Raman, and flow cytometry are used to confirm the degradation. Overall, both GQDs exert little activation on monocytes and macrophages although they decrease the metabolic viability in a dose‐dependent manner. The loss of native GQD structure and crystal lattice provide evidence of their biodegradability. Both the safety and biodegradability of S‐GQDs and N‐GQDs ensure their potential in biomedical applications. [ABSTRACT FROM AUTHOR]

In response to peroxynitrite (ONOO−) generation, myogenic stem satellite cell activator HGF (hepatocyte growth factor) undergoes nitration of tyrosine residues (Y198 and Y250) predominantly on fast IIa and IIx myofibers to lose its binding to the signaling receptor c‐met, thereby disturbing muscle homeostasis during aging. Here we show that rat anti‐HGF monoclonal antibody (mAb) 1H41C10, which was raised in‐house against a synthetic peptide FTSNPEVRnitroY198EV, a site well‐conserved in mammals, functions to confer resistance to nitration dysfunction on HGF. 1H41C10 was characterized by recognizing both nitrated and non‐nitrated HGF with different affinities as revealed by Western blotting, indicating that the paratope of 1H41C10 may bind to the immediate vicinity of Y198. Subsequent experiments showed that 1H41C10‐bound HGF resists peroxynitrite‐induced nitration of Y198. A companion mAb‐1H42F4 presented similar immuno‐reactivity, but did not protect Y198 nitration, and thus served as the control. Importantly, 1H41C10‐HGF also withstood Y250 nitration to retain c‐met binding and satellite cell activation functions in culture. The Fab region of 1H41C10 exerts resistivity to Y250 nitration possibly due to its localization in the immediate vicinity to Y250, as supported by an additional set of experiments showing that the 1H41C10‐Fab confers Y250‐nitration resistance which the Fc segment does not. Findings highlight the in vitro preventive impact of 1H41C10 on HGF nitration‐dysfunction that strongly impairs myogenic stem cell dynamics, potentially pioneering cogent strategies for counteracting or treating age‐related muscle atrophy with fibrosis (including sarcopenia and frailty) and the therapeutic application of investigational HGF drugs. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting the lives of millions of people worldwide. Although the mechanism underlying PD pathogenesis is largely undefined, increasing evidence indicates that oxidative and nitrosative stresses play a crucial role in PD occurrence and development. Among them, the role of oxidative stress has been widely acknowledged, but there is relatively less attention given to nitrosative stress, which is mainly derived from peroxynitrite. In the present review, after briefly introducing the background of PD, we discuss the physiopathological function of peroxynitrite and especially highlight how overloaded peroxynitrite is involved in PD pathogenesis. Then, we summarize the currently reported fluorescence imaging-based peroxynitrite detection probes. Moreover, we specifically emphasize the probes that have been applied in PD research. Finally, we propose perspectives on how to develop a more applicable peroxynitrite probe and leverage it for PD theranostics. Conclusively, the present review broadens the knowledge on the pathological role of peroxynitrite in the context of PD and sheds light on how to develop and utilize fluorescence imaging-based strategies for peroxynitrite detection. [ABSTRACT FROM AUTHOR]

Abstract Acanthopanax senticosus belongs to Araliaceae family and is traditionally used as a tonic. The roots and stems are mainly used as treatments for hypodynamia, rheumatism, and hypertension, but their frequent use may lead to extinction. However, comprehensive and simultaneous analysis of the remaining parts were still limited. There is a need to reorganize them for standardization of functional foods. In this study, 50 phenolic compounds and 82 triterpenoid saponins from the shoots, leaves, fruits, and stems of were characterized using UPLC-QTOF-MS and UPLC-QTRAP-MS/MS. Among them, 52 compounds were newly determined as the cis and malonyl-bound phenolic acids and were found to be structural isomers of Acanthopanax flavonoids and saponins. All compounds were absolutely/relatively quantified, and shoots had the highest content. Peroxynitrite and α-glucosidase inhibitory activities were performed, followed by evaluation of structure-activity relationships. Particularly, hederasaponin B and ciwujianoside B showed remarkable efficacy, which were affected by the C-23 hydroxylation, the C-20(29) double bond, and the presence of rhamnose. These detailed profiling can be used as fundamental data for increasing the utilization of A. senticosus and developing them into functional foods.

