Enhancing microbial superoxide generation and conversion to hydroxyl radicals for enhanced bioremediation using iron-binding ligands.

Harnessing bacteria for superoxide production in bioremediation holds immense promise, yet its practical application is hindered by slow production rates and the relatively weak redox potential of superoxide. This study delves into a cost-effective approach to amplify superoxide production using an Arthrobacter strain, a prevalent soil bacterial genus. Our research reveals that introducing a carbon source along with specific iron-binding ligands, including deferoxamine (DFO), diethylenetriamine pentaacetate (DTPA), citrate, and oxalate, robustly augments microbial superoxide generation. Moreover, our findings suggest that these iron-binding ligands play a pivotal role in converting superoxide into hydroxyl radicals by modulating the electron transfer rate between Fe(III)/Fe(II) and superoxide. Remarkably, among the tested ligands, only DTPA emerges as a potent promoter of this conversion process when complexed with Fe(III). We identify an optimal Fe(III) to DTPA ratio of approximately 1:1 for enhancing hydroxyl radical production within the Arthrobacter culture. This research underscores the efficacy of simultaneously introducing carbon sources and DTPA in facilitating superoxide production and its subsequent conversion to hydroxyl radicals, significantly elevating bioremediation performance. Furthermore, our study reveals that DTPA augments superoxide production in cultures of diverse soils, with various soil microorganisms beyond Arthrobacter identified as contributors to superoxide generation. This emphasizes the universal applicability of DTPA across multiple bacterial genera. In conclusion, our study introduces a promising methodology for enhancing microbial superoxide production and its conversion into hydroxyl radicals. These findings hold substantial implications for the deployment of microbial reactive oxygen species in bioremediation, offering innovative solutions for addressing environmental contamination challenges.

Gelsemium low doses protect against serum deprivation-induced stress on mitochondria in neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.

Effect of 5ß-dihydrotestosterone on vasodilator function and on cell proliferation.

Aging is one of the main factors associated with cardiovascular diseases. Androgens exert beneficial effects on the cardiovascular system and testosterone (TES) replacement therapy improves cardiometabolic risk factors. However, TES is contraindicated in patients with prostate cancer due to its proliferative effects on prostatic tumor cells. Additionally, TES and its reduced metabolites 5α- and 5ß-dihydrotestosterone (5α-DHT and 5ß-DHT) exert vasodilatory effects. Since androgen levels decrease during aging and 5ß-DHT lacks genomic effects, this study is focused on analyzing its effect on vasodilator function and the proliferation rate of prostatic tumor and vascular smooth muscle cells. To study the vascular function, mesenteric arteries from aged-orchidectomized Sprague-Dawley rats were used. Mesenteric segments were divided into one control (without treatment) and three groups with the androgens (10 nM, 30 min) to analyze: acetylcholine- and sodium nitroprusside-induced responses and nitric oxide and superoxide anion production. To analyze cell proliferation, the effect of androgens on cell viability was determined. The results showed that 5ß-DHT improves vasodilator function in arteries from aged-orchidectomized rats and induces antioxidant action, while the proliferation rate of the androgen-dependent prostatic tumor cells remains unaltered. These results make 5ß-DHT a promising therapeutic agent for the treatment of cardiovascular pathologies.

Sexual Dimorphism in Impairment of Acetylcholine-Mediated Vasorelaxation in Zucker Diabetic Fatty (ZDF) Rat Aorta: A Monogenic Model of Obesity-Induced Type 2 Diabetes.

Several reports, including our previous studies, indicate that hyperglycemia and diabetes mellitus exert differential effects on vascular function in males and females. This study examines sex differences in the vascular effects of type 2 diabetes (T2D) in an established monogenic model of obesity-induced T2D, Zucker Diabetic Fatty (ZDF) rats. Acetylcholine (ACh) responses were assessed in phenylephrine pre-contracted rings before and after apocynin, a NADPH oxidase (NOX) inhibitor. The mRNA expressions of aortic endothelial NOS (eNOS), and key NOX isoforms were also measured. We demonstrated the following: (1) diabetes had contrasting effects on aortic vasorelaxation in ZDF rats, impairing relaxation to ACh in females while enhancing it in male ZDF rats; (2) inhibition of NOX, a major source of superoxide in vasculature, restored aortic vasorelaxation in female ZDF rats; and (3) eNOS and NOX4 mRNA expressions were elevated in female (but not male) ZDF rat aortas compared to their respective leans. This study highlights sexual dimorphism in ACh-mediated vasorelaxation in the aorta of ZDF rats, suggesting that superoxide may play a role in the impaired vasorelaxation observed in female ZDF rats.

