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).

Diabetes mellitus is a metabolic disorder caused by a dysfunction in insulin action or secretion, leading to an elevation in blood glucose levels. It is a highly prevalent condition and as a result, the NHS spends 10 % of its entire budget on diabetes mellitus care, that is equivalent to £10 billion a year. Diabetes mellitus has been linked with vascular and neurological complications which may be associated with the progression of neurodegeneration and Alzheimer\'s disease. Chronic hyperglycaemia increases the production of the reactive oxidant species (ROS) such as methylglyoxal (MGO). MGO has been linked with vascular complications, neuropathy and cytotoxicity. The main aim of this study was to investigate the potential beneficial effect of antidiabetic agents such as metformin and dapagliflozin on human brain neuronal cells (SH-SY5Y) treated with MGO. SH-SY5Y cells were cultured in DMEM/F12 media and subjected overnight incubation with one of the following treatment conditions: Control (untreated); MGO (1 μM); MGO (100 μM); metformin (100 μM) + MGO (100 μM); and dapagliflozin (10 μM) + MGO (100 μM). Several assays were conducted to explore the effect of the treatment groups on the SH-SY5Y cells. These included: MTT assay; LDH assay, peroxynitrite fluorescence assay, and laser scanning confocal microscopy. MGO (100 μM) led to significant cell injury and damage and significantly reduced the survival of the cells by approximately 50-75 %, associated with significant increase in peroxynitrite. The addition of metformin (100 μM) or dapagliflozin (10 μM) represented significant protective effects on the cells and prevented the cell damage caused by the high MGO concentration. As a result, the findings of this research reveal that MGO-induced cell damage may partly be mediated by the generation of peroxynitrite, while the antidiabetic agents such as metformin and dapagliflozin prevent brain cell death, which potentially may play prophylactic roles against the risk of dementia in diabetic patients.

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.

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.

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.

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.

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.

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.

Hepatic fibrosis (HF) is caused by persistent inflammation, which is closely associated with hepatic oxidative stress. Peroxynitrite (ONOO-) is significantly elevated in HF, which would be regarded as a potential biomarker for the diagnosis of HF. Research has shown that ONOO- in the Golgi apparatus can be overproduced in HF, and it can induce hepatocyte injury by triggering Golgi oxidative stress. Meanwhile, the ONOO- inhibitors could effectively relieve HF by inhibiting Golgi ONOO-, but as yet, no Golgi-targetable fluorescent probe available for diagnosis and assessing treatment response of HF through sensing Golgi ONOO-. To this end, we reported a ratiometric fluorescent probe, Golgi-PER, for diagnosis and assessing treatment response of HF through monitoring the Golgi ONOO-. Golgi-PER displayed satisfactory sensitivity, low detection limit, and exceptional selectivity to ONOO-. Combined with excellent biocompatibility and good Golgi-targeting ability, Golgi-PER was further used for ratiometric monitoring the Golgi ONOO- fluctuations and screening of ONOO- inhibitors from polyphenols in living cells. Meanwhile, using Golgi-PER as a probe, the overexpression of Golgi ONOO- in HF and the treatment response of HF to the screened rosmarinic acid were precisely visualized for the first time. Furthermore, the screened RosA has a remarkable therapeutic effect on HF, which may be a new strategy for HF treatment. These results demonstrated the practicability of Golgi-PER for monitoring the occurrence, development, and personalized treatment response of HF.

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-.