Selective detection of reactive oxygen species (ROS) is vital for studying their role in brain diseases. Fluorescence probes can distinguish ONOO− species from other ROS; however, their selectivity toward ONOO− species depends on the ONOO− recognition group. Aryl‐boronic acids and esters, which are common ONOO− recognition groups, are not selective for ONOO− over H2O2. In this study, we developed a diaminonaphthalene (DAN)‐protected boronic acid as a new ONOO− recognition group that selectively reacts with ONOO− over H2O2 and other ROS. Three DAN‐protected boronic acid (DANBA)‐based fluorophores that emit fluorescence over visible to near‐infrared (NIR) regions, Cou‐BN, BVP‐BN, and HDM‐BN, and their aryl‐boronic acid‐based counterparts (Cou‐BO, BVP‐BO, and HDM‐BO), were developed. The DANBA‐based probes exhibited enhanced selectivity toward ONOO− over that of their control group, as well as universality in solution assays and in vitro experiments with PC12 cells. The NIR‐emissive HDM‐BN was optimized to delineate in vivo ONOO− levels in mouse brains with Parkinson's disease. This DAN‐protected boronic acid belongs to a new generation of recognition groups for developing ONOO− probes, and this strategy could be extended to other common hydroxyl‐containing dyes to detect ONOO− levels in complex biological systems and processes. [ABSTRACT FROM AUTHOR]

Multidrug resistance to clinical chemotherapeutic drugs severely limits antitumor efficacy and patient survival. The integration of chemotherapy with photothermal therapy (PTT) and reactive nitrogen species has become a major strategy to enhance cancer treatment efficacy. Herein, a multifunctional peroxynitrite (ONOO−) nanogenerator (PBT/NO/Pt) for NIR‐II fluorescence (NIR‐II FL)/NIR‐II photoacoustic (NIR‐II PA) imaging‐guided chemo/NIR‐II PTT/ONOO− combination therapy is reported. The multifunction nanogenerator is developed by co‐loading a pH‐sensitive nitric oxide donor (DETA NONOate) and nicotinamide adenine dinucleotide phosphate oxidases trigger superoxide (O2•−) generator chemotherapy drug (CDDP) to an NIR‐II excitation‐conjugated polyelectrolyte (PNC11BA). PNC11BA has non‐conjugated alkyl chain segments in the polymer backbone and abundant positively charged phenylboronic acid in its side chains, which support the anti‐quenching of NIR‐II FL and the integration of DETA NONOate and CDDP into PBT/NO/Pt. In the acidic tumor microenvironment, the coordination bonds between CDDP and PNC11BA are cleaved, releasing CDDP for chemotherapeutic activity. The simultaneous release of nitric oxide (NO) and O2•− rapidly leads to the in situ generation of the more cytotoxic reactive physiological nitrogen species ONOO−. In vitro and in vivo results prove that PBT/NO/Pt exhibited a markedly ONOO− enhanced chemo–photothermal synergistic therapy for SKOV3/DDP tumor by downregulating the intracellular glutathione and increasing CDDP–DNA adducts. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6–14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology. [ABSTRACT FROM AUTHOR]

The study was based to find out the effect of iron deficiency anemia on glutathione concentration, and the level of peroxynitrite was taken as an indication of the change in oxidative stress levels for patients with iron deficiency anemia. Sixty-one random samples were collected from a specialized laboratory in the city of Mosul, and the samples were from women whose ages ranged from 17 to 50 years. The samples were divided into three groups, the first group included severe anemia, and the second group had a blood test result within the average limits, and the third group was the control group for women without anima. When a comparison was made between the three groups, a significant decrease in the concentration of glutathione was found, followed by a rise in the level of peroxynitrite in the first and the second group when compared with the third group in which the concentration of peroxynitrite decreased and glutathione increased. The study also included conducting biochemical tests that included zinc, copper and iron. The results showed a decrease in zinc in the first and second group compared to the third group, while copper decreased in the first group compared with the second and third. Iron decreased in the first and second groups only. [ABSTRACT FROM AUTHOR]

A near-infrared fluorescent probe (TX-P) for detecting peroxynitrite is constructed. The probe has a near-infrared emission (725 nm), large Stokes shift (125 nm) and excellent sensitivity and selectivity. In addition, TX-P can be used to visualize ONOO− in living cells, image ONOO− in paw edema mice and evaluate anti-inflammatory drugs. [ABSTRACT FROM AUTHOR]

Although the labile iron pool (LIP) biochemical identity remains a topic of debate, it serves as a universal homeostatically regulated and essential cellular iron source. The LIP plays crucial cellular roles, being the source of iron that is loaded into nascent apo-iron proteins, a process akin to protein post-translational modification, and implicated in the programmed cell death mechanism known as ferroptosis. The LIP is also recognized for its reactivity with chelators, nitric oxide, and peroxides. Our recent investigations in a macrophage cell line revealed a reaction of the LIP with the oxidant peroxynitrite. In contrast to the LIP's pro-oxidant interaction with hydrogen peroxide, this reaction is rapid and attenuates the peroxynitrite oxidative impact. In this study, we demonstrate the existence and antioxidant characteristic of the LIP and peroxynitrite reaction in various cell types. Beyond its potential role as a ubiquitous complementary or substitute protection system against peroxynitrite for cells, the LIP and peroxynitrite reaction may influence cellular iron homeostasis and ferroptosis by changing the LIP redox state and LIP binding properties and reactivity. [ABSTRACT FROM AUTHOR]

