Cerebral ischemic stroke is a leading cause of death worldwide. Though timely reperfusion reduces the infarction size, it exacerbates neuronal apoptosis due to oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of antioxidant enzymes. Activating Nrf2 gives a therapeutic approach to ischemic stroke.

Herein we explored flavonoids identified from Polypodium hastatum as Nrf2 activators and their protective effects on PC12 cells injured by oxygen and glucose deprivation/restoration (OGD/R) as well as middle cerebral artery occlusion (MCAO) mice.

The results showed among these flavonoids, AAKR significantly improved the survival of PC12 cells induced by OGD/R and activated Nrf2 in a Keap1-dependent manner. Further investigations have disclosed AAKR attenuated oxidative stress, mitochondrial dysfunction and following apoptosis resulting from OGD/R. Meanwhile, activation of Nrf2 by AAKR was involved in the protective effects. Finally, it was found that AAKR could protect MCAO mice brains against ischemia/reperfusion injury via activating Nrf2.

This investigation could provide lead compounds for the discovery of novel Nrf2 activators targeting ischemia/reperfusion injury.

In health, the gut microbiome functions as a stable ecosystem maintaining overall balance and ensuring its own survival against environmental stressors through complex microbial interaction. This balance and protection from stressors is maintained through interactions both within the bacterial ecosystem as well as with its host. As a consequence, the gut microbiome plays a critical role in various physiological processes including maintaining the structure and function of the gut barrier, educating the gut immune system, and modulating the gut motor, digestive/absorptive, as well as neuroendocrine system all of which are crucial for human health and disease pathogenesis. Pre- and probiotics, widely available and clinically established, offer various health benefits primarily by beneficially modulating the gut microbiome. However, their clinical outcomes can vary significantly due to differences in host physiology, diets, individual microbiome compositions, and other environmental factors. This perspective paper highlights emerging scientific insights into the importance of microbial micronutrient sharing, gut redox balance, keystone species, and the gut barrier in maintaining a diverse and functional microbial ecosystem, and their relevance to human health. We propose a novel approach that targets microbial ecosystems and keystone taxa performance by supplying microbial micronutrients in the form of colon-delivered vitamins, and precision prebiotics [e.g. human milk oligosaccharides (HMOs) or synthetic glycans] as components of precisely tailored ingredient combinations to optimize human health. Such a strategy may effectively support and stabilize microbial ecosystems, providing a more robust and consistent approach across various individuals and environmental conditions, thus, overcoming the limitations of current single biotic solutions.

Nanozymes, a class of nanomaterials mimicking natural enzymatic functions, have gained significant attention due to their exceptional biocatalytic properties and wide-ranging applications in biosensing. The total antioxidant capacity (TAC) can serve as a crucial parameter for assessing food quality, guiding dietary choices, and monitoring health conditions. In recent years, various nanomaterials with peroxidase (POD)-like and oxidase (OXD)-like activity have been widely used for TAC determination. This review discusses the enzyme-mimicking catalytic activities of nanozymes related to TAC determination, the construction principles of nanozyme-based TAC sensors and systematically classifies the application of nanozyme sensors in TAC determination. Furthermore, the potential opportunities and challenges in the development of nanozyme-based sensors are evaluated, aiming to provide valuable insights for researchers in related fields.

Photodynamic therapy (PDT) using photosensitizer (PS)-integrated covered self-expandable metallic stents (SEMS) is proposed a new therapeutic approach for the treatment of palliative malignancies; however, the currently hydrophobic PS reduces the photoreactive effect, which leads to aggregation with low water solubility. In here, an aluminum (III)-phthalocyanine chloride tetrasulfonic acid (Al-PcS4)-integrated silicone-covered self-expanding catheter was successfully fabricated to perform localized PDT. The ratio of MeOH and Al-PcS4 concentrations was optimized to achieve PS coating uniformity. The photodynamic activity of the Al-PcS4-integrated silicone membrane was evaluated through laser exposure on membrane-layered tumor cell lines, tumor xenograft-bearing mice. PDT with the Al-PcS4-integrated membrane successfully generated sufficient cytotoxic singlet oxygen, inducing cell death in the esophageal cancer cell lines. PDT-treated tumor xenograft-bearing mice undergo apoptotic cell death and showed significant tumor regression. Localized PDT using an Al-PcS4-integrated silicone-covered self-expanding catheter was technically successful in the rabbit esophagus without severe complications. Based on the endoscopy, esophagography, histology, and immunohistochemistry, our study verified that localized PDT using the Al-PcS4-integrated silicone-covered self-expanding catheter was effective and safe to evenly induce tissue damage. Al-PcS4-integrated silicone-covered self-expanding catheter has substantial potential for the minimally invasive local therapy in malignant esophageal cancer.