The inhibitory potential of 4,7-dihydroxycoumarin derivatives on ROS-producing enzymes and direct HOO•/o2• - radical scavenging activity - a comprehensive kinetic DFT study.

This study examined the antiradical activity of three synthesized coumarin derivatives: (E)-3-(1-((2-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (A1-OH), (E)-3-(1-((3-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (A2-OH), and (E)-3-(1-((4-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (A3-OH) against HOO•/O2•- radical species. The investigation included electron spin resonance (ESR) measurements and a DFT kinetic study. Thermodynamic and kinetic parameters of antiradical mechanisms-Formal Hydrogen Atom Transfer (f-HAT), Radical Adduct Formation (RAF), Sequential Proton Loss followed by Electron Transfer (SPLET), and Single-Electron Transfer followed by Proton Transfer (SET-PT)-were evaluated using the Quantum Mechanics-based test for Overall Free Radical Scavenging Activity (QM-ORSA) under physiological conditions. ESR results indicated antiradical activity decreased in the sequence A1-OH (58.7%) > A2-OH (57.5%) > A3-OH (53.1%). Kinetic analysis revealed the f-HAT mechanism dominated HOO• inactivation. A newly formulated Sequential Proton Loss followed by Radical Adduct Formation (SPL-RAF) mechanism described interactions with O2•-. The activity toward O2•- was A2-OH (1.26 × 106 M-1s-1) > A3-OH (7.71 × 105 M-1s-1) > A1-OH (4.22 × 105 M-1s-1). Molecular docking and dynamics studies tested inhibitory capability against enzymes producing reactive species: Lipoxygenase (LOX), Myeloperoxidase (MPO), NAD(P)H oxidase (NOX), and Xanthine Oxidase (XOD). Affinity to enzymes decreased in the order: XOD > LOX > NOX > MPO.

Antioxidant Scavenging of the Superoxide Radical by Yerba Mate (Ilex paraguariensis) and Black Tea (Camellia sinensis) Plus Caffeic and Chlorogenic Acids, as Shown via DFT and Hydrodynamic Voltammetry.

We describe the antioxidant capability of scavenging the superoxide radical of several tea and yerba mate samples using rotating ring-disk electrochemistry (RRDE). We directly measured superoxide concentrations and detected their decrease upon the addition of an antioxidant to the electrochemical cell. We studied two varieties of yerba mate, two varieties of black tea from Bangladesh, a sample of Pu-erh tea from China, and two components, caffeic acid and chlorogenic acid. All of these plant infusions and components showed strong antioxidant activities, virtually annihilating the available superoxide concentration. Using density functional theory (DFT) calculations, we describe a mechanism of superoxide scavenging via caffeic and chlorogenic acids. Superoxide can initially interact at two sites in these acids: the H4 catechol hydrogen (a) or the acidic proton of the acid (b). For (a), caffeic acid needs an additional π-π superoxide radical, which transfers electron density to the ring and forms a HO2- anion. A second caffeic acid proton and HO2- anion forms H2O2. Chlorogenic acid acts differently, as the initial approach of superoxide to the catechol moiety (a) is enough to form the HO2- anion. After an additional acidic proton of chlorogenic acid is given to HO2-, three well-separated compounds arise: (1) a carboxylate moiety, (2) H2O2, and a (3) chlorogenic acid semiquinone. The latter can capture a second superoxide in a π-π manner, which remains trapped due to the aromatic ring, as for caffeic acid. With enough of both acids and superoxide radicals, the final products are equivalent: H2O2 plus a complex of the type [X-acid-η-O2], X = caffeic, chlorogenic. Chlorogenic acid (b) is described by the following reaction: 2 O2•- + 2 chlorogenic acid → 2 chlorogenic carboxylate + O2 + H2O2, and so, it acts as a non-enzymatic superoxide dismutase (SOD) mimic, as shown via the product formation of O2 plus H2O2, which is limited due to chlorogenic acid consumption. Caffeic acid (b) differs from chlorogenic acid, as there is no acidic proton capture via superoxide. In this case, approaching a second superoxide to the H4 polyphenol moiety forms a HO2- anion and, later, an H2O2 molecule upon the transfer of a second caffeic acid proton.