The undesirable efficacy of immunotherapy in cancer patients is associated with the inactivation of immune responses in tumor immunosuppressive microenvironment (TIME). The pivotal roles of nitric oxide (NO) and peroxynitrite (ONOO−) in immunoregulation can augment anticancer immunotherapy. Here, ultrasound (US)‐responsive nanoparticles (NPs), denoted as Cu‐PG NPs, are elaborately constructed to achieve incremental NO release for gas therapy and controlled generation of superoxide anion (O2•−) for sonodynamic therapy (SDT), thereby leading to synergistic in situ ONOO− generation and TIME reprogramming. In vitro and in vivo experimental results collectively confirm that US‐activated Cu‐PG NPs effectively regulate immune circulation, which involves multiple steps to ameliorate compromise immunogenicity without systemic toxicity. These steps comprise the initiation of immunogenic cell death in cancer cells, induction of dendritic cells maturation, promotion of cytotoxic T lymphocytes infiltration, and polarization of macrophages toward the pro‐inflammatory M1 phenotype. Importantly, this therapeutic approach reinforces systemic immunity and elicits immune memory to inhibit the proliferation of distant tumors, particularly integration with anti‐PD‐L1 antibodies. This work proposes the synergistic gas therapy and SDT strategy for generating ONOO−, which holds enormous potential in potentiating immunotherapy sensitivity by further facilitating the coordinated remodeling of TIME. [ABSTRACT FROM AUTHOR]

We report on an unexpected reaction between ammonia and potassium ozonide dissolved in liq. NH3 resulting in the formation of peroxynitrite, [ONOO]−, which exclusively happens in the presence of a specific partially fluorinated aniline‐based ammonium cation. High‐resolution structural data of the peroxynitrite anion in cis‐conformation have been obtained. We further studied this molecule anion by single crystal Raman spectroscopy. The cis and trans isomers of peroxynitrite were analysed computationally with respect to their relative energies, the cis‐trans transition barrier and their decomposition pathways to the nitrate anion. By experimentally examining cations decorated with fluorine ligands to different degrees, we demonstrate that fluorine specific interactions play a crucial role in the unexpected formation of peroxynitrite and as a conspicuously structure directing factor for the underlying crystalline solid phases, exhibiting distinct micro‐separations of fluorine and hydrogen enriched regions. [ABSTRACT FROM AUTHOR]

Background: The underlying pathophysiological mechanisms in epilepsy, one of the most common neurological diseases, are still unknown. Oxidative/nitrosative stress is considered a possible mechanism involved in epileptogenesis. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is involved in the pathogenesis of signal regulation, cellular damage and central nervous system conditions in living organisms. In this study, we aimed to compare peoxynitrite (ONOO−), a marker of nitrosative stress, total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI) and 8-hydroxy-2-deoxyguanosine (8-OHdG), DNA damage marker, levels in epileptic patients receiving monotherapy and polytherapy with the healthy control group.Methods: The study included 120 patients with diagnosis of epilepsy and 40 healthy volunteers as controls. The TOS, TAS, OSI, ONOO− and 8-OHdG were studied in all groups.Results: The study group included 30 girls (50%) and 30 boys (50%) receiving monotherapy and 31 girls (51.7%) and boys 49.3%) receiving polytherapy while control group included 19 girls (47.5%) and 21 boys (52.5%). The TOS and OSI values were found to be significantly higher in polytherapy group when compared to monotherapy and control groups). The ONOO− values were found to be significantly lower in polytherapy group when compared to monotherapy and control groups. In addition, ONOO− values were found to be higher in monotherapy group than controls. There was no significant difference in 8-OHdG values between the groups.Conclusions: Significant increases were observed in TOS and OSI parameters in polytherapy group when compared to monotherapy and control groups, suggesting that antiepileptic treatment enhances oxidative stress. Lack of significant difference in 8-OHdG suggested that the treatment is effective in patients and that no DNA damage occurred yet. [ABSTRACT FROM AUTHOR]

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are common food additives for human consumption. We examined multi-organ toxicity of both compounds on Wistar rats orally exposed for 90 days. Rats were divided into three groups: (1) control (saline solution), (2) E171-exposed, and (3) ZnO NPs-exposed. Histological examination was performed with hematoxylin–eosin (HE) staining and transmission electron microscopy (TEM). Ceramide (Cer), 3-nitrotyrosine (NT), and lysosome-associated membrane protein 2 (LAMP-2) were detected by immunofluorescence. Relevant histological changes were observed: disorganization, inflammatory cell infiltration, and mitochondrial damage. Increased levels of Cer, NT, and LAMP-2 were observed in the liver, kidney, and brain of E171- and ZnO NPs-exposed rats, and in rat hearts exposed to ZnO NPs. E171 up-regulated Cer and NT levels in the aorta and heart, while ZnO NPs up-regulated them in the aorta. Both NPs increased LAMP-2 expression in the intestine. In conclusion, chronic oral exposure to metallic NPs causes multi-organ injury, reflecting how these food additives pose a threat to human health. Our results suggest how complex interplay between ROS, Cer, LAMP-2, and NT may modulate organ function during NP damage. [ABSTRACT FROM AUTHOR]

A novel cyclic chalcone fluorescent probe C-PN was synthesized to detect ONOO−. After reaction with peroxynitrite, the double bond of C-PN in the cyclic chalcone structure was disconnected, which caused the change of intramolecular charge transfer (ICT) effect, emitting blue fluorescence and quenching orange red fluorescence. Visible to the naked eye, the color of the probe solution changed. The probe showed low sensitivity (detection limit = 20.2 nm), short response time (less than 60 s) at low concentration of ONOO−, good visibility, and good selectivity and stability for ONOO−. [ABSTRACT FROM AUTHOR]

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.

