Mitochondrial alternative oxidase pathway helps in nitrooxidative stress tolerance in germinating chickpea.

Mitochondrial alternative oxidase (AOX) is an important protein that can help in regulating reactive oxygen species and nitric oxide in plants. The role of AOX in regulation of nitro-oxidative stress in chickpea is not known. Using germinating chickpea as a model system, we investigated the role of AOX in nitro-oxidative stress tolerance. NaCl treatment was used as an inducer of nitro-oxidative stress. Treatment of germinating seeds with 150 mM NaCl led to reduced germination and radicle growth. The AOX inhibitor SHAM caused further inhibition of germination, and the AOX inducer pyruvate improved growth of the radicle under NaCl stress. Isolated mitochondria from germinated seeds under salt stress not only increased AOX capacity but also enhanced AOX protein expression. Measurement of superoxide levels revealed that AOX inhibition by SHAM can enhance superoxide levels, whereas the AOX inducer pyruvate reduced superoxide levels. Measurement of NO by gas phase chemiluminescence revealed enhanced NO generation in response to NaCl treatment. Upon NaCl treatment there was enhanced tyrosine nitration, which is an indicator of nitrosative stress response. Taken together, our results revealed that AOX induced under salinity stress in germinating chickpea can help in mitigating nitro-oxidative stress, thereby improving germination.

Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Novel angiogenesis inhibitors with superoxide anion radical amplification effect: Surmounting the Achilles' heels of angiogenesis inhibitors and photosensitizers.

Angiogenesis inhibitors and photosensitizers are pivotal in tumor clinical treatment, yet their utilization is constrained. Herein, eleven novel angiogenesis inhibitors were developed through hybridization strategy to overcome their clinical limitations. These title compounds boast excitation wavelengths within the "therapeutic window", enabling deep tissue penetration. Notably, they could generate superoxide anion radicals via the Type I mechanism, with compound 36 showed the strongest superoxide anion radical generating capacity. Biological evaluation demonstrated remarkable cellular activity of all the title compounds, even under hypoxic conditions. Among them, compound 36 stood out for its superior anti-proliferative activity in both normoxic and hypoxic environments, surpassing individual angiogenesis inhibitors and photosensitizers. Compound 36 induced cell apoptosis via superoxide anion radical generation, devoid of dark toxicity. Molecular docking revealed that the target-recognizing portion of compound 36 was able to insert into the ATP binding pocket of the target protein similar to sorafenib. Collectively, our results suggested that hybridization of angiogenesis inhibitors and photosensitizers was a potential strategy to address the limitations of their clinical use.

Annual rhythm in immune functions of blood leucocytes in an ophidian, Natrix piscator.

Annual variations in animal's physiological functions are an essential strategy to deal with seasonal challenges which also vary according to the time of year. Information regarding annual adaptations in the immune-competence to cope with seasonal stressors in reptiles is scarce. The present research plan was designed to analyze the presence of circannual immune rhythms in defense responses of the leucocytes in an ophidian, Natrix piscator. Peripheral blood leucocytes were obtained, counted, and superoxide anion production, neutrophil phagocytosis, and nitrite release were tested to assess the innate immune functions. Peripheral blood lymphocytes were separated by centrifugation (utilizing density gradient) and the cell proliferation was measured. The Cosinor rhythmometry disclosed the presence of significant annual rhythms in the number of leucocytes, superoxide anion production, nitric oxide production, and proliferation of stimulated lymphocytes. The authors found that respiratory burst activity and proliferative responses of lymphocytes were crucial immune responses that showed the annual rhythm. It was summarized that the immune function of the N. piscator is a labile attribute that makes the animal competent to cope with the seasonal stressor by adjustment in the potency of response.

Superoxide dismutase nanozymes: current status and future perspectives on brain disease treatment and diagnosis.

Superoxide dismutase (SOD) is an important metalloenzyme that catalyzes the dismutation of superoxide radicals (O2˙-) into hydrogen peroxide (H2O2) and oxygen (O2). However, the clinical application of SOD is severely limited due to its structural instability and high cost. Compared with natural enzymes, nanomaterials with enzyme-like activity, nanoenzymes, are more stable, economical and easy to modify and their activity can be adjusted. Certain nanozymes that exhibit SOD-like activity have been created and shown to help prevent illnesses brought about by oxidative stress. These SOD-like nanozymes offer an important solution to the problems associated with the clinical application of SOD. In this review, we briefly introduce neurodegenerative diseases, present the research progress of SOD-like nanoenzymes in the diagnosis and treatment of brain diseases, review their mechanism of action in the treatment and diagnosis of brain diseases, and discuss the shortcomings of the current research with a view to providing a reference for future research. We expect more highly active SOD-like nanoenzymes to be developed with a wide range of applications in the diagnosis and treatment of brain diseases.

Inhibition of superoxide and iNOS augment cutaneous nitric oxide-dependent vasodilation in non-Hispanic black young adults.

We assessed the combined effect of superoxide and iNOS inhibition on microvascular function in non-Hispanic Black and non-Hispanic White participants (n = 15 per group). Participants were instrumented with four microdialysis fibers: (1) lactated Ringer's (control), (2) 10 µM tempol (superoxide inhibition), (3) 0.1 mM 1400 W (iNOS inhibition), (4) tempol + 1400 W. Cutaneous vasodilation was induced via local heating and NO-dependent vasodilation was quantified. At control sites, NO-dependent vasodilation was lower in non-Hispanic Black (45 ± 9% NO) relative to non-Hispanic White (79 ± 9% NO; p < 0.01; effect size, d = 3.78) participants. Tempol (62 ± 16% NO), 1400 W (78 ± 12% NO) and tempol +1400 W (80 ± 13% NO) increased NO-dependent vasodilation in non-Hispanic Black participants relative to control sites (all p < 0.01; d = 1.22, 3.05, 3.03, respectively). The effect of 1400 W (p = 0.04, d = 1.11) and tempol +1400 W (p = 0.03, d = 1.22) was greater than tempol in non-Hispanic Black participants. There was no difference between non-Hispanic Black and non-Hispanic White participants at 1400 W or tempol + 1400 W sites. These data suggest iNOS has a greater effect on NO-dependent vasodilation than superoxide in non-Hispanic Black participants.

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by human platelets, understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually, identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Singlet Oxygen and Superoxide Anion Radical Detection by EPR Spin Trapping in Thylakoid Preparations.

Reactive oxygen species (ROS) are produced by energy transfer and electron transport in plant chloroplast thylakoids at non-toxic levels under normal growth conditions, but at threatening levels under adverse or fluctuating environmental conditions. Among chloroplast ROS, singlet oxygen and superoxide anion radical, respectively, produced by photosystem II (PSII) and PSI, are known to be the major ROS under several stress conditions. Both are very unlikely to diffuse out of chloroplasts, but they are instead capable of triggering ROS-mediated chloroplast operational retrograde signalling to activate defence gene expression in concert with hormones and other molecular compounds. Therefore, their detection, identification and localization in vivo or in biological preparations is a priority for a deeper understanding of their role in (concurrent) regulation of plant growth and defence responses. Here, we present two EPR spin traps, abbreviated as TEMPD-HCl and DEPMPO, to detect and identify ROS in complex systems, such as isolated thylakoids, together with some hints and cautions to perform reliable spin trapping experiments.

Identification of Superoxide Dismutase (SOD) Isozymes in Plant Tissues.

Superoxide and hydrogen peroxide are reactive oxygen species (ROS) involved in the oxidation of multiple biological molecules and the signaling processes during plant growth and stress response. Thus, control of ROS is fundamental for cell survival and development, with superoxide dismutase (EC 1.15.1.1, SOD) being one of the main enzymes involved. Different isoforms of SOD catalyze the dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2) and oxygen (O2), such as Mn-SODs, Cu,Zn-SODs, and Fe-SODs. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) combined with a specific staining method for SOD activity, the protocol describes the identification of different SOD isozymes, based on their differential inhibition by KCN and H2O2, in different organs and plant species such as pea (Pisum sativum L.) leaves and pepper (Capsicum annuum L.) fruits.

Role of biochar in superoxide-dominated dye degradation in catalyst-activated peroxymonosulphate process.

In recent times, the application of biochar (BC) as an upcoming catalyst for the elimination of recalcitrant pollutants has been widely explored. Here, an iron loaded bamboo biochar activated peroxymonosulphate (PMS) process was tested for removing Congo red (CR) dye from water medium. The catalyst was synthesized using a green synthesis method using neem extracts and characterized using SEM, FTIR, and XRD. The effects of various operating parameters, including solution pH, catalyst dosage, and pollutant dosage, on dye degradation efficiency were examined. The results showed that at the optimized conditions of 300 mg L-1 PMS concentration, 200 mg L-1 catalyst dosage, and pH 6, about 89.7% of CR dye (initial concentration 10 ppm) was removed at 60 min of operation. Scavenging experiments revealed the significant contribution of O2•-, •OH, and 1O2 for dye degradation, with a major contribution of O2•-. The activation of PMS was mainly done by biochar rather than iron (loaded on biochar). The catalyst was highly active even after four cycles.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.

Therapeutic nuclear magnetic resonance and intermittent hypoxia trigger time dependent on/off effects in circadian clocks and confirm a central role of superoxide in cellular magnetic field effects.