PROTECTIVE EFFECT OF A NEW SUPEROXIDE-PRODUCING ENZYME COMPLEX FROM RASPBERRY IN RATS WITH THIRD-DEGREE THERMAL BURNS.

Thermal burns are the most common type of burn injuries. Medical treatment for burns is crucial, especially for third-degree burns and when a significant surface area of the body is affected. One of the most pressing issues in modern medicine is the search for new effective means to accelerate the healing of burn wounds. Oxygen radicals play a significant role in maintaining homeostasis, forming the body's resistance to infection, and ensuring the regeneration of organs and tissues. In this study, a superoxide (O2-)-producing enzyme (SPE) from raspberries was applied (topically to the skin, injected under the wound surface, with solution concentrations of 12.75% and 5%) after a third-degree thermal burn to determine its reparative effects on the skin. To assess the condition of the animals that had suffered burn injuries and the healing process, blood parameters were analyzed, and cytogenetic indices of bone marrow from the femur of the animals were studied: mitotic index, number of polyploid cells, and chromosomal aberrations. When analyzing hematological, cytogenetic, and histological parameters, significant differences were found between the «clean burn¼ groups and the groups in which SPE was used in different concentrations and methods of application. The use of SPE in both concentrations contributed to a reduction in the area of burn wounds compared to a «clean burn¼. The survival rate of animals for 30 days (before the end of the experiment) was 100% when using a 12.75% SPE solution and 50% when using a 5% SPE solution. The use of SPE led to significant differences in hematological parameters from the «clean burn¼ group throughout the entire duration of the experiment, showing a tendency to normalize the parameters. Under the influence of the 12.75% SPE solution, there was a tendency toward normalization of the mitotic index, along with a significant reduction in the percentage of polyploid cells and chromosomal aberrations, which may indicate its beneficial effects. This study found that a 12.75% SPE solution derived from raspberries was more effective and had healing properties on third-degree thermal burns, promoting rapid healing of the burn wound.

A hemicyanine-modified upconversion nanoprobe for NIR-excited evaluating superoxide signaling in drug-induced liver injury.

BACKGROUND: Drug-induced liver injury (DILI) is the most important standard for the entrance of clinical drugs into the pharmaceutical market. The elevation of superoxide anion (O2•-) during drug metabolism can mediate apoptosis of hepatocytes and further generation of liver damage. Therefore, developing an effective imaging method for evaluating O2•- levels during DILI is of great importance. However, current reported O2•- fluorescent probes either use short excitation wavelengths or a single intensity detection system, limiting the accurate quantification of O2•- in deep tissue in vivo. RESULTS: We developed a NIR-excited ratiometric nanoprobe (CyD-UCNPs) by assembly of O2•--sensitive hemicyanine dyes (CyD) on the surface of Tm/Er-codoped upconversion nanoparticles (UCNPs) with the assistance of α-cyclodextrin, which exhibited a robust "turn-on" ratiometric sensing signal. In vitro experiments indicated that CyD-UCNPs respond well to O2•- with high selectivity. Furthermore, by taking advantage of the outstanding optical properties produced by the luminescent resonance energy transfer between the UCNPs and CyD upon the excitation of 980 nm, the ratiometric upconversion luminescence signal of CyD-UCNPs was successfully utilized to monitor the fluctuation of O2•- levels under phorbol-12-myristate-13-acetate (PMA)/cisplatin-induced oxidative stress in living cells, liver tissues, and zebrafish. More importantly, endogenous change in O2•- levels in the liver sites of mice during DILI and its prevention with L-carnitine was visualized using CyD-UCNPs. SIGNIFICANCE: This study provides a ratiometric NIR-excited imaging strategy for investigating the correlation between O2•- levels and DILI and its prevention, which is significant for early diagnosis of DILI and preclinical screening of anti-hepatotoxic drugs in vivo.

Intracellular peroxynitrite perturbs redox balance, bioenergetics, and Fe-S cluster homeostasis in Mycobacterium tuberculosis.

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

Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury.

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km = 2.7 µM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.

Oxidative stress and galectin-3 levels during skin grafting after hand injury: gender differences.