Breast cancer remains one of the leading causes of cancer-related deaths, with therapeutic resistance and limited treatment options posing significant challenges. This study investigated the anticancer properties of isoquinocycline B (IQCB), an anthraquinone derivative obtained from a freshwater sponge microbiome Micromonospora sp. MS-62 (FBCC-B8445), against the MDA-MB-231 human breast cancer cell line. IQCB showed the greatest activity against cytotoxicity with an IC50 values of 9.2 ± 1.0 μM. IQCB treatment led to G0/G1 cell cycle arrest and apoptosis through mitochondrial pathways by suppressing cyclin D1/CDK4 expression, enhancing p27 levels, and reducing phosphorylated Akt levels. Furthermore, IQCB induced oxidative stress by promoting excessive reactive oxygen species (ROS) production, thereby activating JNK and p38-MAPK signaling while simultaneously inhibiting ERK phosphorylation. Apoptotic markers such as PARP cleavage and caspase-3 activation confirmed that mitochondrial-mediated apoptosis was a key mechanism of action. These results highlight the potential of IQCB as a therapeutic candidate for breast cancer and underscore the need for further research to explore its efficacy and mechanisms.

Stroke is a serious cerebrovascular disease that is categorized into two types: ischemic and hemorrhagic. The pathological mechanisms of ischemic stroke are complex and diverse, encompassing processes such as neuroinflammation and apoptosis. The pathological processes of hemorrhagic stroke primarily involve the disruption of the blood-brain barrier and cerebral edema. Western medical treatment methods show certain effectiveness during the acute phase of stroke, but they are limited by a narrow therapeutic window and secondary injuries. Traditional Chinese medicine (TCM) has a long history and unique advantages in treating stroke. Studies confirm that active compounds derived from TCM exert multi-pathway, multi-target effects, significantly improving therapeutic outcomes and reducing adverse reactions. However, due to the complexity of the components in TCM, research on monomeric components still faces challenges. This article reviews the relevant research progress published in domestic and international journals over the past twenty years regarding the mechanisms of action of monomeric components of TCM in the treatment of stroke, aiming to provide insights and references for the clinical application of TCM in stroke treatment and further new drug development.

In the current study, the mussels Mytilus galloprovincialis, exposed to four varying temperatures (17, 20, 23, and 26 °C), were contaminated with 50 μg/L of bisphenol A both with and without Nannochloropsis salina. The toxicity evaluation is determined by quantifying various biomarkers related to oxidative stress, neurotoxicity, and cellular damage. The key findings indicate that the toxicity of bisphenol A is heightened by rising temperature. The impact of bisphenol A is most evident at 26 °C, leading to excessive production of reactive oxygen species, depletion of non-enzymatic antioxidants, and activation of antioxidant enzymes (catalase and glutathione-S-transferase). The rise in malondialdehyde levels confirms lipid peroxidation caused by bisphenol A and intensified by thermal stress. These findings have been supported by strong molecular interactions between bisphenol A and lectin mytilec apo-form and proximal thread matrix protein 1 from M. galloprovincialis following the computational modeling assay. The incorporation of N. salina as a food additive helped, firstly, to mitigate the stress effects and, secondly, resulted in a noticeable enhancement of oxidative balance and filtration ability, along with decreased lipid peroxidation.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating multisystem disorder of unclear etiology that affects many individuals worldwide. One of its hallmark symptoms is prolonged fatigue following exertion, a feature also observed in long COVID, suggesting an underlying dysfunction in energy production in both conditions. Here, mitochondrial dysfunction and its potential pathogenetic role in these disorders are reviewed.

Precise tumor therapy is essential for improving treatment specificity, enhancing efficacy, and minimizing side effects. Targeting organelles is a key strategy for achieving this goal and is a frontier research area attracting a considerable amount of attention. The concept of organelle targeting has a significant effect on the structural design of the nanodrugs employed. Most notably, the intricate interactions among different organelles in a tumor cell essentially create a unified system. Unfortunately, this aspect might have been somewhat overlooked when existing organelle-targeting nanodrugs were designed. In this review, we underscore the synergistic relationship among the various organelles and advocate for a holistic view of organelle-targeting design. Through the integration of biology and material science, recent advancements in organelle targeting, escaping, and collaborating are consolidated to offer fresh perspectives for the development of antitumor nanomedicines.

Oxidative stress plays a pivotal role in the pathophysiology of infectious diseases, contributing to pathogenesis and the appearance of clinical symptoms. However, the involvement of oxidative stress in renal cryptococcosis remains unknown, as do the potential protective effects of rutin. Cryptococcus neoformans is considered the main agent of cryptococcosis, a systemic life-threatening opportunistic fungal disease that affects internal organs. In 2022, the World Health Organization classified it as a critical-priority group on its Fungal Priority Pathogens List. Rutin, a flavonoid with potent antioxidant and antifungal properties, has been proposed as a protective agent. Thus, this study aimed to evaluate whether 50 mg rutin/kg body weight prevents or reduces C. neoformans var. Grubii-induced renal oxidative stress. Renal fungal burden was significantly lower in rats that were treated with rutin and experimentally infected with C. neoformans, compared to those treated with saline solution and experimentally infected. Renal reactive oxygen species (ROS) and protein carbonylation levels were significantly higher in experimentally infected rats compared to uninfected controls, whereas catalase (CAT) and glutathione S-transferase (GST) activities were significantly lower. Treatment with rutin prevented the increase in renal ROS levels and the inhibition of CAT activity elicited by C. neoformans var. Grubii. However, no significant differences were observed in lipid damage or superoxide dismutase activity. This study is the first to demonstrate that C. neoformans var. Grubii infection induces renal oxidative damage in rats by promoting oxidative stress, increasing ROS levels, and impairing antioxidant defenses. Rutin treatment restored redox status in experimental rats through mechanisms involving oxidative stress. The protective effects of rutin against C. neoformans-induced kidney damage may result from its combined ability to scavenge ROS, inhibit protein damage, and enhance the antioxidant system.

Rubus L. is widely distributed throughout the world as a medicinal and food plant with rich values. There is increasing evidence that Rubus L. polysaccharides are important and representative active macromolecules with abundant in vitro and in vivo bioactivities, such as: antioxidant, immunomodulatory, hypoglycemic, anti-inflammatory, anti-tumor, anti-fatigue and so on. Despite significant advancements in research, a comprehensive and systematic review of Rubus L. polysaccharides has been absent. In this paper, we innovatively provide an in-depth overview of the extraction and purification methods, structural characterization, biological activities, possible molecular mechanisms, toxicity and applications of Rubus L. polysaccharides for the first time. In addition, the existing literature was quantitatively analyzed by bibliometrics. The paper also focuses on the challenges and future perspectives of the existing studies with a view to providing new insights and directions for the future development of Rubus L. polysaccharides.

Harnessing the modulation of redox homeostasis represents a promising anticancer strategy. Here, we design and evaluate Se-SN38, a prodrug of the camptothecin (CPT) derivative 7-ethyl-10-hydroxycamptothecin (SN38) with a cyclic five-membered diselenide moiety for redox-triggered activation. We demonstrate that Se-SN38 exhibits superior cytotoxicity in various cancer cell lines over the parent drug SN38 or the control prodrug S-SN38, a sulfur analogue of Se-SN38. This increased potency is attributed to the efficient release of SN38 and induction of oxidative stress, as demonstrated by a significant rise in reactive oxygen species production, along with a marked depletion of cellular total thiols and a decreased GSH/GSSG ratio. Furthermore, Se-SN38 treatment leads to inhibition of thioredoxin reductase activity, disruption of mitochondrial membrane potential, and induction of DNA damage, culminating in apoptosis. These findings suggest that Se-SN38 represents a promising strategy to enhance the therapeutic efficacy of CPT derivatives by exploiting the unique redox-active properties of cyclic five-membered diselenide to induce oxidative stress and apoptosis.

This study investigated the effects of the bloom-forming dinoflagellate Alexandrium tamarense and its potentially associated paralytic shellfish toxins on the early life stages of Larimichthys crocea (large yellow croaker) by integrating physiological effects with transcriptomic analysis to explore the molecular mechanisms underlying these harmful impacts. The results showed that 48-h acute exposure to A. tamarense culture and cell-free filtrate significantly reduced the heart rate in embryos and increased mortality rates in both embryos and larvae. Transcriptome sequencing of the filtrate-exposed group identified 130 differentially expressed genes in the embryo group and 884 in the juvenile group. Further analysis revealed that algal exposure triggered the activation of innate immunity in embryos, as evidenced by the significant upregulation of immune-related cytokines such as CCL20, IL11, and ILRA10. These genes were enriched in the cytokine-cytokine receptor interaction pathway and may induce immune responses through their respective downstream pathways. Additionally, the downregulation of the RNA polymerase and ribosome pathways suggests that protein synthesis was affected during the embryo stress response induced by A. tamarense. In juveniles, genes related to cardiac function, particularly those associated with myocardial contraction and calcium ion regulation, were downregulated after exposure to algal filtrate, further suggesting that A. tamarense, possibly through paralytic shellfish toxins, inhibits the heart function of L. crocea. The findings of this study elucidate the toxicological mechanisms of A. tamarense on the early life stages of L. crocea, providing scientific evidence for the impact of harmful algal blooms on marine life health and offering valuable insights for management strategies in aquaculture.

Iron is a crucial element for living organism in terms of oxygen transport, hematopoiesis, enzymatic activity, mitochondrial respiratory chain function and also immune system function. The human being has evolved a mechanism to regulate body iron. In some rheumatic diseases such as rheumatoid arthritis (RA), systemic lupus erythematous (SLE), systemic sclerosis (SSc), ankylosing spondylitis (AS), and gout, this balanced iron regulation is impaired. Altered iron homeostasis can contribute to disease progression through ROS production, fibrosis, inflammation, abnormal bone homeostasis, NETosis and cell senescence. In this review, we have focused on the iron metabolism in rheumatic disease and its role in disease progression.

Overabundance of reactive oxygen species (oxidative distress) leads to redox homeostasis disturbance and is associated with many pathological conditions. Accumulating evidence suggests that oxidative distress may contribute to osteoporosis. This review thoroughly outlines the relationships among osteoporosis, redox homeostasis, and alcohol addiction, since these relations are not sufficiently known and subsequently summarized. The brain-bone axis plays a crucial role in alcohol-induced damage to the nervous and skeletal systems. Alterations in the nervous system can lead to osteoporosis because the central nervous system is involved in bone remodeling through various neural pathways. Conversely, as an endocrine organ, bone secretes a number of bone-derived factors (osteokines), which can influence brain function and behavior. As a result, osteoporosis is more common in individuals with neurological disorders, and sudden neurological events can rapidly increase the risk of osteoporosis. Excessive alcohol consumption is linked to many neurological complications, as well as osteoporosis, which are manifested by disrupted redox homeostasis, inflammation, neurodegeneration, inhibition of neurogenesis, decreased bone mineral density, impaired bone microarchitecture, altered mineral homeostasis, raising fracture risk, hormonal dysregulation, and altered gut microbiota composition. Compared to men, alcohol dependence has more negative consequences for women, including an increased risk of liver, cardiovascular, metabolic, mental disorders, and breast cancer. Abstinence has been demonstrated to improve bone and brain health in alcohol addiction. The discovery of the brain-bone axis may lead to the development of new therapeutic approaches for alcohol and other substance addictions. Further research is needed in this direction, as many questions remain unanswered.

