Nebulized Inhalation of Peptide-Modified DNA Origami To Alleviate Acute Lung Injury.

Acute lung injury (ALI) is a severe inflammatory lung disease, with high mortality rates. Early intervention by reactive oxygen species (ROS) scavengers could reduce ROS accumulation, break the inflammation expansion chain in alveolar macrophages (AMs), and avoid irreversible damage to alveolar epithelial and endothelial cells. Here, we reported cell-penetrating R9 peptide-modified triangular DNA origami nanostructures (tDONs-R9) as a novel nebulizable drug that could reach the deep alveolar regions and exhibit an enhanced uptake preference of macrophages. tDONs-R9 suppressed the expression of pro-inflammatory cytokines and drove polarization toward the anti-inflammatory M2 phenotype in macrophages. In the LPS-induced ALI mouse model, treatment with nebulized tDONs-R9 alleviated the overwhelming ROS, pro-inflammatory cytokines, and neutrophil infiltration in the lungs. Our study demonstrates that tDONs-R9 has the potential for ALI treatment, and the programmable DNA origami nanostructures provide a new drug delivery platform for pulmonary disease treatment with high delivery efficiency and biosecurity.

Mitochondria-Derived Reactive Oxygen Species Contribute to Synergistic Interaction of Diabetes and Hypertension in Causing Chronic Kidney Injury.

Diabetes (DM) and hypertension (HTN) are major risk factors for chronic kidney injury, together accounting for >70% of end-stage renal disease. The combination of DM and HTN significantly accelerates development of renal injury; however, the underlying mechanisms of this synergy are still poorly understood. This study assessed whether mitochondria (MT) dysfunction is essential in developing renal injury in a rat model with combined DM and HTN. Type 1 DM was induced in Wistar rats by streptozotocin (STZ). HTN was induced six weeks later by inter-renal aorta constriction between the renal arteries, so that right kidneys were exposed to HTN while left kidneys were exposed to normotension. Kidneys exposed to DM or HTN alone had only mild glomerular injury and urinary albumin excretion (UAE). In contrast, kidneys exposed to DM plus 8 weeks HTN had significantly increased UAE and glomerular structural damage with reduced glomerular filtration rate. Marked increases in MT-derived reactive oxygen species (ROS) were also observed in right kidneys exposed to HTN+DM. We further tested whether treatment with MT-targeted antioxidant (MitoTEMPO) after the onset of HTN attenuates renal injury in rats with DM+HTN. Results show that kidneys in DM+AC+MitoTEMPO rats had lower UAE, less glomerular damage, and preserved MT function compared to untreated DM+AC rats. Our studies indicate that MT-derived ROS play a major role in promoting kidney dysfunction when DM is combined with HTN. Preserving MT function might be a potential therapeutic approach to halt the development of renal injury when DM coexists with HTN.

Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues.

BACKGROUND: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. METHODS: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. RESULTS: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. CONCLUSION: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

Carbonized Polymer Dot-Tannic Acid Nanoglue: Tissue Reinforcement with Concurrent Fluorescent Tracking, Insulin Delivery, and Reactive Oxygen Species Regulation for Normal and Diabetic Wound Healing.

Nanotizing biosealant components offer a multitude of chemical functionalities for superior adhesion-cohesion, delivering unique properties for comprehensive wound healing that are otherwise impossible to achieve using commercial variants. For the first time, a two-step controlled hydrothermal pyrolysis is reported to nanotize dopamine, phloroglucinol, and glutaraldehyde into carbon dot (CD) to be subsequently converted into carbonized polymer dot (CPD) with gelatin as a co-substrate. Chemical crosslinking of CD with gelatin through Schiff base formation before the second pyrolysis step ensures a complex yet porous polymeric network. The retention of chemical functionalities indigenous to CD substrates and gelatin along with the preservation of CD photoluminescence in CPD for optical tracking is achieved. A unique nanoformulation is created with the CPD through tannic acid (TA) grafting evolving CPD-TA nanoglue demonstrating ≈1.32 MPa strength in lap shear tests conducted on porcine skin, surpassing traditional bioadhesives. CPD-TA nanoglue uploaded insulin as chosen cargo disbursal at the wound site for healing normal and in vitro diabetic wound models using HEKa cells with extraordinary biocompatibility. Most importantly, CPD-TA can generate reactive oxygen species (ROS) and scavenge simultaneously under ambient conditions (23 W white LED or dark) for on-demand sterilization or aiding wound recovery through ROS scavenging.

