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Nitric oxide (NO) is an essential inorganic signaling molecule produced by constitutive NO synthase (cNOS) in the neurological system. Under pathological conditions, NO rapidly reacts with superoxide (O 2 •- ) to generate peroxynitrite (ONOO ¯ ). Elevated ONOO ¯ concentrations induce nitroxidative stress, potentially contributing to numerous pathological processes as observed in neurodegenerative diseases including Alzheimer's disease (AD). Metalloporphyrin nanosensors, (200-300 nm diameter), were applied to quantify the NO/ONOO ¯ balance produced by a single human neural progenitor cell (hNPC), in situ. These nanosensors, positioned in proximity of 4-5 ± 1 μm from the hNPCs membrane, enabled real-time measurement of NO and ONOO ¯ concentrations following calcium ionophore (CaI) stimulation. The ratio of NO to ONOO ¯ concentration ([NO]/[ONOO ¯ ]) was established for the purpose of quantifying nitroxidative stress levels. Normal hNPCs produced a maximum of 107 ± 1 nmol/L of NO and 451 ± 7 nmol/L of ONOO ¯ , yielding a [NO]/[ONOO ¯ ] ratio of 0.25 ± 0.005. In contrast, the model of the dysfunctional hNPCs, for long-term (48 h) amyloid-beta 42 (Aβ 42 ) exposure significantly altered NO/ONOO ¯ production. The NO level decreased to 14 ± 0.1 nmol/L, while ONOO ¯ increased to 843 ± 0.8 nmol/L, resulting in a 94 % reduction of the [NO]/[ONOO ¯ ] ratio to 0.016 ± 0.0001. The [NO]/[ONOO ¯ ] ratio is determined by this work as a possible biomarker of nNOS efficiency and hNPC dysfunction, with implications for neurodegenerative disorders such as AD. Promising applications in the early medical diagnosis of neurological illnesses, electrochemical metalloporphyrin nanosensors demonstrate efficacy in real-time nitroxidative stress monitoring., Competing Interests: Declaration of competing interest The authors declare no conflicts financial interests or personal relationships in this work., (Copyright © 2024. Published by Elsevier Inc.)

The impact of four clinically significant genetic variants of endothelial nitric oxide synthase (eNOS) polymorphisms on the concentrations of nitric oxide [NO] and peroxynitrite [ONOO - ] has been given scant consideration. This study utilized a [NO]/[ONOO - ] ratio to determine the extent of endothelial dysfunction caused by these variations in the eNOS gene. The single nucleotide polymorphisms (T-786C, C-665T, and Glu298Asp) and a variable number of tandem repeats (intron 4 a/b/c) were genotyped in human umbilical vein endothelial cells (HUVEC), using sanger sequencing and DNA electrophoresis, respectively. Nanosensors were used to determine the maximal [NO] and [ONOO - ], while traditional and low-temperature SDS-PAGE were used to evaluate the expression of eNOS and the eNOS dimer-to-monomer ratio, respectively. The study results indicate that the eNOS haplotype H3 (G T/C C 4a/c allele) may have a protective effect against cardiovascular disease (CVD) with the [NO]/[ONOO - ] ratio higher than 2. However, the eNOS haplotypes H2 (G T/C C 4a/b) and H5 (T T/C C 4b) increase the susceptibility to CVD with [NO]/[ONOO - ] ratio lower than 1. The results suggest that certain eNOS genetic variants may influence susceptibility to cardiovascular disease (CVD) while other variants may have a protective effect., 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: Peroxynitrite (ONOO - ) is a bioactive molecule involved in various biochemical processes, and the abnormal concentration fluctuations of ONOO - in living systems are closely associated with various diseases, including cancer. An important characteristic of the tumor microenvironment is the overexpression of ONOO - , highlighting the significance of specific detection of ONOO - in distinguishing between tumor tissue and normal tissue. A single near-infrared second window (NIR-II) molecular probe integrated fluorescence imaging and photothermal therapy can achieve precise localization and effective ablation of deep-seated tumor tissue. However, it still remains challenges. (87) RESULTS: In this study, we present a probe (BDⅡ-ONOO - ) that integrates NIR-II fluorescence imaging and photothermal therapy for the specific detection of ONOO - . The probe exhibits excellent selectivity, high sensitivity, low toxicity and high biocompatibility. In the presence of ONOO - , the probe can quickly respond to ONOO - and emit NIR-II fluorescence at 900 nm with 7.6-fold change in fluorescence intensity. In addition, the probe BDⅡ-ONOO - exhibits a high photothermal conversion efficiency of 41.6 % under 808 nm laser irradiation in the presence of ONOO - . In vivo imaging results indicate that the probe BDⅡ-ONOO - not only effectively distinguishes tumor tissue from normal tissue but also performs photothermal treatment on 4T1 tumor without apparent biological toxicity. (112) SIGNIFICANCE: This work provides insights for the future development of tumor-specific diagnostic and therapeutic approaches with good biocompatibility and deep tissue penetration. The probe described in this work may be employed as a powerful tool for the diagnosis and precise treatment of tumors in vivo. (44)., 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.)