The study included 40 patients of both genders who underwent skin transplantation after a hand injury. The study aims to evaluate the oxidative stress parameters in patients' blood and serum levels of galectin-3 in order to investigate gender differences pre- and post- skin transplantation. The results of the study suggest a significant increase in superoxide anion radical levels, catalase activity, and reduced glutathione levels in females before skin transplantation. The surgical treatment caused significant increase in superoxide anion radical and hydrogen peroxide levels as prooxidants in males, while superoxide dismutase and catalase activity were also increased 7 days after the procedure. In females, superoxide anion radical and TBARS levels increased after surgical procedure as well as the activity of catalase. Regarding galectin-3 levels, a significant interaction between gender and time was observed (gender×time; p=0.000). Correlation analysis of different oxidative stress markers with gal-3 revealed the existence of a significant negative correlation of superoxide anion radical, catalase, and reduced glutathione with gal-3, but only in female patients. It can be concluded that OS as well as galectin-3 play important roles at least in the first 7 days of the postoperative period.

Sonoinduced Tumor Therapy and Metastasis Inhibition by a Ruthenium Complex with Dual Action: Superoxide Anion Sensitization and Ligand Fracture.

Photoresponsive ruthenium(II) complexes have recently emerged as a promising tool for synergistic photodynamic therapy and chemotherapy in oncology, as well as for antimicrobial applications. However, the limited penetration power of photons prevents the treatment of deep-seated lesions. In this study, we introduce a sonoresponsive ruthenium complex capable of generating superoxide anion (O2•-) via type I process and initiating a ligand fracture process upon ultrasound triggering. Attaching hydroxyflavone (HF) as an "electron reservoir" to the octahedral-polypyridyl-ruthenium complex resulted in decreased highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) energy gaps and triplet-state metal to ligand charge transfer (3MLCT) state energy (0.89 eV). This modification enhanced the generation of O2•- under therapeutic ultrasound irradiation at a frequency of 1 MHz. The produced O2•- rapidly induced an intramolecular cascade reaction and HF ligand fracture. As a proof-of-concept, we engineered the Ru complex into a metallopolymer platform (PolyRuHF), which could be activated by low-power ultrasound (1.5 W cm-2, 1.0 MHz, 50% duty cycle) within a centimeter range of tissue. This activation led to O2•- generation and the release of cytotoxic ruthenium complexes. Consequently, PolyRuHF induced cellular apoptosis and ferroptosis by causing mitochondrial dysfunction and excessive toxic lipid peroxidation. Furthermore, PolyRuHF effectively inhibited subcutaneous and orthotopic breast tumors and prevented lung metastasis by downregulating metastasis-related proteins in mice. This study introduces the first sonoresponsive ruthenium complex for sonodynamic therapy/sonoactivated chemotherapy, offering new avenues for deep tumor treatment.

Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS.

The bactericidal activity of several antibiotics partially relies on the production of reactive oxygen species (ROS), which is generally linked to enhanced respiration and requires the Fenton reaction. Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. Here, we use Bacillus subtilis cells in stationary phase, as a model system of dormant cells, to show that pharmacological induction of membrane depolarization enhances the antibiotics' bactericidal activity and also leads to ROS production. However, in contrast to previous studies, this results primarily in production of superoxide radicals and does not require the Fenton reaction. Genetic analyzes indicate that Rieske factor QcrA, the iron-sulfur subunit of respiratory complex III, seems to be a primary source of superoxide radicals. Interestingly, the membrane distribution of QcrA changes upon membrane depolarization, suggesting a dissociation of complex III. Thus, our data reveal an alternative mechanism by which antibiotics can cause lethal ROS levels, and may partially explain why membrane-targeting antibiotics are effective in eliminating persisters.

Effects of superoxide radical on photosynthesis and K+ and redox homeostasis in quinoa and spinach.