A Polymeric Nanosponge as a Broad-Spectrum Reactive Oxygen Species Scavenger for Acute Kidney Injury Treatment.

Acute kidney injury (AKI) is closely associated with the overproduction of reactive oxygen species (ROS), which can cause multiple organ dysfunctions without timely treatment. However, only supportive treatments are currently available for AKI in clinics. Here, we developed nanomaterials of hyperbranched polyphosphoester (PPE) containing abundant thioether (S-PPE NP) and thioketal bonds (TK-PPE NP). Our data demonstrates that S-PPE NP exhibits an excellent capability of absorbing and scavenging multiple types of ROS, including H2O2, •OH, and •O2-, via thioether oxidation to sulfone or sulfoxide; it was also determined that S-PPE NP efficiently eliminates intracellular ROS, thus preventing cellular damage. Moreover, S-PPE NP was able to efficiently accumulate in the injured kidneys of AKI-bearing mice. As a result, the administration of S-PPE NP provided a superior therapeutic effect in AKI-bearing mice by downregulating ROS- and inflammation-related signaling pathways, thus reducing cell apoptosis. This thioether-containing polymer represents a promising broad-spectrum ROS scavenger that can be used for effective AKI treatments.

Compound 5 alleviated acute kidney injury without affecting the antitumor effect after cisplatin treatment.

Despite being a powerful weapon against cancer cells, cisplatin's therapeutic potential is hampered by numerous adverse reactions, including acute kidney injury (AKI). Compound 5 has 3-SH fragments at the end of the vertical short alkyl side chain, which is an ROS scavenger synthesized. In this study, we evaluated the protective effect of compound 5 on the kidney after cisplatin administration and its mechanism. The results founded that compound 5 can alleviate serum urea nitrogen and serum creatinine induced by cisplatin administration in vivo. In addition, histopathological analysis of the kidneys showed that compound 5 significantly reduced cisplatin-induced (Cis-induced) renal toxicity compared with the cisplatin group. A mechanism study showed that compound 5 significantly reduces NOX4 levels, improves the activity of antioxidant enzymes (SOD and GSH-Px), reduces Malondialdehyde (MDA) levels, increases the total antioxidant level, reduces oxidative stress, and thus reduces kidney tissue damage. At the same time, compound 5 activated the Nrf2 signaling pathway. In addition, it can increase the expression of Bax, reduce the expression of Bcl-2 and caspase-3, a marker of apoptosis, which is beneficial to the survival of kidney cells. Additionally, compound 5 did not interfere with the antitumor effects of cisplatin in in vivo xenotransplantation models.

Oral Metal-Free Melanin Nanozymes for Natural and Durable Targeted Treatment of Inflammatory Bowel Disease (IBD).

Oral antioxidant nanozymes bring great promise for inflammatory bowel disease (IBD) treatment. To efficiently eliminate reactive oxygen species (ROS), various metal-based nanozymes have been developed for the treatment of IBD but their practical applications are seriously impaired by unstable ROS-eliminating properties and potential metal ion leakage in the digestive tract. Here, the authors for the first time propose metal-free melanin nanozymes (MeNPs) with excellent gastrointestinal stability and biocompatibility as a favorable therapy strategy for IBD. Moreover, MeNPs have extremely excellent natural and long-lasting characteristics of targeting IBD lesions. In view of the dominant role of ROS in IBD, the authors further reveal that oral administration of MeNPs can greatly alleviate the six major pathological features of IBD: oxidative stress, endoplasmic reticulum stress, apoptosis, inflammation, gut barrier disruption, and gut dysbiosis. Overall, this strategy highlights the great clinical application prospects of metal-free MeNPs via harnessing ROS scavenging at IBD lesions, offering a paradigm for antioxidant nanozyme in IBD or other inflammatory diseases.

Scavenging of reactive oxygen species effectively reduces Pseudomonas aeruginosa biofilms through disrupting policing.