Endoplasmic reticulum (ER) stress and autophagy (ER-phagy) occurring in nerve cells are crucial physiological processes closely associated with Alzheimer's disease (AD). Visualizing the two processes is paramount to advance our understanding of AD pathologies. Among the biomarkers identified, peroxynitrite (ONOO - ) emerges as a key molecule in the initiation and aggravation of ER stress and ER-phagy, highlighting its significance in the underlying mechanisms of the two processes. In this work, we designed and synthesized an innovative ONOO - -responsive AIEgen-based fluorescent probe (DHQM) with the ability to monitor ER stress and ER-phagy in AD model cells. DHQM demonstrated excellent aggregation-induced emission (AIE) properties, endowing it with outstanding ability for washing-free intracellular imaging. Meanwhile, it exhibited high sensitivity, remarkable selectivity to ONOO - , and exceptional ER-targeting ability. The probe was successfully applied for fluorescence imaging of ER ONOO - fluctuations to assess the ER stress status in aluminum-induced AD model cells. Our findings revealed that aluminum-induced ferroptosis, a regulated cell death process, was pivotal in the excessive ONOO - production, which in turn activated and exacerbated ER stress. Furthermore, the aluminum-stimulated ER-phagy was observed utilizing DHQM, which might be crucial in inhibiting ferroptosis and mitigating aberrant ER stress. Overall, this study not only offers valuable insights into the pathological mechanisms of AD at the ER level but also opens new potential therapeutic avenues targeting these pathways.

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.)

Ulcerative colitis (UC), often referred to as "green cancer", is a chronic inflammatory bowel disease with an unclear etiology, closely associated with the imbalance of hydrogen sulfide (H 2 S) and peroxynitrite (ONOO - ). H 2 S exhibits anti-inflammatory effects at physiological levels, but excessive concentrations can compromise the intestinal barrier, while ONOO - aggravates inflammation. To facilitate the molecular-level monitoring of these compounds in UC, we developed a novel fluorescent probe, BCH , capable of simultaneously detecting H 2 S and ONOO - via distinct fluorescent channels in a cascade mode. BCH features rapid response times (H 2 S: 6 min; ONOO - : 5 min) with well-separated emission peaks (120 nm), minimizing spectral overlap. The probe demonstrates high selectivity and good detection linearity for H 2 S (0-500 μM) and ONOO - (0-100 μM), with detection limits of 1.6 μM for H 2 S and 0.05 μM for ONOO - . BCH has been successfully applied for imaging H 2 S and ONOO - in living cells and zebrafish, enabling the sensitive detection of their fluctuations in a UC mouse model. These findings indicate that BCH is highly promising for studying the roles of H 2 S and ONOO - in UC and assessing their biological functions.

Chemotherapy is the primary treatment option for pancreatic cancer, although nanocarrier-based drug delivery systems often struggle with multiple physiological barriers, limiting their therapeutic efficacy. Here, we developed a pH/reactive oxygen species (ROS) dual-sensitive self-adaptive nanocarrier (DAT CPT ) encapsulating camptothecin (CPT), an analog of the pancreatic chemotherapeutic drug irinotecan (CPT-11), to enhance chemotherapy outcomes in orthotopic pancreatic cancer by addressing multiple physiological barriers. The nanocarrier features a peripherally positively charged arginine (Arg) residue on DAT CPT and is masked with an acid-labile 2,3-dimethylmaleic anhydride (DA) to improve circulation time. In the acidic tumor microenvironment (TME), DA dissociates, exposing arginine to facilitate nanocarrier binding and internalization of DAT CPT . Subsequently, peroxynitrite (ONOO - ) is generated by a cascade reaction between exposed Arg and ROS, which effectively activates matrix metalloproteinases (MMPs) to degrade the dense extracellular matrix (ECM) and enhance the deep accumulation and penetration of DAT CPT . Meanwhile, ONOO - inhibits tumor metastasis by influencing mitochondrial function, preventing adenosine triphosphate (ATP) production, and inhibiting ATP-dependent tumor-derived microvesicles (TMVs). This study presents a promising strategy to develop efficient nanocarriers to address multiple physiological barriers in antipancreatic cancer therapy.

pH and peroxynitrite (ONOO - ) are two critical biomarkers to unveil the corresponding status of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are closely related to Alzheimer's disease (AD). Simultaneously monitoring pH and ONOO - fluctuations in the ER and mitochondria during AD progression is pivotal for clarifying the interplay between the disorders of the two organelles and revealing AD pathogenesis. Herein, we designed and synthesized a dual-channel fluorescent probe (DCFP) to visualize pH and ONOO - in the ER and mitochondria. DCFP possessed excellent sensitivity and selectivity to pH and ONOO - without spectral crosstalk and was utilized in monitoring the two analytes within AD model cells and larval zebrafish. Importantly, DCFP could preferentially target mitochondria in normal cells and be enriched in the ER after mitochondrial depolarization. With the aid of DCFP, the slower acidification rate of the ER than that of mitochondria induced by Aβ oligomers (AβOs) was first identified, which could be ascribed to the relief of the AβOs-triggered ER stress through the Ca 2+ migration from the ER to mitochondria. Moreover, continuous exposure to AβOs led to mitochondrial Ca 2+ overload, accelerating the acidification and ONOO - overproduction within mitochondria. As a result, intracellular oxidative stress levels were elevated, further exacerbating ER stress and aggravating ER acidification in turn. The advanced understanding of the potential interplay between the ER and mitochondria in this work may offer new insights and methodologies for studying AD pathogenesis. The DCFP developed in this work could also be employed to study other diseases related to ER stress and mitochondrial dysfunction.