Methyl viologen (MV), also known as paraquat, is a widely used herbicide but has also been reported as highly toxic to different life forms. The mode of its operation is related to superoxide radical (O2.-) production and consequent oxidative damage. However, besides the damage to key macromolecules, reactive oxygen species (ROS; to which O2.- belongs) are also known as regulators of numerous ion transport systems located at cellular membranes. In this study, we used MV as a tool to probe the role of O2.- in regulating membrane-transport activity and systemic acquired tolerance in halophytic Chenopodium quinoa and glycophytic spinach plants. Both plant species showed growth reduction in terms of reduced shoot length, lower shoot fresh and dry weight, photosynthesis rate, and chlorophyll contents; however, quinoa showed less reduction in growth compared with spinach. This whole plant response was further examined by measuring the ion concentration, gene expression of ion transporters, activation of antioxidants, and osmolyte accumulation. We observed that at the mechanistic level, the differences in growth in response to MV were conferred by at least four complementary physiological mechanisms: (1) higher K+ loss from spinach leaves resulted from higher expression of MV-induced plasma membrane-based depolarization-activated K+ efflux GORK channel, (2) higher activation of high-affinity K+ uptake transporter HAK5 in quinoa, (3) higher antioxidant production and osmolyte accumulation in quinoa as compared with spinach, and (4) maintaining a higher rate of photosynthesis due to higher chlorophyll contents, and efficiency of photosystem II and reduced ROS and MDA contents. Obtained results also showed that MV induced O2.- significantly reduced N contents in both species but with more pronounced effects in glycophytic spinach. Taken together this study has shown the role of O2.- in regulating membrane ion transport and N metabolism in the leaves of halophyte vs. glycophyte in the context of oxidative stress tolerance.

Algae promotes the biogenic oxidation of Mn(II) by accelerated extracellular superoxide production.

Microbial manganese (Mn) oxidation, predominantly occurs within the anaerobic-aerobic interfaces, plays an important role in environmental pollution remediation. The anaerobic-aerobic transition zones, notably riparian and lakeside zones, are hotspots for algae-bacteria interactions. Here, we adopted a Mn(II)-oxidizing bacterium Pseudomonas sp. QJX-1 to investigate the impact of algae on microbial Mn(II) oxidation and verify the underlying mechanisms. Interestingly, we achieved a remarkable enhancement in bacterial Mn(II)-oxidizing activity within the algae-bacteria co-culture, despite the inability to oxidize Mn(II) for the algae used in this study. In addition, the bacterial density almost remains constant in the presence of algal cells. Therefore, the increased Mn(II) oxidation by QJX-1 in the presence of algae cannot be due to the increased biomass. Within this co-culture system, the Mn(II) oxidation rate surged to an impressive 0.23 mg/L/h, in stark contrast to 0.02 mg/L/h recorded within pure QJX-1 system. The presence of algae could inhibit the Fe-S cluster activity of QJX-1 by the produced active substance in co-culture, and result in the acceleration of extracellular superoxide production due to the impairment of electron transfer functions located in QJX-1 cell membranes. Moreover, elevated peroxidase gene expression and heightened extracellular catalase activity not only expedited Mn(II) ions oxidation but also facilitated conversion of intermediate Mn(III) ions into microbial Mn oxides, achieved through the degradation of hydrogen peroxide. Therefore, the acceleration of extracellular superoxide production and the decomposition of hydrogen peroxide are identified as the principal mechanisms behind the observed enhancement in Mn(II) oxidation within algae-bacteria co-cultures. Our findings highlight the need to consider the effect of algae on microbial Mn(II) oxidation, which plays an important role in the environmental pollution remediation.

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition.

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting superoxide (O2●-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). Human MnSOD has evolved to be highly product inhibited to limit the formation of H2O2, a freely diffusible oxidant and signaling molecule. The product-inhibited complex is thought to be composed of a peroxide (O22-) or hydroperoxide (HO2-) species bound to Mn ion and formed from an unknown PCET mechanism. PCET mechanisms of proteins are typically not known due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the mechanism, we combine neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states of the enzyme to reveal the positions of all the atoms, including hydrogen, and the electronic configuration of the metal ion. The data identifies the product-inhibited complex, and a PCET mechanism of inhibition is constructed.

Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

Ile-Pro-Pro attenuates sympathetic activity and hypertension.