Influenza viruses are deadly respiratory pathogens of special importance due to their long history of global pandemics. During influenza virus infections, the host responds by producing interferons, which activate interferon-stimulated genes (ISGs) inside target cells. One of these ISGs is inducible nitric oxide synthase (iNOS). iNOS produces nitric oxide (NO) from arginine and molecular oxygen inside the cell. NO can react with superoxide radicals to form reactive nitrogen species, principally peroxynitrite. While much work has been done studying the many roles of nitric oxide in influenza virus infections, the direct effect of peroxynitrite on influenza virus proteins has not been determined. Manipulations of NO, either by knocking out iNOS or chemically inhibiting NO, produced no change in virus titers in mouse models of influenza infection. However, peroxynitrite has a known antimicrobial effect on various bacteria and parasites, and the reason for its lack of antimicrobial effect on influenza virus titers in vivo remains unclear. Therefore, we wished to test the direct effect of nitration of influenza virus proteins. We examined the impact of nitration on virus infectivity, replication, and immunogenicity. We observed that the nitration of influenza A virus proteins decreased virus infectivity and replication ex vivo. We also determined that the nitration of influenza virus hemagglutinin protein can reduce antibody responses to native virus protein. However, our study also suggests that nitration of influenza virus proteins in vivo is likely not extensive enough to inhibit virus functions substantially. These findings will help clarify the role of peroxynitrite during influenza virus infections. IMPORTANCE Nitric oxide and peroxynitrite produced during microbial infections have diverse and seemingly paradoxical functions. While nitration of lung tissue during influenza virus infection has been observed in both mice and humans, the direct effect of protein nitration on influenza viruses has remained elusive. We addressed the impact of nitration of influenza virus proteins on virus infectivity, replication, and immunogenicity. We observed that ex vivo nitration of influenza virus proteins reduced virus infectivity and immunogenicity. However, we did not detect nitration of influenza virus hemagglutinin protein in vivo. These results contribute to our understanding of the roles of nitric oxide and peroxynitrite in influenza virus infections.

Introduction: CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. Methods: In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. Results and discussion: We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy. [ABSTRACT FROM AUTHOR]

An AIE-based fluorescent probe was designed to evaluate peroxynitrite levels in complex biological samples. The newly synthesized hydrazone-conjugated probe fluoresces strongly in the presence of peroxynitrite. Clinically, the peroxynitrite levels can be measured in human serum and cellular mitochondria with an LOD of 6.5 nM by fluorescence imaging in vitro. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate–glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S -nitrosation, persulfidation, and S -sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry. [ABSTRACT FROM AUTHOR]

Background: Peroxynitrite (ONOO−) is an oxidant linked with several human pathologies. Apigenin, a natural flavonoid known for its health benefits, remains unexplored in relation to ONOO− effects. This study investigated the potential of apigenin to structurally protect fibrinogen, an essential blood clotting factor, from ONOO−-induced damage. Methods: Multi-approach analyses were carried out where fibrinogen was exposed to ONOO− generation while testing the efficacy of apigenin. The role of apigenin against ONOO−-induced modifications in fibrinogen was investigated using UV spectroscopy, tryptophan or tyrosine fluorescence, protein hydrophobicity, carbonylation, and electrophoretic analyses. Results: The findings demonstrate that apigenin significantly inhibits ONOO−-induced oxidative damage in fibrinogen. ONOO− caused reduced UV absorption, which was reversed by apigenin treatment. Moreover, ONOO− diminished tryptophan and tyrosine fluorescence, which was effectively restored by apigenin treatment. Apigenin also reduced the hydrophobicity of ONOO−-damaged fibrinogen. Moreover, apigenin exhibited protective effects against ONOO−-induced protein carbonylation. SDS-PAGE analyses revealed that ONOO−treatment eliminated bands corresponding to fibrinogen polypeptide chains Aα and γ, while apigenin preserved these changes. Conclusions: This study highlights, for the first time, the role of apigenin in structural protection of human fibrinogen against peroxynitrite-induced nitrosative damage. Our data indicate that apigenin offers structural protection to all three polypeptide chains (Aα, Bβ, and γ) of human fibrinogen. Specifically, apigenin prevents the dislocation or breakdown of the amino acids tryptophan, tyrosine, lysine, arginine, proline, and threonine and also prevents the exposure of hydrophobic sites in fibrinogen induced by ONOO−. [ABSTRACT FROM AUTHOR]