Peroxynitrite (ONOO - ) is a highly reactive nitrogen species that can cause significant damage to proteins, lipids, and DNA. Various enzymes, including metalloenzymes, play crucial roles in reducing ONOO - concentrations to protect cellular components. While the interaction of ONOO - with heme proteins is well known, the reduction by Cu-containing proteins is less studied. Amyloid precursor protein (APP), implicated in Alzheimer's disease, has an E2 domain that binds copper ions with a dissociation constant of K D ∼ 10 -12 M and is proposed to be involved in iron homeostasis, copper trafficking, and oxidative stress response. Our recent studies using EXAFS, UV-Vis, and EPR spectroscopy revealed a previously unidentified labile water ligand in the Cu(II) site of the E2 domain, suggesting reactivity with anionic substrates like ONOO - . Experimental data showed that Cu(I)-E2 reduces ONOO - at a significant rate (1.1 × 10 5 M -1 s -1 ), comparable to native peroxynitrite scavengers, while maintaining active site integrity through multiple redox cycles. This study further investigates the mechanism of ONOO - reduction by Cu(I)-E2 using the Griess assay, demonstrating that reduction occurs via single electron transfer, forming nitrite and nitrate. This process aligns with previous findings that Cu(I)-E2 is oxidized to Cu(II)-E2 upon ONOO - reduction. Mutations at Lys435, affecting secondary sphere interactions, revealed that factors beyond electrostatics are involved in substrate recruitment. MD simulations suggest that steric hindrance from a newly formed hydrogen bond also plays a role. Understanding ONOO - reduction by the E2 domain of APP expands our knowledge of copper proteins in mitigating oxidative stress and elucidates their physiological and pathological roles, particularly in Alzheimer's disease.

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.)

Tendinopathy is one of the most prevalent musculoskeletal disorders, significantly affecting the quality of life of patients. Treatment outcomes can be improved with an early diagnosis and timely targeted interventions. Increasing evidence indicates that ROS and endoplasmic reticulum (ER) stress play key roles in modulating the differentiation processes of tendon-derived stem cells (TDSCs), thereby contributing to the initiation and progression of tendinopathy. However, the relationship between ONOO - and the differentiation process, as well as the various stages of tendinopathy, remains unexplored. Herein, we developed two highly specific and sensitive fluorescent probes ( Rod-Cl and Rod-Br ) for detecting ONOO - in the ER. Rod-Br can detect basal levels of ONOO - in the ER of TDSCs and measure ONOO - levels in primary TDSCs stimulated by interleukin-1β over various durations, allowing for comparisons between chondrogenic and osteogenic differentiation and ER stress levels. Additionally, we examined ONOO - variations in different stages of tendinopathy and treatment rat models in vivo and discussed the potential mechanisms. This research provides a robust tool for analyzing ONOO - dynamics in the tenogenic and osteogenic differentiation of TDSCs, offering new insights into the pathophysiology and treatment of tendinopathy.

Mitochondria are the energy factory of cells and can be easily damaged by reactive oxygen species (ROS) because of the frequent occurrence of oxidative stress. Abnormality in mitophagy is often associated with many diseases including inflammation, cancer, and aging. While previously developed fluorescent probes mainly focus on detecting just ROS or mitophagy, quite rare studies have endeavored to comprehensively capture the entire mitophagic process, encompassing both the production of ROS and the induction of mitophagy. Herein, we report a new ratiometric fluorescent probe NA-DP for tracking peroxynitrite (ONOO - ) as well as the subsequent oxidative stress-induced mitophagy. To a naphthalimide-based dye, an ONOO - -responsive diphenyl phosphinate moiety and the mitochondria-targeting triphenylphosphonium group were attached. The probe showed a highly selective response to ONOO - through an addition-elimination reaction with diphenyl phosphinate. Owing to its outstanding pH stability and organelle-targeting ability, NA-DP was successfully used to detect mitophagy induced by oxidative stress after the generation of ONOO - . In the meantime, the probe was also used to track starvation-induced mitophagy and indicate that starvation-induced mitophagy is independent of ONOO - . Therefore, NA-DP has the ability to precisely track oxidative stress-induced mitophagy by distinguishing it from starvation-induced mitophagy. This study offers a new chemical tool to study the relationship between ROS generation and mitophagy.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases including cancer in a chronic setting. Peroxynitrite (PN) is a major reactive nitrogen species generated during inflammation. It produces various DNA lesions including 8-nitro-guanine which spontaneously converts into abasic sites, resulting in DNA strand breakage, and is suspected to be mutagenic. Here, we report the discovery of a previously unrecognized function of the human repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate spontaneously reacts with 8-nitro-guanine in DNA to form a stable DNA-protein cross-link (DPC). Interestingly, the process of DPC formation provided protection from PN-mediated genome instability, growth arrest, and apoptosis. The Cys145 mutant of hAGT failed to form a DPC and was unable to protect cells from inflammation-associated PN-mediated cytotoxicity. Gel-shift, dot blot, and UV-vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual-function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs are known to be detrimental to the cell, recently, multiple pathways have been identified in normal cells for their repair.

Peroxynitrite (ONOO - ) is a critical biomarker associated with a wide array of diseases including cancer, inflammatory conditions, and neurodegenerative disorders. This study introduces an innovative chemiluminescence nanoprobe (CLNP) based on a bicyclic dioxetane structure, designed for highly sensitive and specific in vivo imaging of ONOO - . Our CLNP demonstrates exceptional capabilities in generating high-contrast imaging of disease lesions, with applications verified across tumor models, acute inflammation, and acute liver injury scenarios. Key findings highlight the probe's rapid response to oxidative species, superior tissue penetration, and high signal-to-noise ratio, underscoring its potential for real-time diagnostic applications. This work represents an important advance in the field of diagnostic imaging using CL probes, offering promising avenues for the early detection and treatment of ONOO - -related pathologies.

The cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L-tryptophan to 4-nitro-L-tryptophan, using nitric oxide (NO) and dioxygen (O 2 ) as co-substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)-peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)Fe III (O 2 - ⋅)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4-nitroindole, the product analogous to that obtained with TxtE. Moreover, 15 N and 18 O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings., (© 2024 Wiley-VCH GmbH.)

Overcoming the resistance of tumor cells to apoptosis and immunosuppression is an important challenge to improve tumor immunotherapy. Non-apoptotic death mode of ferroptosis has been regarded as a new strategy to enhance tumor immunotherapy against drug-resistant cancers. The lethal accumulation of lipid peroxides (LPO) determines the progress of ferroptosis. The high susceptibleness of ferroptosis provides an opportunity for combating triple-negative breast cancer. Reactive nitrogen species (RNS) produced by nitric oxide (NO) and reactive oxygen species (ROS) is more lethal than ROS for tumor cells. Herein, an RNS-mediated immunotherapy strategy for inducing ferroptosis pathway is proposed by improving LPO accumulation, and constructed a multifunctional liposome (Lipo-MT-SNAP) comprised of peroxynitrite (ONOO - ) generator, tumor targeted group, inhibiting glutathione peroxidase 4 (GPX4), and basic units (dipalmitoyl phosphatidylcholine and cholesterol). The significant enhancement of LPO resulted from the intense oxidative damage of ONOO - impaired synthesis of GPX4 by depleting glutathione, which further amplified ferroptosis and triggered immunogenic cell death. In vivo, RNS-mediated photoimmunotherapy can promote polarization of M2 to M1 macrophages and dendritic cells maturation, further infiltrate T cells, regulate the secretion of inflammatory factors, and reprogram the tumor microenvironment. The powerful RNS-mediated ferroptosis induces strong immunogenicity and effectively inhibit tumor proliferation., (© 2024 Wiley‐VCH GmbH.)

Cerebral ischemia-reperfusion injury produces excessive reactive oxygen and nitrogen species, including superoxide, nitric oxide, and peroxynitrite (ONOO - ). We recently developed a new ONOO - -triggered metal-free carbon monoxide donor (PCOD585), exhibiting a notable neuroprotective outcome on the rat middle cerebral artery occlusion model and rendering an exciting intervention opportunity toward ischemia-induced brain injuries. However, its therapeutic mechanism still needs to be addressed. In the pharmacological study, we found PCOD585 inhibited neuronal Bcl2/Bax/caspase-3 apoptosis pathway in the peri-infarcted area of stroke by scavenging ONOO - . ONOO - scavenging further led to decreased Acyl-CoA synthetase long-chain family member 4 and increased glutathione peroxidase 4, to minimize lipoperoxidation. Additionally, the carbon monoxide release upon the ONOO - reaction with PCOD585 further inhibited the neuronal Iron-dependent ferroptosis associated with ischemia-reperfusion. Such a synergistic neuroprotective mechanism of PCOD585 yields as potent a neuroprotective effect as Edaravone. Additionally, PCOD585 penetrates the blood-brain barrier and reduces the degradation of zonula occludens-1 by inhibiting matrix metalloproteinase-9, thereby protecting the integrity of the blood-brain barrier. Our study provides a new perspective for developing multi-functional compounds to treat ischemic stroke., Competing Interests: Declarations. Ethics Approval: The animal experiment was conducted by the national and institutional guidelines on ethics and biosafety, and the protocol was approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (License ID: 201907W079). Consent to Participate: Not applicable. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Inflammation of the ileum, or ileitis, is commonly caused by Crohn's disease (CD) but can also accompany ulcerative colitis (backwash ileitis), infections or drug-related damage. Oxidative tissue injury triggered by reactive oxygen species (ROS) is considered part of the ileitis etiology. However, not only elevated ROS but also permanently decreased ROS are associated with inflammatory bowel disease (IBD). While very early onset IBD (VEO-IBD) is associated with a spectrum of NOX1 variants, how NOX1 inactivation contributes to disease development remains ill-defined. Besides propagating signaling responses, NOX1 provides superoxide for peroxynitrite formation in the epithelial barrier. Here we report that NOX4, an H 2 O 2 -generating NADPH oxidase with documented tissue protective effects in the intestine and other tissues, limits the generation of ileal peroxynitrite by NOX1/NOS2. Deletion of NOX4 leads to persistent peroxynitrite excess, hyperpermeability, villus blunting, muscular hypertrophy, chemokine/cytokine upregulation and dysbiosis. Conversely, SAMP1/YitFc mice, a CD-like ileitis model, showed age-dependent NOX1/NOS2 downregulation preventing ileal peroxynitrite formation in homeostasis and LPS-induced acute inflammation. Deficiency in NOX1 correlated with the upregulation of antimicrobial peptides, suggesting that ileal peroxynitrite acts as chemical barrier and microbiota modifier in the ileum., 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 The Author(s). Published by Elsevier Inc. All rights reserved.)