Isoleucine-proline-proline (Ile-Pro-Pro, IPP) is a natural food source tripeptide that inhibits angiotensin-converting enzyme (ACE) activity. The aim of this study was to determine the central and peripheral roles of IPP in attenuating sympathetic activity, oxidative stress and hypertension. Male Sprague-Dawley rats were subjected to sham-operated surgery (Sham) or two-kidney one-clip (2K1C) surgery to induce renovascular hypertension. Renal sympathetic nerve activity and blood pressure were recorded. Bilateral microinjections of IPP to hypothalamic paraventricular nucleus (PVN) attenuated sympathetic activity (-16.1 ± 2.5%, P < 0.001) and hypertension (-8.7 ± 1.5 mmHg, P < 0.01) in 2K1C rats by inhibiting ACE activity and subsequent angiotensin II and superoxide production in the PVN. Intravenous injections of IPP also attenuated sympathetic activity (-15.1 ± 2.1%, P < 0.001) and hypertension (-16.8 ± 2.3 mmHg, P < 0.001) via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. The duration of the effects of the intravenous IPP was longer than those of the PVN microinjection, but the sympatho-inhibitory effect of intravenous injections occurred later than that of the PVN microinjection. Intraperitoneal injection of IPP (400 pmol/day for 20 days) attenuated hypertension and vascular remodeling via inhibiting ACE activity and oxidative stress in both PVN and arteries of 2K1C rats. These results indicate that IPP attenuates hypertension and sympathetic activity by inhibiting ACE activity and oxidative stress. The sympathoinhibitory effect of peripheral IPP is mainly caused by the ACE inhibition in PVN, and the antihypertensive effect is related to the sympathoinhibition and the arterial ACE inhibition. Long-term intraperitoneal IPP therapy attenuates hypertension, oxidative stress and vascular remodeling.

The Effect of Botulinum Toxin A on the NADPH Oxidase System and Ischemia-Reperfusion Injury.

BACKGROUND: Despite the increasing popularity of various materials for ischemia-reperfusion (I/R) injury mitigation, research on botulinum toxin type A (BoNTA) remains limited. This study assesses BoNTA's efficacy in protecting flaps from I/R injury by inhibiting the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system and reducing reactive oxygen species (ROS) production. METHODS: Seventy-six Sprague-Dawley rats were studied. We examined the effects of BoNTA on superoxide production in four rats using a lucigenin-enhanced chemiluminescence assay (LECL). Another group of 60 rats had their superficial inferior epigastric artery (SIEA) flaps treated with either BoNTA or saline and clamped for 0, 1, and 4 hours before reperfusion. Flap survival and histological outcomes were assessed five days post-operation. ROS production in SIEA flaps and femoral vessels was analyzed in 12 additional rats, post-I/R injury. RESULTS: The LECL results showed that the BoNTA group had significantly lower superoxide production compared to controls, with notable reductions at 4 hours. While no significant differences were noted at the 0 and 1-hour marks, the 4-hour mark showed significant protective effects in BoNTA-treated groups. The survival rate was 90% for BoNTA-treated rats versus 60% for controls ( P = 0.028). Significant reductions in ROS were also observed in the 4-hour I/R group. CONCLUSIONS: BoNTA effectively protects against I/R injury by inhibiting the NADPH oxidase system and reducing ROS levels. These results support further investigation into the specific mechanisms of NADPH oxidase inhibition by BoNTA and its potential clinical applications, given its safety profile. CLINICAL RELEVANCE STATEMENT: The findings from the present study are expected to provide a basis for clinical studies regarding this use of BoNTA.

The protective effect of imatinib against pancreatic ß-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress.

Glucocorticoids (GCs) are known to stimulate pancreatic beta (ß)-cell apoptosis via several mechanisms, including oxidative stress. Our previous study suggested an increase in dexamethasone-induced pancreatic ß-cell apoptosis via a reduction of glutathione S-transferase P1 (GSTP1), which is an antioxidant enzyme. Imatinib, which is a tyrosine kinase inhibitor, also exerts antioxidant effect. This study aims to test our hypothesis that imatinib would prevent pancreatic ß-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress. Our results revealed that dexamethasone significantly increased apoptosis in INS-1 cells when compared to the control, and that imatinib significantly decreased INS-1 cell apoptosis induced by dexamethasone. Moreover, dexamethasone significantly increased superoxide production in INS-1 cells when compared to the control; however, imatinib, when combined with dexamethasone, significantly reduced superoxide production in INS-1 cells. Dexamethasone significantly decreased GSTP1, p-ERK1/2, and BCL2 protein expression, but significantly increased p-JNK, p-p38, and BAX protein expression in INS-1 cells-all compared to control. Importantly, imatinib significantly ameliorated the effect of dexamethasone on the expression of GSTP1, p-ERK1/2, p-JNK, p-p38 MAPK, BAX, and BCL2. Furthermore-6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), which is a GSTP1 inhibitor, neutralized the protective effect of imatinib against pancreatic ß-cell apoptosis induced by dexamethasone. In conclusion, imatinib decreases pancreatic ß-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress.