Lower Reactive Oxygen Species Production Research Articles

Tobacco NtUBC1 and NtUBQ2 enhance salt tolerance by reducing sodium accumulation and oxidative stress through proteasome activation in Arabidopsis

The ubiquitin/proteasome system plays a crucial role in the regulation of plant responses to environmental stress. Here, we studied the involvement of the UBC1 and UBQ2 genes encoding a ubiquitin conjugating enzyme (E2) and ubiquitin extension protein, respectively, in the response to salt stress. Our results showed that the constitutive expression of tobacco NtUBC1 and NtUBQ2 in Arabidopsis thaliana improved salt tolerance, along with the lower Na+ level and higher K+/Na+ ratio compared to control plants. Moreover, the expression levels of sodium transporters, including AtHKT1 (High-Affinity K+ Transporter1) and AtSOS1 (Salt Overly Sensitive 1), were higher in NtUBC1- and NtUBQ2-Arabidopsis. However, the transcript level of AtNHX1 (Na+/H+ Exchanger 1) was similar between control and transgenic plants. After salt exposure, the activity of the 26S proteasome markedly increased in NtUBC1- and NtUBQ2-expressing plants; however, ubiquitinated protein levels decreased compared to control plants. Furthermore, higher activity of antioxidant enzymes and lower ROS production were observed in UBC1- and UBQ2-expressing plants. We further challenged atubc1, atubc2, and atubq2 single mutants and atubc1ubc2 double mutant lines with salt stress; interestingly, the salt sensitivity and sodium levels of the studied mutants were enhanced, while the potassium levels were reduced. However, the atubc1ubc2 double mutant illustrated a more severe phenotype than the single mutants, probably due to the redundant function of UBC1 and UBC2 in Arabidopsis. Taken together, NtUBC1 and NtUBQ2 enhance salt tolerance by enhancing 26S proteasome activity and reducing Na+ accumulation, ROS, and ubiquitinated/salt-denatured proteins.

Enhanced ROS Production in Mitochondria from Prematurely Aging mtDNA Mutator Mice.

An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process ("the mitochondrial hypothesis of aging"). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower - not higher - than in wildtype, thus apparently invalidating the "mitochondrial hypothesis of aging". We have here re-examined ROS production rates in mtDNA-mutator mice mitochondria. Using traditional conditions for measuring ROS (succinate in the absence of rotenone), we indeed found lower ROS in the mtDNA-mutator mitochondria compared to wildtype. This ROS mainly results from reverse electron flow driven by the membrane potential, but the membrane potential reached in the isolated mtDNA-mutator mitochondria was 33 mV lower than that in wildtype mitochondria, due to the feedback inhibition of succinate oxidation by oxaloacetate, and to a lower oxidative capacity in the mtDNA-mutator mice, explaining the lower ROS production. In contrast, in normal forward electron flow systems (pyruvate (or glutamate) + malate or palmitoyl-CoA + carnitine), mitochondrial ROS production was higher in the mtDNA-mutator mitochondria. Particularly, even during active oxidative phosphorylation (as would be ongoing physiologically), higher ROS rates were seen in the mtDNA-mutator mitochondria than in wildtype. Thus, when examined under physiological conditions, mitochondrial ROS production rates are indeed increased in mtDNA-mutator mitochondria. While this does not prove the validity of the mitochondrial hypothesis of aging, it may no longer be said to be negated in this respect. This paper is dedicated to the memory of Professor Vladimir P. Skulachev.

Epigen enhances the developmental potential of in vitro fertilized embryos by improving cytoplasmic maturation

Numerous growth factors contribute to oocyte maturation and embryonic development in vivo; however, only a few are understood. One such factor is epigen, a new member of the epidermal growth factor (EGF) family that is secreted by the granulosa cells of immature oocytes. We hypothesized that epigen may play a role in oocyte maturation, specifically in the nuclear and cytoplasmic aspects. This study aimed to investigate the effects of epigen on porcine oocyte maturation and embryo development in vitro. In this study, three different concentrations of epigen (3, 6, and 30 ng/mL) were added to tissue culture medium-199 (TCM-199) during in vitro maturation of porcine oocytes. A control group that did not receive epigen supplementation was also included. Mature porcine oocytes were fertilized, and the resulting zygotes were cultured until day 7. The levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured in the in vitro matured oocytes. At the same time, the expression patterns of genes related to apoptosis were detected in day 7 blastocysts (BLs) using real-time quantitative PCR Apoptosis was detected by annexin-V assays in mature oocytes. Data were analyzed using ANOVA and Duncan's test on SPSS, and results are presented as mean ± SEM. The group that received 6 ng/mL epigen had a significantly lower rate of germinal vesicle breakdown (GVBD) than the control group without affecting the nuclear maturation among the experimental groups. Among the treatment groups, the 6 ng/mL epigen group showed significantly higher levels of intracellular GSH and lower ROS production. Supplementation with 6 ng/mL epigen significantly improved blastocyst (BL) formation rates compared to those in the control and 3 ng/mL groups. Additionally, the blastocyst expansion rate was significantly higher with epigen supplementation (6 ng/mL). In the fertilization experiment, the group supplemented with 6 ng/mL epigen exhibited significantly higher levels of monospermy and fertilization efficiency and lower levels of polyspermy than the control group. This study indicated that adding epigen at a concentration of 6 ng/mL can significantly enhance the developmental potential of porcine oocytes fertilized in vitro. Specifically, the study found that epigen improves cytoplasmic maturation, which helps prevent polyspermy and emulates monospermic penetration.

Dye-augmented bandgap engineering of a degradable cascade nanoreactor for tumor immune microenvironment-enhanced dynamic phototherapy of breast cancer

Non-invasive precision tumor dynamic phototherapy has broad application prospects. Traditional semiconductor materials have low photocatalytic activity and low reactive oxygen species (ROS) production rate due to their wide band gap, resulting in unsatisfactory phototherapy efficacy for tumor treatment. Employing the dye-sensitization mechanism can significantly enhance the catalytic activity of the materials. We develop a multifunctional nanoplatform (BZP) by leveraging the benefits of bismuth-based semiconductor nanomaterials. BZP possesses robust ROS generation and remarkable near-infrared photothermal conversion capabilities for improving tumor immune microenvironment and achieving superior phototherapy sensitization. BZP produces highly cytotoxic ROS species via the photocatalytic process and cascade reaction, amplifying the photocatalytic therapy effect. Moreover, the simultaneous photothermal effect during the photocatalytic process facilitates the improvement of therapeutic efficacy. Additionally, BZP-mediated phototherapy can trigger the programmed death of tumor cells, stimulate dendritic cell maturation and T cell activation, modulate the tumor immune microenvironment, and augment the therapeutic effect. Hence, this study demonstrates a promising research paradigm for tumor immune microenvironment-improved phototherapy. Statement of significanceThrough the utilization of dye sensitization and rare earth doping techniques, we have successfully developed a biodegradable bismuth-based semiconductor nanocatalyst (BZP). Upon optical excitation, the near-infrared dye incorporated within BZP promptly generates free electrons, which, under the influence of the Fermi energy level, undergo transfer to BiF3 within BZP, thereby facilitating the effective separation of electron–hole pairs and augmenting the catalytic capability for reactive oxygen species (ROS) generation. Furthermore, a cascade reaction mechanism generates highly cytotoxic ROS, which synergistically depletes intracellular glutathione, thereby intensifying oxidative stress. Ultimately, this dual activation strategy, combining oxidative and thermal damage, holds significant potential for tumor immunotherapy.

The protective effects of empagliflozin on DNA oxidative changes in a model of vascular endothelial and smooth muscle cells damaged by oxidized cholesterol

BackgroundDiabetes patients often suffer chronic vascular complications resulting from endothelial dysfunction, smooth muscle cell (SMC) proliferation, inflammation and disturbed oxidative balance. Empagliflozin is one of three approved sodium-glucose cotransporter 2 (SGLT2) inhibitors for type 2 diabetes mellitus. The aim of this studywas to determine the protective and repairing effect of EMPA in a model of vascular endothelial and SMC damage with 25-hydroxycholesterol (25-OHC). MethodsHuman umbilical vascular endothelial cells (HUVECs) and SMCs were treated with compounds which induce DNA single-strand breaks (SSBs) and subjected to comet assay. Oxidative DNA damage was detected using endonuclease III (Nth) or human 8 oxoguanine DNA glycosylase (hOOG1). Reactive oxygen species (ROS) formation was determined by the fluorescence of a 6-carboxy-2′,7′-dichlorodihydrofluoresce probe in diacetate (H2DCFDA). Results25-OHC-stimulated SMCs showed greater resistance to ROS generation and DNA damage compared to HUVECs. In both experimental models, EMPA treatment was associated with lower ROS production and DNA damage, including oxidative damage to purines and pyrimidines. This effect was not dose-dependent. EMPA was found to counteract this DNA damage by inhibiting ROS production. ConclusionsIt appears that the EMPA induced indirect repair of DNA by inhibiting ROS production.

Gynura divaricata (L.) DC. promotes diabetic wound healing by activating Nrf2 signaling in diabetic rats

The ethnopharmacological significanceDiabetic chronic foot ulcers pose a significant therapeutic challenge as a result of the oxidative stress caused by hyperglycemia. Which impairs angiogenesis and delays wound healing, potentially leading to amputation. Gynura divaricata (L.) DC. (GD), a traditional Chinese herbal medicine with hypoglycemic effects, has been proposed as a potential therapeutic agent for diabetic wound healing. However, the underlying mechanisms of its effects remain unclear. Aim of the studyIn this study, we aimed to reveal the effect and potential mechanisms of GD on accelerating diabetic wound healing in vitro and in vivo. Materials and methodsThe effects of GD on cell proliferation, apoptosis, reactive oxygen species (ROS) production, migration, mitochondrial membrane potential (MMP), and potential molecular mechanisms were investigated in high glucose (HG) stimulated human umbilical vein endothelial cells (HUVECs) using CCK-8, flow cytometry assay, wound healing assay, immunofluorescence, DCFH-DA staining, JC-1 staining, and Western blot. Full-thickness skin defects were created in STZ-induced diabetic rats, and wound healing rate was tracked by photographing them every day. HE staining, immunohistochemistry, and Western blot were employed to investigate the effect and molecular mechanism of GD on wound healing in diabetic rats. ResultsGD significantly improved HUVEC survival, decreased apoptosis, lowered ROS production, restored MMP, improved migration ability, and raised VEGF expression. The use of Nrf2-siRNA completely abrogated these effects. Topical application of GD promoted angiogenesis and granulation tissue growth, resulting in faster healing of diabetic wounds. The expression of VEGF, CD31, and VEGFR was elevated in the skin tissue of diabetic rats after GD treatment, which upregulated HO-1, NQO-1, and Bcl-2 expression while downregulating Bax expression via activation of the Nrf2 signaling pathway. ConclusionThe findings of this study indicate that GD has the potential to serve as a viable alternative treatment for diabetic wounds. This potential arises from its ability to mitigate the negative effects of oxidative stress on angiogenesis, which is regulated by the Nrf2 signaling pathway. The results of our study offer valuable insights into the therapeutic efficacy of GD in the treatment of diabetic wounds, emphasizing the significance of directing interventions towards the Nrf2 signaling pathway to mitigate oxidative stress and facilitate the process of angiogenesis.

Recording changes in biochemical parameters <i>in vivo</i> in the ischemic stroke model

Stroke is a significant and socially insidious disease that ranks second among fatal diseases according to the World Health Organization [1]. Understanding the molecular mechanisms behind the pathogenesis of this ailment will enable the development of more effective preventative measures and treatment strategies to minimize the negative consequences of stroke. Despite the abundance of experimental data, most of which were acquired indirectly, the study of the dynamics of biochemical parameters in brain tissue in real time during the acute phase of ischemic stroke is difficult. The use of genetically-encoded sensors creates novel possibilities for monitoring alterations in different biochemical and metabolic parameters in vivo tissues. In this study, we evaluated pH changes, hydrogen peroxide production (an important type of biologically active ROS), and polysulfide synthesis in various types of brain tissue cells of SHR rats during the development of ischemic stroke in real time using sensors such as SypHer3s (for pH detection), HyPer7 (for H2O2 detection), and PersIc (for polysulfide detection). Middle cerebral artery occlusion was used to simulate an ischemic stroke. The in vivo sensor signals were registered with a fiber optic setup that was created in the laboratory of spectroscopy and nonlinear optics at Moscow State University. The studies revealed that in the acute phase of stroke, acidosis occurred in the cytoplasm of neurons in the caudate nucleus, the epicenter of ischemia. The pH mutated from 7.25±0.08 to 6.7±0.15 within the first few seconds after arterial occlusion initiation. A gradual increase in pH was observed after the initial drop, which persisted throughout reperfusion but did not return to the original value in all animals. In the penumbra zone, a wave-like shift in sensor signal was detected, whereas no change in sensor signal was noted in the healthy hemisphere. Investigation of the dynamics of H2O2 formation in the mitochondrial matrix of caudate neurons revealed minimal sensor oxidation during ischemia/reperfusion in the acute phase of stroke, indicating low ROS production. Nevertheless, a substantial increase in the sensor signal was detected after 24 hours following the surgery. Thus, the confirmation of oxidative stress development in the affected hemisphere differed from the commonly accepted view in terms of its dynamics. Previously, it was believed that excessive production of H2O2 leading to oxidative stress and related brain cell death occurred primarily in the acute phase. However, a comparison of hydrogen peroxide production dynamics in neurons and astrocytes revealed differences between these cell populations. It was discovered that as early as 12 hours after middle cerebral artery occlusion, the sensor signal in astrocytes increased more intensely than in neurons. This trend persisted until the end of the measurements, 40 hours after surgery. The observed distinctions may stem from glial cells’ protective function in counteracting the harmful consequences of hydrogen peroxide on neurons, along with their contribution to maintaining the myelin structure in the brain. Additionally, the role of astrocytes in neuroinflammation development is noteworthy. Reactive sulfur species, in addition to reactive oxygen species, appear to be significant contributors to the development of pathological processes. The PersIc sensor signal measurement did not show any disparities between the caudate nucleus of the healthy hemisphere and the hemisphere affected by stroke development in terms of polysulfide and persulfide appearance detection. However, the area surrounding the core infarction is noteworthy due to the observed bouts of acidosis using the SypHer3s sensor. Our findings suggest a potential association between these bouts, spreading depolarization, changes in calcium concentration, and the development of neuroinflammation. These reactions may ultimately lead to the synthesis of polysulfides, known modulators of inflammatory reactions. Thus, our data provides valuable additions to the existing knowledge on metabolic changes that take place during the progression of ischemic brain injury.

Race and sex differences in ROS production and SOD activity in HUVECs.

Black individuals and men are predisposed to an earlier onset and higher prevalence of hypertension, compared with White individuals and women, respectively. Therefore, the influence of race and sex on reactive oxygen species (ROS) production and superoxide dismutase (SOD) activity following induced inflammation was evaluated in female and male human umbilical vein endothelial cells (HUVECs) from Black and White individuals. It was hypothesized that HUVECs from Black individuals and male HUVECs would exhibit greater ROS production and impaired SOD activity. Inflammation was induced in HUVEC cell lines (n = 4/group) using tumor necrosis factor-alpha (TNF-α, 50ng/ml). There were no between group differences in ROS production or SOD activity in HUVECs from Black and White individuals, and HUVECs from Black individuals exhibited similar SOD activity at 24hr compared with 4hr of TNF-α treatment (p>0.05). However, HUVECs from White individuals exhibited significantly greater SOD Activity (p<0.05) at 24hr as compared to 4hr in the control condition but not with TNF-α treatment (p>0.05). Female HUVECs exhibited significantly lower ROS production than male HUVECs in the control condition and following TNF-α induced inflammation (p<0.05). Only female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF-α induced inflammation (p<0.05). HUVECs from White individuals alone exhibit blunted SOD activity when comparing control and TNF-α conditions. Further, compared to female HUVECs, male HUVECs exhibit a pro-inflammatory state.

In Vivo Aggregation of Clearable Bimetallic Nanoparticles with Interlocked Surface Motifs for Cancer Therapeutics Amplification.

Although renal-clearable luminescent metal nanoparticles (NPs) have been widely developed, their application to efficient cancer therapy is still limited due to low reactive oxygen species (ROS) production. Here, a novel system of clearable mercaptosuccinic acid (MSA) coated Au-Ag bimetallic NPs is designed to enhance ROS production. The results show that the strong COO-Ag coordination bonds between the carboxylic acid groups of MSA and Ag atoms on the Au-Ag bimetallic NPs could construct high-rigidity interlocked surface motifs to restrict the intrananoparticle motions for enhanced ROS generation. Moreover, bimetallic NPs exhibit pH-responsive self-assembly capability under the acidic environment inside lysosomes of cancer cells at both in vitro and in vivo, restricting the internanoparticle motions to further boost ROS production. The well-designed bimetallic NPs show high tumor targeting efficiency, fast elimination from the body through rapid liver biotransformation, and extensive destruction to cancer cells, resulting in good security and prominent therapeutic performance.

Heavy metals do not induce ROS production by mitochondrial respirasome

Since the discovery of the respirasome constituted by complexes I, III2, and IV, its precise participation in mitochondrial bioenergetics is poorly understood. We previously determined a higher NADH:DBQ oxidoreductase activity coupled to a lower ROS production by the respirasome than the free complex I. Toxicological studies suggest that respiratory complexes are heavy metals target during mitochondrial intoxication increasing ROS production, reducing ATP synthesis and cell viability; however, the inhibition of respiratory complexes activities by heavy metals is still unknown. Here we showed a putative deactivation of the respirasomal-complex I by seven of the most toxicologically relevant heavy metals, without increasing the ROS production. Contrastingly, the free complex I was more resistant to heavy metals but was 30 times more ROS-producing. These results underlie the preventive role of the respirasome in mitochondrial electron leak and ROS production and recall its disassembled in some pathologies which involve mitochondrial damage and oxidative stress.

N-3 PUFA-Enriched Diet Preserves Skeletal Muscle Mitochondrial Function and Redox State and Prevents Muscle Mass Loss in Mice with Chronic Heart Failure.

Rationale and Methods: Skeletal muscle derangements, potentially including mitochondrial dysfunction with altered mitochondrial dynamics and high reactive oxygen species (ROS) generation, may lead to protein catabolism and muscle wasting, resulting in low exercise capacity and reduced survival in chronic heart failure (CHF). We hypothesized that 8-week n-3-PUFA isocaloric partial dietary replacement (Fat = 5.5% total cal; EPA + DHA = 27% total fat) normalizes gastrocnemius muscle (GM) mitochondrial dynamics regulators, mitochondrial and tissue pro-oxidative changes, and catabolic derangements, resulting in preserved GM mass in rodent CHF [Myocardial infarction (MI)-induced CHF by coronary artery ligation, left-ventricular ejection fraction <50%]. Results: Compared to control animals (Sham), CHF had a higher GM mitochondrial fission-fusion protein ratio, with low ATP and high ROS production, pro-inflammatory changes, and low insulin signalling. n-3-PUFA normalized all mitochondrial derangements and the pro-oxidative state (oxidized to total glutathione ratio), associated with normalized GM cytokine profile, and enhanced muscle-anabolic insulin signalling and prevention of CHF-induced GM weight loss (all p < 0.05 vs. CHF and p = NS vs. S). Conclusions:n-3-PUFA isocaloric partial dietary replacement for 8 weeks normalizes CHF-induced derangements of muscle mitochondrial dynamics regulators, ROS production and function. n-3-PUFA mitochondrial effects result in preserved skeletal muscle mass, with potential to improve major patient outcomes in clinical settings.

The protective role of tetraploidy and nanoparticles in arsenic-stressed rice: Evidence from RNA sequencing, ultrastructural and physiological studies

Genome doubling in plants induces physiological and molecular changes to withstand environmental stress. Diploid rice (D-2x) and its tetraploid (T-4x) plants were treated with 25 μM Arsenic (As) and 15 mg L−1 TiO2 nanoparticles (NPs), and results indicated decreased growth and photosynthetic activity with high accumulation of reactive oxygen species (ROS) due to the As-toxicity in rice lines, significantly in D-2x rice plants. The treatment of As-contaminated rice with TiO2 NPs resulted in increased root length (8.17%) and chlorophyll AB (13.28%) and decreased electrolyte leakage (21.76%) and H2O2 (17.65%) contents than its counterpart diploid rice. Moreover, TiO2 NPs improved the activity of peroxidase, catalase, glutathione, and superoxide dismutase and reduced lipid peroxidation due to lower ROS production in D-2x and T-4x under As toxicity. Transcriptome analysis revealed abrupt changes in the expression levels of key signaling heat shock proteins, tubulin, aquaporins, As, and metal transporters under As toxicity in T-4x and D-2x lines. The KEGG and GO studies highlighted the striking distinctions between rice lines under As-stress in glutathione metabolism, H2O2 catabolic process, MAPK signaling pathway, and carotenoid biosynthesis terms, revealing consistency between physiological and molecular results. Root cells from D-2x rice were significantly more distorted by As poisoning than those from 4x rice, and cell organelles, such as mitochondria and endoplasmic reticulum, were changed or deformed. These findings proved the superiority of tetraploid rice lines over their diploid counterpart in coping with As-stress.

Improvement of Antioxidant Defences in Keratinocytes Grown in Physioxia: Comparison of 2D and 3D Models.

Keratinocytes prevent skin photoaging by ensuring the defence against oxidative stress, an excessive production of reactive oxygen species (ROS). They are localized within the epidermis where the oxygen level (1-3% O2), named physioxia, is low compared to other organs. Oxygen is essential for life but also generates ROS. Most of the in vitro studies on keratinocyte antioxidant capacities are performed under atmospheric oxygen, named normoxia, which is very far from the physiological microenvironment, thus submitting cells to an overoxygenation. The present study is aimed at investigating the antioxidant status of keratinocyte grown under physioxia in both 2D and 3D models. First, we show that the basal antioxidant profiles of keratinocytes display important differences when comparing the HaCaT cell line, primary keratinocytes (NHEK), reconstructed epidermis (RHE), and skin explants. Physioxia was shown to promote a strong proliferation of keratinocytes in monolayers and in RHE, resulting in a thinner epidermis likely due to a slowdown in cell differentiation. Interestingly, cells in physioxia exhibited a lower ROS production upon stress, suggesting a better protection against oxidative stress. To understand this effect, we studied the antioxidant enzymes and reported a lower or equivalent level of mRNA for all enzymes in physioxia conditions compared to normoxia, but a higher activity for catalase and superoxide dismutases, whatever the culture model. The unchanged catalase amount, in NHEK and RHE, suggests an overactivation of the enzyme in physioxia, whereas the higher amount of SOD2 can explain the strong activity. Taken together, our results demonstrate the role of oxygen in the regulation of the antioxidant defences in keratinocytes, topic of particular importance for studying skin aging. Additionally, the present work points out the interest of the choice of both the keratinocyte culture model and the oxygen level to be as close as possible to the in situ skin.

Unravelling the pathway determining the CO2 selectivity in photocatalytic toluene oxidation on TiO2 with different particle size

The rapid intermediate species accumulation on TiO2 induces the deactivation and is a long-standing unsolved issue in the photocatalytic oxidation of volatile organic compounds (VOCs). Exploring efficient pathway to achieve deep oxidation is of crucial significance. In this study, we observed that TiO2 with small particle size (S-TiO2) had high toluene conversion but low CO2 mineralization, while TiO2 with large particle size (L-TiO2) had low toluene conversion but high CO2 mineralization. The characterization results showed that S-TiO2 had a larger specific surface area and more surface hydroxyl groups than L-TiO2, which accounted for its higher toluene conversion. Additionally, low toluene adsorption but a larger quantity of reactive oxygen species (ROS) was observed on L-TiO2. Our findings further revealed that toluene on S-TiO2 was initially oxidized to benzene-ring-containing intermediate species by ROS. However, the accumulation of these species suppressed the ROS production and blocked the ring-opening reaction, resulting in low CO2 selectivity. In contrast, nearly no intermediate species was accumulated on L-TiO2 due to the low toluene adsorption and abundant ROS production, which allowed the photocatalytic oxidation of toluene on L-TiO2 to follow the direct ring-opening pathway and achieve higher CO2 selectivity. This study provides new insight into the mechanism of the photocatalytic oxidation of VOCs.

Hydrogen Sulfide Gas Amplified ROS Cascade: FeS@GOx Hybrid Nanozyme Designed for Boosting Tumor Chemodynamic Immunotherapy.

Chemodynamic immunotherapy that utilizes catalysts to produce reactive oxygen species (ROS) for killing tumor cells and arousing antitumor immunity, has received considerable attention. However, it is still restricted by low ROS production efficiency and insufficient immune activation, due to intricate redox homeostasis in tumor microenvironment (TME). Herein, a metalloprotein-like hybrid nanozyme (FeS@GOx) is designed by in situ growth of nanozyme (ferrous sulfide, FeS) in natural enzyme (glucose oxidase, GOx) to amplify ROS cascade for boosting chemodynamic immunotherapy. In FeS@GOx, GOx allows the conversion of endogenous glucose to gluconic acid and hydrogen peroxide, which provides favorable increasing hydrogen peroxide for subsequent Fenton reaction of FeS nanozymes, thus reinforcing the ROS production. Notably, H2 S release is activated by the gluconic acid generation-related pH decrease, which can suppress the activity of endogenous thioredoxin reductase and catalase to further inhibit the ROS elimination. Thus, FeS@GOx can sustainably amplify ROS accumulation and perturb intracellular redox homeostasis to improve chemodynamic therapy and trigger robust immunogenic cell death for effective immunotherapy combined with immune checkpoint blockade. This work proposes a feasible H2 S amplified ROS cascade strategy employing a bioinspired hybrid nanozyme, providing a novel pathway to multi-enzyme-mediated TME modulation for precise and efficient chemodynamic immunotherapy. This article is protected by copyright. All rights reserved.

Afu-Emi1 Contributes to Stress Adaptation and Voriconazole Susceptibility in Aspergillus fumigatus.

Invasive aspergillosis (IA) is the second most common invasive fungal disease and is associated with high mortality rates. Aspergillus fumigatus is the predominant causal agent of this life-threatening infection. Triazoles are still the cornerstone of antifungal treatment, and voriconazole remains the first-line choice. However, voriconazole resistance has been increasingly reported, which results in significantly higher mortality rates for IA and is particularly problematic. In the present study, we report the identification and functional study of a protein with previously unknown function that is encoded by the gene designated Afu-emi1 (AFUA_1G07360). High-throughput gene replacement technology was applied to construct the knockout ΔAfu-emi1 strain and a revertant strain. The MICs for azoles, including posaconazole, itraconazole, and voriconazole, were evaluated via the broth microdilution method and E-tests, which revealed that disruption of Afu-emi1 resulted in 4-fold increased susceptibility to voriconazole. Colony growth in the presence of oxidants, namely, H2O2 and menadione, and osmotic pressure-altering agents, namely, NaCl and d-sorbitol, was measured. The Afu-emi1 mutant strain exhibited a significant growth defect under oxidative and osmotic stress. The reactive oxygen species (ROS) production levels with or without voriconazole pretreatment were determined, and the Afu-emi1 mutant strain exhibited significantly lower ROS production levels. The effects of Afu-emi1 disruption on voriconazole susceptibility, growth under stress, and ROS production were restored in the revertant strain. In addition, the expression of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b in the ΔAfu-emi1 strain was significantly reduced. In conclusion, deletion of the gene Afu-emi1 resulted in increased voriconazole susceptibility, attenuated ability for oxidative and osmotic stress adaptation, decreased ROS production, and downregulation of cyp51A, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1b expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. IMPORTANCE Voriconazole is the first-line choice for IA, a life-threatening disease. Therefore, voriconazole resistance has become particularly problematic. Disruption of Afu-emi1 resulted in increased susceptibility to voriconazole, a significant growth defect under oxidative and osmotic stress, and downregulation of target enzyme Cyp51A and efflux pump expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation and cyp51A and efflux pump expression in this medically important fungus. Targeting Afu-Emi1 might help to enhance azole therapeutic efficacy and impede azole resistance.

NCF1-dependent production of ROS protects against lupus by regulating plasmacytoid dendritic cell development and functions.

Low capacity to produce reactive oxygen species (ROS) due to mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aim to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using a set of cell-specific Cre-deleter, the human NCF1-339 variant knock-in, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbates both pristane-induced lupus and a newly established Yaa-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFNα levels and expression of IFN-stimulated genes. Mechanistic studies reveal that ROS deficiency enhances pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive STING/IFNα/JAK1/STAT1 cascade further augments type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protects against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.

Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.

Liposomes loaded with dual clinical photosensitizers for enhanced photodynamic therapy of cervical cancer.

Photodynamic therapy (PDT) has become a potential anti-cancer strategy owing to its negligible invasiveness, low toxicity, and high selectivity. The photosensitizer (PS) plays an indispensable role in PDT. Herein, a novel type of PS (Ce6-MB@Lips) which can be excited by a near-infrared (NIR) laser was designed and synthesized. Methylene blue (MB) and Chlorin e6 (Ce6), two organic dyes approved by the Food and Drug Administration (FDA), were used to prepare Ce6-MB@Lips by thin-film dispersion method, which improve the water solubility of Ce6 and reduce the cytotoxicity of MB. The Ce6-MB@Lips were shown to have a spherical nanostructure with an average particle size of 160.3 nm and excellent water solubility. Then the optical properties of Ce6-MB@Lips were further studied. Ce6-MB@Lips showed absorption peaks at 413 nm/670 nm and fluorescence peak at 697 nm. Compared with Ce6@Lips and MB@Lips, Ce6-MB@Lips showed better stability, stronger fluorescence intensity, and higher singlet oxygen (1O2) generation ability. Cell experimental analysis exhibited that the stable Ce6-MB@Lips showed low cytotoxicity, high phototoxicity and high reactive oxygen species (ROS) production capacity. After effective cell internalization, the prepared Ce6-MB@Lips showed excellent ability to promote tumor cell apoptosis in vitro. The Ce6-MB@Lips could be a promising candidate for PDT of cervical cancer.

Resfriamento de sêmen suíno em um diluente suplementado com isoespintanol

ABSTRACT: Spermatozoa experience oxidative, osmotic, chemical, and thermal stresses when cooled, which degrade the quality and fertilizing capacity of the cells. Adding antioxidants to the sperm extender mitigates these alterations. This study evaluated the effect of isoespintanol (ISO) on boar semen subjected to cooling. Fifteen ejaculates from five boars (Susscrofadomestica) were extended in Beltsville thawing solution (BTS) supplemented with 0 µM (control), 5 µM (ISO5), 10 µM (ISO10), 15 µM (ISO15), 20 µM (ISO20), 25 µM (ISO25), and 30 µM (ISO30) of ISO, which were then cooled for five days at 16 °C. Sperm kinetics, total motility (TM), and progressive motility (PM) were evaluated every 24 h using an IVOS computer-assisted sperm analysis (CASA) system. On day 1 and day 5 of cooling, a hypoosmotic test, spectrofluorometry, and flow cytometry were performed to evaluate the following: membrane functionality, measured as a function of hypoosmotic swelling (HOS); total antioxidant capacity (TAC); reactive oxygen species (ROS); and mitochondrial membrane potential (Δ¥M). Regression analysis and comparison of means using the Duncan test were performed. The ISO added had a slight impact on sperm motility, as evidenced by a reduction in TM at 24 h of cooling (but not prior) with the addition of 20 µM of ISO. Similarly, no effect of the ISO on the kinetics and functional integrity of the sperm membrane was observed at 96 h of cooling; however, the regression coefficients indicated that the ISO lowered the rate of decrease in sperm motility and the proportion of rapid spermatozoa relative to the concentration of ISO used. The ISO did not affect the TAC of the cooled semen; however, different concentrations of ISO lowered ROS production in the semen after 96 h of cooling. ISO also impacted the Δ¥M of the spermatozoa at 0 h of cooling, increasing the proportion of low Δ¥M cells and decreasing the proportion of high Δ¥M cells. In conclusion, ISO can reduce the loss of quality and oxidative stress occurring in boar semen during cooling and can modulate the mitochondrial activity of sperm.