The widespread use of QDs raises health and environmental concerns, and the ROS induced oxidative stress is reported as the main mechanism of QDs toxicity. Cytochrome P450 (CYP450) superfamily, the primary enzyme system for metabolizing external compounds in the liver, also generates reactive oxygen species (ROS), making it crucial for detoxification and ROS production. Therefore, we investigated whether QDs could cause liver tissue damage by affecting the activity of CYP450 isoenzymes (CYP1A2, CYP2E1, CYP2D2, and CYP3A1) in liver microsomes, thereby altering ROS generation. This mechanism has not been previously reported. Our experiments indicate that CdTe QDs exhibit a dose/time-dependent relationship with the enzymatic activities of CYP1A2 and CYP2E1, which are closely related to ROS generation. However, an inconsistency was observed between the data for CYP2E1 activity in vivo and in vitro due to the complexity of in vivo regulatory factors. More importantly, in vitro experiments have shown that CdTe QDs can significantly promote the enzymatic activity of CYP1A2. Therefore, we speculate that CdTe QDs may induce ROS generation by enhancing CYP450 enzyme activities. In addition, molecular docking experiments were conducted to illustrate the impact of CdTe QDs on the structure of CYP1A2, leading to functional change (i.e., enzyme activity). These findings suggest a novel mechanism by which CdTe QDs regulate CYP450 activities in liver microsomes, particularly CYP1A2. This may represent a crucial pathway through which CdTe QDs induce excessive ROS generation, leading to oxidative stress and liver damage.

The prevalence of autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by impairments in social communication and restricted/repetitive behavioral patterns, has increased significantly over the past few decades. The etiology of ASD involves a highly complex interplay of genetic, neurobiological, and environmental factors, contributing to significant heterogeneity in its clinical phenotype. In the evolving landscape of ASD research, increasing evidence suggests that oxidative stress, resulting from both intrinsic and extrinsic factors, may be a crucial pathophysiological driver in ASD, influencing neurodevelopmental processes that underlie behavioral abnormalities. Elevated levels of oxidative stress biomarkers, including lipid peroxides, protein oxidation products, and DNA damage markers, alongside deficient antioxidant enzyme activity, have been consistently linked to ASD. This may be attributed to dysregulated activity of nuclear factor erythroid 2-related factor 2 (NRF2), a pivotal transcription factor that maintains cellular redox homeostasis by orchestrating the expression of genes involved in antioxidant defenses. Here, we summarize the converging evidence that redox imbalance in ASD may result from NRF2 dysregulation, leading to reduced expression of its target genes. We also highlight the most promising antioxidant compounds under investigation, which may restore NRF2 activity and ameliorate ASD behavioral symptoms.

Chronic rhinosinusitis (CRS) is inflammation of the sinuses and nasal passages that lasts for >3 months. Its pathogenesis is complex, treatment is difficult, and it has multiple effects on patients. Although surgical treatment can effectively relieve the symptoms, the recurrence rate is high, and there are postoperative complications such as infection. At present, nasal spray hormone, antibiotics and other western drugs are used in clinical treatment, but there are drug dependence and toxic side effects. However, traditional Chinese medicine (TCM) has made remarkable progress in the treatment and promotion of postoperative recovery, guided by its unique TCM theory, and has little toxic and side effects, providing more treatment options for patients.

The review aims to elucidate the mechanism of CRS from the aspects of traditional medicine and modern medicine, and evaluate the influence of TCM compound, components of TCM, TCM nasal irrigation, TCM fumigation and other auxiliary treatment methods on CRS, providing a new perspective for the application of TCM in CRS.

We conducted the literature retrieval with PubMed, Web of Science, Google Scholar and CNKI databases in a systematic manner (up to July 2024). The keywords included "sinusitis", "chronic rhinosinusitis", "nasal polyps", "herbal medicine", "medicinal plants", "traditional Chinese medicine", "oxidative stress", "pathogenic microbial", "anatomic structure" and so on. The obtained literatures were comprehensively sorted out. For image creation, Figdraw 2.0 was methodically employed.

The pathogenesis of CRS involves various interaction mechanisms such as bacterial biofilm formation, oxidative stress injury and impaired ciliary mucosa clearance. It is worth noting that TCM exerts therapeutic effects by targeting the above-mentioned pathological processes. Clinical studies have confirmed that TCM comprehensive therapy can significantly improve sinus symptom score, accelerate postoperative mucosal epithelialization, and promote postoperative rehabilitation of CRS. We also discussed the toxic side effects and clinical applications of related drugs.

In TCM, CRS is classified under the diagnostic category of Bi Yuan. Its pathogenesis is attributed to exogenous invasion of the six climatic pathogens (Liu Yin: wind, cold, summer heat, dampness, dryness, and fire), spleen-stomach qi deficiency, internal damp-heat accumulation, and qi-blood stasis. Guided by TCM principles, therapeutic strategies are individualized through syndrome differentiation, which tailors interventions to the patient's unique clinical manifestations. Therapeutic modalities include oral herbal formulations (e.g., decoctions or granules), acupuncture, and acupoint application. These approaches aim to restore physiological balance by harmonizing yin and yang, resolving meridian obstructions, and enhancing lung qi circulation to alleviate nasal congestion and improve ventilation.

Sprint interval training (SIT) is a time-efficient type of endurance training that involves large type 2 muscle fibre recruitment. Effective antioxidant supplementation may mitigate positive training adaptations by limiting the oxidant challenge. Our aim was to test whether SIT affects type 2 more than type 1 muscle fibres, and whether the muscular training response is mitigated by antioxidant treatment. Young men performed three weekly SIT sessions (4-6 × 30 s all-out cycling) for 3 weeks while treated with antioxidants (vitamin C, 1 g day-1; vitamin E, 235 mg day-1) or placebo. Vastus lateralis biopsies were taken to measure (i) activation of genes for reactive oxygen/nitrogen species (ROS) sensors and inflammatory mediators with quantitative RT-PCR and (ii) fibre type-specific proteome adaptations using MS-based proteomics. Vitamin treatment decreased the upregulation of genes for ROS sensors and inflammatory regulators during the first SIT session. The 3 weeks of SIT caused generally larger proteome adaptations in type 2 than in type 1 fibres, and this included larger increases in abundance of proteins involved in mitochondrial energy production. Vitamin treatment blunted the SIT-induced proteome adaptations, whereas it did not affect the training-induced improvement in maximal cycling performance. In conclusion, (i) the large type 2 fibre recruitment and resulting proteome adaptations are instrumental to the effectiveness of SIT and (ii) antioxidant supplementation counteracts positive muscular adaptations to SIT, which would blunt any improvement in submaximal endurance performance, whereas it does not affect the improvement in maximal cycling performance, where O2 delivery to muscle would be limiting. KEY POINTS: Sprint interval training (SIT) is a time-efficient type of endurance training that involves large recruitment of fast-twitch muscle fibres. Treatment with antioxidants may mitigate the positive effects of endurance training. Fibre type-specific proteomics performed on muscle biopsies obtained from young men before and after 3 weeks of SIT showed larger training effects in fast- than in slow-twitch fibres. Antioxidant treatment in the form of vitamin C and E pills counteracted the positive muscular adaptations to the 3 weeks of SIT. These results increase our understanding of why SIT is an effective endurance training regime and provide further evidence against the common belief that antioxidant supplements are beneficial in a physical exercise context.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of lung parenchymal scarring that is triggered by repeated microinjury to a vulnerable alveolar epithelium. It is increasingly recognised that cellular ageing, whether physiological or accelerated due to telomere dysfunction, renders the epithelium less able to cope with injury and triggers changes in epithelial behaviour that ultimately lead to the development of disease.

This review aims to highlight how, with increasing age, the alveolar epithelium becomes vulnerable to exogenous insults. We discuss the downstream consequences of alveolar epithelial dysfunction on epithelial phenotype, alveolar repair and pro-pathogenic interactions with other alveolar niche-resident cell types which drive IPF pathogenesis.

We highlight how a wide array of cellular mechanisms that maintain cellular homeostasis become dysfunctional with ageing. Waning replicative capacity, genomic stability, mitochondrial function, proteostasis and metabolic function all contribute to a phenotype of vulnerability to 'second hits'. We discuss how in IPF the alveolar epithelium becomes dysfunctional, highlighting changes in repair capacity and fundamental cellular phenotype and how interactions between abnormal epithelium and other alveolar niche-resident cell types perpetuate disease.

The ageing epithelium is a vulnerable epithelium which, with the cumulative effects of environmental exposures, fundamentally changes its behaviour towards stalled differentiation, failed repair and profibrotic signalling. Further dissection of aberrant epithelial behaviour, and its impact on other alveolar cell types, will allow identification of novel therapeutic targets aimed at earlier pathogenic events.

The ubiquity and abundance of plastic debris is one of the most significant challenges facing marine environments. Recent research has demonstrated that microplastics are consumed by marine organisms from a wide variety of trophic levels. However, little is known about the sub-lethal effects of microplastic exposure on the health of coral reef fishes or their impacts on predator-prey interactions. To examine this, we exposed juvenile Ambon damselfish (Pomacentrus amboinensis) to one of two types of polystyrene plastic particles (virgin, or with the plasticizer di(2-ethylhexyl) phthalate - DEHP), and a control (no plastic). After 2 days (6 exposures), the initiation of antioxidant metabolism and oxidative defence was quantified. We also exposed the treated juvenile fish to a piscivorous fish (Pseudochromis fuscus) in mesocosms over a 22-h period to investigate whether microplastic exposure affected prey survival. Biomarkers associated with oxidative damage and antioxidant metabolism indicated that microplastic exposure had a negative effect on the health of P. amboinensis. Additionally, P. amboinensis exposed to DEHP microplastics showed the greatest levels of oxidative stress and damage, however the magnitude of this was dependent on the number of ingested particles. Interestingly, survival of P. amboinensis did not differ among plastic treatments during mesocosm survival trials. These results highlight that while studies may not find immediate lethal consequences to plastic ingestion, there may be more subtle sub-lethal costs that may have ecologically important consequences at later life stages, through energetic impacts on growth and energy allocation.

Androgenetic alopecia is associated with testosterone-mediated anagen-to-catagen transition and matrix keratinocyte apoptosis in hair follicle cells. Activation of Nox isozymes is involved in testosterone-mediated keratinocyte apoptosis, leading to androgenetic alopecia. This indicates that Nox isozymes can serve as therapeutic targets for androgenetic alopecia. The isolated compounds from natural products were screened to evaluate their ROS-inhibition efficacy and it was found that 1'S-1'-acetoxychavicol acetate (ACA, 26), a natural compound isolated from Alpinia galanga (L.) Willd. (Zingiberaceae), exhibits inhibitory activity on Nox isozymes. Nox inhibition by ACA suppressed testosterone-dependent H2O2 generation and cell death in keratinocytes. Incubation with ACA in human hair follicle organ culture mitigated testosterone-dependent suppression of hair growth. We validated that ACA regulates androgenetic alopecia in a mouse model. Local application of ACA on the dorsal skin in an androgenetic alopecia model of C57BL/6 mice significantly suppressed testosterone-induced hair loss in a dose-dependent manner. Moreover, hair follicle length in ACA-treated mice was enhanced compared to that in control mice. These findings provide a molecular mechanism in which ACA inhibits Nox activity in hair follicle cells, indicating its potential as an effective treatment of AGA.

A catalase-assisted peroxide quenching strategy is established by the injection of catalase, which consumes intracellular hydrogen peroxide generated from oxygen stress, into a living cell using a nanopipette. Accordingly, the amounts of reactive oxygen intermediates (ROIs) are quantified at the single cell level.

Reactive oxygen species (ROS), regulators of cellular behaviors ranging from signaling to cell death, have complex production and control mechanisms to maintain a dynamic redox balance under physiological conditions. Redox imbalance is frequently observed in tumor cells, where ROS within tolerable limits promote oncogenic transformation, while excessive ROS induce a range of regulated cell death (RCD). As such, targeting ROS-mediated regulated cell death as a vulnerability in cancer. However, the precise regulatory networks governing ROS-mediated cancer cell death and their therapeutic applications remain inadequately characterized. In this Review, we first provide a comprehensive overview of the mechanisms underlying ROS production and control within cells, highlighting their dynamic balance. Next, we discuss the paradoxical nature of the redox system in tumor cells, where ROS can promote tumor growth or suppress it, depending on the context. We also systematically explored the role of ROS in tumor signaling pathways and revealed the complex ROS-mediated cross-linking networks in cancer cells. Following this, we focus on the intricate regulation of ROS in RCD and its current applications in cancer therapy. We further summarize the potential of ROS-induced RCD-based therapies, particularly those mediated by drugs targeting specific redox balance mechanisms. Finally, we address the measurement of ROS and oxidative damage in research, discussing existing challenges and future prospects of targeting ROS-mediated RCD in cancer therapy. We hope this review will offer promise for the clinical application of targeting oxidative stress-mediated regulated cell death in cancer therapy.

Maternal conditions during pregnancy can influence the long-term health of offspring. In particular, maternal malnutrition (MM), such as protein restriction, affects the development of several organs, including the male reproductive system. This study examined how a low-protein maternal diet impacts the structure and function of the dorsolateral prostate (DLP) in aging male rats. Male offspring were divided into two groups: A control group (CTR), whose mothers received a normal protein diet (17%) during pregnancy and lactation, and a low-protein group (GLLP), whose mothers received a low-protein diet (6%) during the same period. At 540 days of age, the offspring were euthanized, and the DLPs were collected for analysis. The GLLP group showed significant structural changes in the DLP, including increased epithelial and reduced stromal compartments. These rats also had lower levels of probasin (a prostate-specific protein), along with a higher number of mast cells, CD68 + macrophages, and IL-10 protein expression, indicating inflammation. Antioxidant balance was disrupted: Glutathione (GSH) levels increased, while catalase (CAT) and superoxide dismutase (SOD) decreased. The expression of SIRT1, a protein linked to aging and oxidative stress control, was reduced. In silico analysis using human prostate cancer data (PRAD-TCGA) revealed that biological pathways related to oxidative stress, immune response, and tissue remodeling were disrupted in both the rat model and human prostate cancer. In summary, maternal protein restriction leads to long-term changes in the dorsolateral prostate of aging male offspring, including inflammation, oxidative stress, and tissue remodeling. The reduced expression of SIRT1 may play a key role in these effects.