Peroxynitrite (ONOO - ) is a quintessential reactive oxygen species (ROS) and reactive nitrogen species (RNS), renowned for its potent oxidizing and nitrifying capabilities. Under normal physiological conditions, a baseline level of ONOO - is present within the body. However, its production escalates significantly in response to oxidative stress. ONOO - is highly reactive with various biomolecules in vivo , particularly proteins, lipids, and nucleic acids, thereby playing a role in a spectrum of physiological and pathological processes, such as inflammation, cancer, neurodegenerative diseases, and cardiovascular diseases. Consequently, detecting ONOO - in vivo is of paramount importance for understanding the etiology of various diseases and facilitating early diagnosis. Fluorescent probes have become a staple in the identification of biomolecules due to their ease of use, convenience, and superior sensitivity and specificity. This review highlights the recent advancements in the development of fluorescent probes for the detection of ONOO - in diverse disease models and provides an in-depth examination of their design and application.

Radiotherapy, employing high-energy rays to precisely target and eradicate tumor cells, plays a pivotal role in the treatment of various malignancies. Despite its therapeutic potential, the effectiveness of radiotherapy is hindered by the tumor's inherent low radiosensitivity and the immunosuppressive microenvironment. Here we present an innovative approach that integrates peroxynitrite (ONOO - )-mediated radiosensitization with the tumor-associated neutrophils (TANs) polarization for the reversal of immunosuppressive tumor microenvironment (TME), greatly amplifying the potency of radiotherapy. Our design employs X-ray-activated lanthanide-doped scintillators (LNS) in tandem with photosensitive NO precursor to achieve in-situ ONOO - generation. Concurrently, the co-loaded TGF-β inhibitor SB525334, released from the LNS-RS nanoplatform in response to the overexpressed GSH in tumor site, promotes the reprogramming of TANs from N2 phenotype toward N1 phenotype, effectively transforming the tumor-promoting microenvironment into a tumor-inhibiting state. This 'one-two punch' therapy efficiently trigger a robust anti-tumor immune response and exert potent therapeutic effects in orthotopic colorectal cancer and melanoma mouse model. Meanwhile, it also significantly prevents liver metastasis and recurrence in metastatic colorectal cancer. The development of X-ray-controlled platforms capable of activating multiple therapeutic modalities may accelerate the clinical application of radiotherapy-based collaborative therapy., 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.)

Drug-induced liver injury (DILI) is a crucial factor that poses a significant threat to human health. DILI process leads to the changes of reactive oxygen species and reactive nitrogen species content in cells, which leads to oxidative and nitrosative stress in cells. However, the high reactivity of hypochlorous acid (HOCl) and peroxynitrite (ONOO⁻), combined with a lack of in situ imaging techniques, has hindered a detailed understanding of their roles in DILI. Therefore, this paper reports a novel sequence-activatable dual-locked molecular probe HA-P3 for the identification and imaging of two DILI-related biomarkers. First, HA-P3 selectively reacts with reactive oxygen species HOCl to leave the recognition receptor diethyl thiocarbamate to form HA-P2. Subsequently, HA-P2 reacts with ONOO⁻, liberating the fluorophore 4-hydroxy-1,8-naphthalimide, which emits a strong fluorescence signal. The two-step reaction effectively reduces the probability of false positive in predicting DILI. HA-P3 achieved the sensitive detection of HOCl and ONOO - in different cells and zebrafish. Furthermore, HA-P3 can distinguish between normal liver cells and hepatoma cells and monitored the elevated levels of HOCl and ONOO⁻ during acetaminophen (APAP)-induced cellular damage. It is worth noting that in the APAP-induced mouse model, the positive correlation between HOCl and ONOO - and DILI was revealed, providing strong direct evidence for the relationship between oxidative/nitrosative stress and 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.)