Biofilm formation is likely to contribute greatly to antibiotic resistance in bacteria and therefore the efficient removal of bacterial biofilms needs addressing urgently. Here, we reported that the supplement of non-inhibitory concentration of N-acetyl-L-cysteine (NAC), a common reactive oxygen species (ROS) scavenger, can significantly reduce the biomass of mature Pseudomonas aeruginosa biofilms (corroborated by crystal violet assay and laser scanning confocal microscopy). 1 mM NAC increased the cheater (ΔlasR mutant) frequency to 89.4 ± 1.5% in the evolved PAO1 after the 15-day treatment. Scavenging of ROS by NAC induced the collapse of P. aeruginosa biofilms, but it did not alter quorum sensing-regulated genes expression (e.g., hcnC and cioAB) and hydrogen cyanide production. The replenishment of public good protease contributed to the recovery of biofilm biomass, indicating the role of disrupting policing in biofilm inhibition. Furthermore, 7 typical ROS scavengers (e.g., superoxide dismutase, catalase and peroxidase, etc.) also effectively inhibited mature P. aeruginosa biofilms. This study demonstrates that scavenging of ROS can promote the selective control of P. aeruginosa biofilms through policing disruption as a targeted biofilm control strategy in complex water environments.

The multifaceted role of reactive oxygen species in tumorigenesis.

Redox homeostasis is an essential requirement of the biological systems for performing various normal cellular functions including cellular growth, differentiation, senescence, survival and aging in humans. The changes in the basal levels of reactive oxygen species (ROS) are detrimental to cells and often lead to several disease conditions including cardiovascular, neurological, diabetes and cancer. During the last two decades, substantial research has been done which clearly suggests that ROS are essential for the initiation, progression, angiogenesis as well as metastasis of cancer in several ways. During the last two decades, the potential of dysregulated ROS to enhance tumor formation through the activation of various oncogenic signaling pathways, DNA mutations, immune escape, tumor microenvironment, metastasis, angiogenesis and extension of telomere has been discovered. At present, surgery followed by chemotherapy and/or radiotherapy is the major therapeutic modality for treating patients with either early or advanced stages of cancer. However, the majority of patients relapse or did not respond to initial treatment. One of the reasons for recurrence/relapse is the altered levels of ROS in tumor cells as well as in cancer-initiating stem cells. One of the critical issues is targeting the intracellular/extracellular ROS for significant antitumor response and relapse-free survival. Indeed, a large number of FDA-approved anticancer drugs are efficient to eliminate cancer cells and drug resistance by increasing ROS production. Thus, the modulation of oxidative stress response might represent a potential approach to eradicate cancer in combination with FDA-approved chemotherapies, radiotherapies as well as immunotherapies.

Involvement of Reactive Oxygen Species in ABA-Induced Increase in Hydraulic Conductivity and Aquaporin Abundance.

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H2O2 production by ABA-treated plants for the effect of this hormone on AQP abundance.

Will the Bacteria Survive in the CeO2 Nanozyme-H2O2 System?

As one of the nanostructures with enzyme-like activity, nanozymes have recently attracted extensive attention for their biomedical applications, especially for bacterial disinfection treatment. Nanozymes with high peroxidase activity are considered to be excellent candidates for building bacterial disinfection systems (nanozyme-H2O2), in which the nanozyme will promote the generation of ROS to kill bacteria based on the decomposition of H2O2. According to this criterion, a cerium oxide nanoparticle (Nanoceria, CeO2, a classical nanozyme with high peroxidase activity)-based nanozyme-H2O2 system would be very efficient for bacterial disinfection. However, CeO2 is a nanozyme with multiple enzyme-like activities. In addition to high peroxidase activity, CeO2 nanozymes also possess high superoxide dismutase activity and antioxidant activity, which can act as a ROS scavenger. Considering the fact that CeO2 nanozymes have both the activity to promote ROS production and the opposite activity for ROS scavenging, it is worth exploring which activity will play the dominating role in the CeO2-H2O2 system, as well as whether it will protect bacteria or produce an antibacterial effect. In this work, we focused on this discussion to unveil the role of CeO2 in the CeO2-H2O2 system, so that it can provide valuable knowledge for the design of a nanozyme-H2O2-based antibacterial system.

ROS signaling cascades: dual regulations for osteoclast and osteoblast.

Accumulating evidence indicates that intracellular reactive oxygen species (ROS) production is highly involved in bone homeostasis by intervening osteoclast or osteoblast differentiation. Interestingly, ROS that are known as oxidizing agents exert dose-dependent biphasic properties in bone remodeling, including preventing osteoblast activity but accelerating osteoclast resorption. ROS mainly composed of superoxide anion radical, hydroxyl radical, nitric oxide, and two-electron reduction product hydrogen peroxide, which are important components to regulate bone cell metabolism and function in mammal skeleton. These free radicals can be partly produced in bone and boosted in an inflammation state. Although numerous researches have emphasized the impacts of ROS on bone cell biology and verified the mechanism of ROS signaling cascades, the recapitulatory commentary is necessary. In this review article, we particularly focus on the regulation of the intracellular ROS and its potential mechanism impacting on cell-signaling transduction in osteoclast and osteoblast differentiation for preferable understanding the pathogenesis and searching for novel therapeutic protocols for human bone diseases.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.

Comparative study of the differential cell death protecting effect of various ROS scavengers.

Oxidative stress has for a long time been associated with cell death, especially classical necrosis, however, its role in other cell death pathways is less clear. Here, we evaluated in a comparative way, the effect of four different reactive oxygen species (ROS) scavengers, N-acetyl-L-cysteine (NAC), α-tocopherol and two superoxide dismutase mimetics, n(III)tetrakis(4-benzoic acid)porphyrin chloride, and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol), in four different cell death models, including menadione-triggered necrosis, staurosporine-induced apoptosis and tumor necrosis factor (TNF)-induced apoptosis and necroptosis. While menadione-triggered necrosis was completely prevented by the classical ROS scavenger NAC and to a substantial amount by the other scavengers, ROS targeting was found to have a marginal effect on the other cell death modalities investigated. Despite its side-effects at higher concentrations, Tempol was able to substantially prevent TNF-induced apoptosis and to a somewhat lesser extent TNF-induced necroptosis. However, this seems to be separated from its ROS-scavenging function.

Inhalable gadofullerenol/[70] fullerenol as high-efficiency ROS scavengers for pulmonary fibrosis therapy.

Pulmonary fibrosis has become a fatal disease for its high incidence and few effective drugs available in clinic. In this study, gadofullerenol (GF-OH) and [70] fullerenol (C70-OH) nanoparticles (NPs) prepared by a one-pot reaction were designed as nanomedicines to treat this fatal disease. It was revealed that the inhalation of gadofullerenols and [70] fullerenols substantially alleviates the collagen deposition induced by acute lung injury. Based on detailed studies of oxidative stress parameters and transforming growth factor-ß1 (TGF-ß1), we demonstrated they owned the antioxidant and anti-inflammatory functions for the modulation of ROS-mediated inflammation process. Thus the therapeutic effect may be associated with synergistic mechanism of scavenging free radicals and indirectly modulating TGF-ß1 expression. Moreover, GF-OH NPs were observed to show the superiority to C70-OH NPs both in vitro and in vivo due to the structural distinction. These results suggest the inhalable fullerenols are highly potential for clinical therapy of pulmonary fibrosis.

Transcripts encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles.

PURPOSE: To study the presence and distribution of genes encoding free radical scavengers in human granulosa cells from primordial and primary ovarian follicles. METHODS: A class comparison study on existing granulosa cell transcriptome from primordial (n = 539 follicles) and primary (n = 261) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy was performed and interrogated. RESULTS: In granulosa cells from primordial follicles, 30 genes were annotated 'mitochondrial dysfunction' including transcripts (PRDX5, TXN2) encoding enzymatic free radical scavengers peroxiredoxin 5 and thioredoxin 2. Several apoptosis regulation genes were noted (BCL2, CAS8, CAS9, AIFM1). In granulosa cells from primary follicles, mitochondrial dysfunction signalling pathway was annotated. High expression of transcripts encoding the free radical scavenger peroxiredoxin 3, as well as anti-apoptotic enzyme BCL2, was found. Interestingly, PARK7 encoding the deglycase (DJ-1) protein was expressed in granulosa cells from primary follicles. DJ-1 is implicated in oxidative defence and functions as a positive regulator of the androgen receptor and as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signalling pathway suppressor PTEN. CONCLUSIONS: The results indicate extensive energy production and free radical scavenging in the granulosa cells of primordial follicles with potential implications for ovarian ageing, cigarette smoking, premature ovarian failure and polycystic ovarian syndrome. Furthermore, DJ-1 may be involved in androgen responsiveness and the regulation of follicle growth via PI3K/PTEN/AKT signalling pathway regulation in the granulosa cells of primary follicles. The involvement of mitochondrial free radical production in the age-related decline of competent oocytes is becoming apparent.

Palmitic Acid Curcumin Ester Facilitates Protection of Neuroblastoma against Oligomeric Aß40 Insult.

BACKGROUND/AIMS: The generation of reactive oxygen species (ROS) caused by amyloid-ß (Aß) is considered to be one of mechanisms underlying the development of Alzheimer's disease. Curcumin can attenuate Aß-induced neurotoxicity through ROS scavenging, but the protective effect of intracellular curcumin on neurocyte membranes against extracellular Aß may be compromised. To address this issue, we synthesized a palmitic acid curcumin ester (P-curcumin) which can be cultivated on the cell membrane and investigated the neuroprotective effect of P-curcumin and its interaction with Aß. METHODS: P-curcumin was prepared through chemical synthesis. Its structure was determined via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). An MTT assay was used to assess Aß cytotoxicity and the protective effect of P-curcumin on SH-SY5Y cells. The effect of P-curcumin on Aß-induced ROS production in vitro and in vivo were assessed based on changes in dichlorofluorescein (DCF) fluorescence. A spectrophotometric method was employed to detect lipid peroxidation. To mimic the interaction of P-curcumin on cell membranes with Aß, liposomes were prepared by thin film method. Finally, the interactions between free P-curcumin and P-curcumin cultivated on liposomes and Aß were determined via spectrophotometry. RESULTS: A novel derivative, palmitic acid curcumin ester was prepared and characterized. This curcumin, cultivated on the membranes of neurocytes, may prevent Aß-mediated ROS production and may inhibit the direct interaction between Aß and the cellular membrane. Furthermore, P-curcumin could scavenge Aß-mediated ROS as curcumin in vitro and in vivo, and had the potential to prevent lipid peroxidation. Morphological analyses showed that P-curcumin was better than curcumin at protecting cell shape. To examine P-curcumin's ability to attenuate direct interaction between Aß and cell membranes, the binding affinity of Aß to curcumin and P-curcumin was determined. The association constants for free P-curcumin and curcumin were 7.66 × 104 M-1 and 7.61 × 105 M-1, respectively. In the liposome-trapped state, the association constants were 3.71 × 105 M-1 for P-curcumin and 1.44× 106 M-1 for curcumin. With this data, the thermodynamic constants of P-curcumin association with soluble Aß (ΔH, ΔS, and ΔG) were also determined. CONCLUSION: Cultivated curcumin weakened the direct interaction between Aß and cell membranes and showed greater neuroprotective effects against Aß insult than free curcumin.

Characterization of a vacuolar H+-ATPase G subunit gene from Juglans regia (JrVHAG1) involved in mannitol-induced osmotic stress tolerance.

KEY MESSAGE: JrVHAG1 is an important candidate gene for plant osmotic tolerance regulation. Vacuolar H+-ATPase (V-ATPase) is important for plant responses to abiotic stress; the G subunit is a vital part of V-ATPase. In this study, a G subunit of V-ATPase was cloned from Juglans regia (JrVHAG1) and functionally characterized. JrVHAG1 transcription was induced by mannitol that increasing 17.88-fold in the root at 12 h and 19.16-fold in the leaf at 96 h compared to that under control conditions. JrVHAG1 was overexpressed in Arabidopsis and three lines (G2, G6, and G9) with highest expression levels were selected for analysis. The results showed that under normal conditions, the transgenic and wild-type (WT) plants displayed similar germination, biomass accumulation, reactive oxygen species (ROS) level, and physiological index. However, when treated with mannitol, the fresh weight, root length, water-holding ability, and V-ATPase, superoxide dismutase, and peroxidase activity of G2, G6, and G9 were significantly higher than those of WT. In contrast, the ROS and cell damage levels of the transgenic seedlings were lower than those of WT. Furthermore, the transcription levels of V-ATPase subunits, ABF, DREB, and NAC transcription factors (TFs), all of which are factors of ABA signaling pathway, were much higher in JrVHAG1 transgenic plants than those in WT. The positive induction of JrVHAG1 gene under abscisic acid (ABA) treatments in root and leaf tissues indicates that overexpression of JrVHAG1 improves plant tolerance to osmotic stress relating to the ABA signaling pathway, which is transcriptionally activated by ABF, DREB, and NAC TFs, and correlated to ROS scavenging and V-ATPase activity.