Afterglow imaging holds great promise for ultrasensitive bioimaging due to its elimination of autofluorescence. Self‐sustaining afterglow molecules (SAMs), which enable all‐in‐one photon sensitization, chemical defect formation and afterglow generation, possess a simplified, reproducible, and efficient superiority over commonly used multi‐component systems. However, there is a lack of SAMs, particularly those with much brighter near‐infrared (NIR) emission and structural flexibility for building high‐contrast activatable imaging probes. To address these issues, this study for the first time reports a methylene blue derivative‐based self‐sustaining afterglow agent (SAN‐M) with brighter NIR afterglow chemiluminescence peaking at 710 nm. By leveraging the structural flexibility and tunability, an activatable nanoprobe (SAN‐MO) is customized for simultaneously activatable fluoro‐photoacoustic and afterglow imaging of peroxynitrite (ONOO−), notably with a superior activation ratio of 4523 in the afterglow mode, which is at least an order of magnitude higher than other reported activatable afterglow systems. By virtue of the elimination of autofluorescence and ultrahigh activation contrast, SAN‐MO enables early monitoring of the LPS‐induced acute inflammatory response within 30 min upon LPS stimulation and precise image‐guided resection of tiny metastatic tumors, which is unattainable for fluorescence imaging. [ABSTRACT FROM AUTHOR]

Visual loss in acute optic neuritis is typically attributed to axonal conduction block due to inflammatory demyelination, but the mechanisms remain unclear. Recent research has highlighted tissue hypoxia as an important cause of neurological deficits and tissue damage in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) and, here, we examine whether the optic nerves are hypoxic in experimental optic neuritis induced in Dark Agouti rats. At both the first and second peaks of disease expression, inflamed optic nerves labelled significantly for tissue hypoxia (namely, positive for hypoxia inducible factor-1α (HIF1α) and intravenously administered pimonidazole). Acutely inflamed nerves were also labelled significantly for innate markers of oxidative and nitrative stress and damage, including superoxide, nitric oxide and 3-nitrotyrosine. The density and diameter of capillaries were also increased. We conclude that in acute optic neuritis, the optic nerves are hypoxic and come under oxidative and nitrative stress and damage. Tissue hypoxia can cause mitochondrial failure and thus explains visual loss due to axonal conduction block. Tissue hypoxia can also induce a damaging oxidative and nitrative environment. The findings indicate that treatment to prevent tissue hypoxia in acute optic neuritis may help to restore vision and protect from damaging reactive oxygen and nitrogen species. [ABSTRACT FROM AUTHOR]

Abstract Multidrug resistance to clinical chemotherapeutic drugs severely limits antitumor efficacy and patient survival. The integration of chemotherapy with photothermal therapy (PTT) and reactive nitrogen species has become a major strategy to enhance cancer treatment efficacy. Herein, a multifunctional peroxynitrite (ONOO−) nanogenerator (PBT/NO/Pt) for NIR‐II fluorescence (NIR‐II FL)/NIR‐II photoacoustic (NIR‐II PA) imaging‐guided chemo/NIR‐II PTT/ONOO− combination therapy is reported. The multifunction nanogenerator is developed by co‐loading a pH‐sensitive nitric oxide donor (DETA NONOate) and nicotinamide adenine dinucleotide phosphate oxidases trigger superoxide (O2•−) generator chemotherapy drug (CDDP) to an NIR‐II excitation‐conjugated polyelectrolyte (PNC11BA). PNC11BA has non‐conjugated alkyl chain segments in the polymer backbone and abundant positively charged phenylboronic acid in its side chains, which support the anti‐quenching of NIR‐II FL and the integration of DETA NONOate and CDDP into PBT/NO/Pt. In the acidic tumor microenvironment, the coordination bonds between CDDP and PNC11BA are cleaved, releasing CDDP for chemotherapeutic activity. The simultaneous release of nitric oxide (NO) and O2•− rapidly leads to the in situ generation of the more cytotoxic reactive physiological nitrogen species ONOO−. In vitro and in vivo results prove that PBT/NO/Pt exhibited a markedly ONOO− enhanced chemo–photothermal synergistic therapy for SKOV3/DDP tumor by downregulating the intracellular glutathione and increasing CDDP–DNA adducts.

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.

AIM: To directly quantify peroxynitrite (ONOO-) using a highly sensitive fluorescence resonance energy transfer probe RN-NA, investigate the association between ONOO- and primary open angle glaucoma (POAG), and clarify whether RN-NA could be used as a potential tool for POAG diagnosis. METHODS: Plasma and aqueous humor (AH) samples were collected from POAG patients (n=100, age: 59.70±6.87y) and age-related cataract (ARC) patients (n=100, age: 61.15±4.60y) admitted to our hospital. Next, RN-NA was used to detect ONOO- in plasma and AH samples, and the relationship between ONOO- level and POAG was analyzed using binary logistic regression. Besides, Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO- with the patients' age, intraocular pressure (IOP), and mean deviation of visual field testing. The ONOO- scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine (SIN-1)-induced ocular hypertension in mice (n=7, 6-8wk). Finally, the IOP and ONOO- in both eyes were measured 30min after the last drug treatment. RESULTS: ONOO- levels of AH and plasma were significantly higher in the POAG group than in the ARC group (P