Cantharidin (CTD) serves as the principal bioactive compound in traditional Chinese medicine Mylabris, commonly employed in cancer treatment. Nevertheless, the clinical application of CTD is partly restricted by hepatotoxicity, and the toxicology mechanism is not fully elucidated. This study aims to explore the potential mechanism of CTD-induced hepatoxicity by targeted metabolomics-based UPLC-QTOF-MS/MS analysis and 16S rRNA sequencing. Studies have shown that the administration of CTD could lead to elevated serum biochemical indices including ALT and AST. Notably, dilatation of the liver central vein, hepatocellular necrosis, and slight vacuoles in rats were observed after CTD intervention. Fecal metabolomics found CTD could up-regulate 10 and down-regulate 33 metabolites, and metabolic pathway enrichment found that CTD could disrupt 2 metabolic pathways, including Arginine biosynthesis metabolism and β-Alanine metabolism. 16S rRNA gene sequencing analysis showed that CTD could increase the abundance of Turicibacter and Clostridium sensu stricto 1, but decrease the amounts of Prevotella 1. Our correlation analyses showed that alterations in the gut microbiota induced by CTD in rats may have impacted changes in the associated hepatic amino acid metabolism pathway. And the mechanism of action of CTD-induced hepatotoxicity may be related to inflammation, oxidative stress, impaired glucose metabolism and reduced hepatic glycogen storage. These findings will offer novel insights for the prevention and treatment of CTD-induced hepatotoxicity.

Metabolic associated steatohepatitis characterized by lipid accumulation, inflammation and fibrosis, is a growing global health issue, contributing to severe liver-related mortality. With limited effective treatments available, there is an urgent need for novel therapeutic strategies. Moringa oleifera, rich in antioxidants, offers potential for combating steatohepatitis, but its cytotoxicity presents challenges. Aloe vera, renowned for its cytocompatibility and anti-inflammatory effects, shows promise in mitigating these risks. Using infrared spectrometry and mass spectrometry, we identified 1586 metabolites from both plants across 84 chemical classes. By encapsulating these phytochemicals in nanoparticles, we achieved increased solubility, cytocompatibility, and gene modulation to hepatic stellate cells affected by steatohepatitis. Chemoinformatic analysis revealed bioactive metabolites, including hesperetin analogs, known to inhibit TGF-β. Our results demonstrate that these nanoparticles not only improved gene expression modulation related to metabolic associated steatohepatitis, particularly TGF-β and COL1A1, but also outperformed free compounds, highlighting their potential as a novel therapeutic approach.

Rheumatoid arthritis (RA) is characterized by massive intra-articular infiltration of pro-inflammatory macrophages, leading to articular immune dysfunction, severe synovitis, and ultimately joint erosion. Comprehensive remodeling of articular immune homeostasis and bone homeostasis is essential for alleviating RA. Here we report on Spirulina platensis (SP), a natural microorganism commercially farmed worldwide as a food, as an efficient regulator of both synovial inflammation and osteoclast differentiation in male RA mouse models. SP reduces excessive reactive oxygen species and downregulates pro-inflammatory cytokines in synovial macrophages. Moreover, SP reprograms pro-inflammatory M1-like macrophages to anti-inflammatory M2-like phenotype, suppressing synovitis and remodeling redox balance. Notably, SP inhibits osteoclast activation effectively and blocks the progression of bone erosion. SP is then engineered with macrophage membranes (SP@M) to enable immune evasion and RA-targeting in vivo. SP@M increases LC3-mediated autophagy as well as strengthens ubiquitin-mediated proteasomal degradation toward KEAP1, which promotes the expression and nuclear translocation of NRF2. The NRF2 further activates antioxidant enzymes to terminate macrophages-initiated pathological cascades and reestablish intra-articular immune homeostasis, conferring a bone recovery and chondroprotective effect in collagen-induced arthritis mouse models. This work shows the therapeutic activity of FDA-approved functional food of SP in suppressing synovial inflammation and osteoclast differentiation, offering an off-the-shelf strategy for RA treatment.

Androgenic alopecia is one of the most common chronic problems for dermatologists worldwide. Some Chinese herbal extracts have been shown to promote hair growth, but the active ingredients are difficult to enter the dermis. Therefore, delivering the active ingredients into the dermis becomes a key factor. Herein, Platycladus orientalis leaf extract (PO-ex) was obtained using ethanol as a solvent, and then hyaluronic acid methacrylate/hyaluronic acid (HAMA/HA) hydrogel was loaded with PO-ex to prepare hyaluronic acid microneedles (PO-ex MN). The double cross-linked HAMA/HA provides sufficient mechanical strength to pierce the stratum corneum and deliver PO-ex into the dermis; PO-ex can effectively improve the environment for hair follicle cell proliferation by removing reactive oxygen free radicals; in addition, the self-repair reaction caused by microneedle mechanical stimulation activates the Wnt/β-catenin pathway associated with trauma repair and promotes hair follicle growth. PO-ex MN is a potential therapeutic strategy for the treatment of androgenic alopecia.

Flaxseed meal is recognized as a rich source of high-quality protein, and this study aims to explore its potential as a source of bioactive peptides. Hydrolysates were prepared via ultrasound-assisted enzymatic hydrolysis. Enzymatic hydrolysates from hot-pressed flaxseed meal showed higher biological activity than those from cold-pressed samples. Three antioxidant peptides, namely PFFWLHHT, HCLEFLSPRF, and ALTMPHNW, were identified and screened from the purified fractions of hot-pressed flaxseed meal using reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and in silico analysis. These peptides displayed scavenging activity against DPPH radicals (IC50 = 0.520, 1.190, and 1.036 mM) and hydroxyl radicals (IC50 = 4.909, 6.471, and 7.076 mM). Molecular docking studies indicated that three peptides could inhibit myeloperoxidase (MPO) activity by occupying the entrance of the active cavity, with their binding affinities measured at -12.2, -9.1 and - 11 kcal/mol, respectively. These findings could lay a foundation for extracting natural antioxidant peptides from flaxseed meals.