In planta characterization of a tau class glutathione S-transferase gene from Juglans regia (JrGSTTau1) involved in chilling tolerance.

KEY MESSAGE: JrGSTTau1 is an important candidate gene for plant chilling tolerance regulation. A tau subfamily glutathione S-transferase (GST) gene from Juglans regia (JrGSTTau1, GeneBank No.: KT351091) was cloned and functionally characterized. JrGSTTau1 was induced by 16, 12, 10, 8, and 6 °C stresses. The transiently transformed J. regia showed much greater GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POD) activities and lower H2O2, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) rate than prokII (empty vector control) and RNAi::JrGSTTau1 under cold stress, indicating that JrGSTTau1 may be involved in chilling tolerance. To further confirm the role of JrGSTTau1, JrGSTTau1 was heterologously expressed in tobacco, transgenic Line5, Line9, and Line12 were chosen for analysis. The germinations of WT, Line5, Line9, and Line12 were similar, but the fresh weight, primary root length, and total chlorophyll content (tcc) of the transgenic lines were significantly higher than those of WT under cold stress. When cultivated in soil, the GST and SOD activities of transgenic tobacco were significantly higher than those of WT; however, the MDA and H2O2 contents of WT were on average 1.47- and 1.96-fold higher than those of Line5, Line9, and Line12 under 16 °C. The DAB, Evans blue, and PI staining further confirmed these results. Furthermore, the abundances of NtGST, MnSOD, NtMAPK9, and CDPK15 were elevated in 35S::JrGSTTau1 tobacco compared with WT. These results suggested that JrGSTTau1 improves the plant chilling tolerance involved in protecting enzymes, ROS scavenging, and stress-related genes, indicating that JrGSTTau1 is a candidate gene for the potential application in molecular breeding to enhance plant abiotic stress tolerance.

Mitochondria-targeted ROS scavenger improves post-ischemic recovery of cardiac function and attenuates mitochondrial abnormalities in aged rats.

Mitochondria-generated reactive oxygen species (ROS) play a crucial role in the pathogenesis of aging and age-associated diseases. In this study, we evaluated the effects of XJB-5-131 (XJB), a mitochondria-targeted ROS and electron scavenger, on cardiac resistance to ischemia-reperfusion (IR)-induced oxidative stress in aged rats. Male adult (5-month old, n=17) and aged (29-month old, n=19) Fischer Brown Norway (F344/BN) rats were randomly assigned to the following groups: adult (A), adult+XJB (AX), aged (O), and aged+XJB (OX). XJB was administered 3 times per week (3mg/kg body weight, IP) for four weeks. At the end of the treatment period, cardiac function was continuously monitored in excised hearts using the Langendorff technique for 30 min, followed by 20 min of global ischemia, and 60-min reperfusion. XJB improved post-ischemic recovery of aged hearts, as evidenced by greater left ventricular developed-pressures and rate-pressure products than the untreated, aged-matched group. The state 3 respiration rates at complexes I, II and IV of mitochondria isolated from XJB-treated aged hearts were 57% (P<0.05), 25% (P<0.05) and 28% (P<0.05), respectively, higher than controls. Ca(2+)-induced swelling, an indicator of permeability transition pore opening, was reduced in the mitochondria of XJB-treated aged rats. In addition, XJB significantly attenuated the H2O2-induced depolarization of the mitochondrial inner membrane as well as the total and mitochondrial ROS levels in cultured cardiomyocytes. This study underlines the importance of mitochondrial ROS in aging-induced cardiac dysfunction and suggests that targeting mitochondrial ROS may be an effective therapeutic approach to protect the aged heart against IR injury.