Peroxynitrite (ONOO - ) is a transient and reactive oxidant with significant roles in numerous biological processes. Research has established a correlation between excessive mitochondrial ONOO - production and various diseases. As such we developed a mitochondria-targeting, fluorescence-based ratiometric probe using a boronate group for ONOO - recognition and coumarin as the fluorophore. The probe exhibited a 615 nm far-red emission, and a new fluorescence emission at 475 nm developed upon adding ONOO - .The probe possessed high sensitivity and selectivity for ONOO - detection with distinct ratiometric fluorescent output and a low detection limit of 11 nM. Significantly, Probe 1 could monitor ONOO - level fluctuations in biological systems due to its superior spectral properties and low toxicity. Notably, probe 1 has been effectively used for imaging in live HepG2 cells, zebrafish, Arabidopsis thaliana, and liver injury mouse tissues., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Revascularization is crucial for treating myocardial infarction (MI). Nitric oxide (NO), at an appropriate concentration, is recognized as an ideal and potent pro-angiogenic factor. However, the application of NO in the treatment of MI is limited. Improper NO supplementation is harmful to revascularization because NO is converted into harmful peroxynitrite (ONOO - ) in MI tissues with high reactive oxygen species (ROS) levels. We overcome these obstacles by embedding biliverdin and NO into Prussian blue (PB) nanolattices to obtain an ONOO - -free NO-embedded nanomedicine (OFEN). Unlike previous NO donors, OFEN provides NO stably and spontaneously for a longer time (>7 days), which makes it possible to maintain a stable concentration of NO, suitable for angiogenesis, through dose optimization. More importantly, based on the synergy between PB and biliverdin, OFEN converts ROS into beneficial O 2 and inhibits the production of ONOO - from the source. OFEN specifically targets MI tissues and achieves sustained and stable NO delivery at the MI site. OFEN effectively promotes revascularization in the MI tissue, significantly reduces myocardial death and fibrosis, and ultimately promotes the complete recovery of cardiac function. Our strategy provides a promising approach for the treatment of myocardial and other ischemic diseases.

Ulcerative colitis (UC) is an inflammatory disease that leads to the overexpression of peroxynitrite (ONOO - ). Up to now, it has been a challenge to design a colon-targeted fluorescent probe with a large emission shift that can detect and image ONOO - in UC mice. Here, a fluorophore (pyran-coumarin) is linked with cholic acid to develop a colon-targeted fluorescent probe ( CPC ) for the detection of ONOO - . The fluorescent probe showed strong near-infrared emission at 725 nm. After reacting with ONOO - , it can be observed that the fluorescence intensity at 725 nm decreases obviously, and the signal at 490 nm increases clearly with an obvious emission shift (235 nm). This response mechanism causes the probe to have good sensitivity and selectivity. Additionally, the probe CPC shows good targeting ability for the cells with overexpressingTGR5 receptors and displays good results for detecting exogenous and endogenous ONOO - in the cells. More importantly, the probe CPC can be especially assembled in the colon site to distinguish normal and UC mice and provide significant information for the diagnosis of UC.

Peroxynitrite (ONOO - ), as an important reactive nitrogen species, plays a pivotal role in the regulation of intracellular redox homeostasis, signal transduction, cell growth and metabolism, and other physiological processes. Organelles are important for regulating ONOO - , and the dysregulation of ONOO - in organelles is closely related to various diseases. Therefore, it is essential to monitor ONOO - in cellular organelles, including mitochondria, lysosome, endoplasmic reticulum (ER), Golgi apparatus, and lipid droplets. However, the latest advances in organelle-targeting ONOO - fluorescent probes have not been reviewed systematically. In this review, we focus on the design, sensing mechanism, and organelle-targeting imaging applications of ONOO - fluorescent probes that were reported since 2018. This review will help to facilitate the comprehension of organelle-targeting fluorescent probes for the detection of ONOO - .

Monitoring peroxynitrite (ONOO - ) fluctuations is particularly important for assessing pathological progression and oxidative damage due to their crucial role in maintaining the redox balance of organisms. However, due to the lack of efficient tools for differentially monitoring ONOO - fluctuations at different concentration ranges in vivo, the precise detection of endogenous ONOO - fluctuations under pathological conditions in living systems remains challenging. Herein, we rationally designed a double-enhanced emission cascade activatable near-infrared (NIR) fluorescent probe ( B-TCF ) for the measurement of ONOO - , which consists of a borate ester response group and a malononitrile hemicyanine fluorophore. Especially, after sequential oxidative hydrolysis of the borate ester group and xanthene skeleton, B-TCF exhibited a sequentially double-enhanced NIR emission response at 776 and 625 nm for different ONOO - concentration ranges. Moreover, B-TCF revealed excellent and promising performance for ONOO - in terms of high selectivity, sensitivity, and reaction rate ( k = 28.2 M -1 s -1 ). Motivated by the two-step emission signal enhancement and large wavelength shift in the NIR region, B-TCF enabled discriminative imaging of ONOO - with the low and high concentrations in living cells. Importantly, B-TCF was successfully applied for assessing the pathological progression of isoniazid and acetaminophen-induced liver damage in vivo by detecting the endogenous different ONOO - levels. Overall, this study not only demonstrates the first double-enhanced emission cascade activatable NIR fluorescent probe for measuring the dynamic variation of ONOO - in related diseases but also shows great potential as an effective molecular tool for evaluating the various stages of drug-induced liver damage.

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 BaTiO 3 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.