Plant-based diets that replace animal-based proteins with plant-based proteins have received increased attention for cardiovascular protection. Nitric oxide (NO) plays an essential role in the maintenance of endothelial function. However, under higher oxidative stress, NO generation produces peroxynitrite, a powerful oxidant and vasoconstrictor. Diet-replaced protein sources has been reported to decrease oxidative stress. However, the effects of plant-based protein on NO and peroxynitrite have not yet been clarified. Therefore, this study aimed to compare the effects of plant- and animal-based-protein meals for a day on NO, peroxynitrite, and NO/peroxynitrite balance. A crossover trial of two meal conditions involving nine healthy men was performed. Participants ate standard meals during day 1. On day 2, baseline measurements were performed and the participants were provided with plant-based-protein meals or animal-based-protein meals. The standard and test meals consisted of breakfast, lunch, and dinner and were designed to be isocaloric. Plant-based-protein meals contained no animal protein. Blood samples were collected in the morning after overnight fasting before and after the test meals consumption. In the plant-based-protein meal condition, serum NOx levels (the sum of serum nitrite and nitrate) significantly increased, while serum peroxynitrite levels did not change significantly. Animal-based-protein meals significantly increased serum peroxynitrite levels but showed a trend of reduction in the serum NOx levels. Furthermore, serum NO/peroxynitrite balance significantly increased after plant-based-protein meals consumption, but significantly decreased after animal-based-protein meals consumption. These results suggest that, compared with animal-based-protein meals, plant-based-protein meals increase NO levels and NO/peroxynitrite balance, which reflects increased endothelial function.

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 aim of this study was to assess the production of reactive nitrogen species in the hearts of rats subjected to surgical trauma simulated against the background of post-traumatic stress disorder (PTSD). Materials and methods.The study was conducted on 42 white Wistar rats weighing 210-230 g, then divided into 6 groups: Group 1 consisted of intact animals, Group 2 included animals exposed to the induction of the PTSD model through single-prolonged stress (SPS), Group 3 – rats subjected to a sham operation, Group 4 – animals undergoing laparotomy, Group 5 – rats undergoing a sham surgical operation following SPS, and Group 6 – animals undergoing laparotomy under modeled SPS. NO synthase activity in the heart homogenate was determined spectrophotometrically. Results. The formation of peroxynitrite was assessed by the content of peroxynitrite of alkaline and alkaline-earth metals. SPS exposure significantly increases the production of reactive nitrogen species in the hearts of rats, in particular, it enhances NO synthase activity by activating the inducible isoform and reduces the activity of constitutive NO synthases, which is accompanied by the growth in the concentration of peroxynitrites. On the 7th day after laparotomy against the background of the experimental PTSD model, the indicators of nitrosative stress in the hearts of rats (total and inducible NO synthase activity and peroxynitrite concentration) significantly exceeded their values in the groups subjected to a single laparotomy and to a sham operation against the background of simulated SPS.

Aim. The aim of the study was to establish the changes in nitric oxide production and metabolism in rat heart during combined influence of organism stimulation with bacterial lipopolysaccharide (LPS) and modeling of metabolic syndrome (MetS). Materials and methods. The study was conducted on 24 mature male Wistar rats weighing 200–260 g. Experiment lasted 60 days. The animals were divided into 4 groups of 6 animals each: control group, MetS group, LPS stimulation group, LPS + MetS group. MetS was reproduced by using a 20 % fructose solution as the only source of drinking water. LPS of Salmonella typhi was administered at a dose of 0.4 μg/kg intraperitoneally. Animals from LPS + MetS group received a 20 % fructose solution as the only source of drinking water and were administered LPS. In 10 % tissue homogenate of rat heart we studied: total activity of NO-synthases (NOS), activity of constitutive (cNOS) and inducible (iNOS) isoforms, activity of nitrate (NaR) and nitrite (NiR) reductases, concentration of peroxynitrites (ONOO-), nitrites, nitrosothiols and hydrogen sulfide. Results. Combination of MetS and stimulation of organism with LPS led to increase in total NOS activity by 32.72 % compared to control group. Activity of cNOS did not change compared to control group. Activity of iNOS increased by 33.76 %. Arginase activity decreased by 23.53 %. NaR activity and NiR activity were increased by 86.67 % and by 149.29 %, respectively. Combination of MetS and stimulation of organism with LPS led to decrease in nitrite and nitrosothiols concentration by 38.73 % and by 54.79 %, respectively. Under these conditions concentration of ONOOelevated by 398.0 % compared to control group. Concentration of H S decreased by 27.56 %. Conclusions. Combination of metabolic syndrome and stimulation of organism with bacterial lipopolysaccharide leads to prevalence of peroxynitrite formation during increased nitric oxide production NO-synthase-dependent and nitrate-nitrite-NO pathways in rat heart.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting the lives of millions of people worldwide. Although the mechanism underlying PD pathogenesis is largely undefined, increasing evidence indicates that oxidative and nitrosative stresses play a crucial role in PD occurrence and development. Among them, the role of oxidative stress has been widely acknowledged, but there is relatively less attention given to nitrosative stress, which is mainly derived from peroxynitrite. In the present review, after briefly introducing the background of PD, we discuss the physiopathological function of peroxynitrite and especially highlight how overloaded peroxynitrite is involved in PD pathogenesis. Then, we summarize the currently reported fluorescence imaging-based peroxynitrite detection probes. Moreover, we specifically emphasize the probes that have been applied in PD research. Finally, we propose perspectives on how to develop a more applicable peroxynitrite probe and leverage it for PD theranostics. Conclusively, the present review broadens the knowledge on the pathological role of peroxynitrite in the context of PD and sheds light on how to develop and utilize fluorescence imaging-based strategies for peroxynitrite detection.

Oxidative stress can be associated with hyperoxia and hypoxia and is characterized by an increase in reactive oxygen (ROS) and nitrogen (RNS) species generated by an underlying disease process or by supplemental oxygen that exceeds the neutralization capacity of the organ system. ROS and RNS acting as free radicals can inactive several enzymes and vasodilators in the nitric oxide pathway promoting pulmonary vasoconstriction resulting in persistent pulmonary hypertension of the newborn (PPHN). Studies in animal models of PPHN have shown high ROS/RNS that is further increased by hyperoxic ventilation. In addition, antioxidant therapy increased PaO2 in these models, but clinical trials are lacking. We recommend targeting preductal SpO2 between 90 and 97%, PaO2 between 55 and 80 mmHg and avoiding FiO2 > 0.6-0.8 if possible during PPHN management. This review highlights the role of oxidative and nitrosative stress markers on PPHN and potential therapeutic interventions that may alleviate the consequences of increased oxidant stress during ventilation with supplemental oxygen.

Macrophages count on two O 2 -consuming enzymes to form reactive radical species: NAPDH oxidase 2 (Nox2) and nitric oxide synthase 2 (inducible isoform, iNOS) that produce superoxide radical (O 2 •−) and nitric oxide (•NO), respectively. If formed simultaneously, the diffusion-controlled reaction of O 2 •− and •NO yields peroxynitrite, a potent cytotoxic oxidant. In human tissues and cells, the oxygen partial pressure (pO 2) normally ranges within 2–14 %, with a typical average pO 2 value for most tissues ca. 5 %. Given that O 2 is a substrate for both Nox2 and iNOS, its tissue and cellular concentration can affect O 2 •− and •NO production. Also, O 2 is a modulator of the macrophage adaptative response and may influence iNOS expression in a hypoxia inducible factor 1-α (HIF1α-)-dependent manner. However, most of the reported experiments in cellula , analyzing the formation and effects of O 2 •− and •NO during macrophage activation and cytotoxicity towards pathogens, have been performed in cells exposed to atmospheric air supplemented with 5 % CO 2 ; under these conditions, most cells are exposed to supraphysiologic oxygen tensions (ca. 20 % O 2) which are far from the physiological pO 2. Here, the role of O 2 as substrate in the oxidative response of J774A.1 macrophages was explored upon exposure to different pO 2 and O 2 •− and •NO formation rates were measured, obtaining a K M of 26 and 42 μM O 2 for Nox2 and iNOS, respectively. Consequently, peroxynitrite formation was influenced by pO 2 , reaching a maximum at ≥ 10 % O 2 , but even at levels as low as 2 % O 2 , a substantial formation rate of this oxidant was detected. Indeed, the cytotoxic capacity of immunostimulated macrophages against the intracellular parasite T. cruzi was significant, even at low pO 2 values, confirming the role of peroxynitrite as a potent oxidizing cytotoxin within a wide range of physiological oxygen tensions. [Display omitted] • Superoxide radical and nitric oxide formation in macrophages depend on pO 2 and their production rates could vary among different tissues. • Peroxynitrite production in macrophages is influenced by pO 2 , with 50 % of maximal rate under 5 % O 2. • The peroxynitrite-dependent cytotoxic capacity of macrophages is maintained even at the low physiological O 2 levels found in tissues. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H 2 O 2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H 2 O 2 - and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation. [Display omitted] • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an oxidant-sensitive cysteine (Cys) in its active site. • Effects of macromolecular crowding on GAPDH oxidation by H 2 O 2 and SIN-1 was investigated. • Dextran differentially affects H 2 O 2 - and SIN-1-mediated GAPDH cross-linking. • Higher yields of active site Cys-derived sulfonic acids were detected with crowding. [ABSTRACT FROM AUTHOR]