Cellular magnetic field effects are assumed to base on coherent singlet-triplet interconversion of radical pairs that are sensitive to applied radiofrequency (RF) and weak magnetic fields (WEMFs), known as radical pair mechanism (RPM). As a leading model, the RPM explains how quantum effects can influence biochemical and cellular signalling. Consequently, radical pairs generate reactive oxygen species (ROS) that link the RPM to redox processes, such as the response to hypoxia and the circadian clock. Therapeutic nuclear magnetic resonance (tNMR) occupies a unique position in the RPM paradigm because of the used frequencies, which are far below the range of 0.1-100 MHz postulated for the RPM to occur. Nonetheless, tNMR was shown to induce RPM like effects, such as increased extracellular H2O2 levels and altered cellular bioenergetics. In this study we compared the impact of tNMR and intermittent hypoxia on the circadian clock, as well as the role of superoxide in tNMR induced ROS partitioning. We show that both, tNMR and intermittent hypoxia, exert on/off effects on cellular clocks that are dependent on the time of application (day versus night). In addition, our data provide further evidence that superoxide plays a central role in magnetic signal transduction. tNMR used in combination with scavengers, such as Vitamin C, led to strong ROS product redistributions. This discovery might represent the first indication of radical triads in biological systems.

Simple, ultrasensitive detection of superoxide anion radical mutations in melanoma mice with SERS microneedles.

Elevated levels of superoxide anion radicals (O2·-) have been implicated in the pathogenesis of a variety of diseases, such as cancer, inflammatory diseases and autoimmune diseases. To determine the O2·- concentration for assisting disease detection, a method based on surface-enhanced Raman scattering (SERS) combined with transparent polymer microneedles has been developed. Photocrosslinked NOA61 is used to prepare microneedles with sulfhydryl group, which can contribute to anchor gold nanoparticles (Au NPs) functionalized by p-mercaptobenzoic acid (PATP). This work successfully constructed SERS microneedles for in situ detection. A REDOX reaction occurred between PATP and O2·-, resulting in the formation of dimethylaminoborane (DMAB) and a subsequent change in Raman signal. Based on the quantitative relationship between the change of peak area ratio at 1042 cm-1 and 1077 cm-1 and the concentration change of O2·-, a standard curve with a linear range of 0-480 ng/mL was constructed. The SERS microneedles were effectively employed to track melanoma progression in mice, establishing a fundamental correlation between O2·- concentration and melanoma stage, as confirmed by ELISA. The benefits of this approach, including convenience, in situ applicability, and low cost, are anticipated to offer novel insights for non-invasive in situ detection, potentially enhancing disease monitoring and diagnosis.

Role of reactive carbonyls and superoxide radicals in protein damage by cigarette smoke extracts: Comparison of Heat-not-Burn e-cigarettes to conventional cigarettes.

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single "Heat-not-Burn" e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from "Heat-Not-Burn" e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.

A model of mitochondrial superoxide production during ischaemia-reperfusion injury for therapeutic development and mechanistic understanding.

Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an in vitro model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential.

A study on the anti-senescent effects of flavones derived from Prinsepia utilis Royle seed residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Prinsepia utilis Royle, also known as the Anas fruit, is a unique perennial woody oil plant from Yunnan Province, China. In the ancient texts of Dongba sutras and Yunnan Southern Materia Medica, it has been documented that the local Naxi, Tibetan, and Mosuo communities extensively utilize the root and leaf fruits of green thorns for various purposes. These include treating mild-to-moderate specific dermatitis, moisturising the skin, providing protection against UV damage, aiding childbirth in pregnant women, safeguarding stomach health, reducing the risk of arteriosclerosis, and delaying aging. AIM OF THE STUDY: In this study, leftover residues from oil extraction were efficiently reused, and flavonoids were identified during subsequent extraction and separation processes. The anti-senescent effects of flavonoids in P. utilis Royle have not been systematically studied. Therefore, the objective of this study was to explore the anti-senescent properties of the flavonoids obtained from P. utilis Royle. METHODS: First, HPLC and other analytical techniques were used to identify the components of the P. utilis Royle flavonoid (PURF). Next, DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase were initially detected using in vitro biochemical assays. To examine its antioxidant properties, a zebrafish model was used, and to confirm its anti-senescent effects, a d-galactose-induced mouse aging model was employed. The anti-senescent mechanism of PURF was examined using a natural senescence HFF model. Furthermore, the anti-senescent target was confirmed using a 3D full T-Skin™ model. RESULTS: In vitro biochemical assays demonstrated that flavones exhibited potent antioxidant activity and anti-senescent potential by inhibiting DPPH, hydroxyl radicals, superoxide anion O2-, collagenase, and elastase. It significantly enhanced the antioxidant effect on zebrafish while suppressing ROS and inflammatory injury, up-regulating COL1A1, COL3A1, AMPK, and mTOR gene expression and down-regulating MMP-9, TGF-ß, p21, and p16 gene expression suggesting its potential anti-senescent ability. Findings from the D-galactose-induced aging mouse model showed that PURF greatly increased SOD levels, while simultaneously decreasing HYP and MDA levels. In addition, when PURF was given to the HFF cell and 3D full T-Skin™ model, consistent trends were observed in gene and protein expression, with up-regulation of COL1A1, COL3A1, AMPK, and mTOR genes and down-regulation of TGF-ß, MMP-1, MMP-9, p21, and p16 genes. Therefore, these preliminary findings indicate that flavones can modulate AMPK/mTOR/TGF-ß signalling pathways to exert its influence. CONCLUSION: The kernel residue of natural P. utilis Royle oil extracted from Yunnan province was previously considered agricultural waste, but we successfully extracted and isolated its flavonoid components. Our preliminary studies demonstrated its potential as an environmentally friendly anti-senescent raw material.

Reactive oxygen species augment contractile responses of saphenous artery in 10-15-day-old but not adult rats: Substantial role of NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases (NOX, a key source of ROS in vascular cells) are involved in the regulation of vascular tone, but this has been explored mainly for adult organisms. Importantly, the mechanisms of vascular tone regulation differ significantly in early postnatal ontogenesis and adulthood, while the vasomotor role of ROS in immature systemic arteries is poorly understood. We tested the hypothesis that the functional contribution of NADPH oxidase-derived ROS to the regulation of peripheral arterial tone is higher in the early postnatal period than in adulthood. We studied saphenous arteries from 10- to 15-day-old ("young") and 3- to 4-month-old ("adult") male rats using lucigenin-enhanced chemiluminescence, quantitative PCR, Western blotting, and isometric myography. We demonstrated that both basal and NADPH-stimulated superoxide anion radical (O2•-) production was significantly higher in the arteries from young in comparison to adult rats. Importantly, pan-inhibitor of NADPH oxidase VAS2870 (10 µM) reduced NADPH-induced O2•- production in arteries of young rats. Saphenous arteries of both young and adult rats demonstrated high levels of Nox2 and Nox4 mRNAs, while Nox1 and Nox3 mRNAs were not detected. The protein contents of NOX2 and NOX4 were significantly higher in arterial tissue of young compared to adult animals. Moreover, VAS2870 (10 µM) had no effect on methoxamine-induced contractile responses of adult arteries but decreased them significantly in young arteries; such effect of VAS2870 persisted after removal of the endothelium. Finally, NOX2 inhibitor GSK2795039 (10 µM), but not NOX1/4 inhibitor GKT137831 (10 µM) weakened methoxamine-induced contractile responses of arteries from young rats. Thus, ROS produced by NOX2 have a pronounced contractile influence in saphenous artery smooth muscle cells of young, but not adult rats, which is associated with the increased vascular content of NOX2 protein at this age.

TEMPO-conjugated tobacco mosaic virus as a magnetic resonance imaging contrast agent for detection of superoxide production in the inflamed liver.

Superoxide, an anionic dioxygen molecule, plays a crucial role in redox regulation within the body but is implicated in various pathological conditions when produced excessively. Efforts to develop superoxide detection strategies have led to the exploration of organic-based contrast agents for magnetic resonance imaging (MRI). This study compares the effectiveness of two such agents, nTMV-TEMPO and kTMV-TEMPO, for detecting superoxide in a mouse liver model with lipopolysaccharide (LPS)-induced inflammation. The study demonstrates that kTMV-TEMPO, with a strategically positioned lysine residue for TEMPO attachment, outperforms nTMV-TEMPO as an MRI contrast agent. The enhanced sensitivity of kTMV-TEMPO is attributed to its more exposed TEMPO attachment site, facilitating stronger interactions with water protons and superoxide radicals. EPR kinetics experiments confirm kTMV-TEMPO's faster oxidation and reduction rates, making it a promising sensor for superoxide in inflamed liver tissue. In vivo experiments using healthy and LPS-induced inflamed mice reveal that reduced kTMV-TEMPO remains MRI-inactive in healthy mice but becomes MRI-active in inflamed livers. The contrast enhancement in inflamed livers is substantial, validating the potential of kTMV-TEMPO for detecting superoxide in vivo. This research underscores the importance of optimizing contrast agents for in vivo imaging applications. The enhanced sensitivity and biocompatibility of kTMV-TEMPO make it a promising candidate for further studies in the realm of medical imaging, particularly in the context of monitoring oxidative stress-related diseases.