This study describes the synthesis and characterization of a novel near-infrared (NIR) fluorescent probe RBNE based on a hybrid rhodamine dye, which shows excellent optical capability for detecting and imaging ONOO - in necrotizing enterocolitis (NEC) mouse model. The probe RBNE undergoes hydrazine redox-process, and subsequently the spirocyclic structure's opening, resulting in a turn-on fluorescence emission with the presence of ONOO - , which exhibits several excellent features, including a significant Stokes shift of 108 nm, near-infrared emission at 668 nm, a lower detection limit of 56 nM, low cytotoxicity, and excellent imaging ability for ONOO - both in vitro and in vivo. The presented study introduces a novel optical tool that has the potential to significantly advance our understanding of peroxynitrite (ONOO - ) behaviors in necrotizing enterocolitis (NEC)., 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.)

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.)

Drug-induced liver injury (DILI) poses a severe threat to public health. Endoplasmic reticulum (ER) stress contributes significantly to DILI pathogenesis, with peroxynitrite (ONOO - ) identified as a pivotal indicator. However, the temporal and spatial fluctuations of ONOO - associated with ER stress in the pathogenesis of DILI remain unclear. Herein, a novel ER-specific near-infrared (NIR) probe (QM-ONOO) with aggregation-induced emission (AIE) features for monitoring ONOO - fluctuations in DILI was elaborately constructed. QM-ONOO exhibited excellent ER-targeting specificity, a large Stoke's shift, and a low detection limit (26.9 nM) toward ONOO - . QM-ONOO performed well in imaging both exogenous and endogenous ONOO - in HepG2 cells. Furthermore, molecular docking calculations validated the ER-targeting mechanism of QM-ONOO. Most importantly, using this probe allowed us to intuitively observe the dynamic fluctuations of ONOO - during the formation and remediation processes of DILI in the acetaminophen (APAP)-induced mouse model. Consequently, this work provides a promising tool for in-depth research of ONOO - associated pathological processes in 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.)

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 FTSNPEVR nitro Y 198 EV, 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., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD + as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S -nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO - ) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO - (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO - can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.

Chemodynamic therapy (CDT) can induce cancer cell death through hydroxyl radicals (·OH) generated from Fenton or Fenton-like reactions. Compared with traditional therapies, CDT effectively overcomes inevitable drug resistance and exhibits low side effects. However, clinical application still faces challenges, primarily due to insufficient ·OH generation and the short-lifetime of ·OH in vivo. To address these challenges, we developed a peroxynitrite (ONOO - )-based CDT nanodrug (DOX@PMOF) composed of MOF-199, NO donor (PArg), and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) activator (doxorubicin, DOX). In DOX@PMOF, MOF-199 serves as both a carrier for loading DOX and a source of Cu + for triggering CDT. Upon uptake by cancer cells, the high concentration of glutathione (GSH) reduces MOF-199 to Cu + , which then reacts with H 2 O 2 to generate ·OH. Moreover, the released DOX upregulates NOX4 expression, leading to the elevated H 2 O 2 level and thereby promoting a high-efficiency Fenton-like reaction for sufficient ·OH generation. Subsequently, PArg generates abundant NO in response to the tumor microenvironment, leading to a cascade of NO and ·OH for the in situ synthesis of ONOO - . ONOO - is more toxic and has a longer lifetime and diffusion distance than ·OH, resulting in a more effective CDT treatment. To further enhance the in vivo therapeutic effect, we coated DOX@PMOF with a homologous cell membrane to form an active tumor-targeting nanodrug (DOX@MPMOF), which has demonstrated the ability to effectively inhibit tumor growth and metastasis while exhibiting good biosafety.

Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO - in stimulated cells, both ex vivo and in vivo, using murine models of acute lung injury (ALI). For complete details on the use and execution of this protocol, please refer to Gutierrez and Aggarwal et al. 1 ., Competing Interests: Declaration of interests E.C.I. is an inventor in a patent application filed by Rutgers University on the subject of this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide ( • NO) and superoxide (O 2 •- ) produced by phagocytes contributes to its success as a human pathogen. Recombination of • NO and O 2 •- generates peroxynitrite (ONOO - ), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward • NO and O 2 •- 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 O 2 •- , 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)., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Migraine research has highlighted the pivotal role of nitric oxide (NO) in migraine pathophysiology. Nitric oxide donors such as glyceryl trinitrate (GTN) induce migraine attacks in humans, whereas spontaneous migraine attacks can be aborted by inhibiting NO production. The present study aimed to investigate how GTN triggers migraine through its three nitric oxide synthase (NOS) isoforms (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) via a suspected feed-forward phenomenon., Methods: Migraine-relevant hypersensitivity was induced by repeated injection of GTN in an in vivo mouse model. Cutaneous tactile sensitivity was assessed using von Frey filaments. Signaling pathways involved in this model were dissected using non-selective and selective NOS inhibitors, knockout mice lacking eNOS or nNOS and their wild-type control mice. Also, we tested a soluble guanylate cyclase inhibitor and a peroxynitrite decomposition catalyst (N total  = 312)., Results: Non-selective NOS inhibition blocked GTN-induced hypersensitivity. This response was partially associated with iNOS, and potentially nNOS and eNOS conjointly. Furthermore, we found that the GTN response was largely dependent on the generation of peroxynitrite and partly soluble guanylate cyclase., Conclusions: Migraine-relevant hypersensitivity induced by GTN is mediated by a possible feed-forward phenomenon of NO driven mainly by iNOS but with contributions from other isoforms. The involvement of peroxynitrite adds to the notion that oxidative stress reactions are also involved., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Reactive oxygen and nitrogen species are small reactive molecules derived from elements in the air─oxygen and nitrogen. They are produced in biological systems to mediate fundamental aspects of cellular signaling but must be very tightly balanced to prevent indiscriminate damage to biological molecules. Small molecule probes can transmute the specific nature of each reactive oxygen and nitrogen species into an observable luminescent signal (or even an acoustic wave) to offer sensitive and selective imaging in living cells and whole animals. This review focuses specifically on small molecule probes for superoxide, hydrogen peroxide, hypochlorite, nitric oxide, and peroxynitrite that provide a luminescent or photoacoustic signal. Important background information on general photophysical phenomena, common probe designs, mechanisms, and imaging modalities will be provided, and then, probes for each analyte will be thoroughly evaluated. A discussion of the successes of the field will be presented, followed by recommendations for improvement and a future outlook of emerging trends. Our objectives are to provide an informative, useful, and thorough field guide to small molecule probes for reactive oxygen and nitrogen species as well as important context to compare the ecosystem of chemistries and molecular scaffolds that has manifested within the field.

There are no licensed vaccines for human cytomegalovirus (HCMV), and current antiviral drugs that target viral proteins are toxic and prone to resistance. Targeting host pathways essential for virus replication provides an alternate strategy that may reduce opportunities for drug resistance to occur. Oxidative stress is triggered by numerous viruses including HCMV. Peroxynitrite is a reactive nitrogen species that is formed during oxidative stress. Herein, we identified that HCMV rapidly induces the generation of intracellular peroxynitrite upon infection in a manner partially dependent upon xanthine oxidase generation. Peroxynitrite promoted HCMV infection in both cell-free and cell-associated infection systems in multiple cell types. Inhibiting peroxynitrite within the first 24 hours of infection prevented HCMV replication and peroxynitrite promoted cell entry and pp65 translocation into the host cell nuclei. Furthermore, using the murine cytomegalovirus model, we demonstrated that antagonizing peroxynitrite significantly reduces cytomegalovirus replication and pathogenesis in vivo . Overall, our study highlights a proviral role for peroxynitrite in CMV infection and implies that RNS and/or the mechanisms that induce their production could be targeted as a novel strategy to inhibit HCMV infection., Importance: Human cytomegalovirus (HCMV) causes significant disease in individuals with impaired or immature immune systems, such as transplant patients and after congenital infection. Antiviral drugs that target the virus directly are toxic and are susceptible to antiviral drug resistance due to virus mutations. An alternate strategy is to target processes within host cells that are required by the virus for replication. Herein, we show that HCMV infection triggers a highly reactive molecule, peroxynitrite, during the initial stages of infection. Peroxynitrite was required for the initial entry of the virus into the cell and promotes virus replication in multiple cell types, suggesting a broad pro-viral function. Importantly, targeting peroxynitrite dramatically inhibited cytomegalovirus replication in cells in the laboratory and in mice, suggesting that therapeutic targeting of this molecule and/or the cellular functions it regulates could represent a novel strategy to inhibit HCMV infection., Competing Interests: The authors declare no conflict of interest.

Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of β-amyloid (Aβ), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aβ nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aβ 1-42 . Photonitrated Aβ 1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aβ 1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aβ 1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aβ occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aβ 1-42 .

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., 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.)

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome.

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.

Background: Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium., Methods and Results: In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold ( P <0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio ( P <0.05)., Conclusions: These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.

Ferroptosis is a type of lipid peroxidation-induced apoptosis brought on by imbalances in iron metabolism and redox. It involves both the thiol-associated anti-ferroptosis pathway and the excessive buildup of reactive oxygen species (ROS), which stimulates the ferroptosis pathway. Determining the precise control mechanism of ferroptosis requires examining the dynamic connection between reactive sulfur species (RSS) and ROS. Cysteine (Cys) and peroxynitrite (ONOO - ) are highly active redox species in organisms and play dynamic roles in the ferroptosis process. In this study, a coumarin dye was conjugated with specific response sites for Cys and ONOO - , enabling the simultaneous detection of Cys and ONOO - through the green and red fluorescence channels, respectively (λ em  = 498 nm for Cys and λ em  = 565 nm for ONOO - ). Using the probe LXB, we monitored the changes in Cys and ONOO - levels in the ferroptosis pathway induced by erastin. The results demonstrate a significant generation of ONOO - and a noticeable decrease in intracellular Cys levels at the beginning upon erastin treatment and finally maintains a relatively low level. This study presents the first probe to investigate the intracellular redox modulation and control between Cys and ONOO - during ferroptosis, providing valuable insights into the potential mutual correlation between Cys and ONOO - in this process., 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.)

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.)

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.

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.)