Reactive oxygen and nitrogen species (RONS) are a range of chemical individuals produced by living cells that contribute to the proper functioning of organisms. Cells under oxidative and nitrative stress show excessive production of RONS (including hydrogen peroxide, H 2 O 2 , hypochlorous acid, HOCl, and peroxynitrite, ONOO−) which may result in a damage proteins, lipids, and genetic material. Thus, the development of probes for in vivo detection of such oxidants is an active area of research, focusing on molecular redox sensors, including boronate-caged fluorophores. Here, we report a boronate-based styryl probe with a cationic pyridinium moiety (BANEP+) for the fluorescent detection of selected biological oxidants in vitro and in vivo. We compare the chemical reactivity of the BANEP+ probe toward H 2 O 2 , HOCl, and ONOO− and examine the influence of the major intracellular non-enzymatic antioxidant molecule, glutathione (GSH). We demonstrate that, at the physiologically relevant GSH concentration, the BANEP+ probe is efficiently oxidized by peroxynitrite, forming its phenolic derivative HNEP+. GSH does not affect the fluorescence properties of the BANEP+ and HNEP+ dyes. Finally, we report the identification of a novel type of molecular marker, with the boronate moiety replaced by the iodine atom, formed from the probe in the presence of HOCl and iodide anion. We conclude that the reported chemical reactivity and structural features of the BANEP+ probe may be a basis for the development of new red fluorescent probes for in vitro and in vivo detection of ONOO−. [Display omitted] • Pyridinium-linked styryl-based boronate fluorescent probe for detection of ONOO−. • Iodide ion influences reaction between boronate probes and biological oxidants. • Iodinated dye as a marker for boronate probe and HOCl reaction in the presence of I−. • Glutathione may change products formed in reaction of boronate probe with ONOO−. [ABSTRACT FROM AUTHOR]

Radiotherapy (RT) resistance is an enormous challenge in glioblastoma multiforme (GBM) treatment, which is largely associated with DNA repair, poor distribution of reactive radicals in tumors, and limited delivery of radiosensitizers to the tumor sites. Inspired by the aberrant upregulation of RAD51 (a critical protein of DNA repair), scavenger receptor B type 1 (SR‐B1), and C‐C motif chemokine ligand 5 (CCL5) in GBM patients, a reduction‐sensitive nitric oxide (NO) donor conjugate of gemcitabine (RAD51 inhibitor) (NG) is synthesized as radio‐sensitizer and a CCL5 peptide‐modified bioinspired lipoprotein system of NG (C‐LNG) is rationally designed, aiming to preferentially target the tumor sites and overcome the RT resistance. C‐LNG can preferentially accumulate at the orthotopic GBM tumor sites with considerable intratumor permeation, responsively release the gemcitabine and NO, and then generate abundant peroxynitrite (ONOO−) upon X‐ray radiation, thereby producing a 99.64% inhibition of tumor growth and a 71.44% survival rate at 120 days in GL261‐induced orthotopic GBM tumor model. Therefore, the rationally designed bioinspired lipoprotein of NG provides an essential strategy to target GBM and overcome RT resistance. [ABSTRACT FROM AUTHOR]

Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling‐receptor c‐met. Here, we show that with aging, extracellular HGF undergoes tyrosine‐residue (Y) nitration and loses c‐met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c‐met‐binding domains. Direct‐immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct in vivo evidence for age‐related increases in nitration of ECM‐bound HGF, preferentially stained for anti‐nitrated Y198 and Y250‐HGF mAbs (raised in‐house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age‐related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty). [ABSTRACT FROM AUTHOR]

Schematic representation of multiple ROS-generation-based tumor-therapy approach of AgNP. [Display omitted] • A simple mixing procedure has been utilized to synthesize bimetallic silver nitroprusside (AgNP) as CDT agent together with peroxynitrite generator. • AgNP decomposes more endogenous H 2 O 2 to produce highly toxic radicals. • AgNP are involved in the inhibition of AKT pathway, which is involved in cell survival, angiogenesis, proliferation, and apoptosis. • AgNP showed biocompatible nature towards normal mouse liver and ovarian organoids while are toxic on PDTO through altering the redox homeostasis. 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 H 2 O 2 and produce reactive oxygen species (ROS) to attain a therapeutic outcome. 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. 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. AgNP have an effective ability to decompose endogenous H 2 O 2 in cells. The decomposed endogenous H 2 O 2 generates several different types of reactive species (•OH, O 2 •−) 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). 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. [ABSTRACT FROM AUTHOR]

Uric Acid (UA) is the final product of the purine nucleoside metabolism, acts physiologically as plasma antioxidant and is a known peroxynitrite scavenger. Several studies have reported lower serum concentrations of UA in Multiple sclerosis (MS) patients. In this study, we examined the effect of UA administration on severe protracted relapsing remitting experimental autoimmune encephalomyelitis (SPR-EAE) in Dark Agouti (DA) rats, an animal model of MS. Compared to placebo, daily UA treatment for either 12 or 23 days after immunization significantly reduced the clinical course and severity of disease. It also suppressed all aspects of inflammation, nitric oxide and peroxynitrite formation, axonal damage and demyelination. These findings suggest that UA might be beneficial in MS treatment by preventing tissue and molecular alterations due to its capacity to neutralise the toxic effect of peroxynitrite. [ABSTRACT FROM AUTHOR]