Most DNA damage caused by oxidative metabolism consists of single lesions that can accumulate in tissues. This review focuses on two classes of lesions: the two 8-oxopurine (8-oxo-Pu) lesions that are repaired by the base excision repair (BER) enzyme and the four 5',8-cyclopurine (cPu) lesions that are repaired exclusively by the nucleotide excision repair (NER) enzyme. The aim is to correlate the simultaneous quantification of these two classes of lesions in the context of neurological disorders. The first half is a summary of reactive oxygen species (ROS) with particular attention to the pathways of hydroxyl radical (HO•) formation, followed by a summary of protocols for the quantification of six lesions and the biomimetic chemistry of the HO• radical with double-stranded oligonucleotides (ds-ODN) and calf thymus DNA (ct-DNA). The second half addresses two neurodegenerative diseases: xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The quantitative data on the six lesions obtained from genomic and/or mitochondrial DNA extracts across several XP and CS cell lines are discussed. Oxidative stress contributes to oxidative DNA damage by resulting in the accumulation of cPu and 8-oxo-Pu in DNA. The formation of cPu is the postulated culprit inducing neurological symptoms associated with XP and CS.

Male-factor infertility accounts for nearly half of all infertility cases, and mounting evidence points to oxidative stress as a pivotal driver of sperm dysfunction, genetic instability, and epigenetic dysregulation. In particular, the oxidative DNA lesion 8-hydroxy-2'-deoxyguanosine (8-OHdG) has emerged as a central mediator at the interface of DNA damage and epigenetic regulation. We discuss how this lesion can disrupt key epigenetic mechanisms such as DNA methylation, histone modifications, and small non-coding RNAs, thereby influencing fertilization outcomes, embryo development, and offspring health. We propose that the interplay between oxidative DNA damage and epigenetic reprogramming is further exacerbated by aging in both the paternal and maternal germlines, creating a "perfect storm" that increases the risk of heritable (epi)mutations. The consequences of unresolved oxidative lesions can thus persist beyond fertilization, contributing to transgenerational health risks. Finally, we explore the promise and potential pitfalls of antioxidant therapy as a strategy to mitigate sperm oxidative damage. While antioxidant supplementation may hold significant therapeutic value for men with subfertility experiencing elevated oxidative stress, a careful, personalized approach is essential to avoid reductive stress and unintended epigenetic disruptions. Recognizing the dual role of oxidative stress in shaping both the genome and the epigenome underscores the need for integrating redox biology into reproductive medicine, with the aim of improving fertility treatments and safeguarding the health of future generations.

Background: Maillard reaction products (MRPs) are known for their antioxidant properties; however, their effects on muscle cells remain unclear. This study aims to elucidate the effects of MRPs on muscle hypertrophy and atrophy in C2C12 myotubes. Methods: MRPs were prepared by heating L-lysine and D-glucose, and their antioxidant activity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Subsequently, mouse C2C12 myoblasts were cultured with MRPs until myotubes formed, and their diameters were measured to assess hypertrophic and atrophic changes. Akt phosphorylation was evaluated by Western blotting, and gene expression levels were analyzed via quantitative PCR. Results: The prepared MRPs exhibited high antioxidant activity in the DPPH radical scavenging assay. MRP treatment significantly increased the average myotube diameter by approximately 40% and enlarged the largest myotube diameter by up to 80%, potentially mediated by enhanced Akt phosphorylation. Under dexamethasone-induced atrophy, MRPs modestly attenuated the reduction in myotube diameter by approximately 20%, although the effect was not statistically significant, and did not significantly alter the fusion index either. Quantitative PCR analysis revealed that MRP treatment significantly reduced the mRNA expression of Nfe2l2, a key regulator of antioxidant response, whereas it had no notable effects on the expression of genes related to myoblast proliferation (Myod1), differentiation (Myog), hypertrophy (Igf1), atrophy (Foxo1 and Trim63), and oxidative stress (Cat, Gclc, and Nqo1). Conclusions: Our findings suggested that MRPs possess antioxidant activity and promote myotube hypertrophy via Akt signaling. This study highlighted the potential of MRPs as functional ingredients for promoting muscle health, though further in vivo studies are required to validate their physiological relevance.

Cardiomyopathies comprise a heterogeneous group of cardiac disorders characterized by structural and functional abnormalities in the absence of significant coronary artery disease, hypertension, valvular disease, or congenital defects. Major subtypes include hypertrophic, dilated, arrhythmogenic, and stress-induced cardiomyopathies. Oxidative stress (OS), resulting from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses, has emerged as a key contributor to the pathogenesis of these conditions. ROS-mediated injury drives inflammation, protease activation, mitochondrial dysfunction, and cardiomyocyte damage, thereby promoting cardiac remodeling and functional decline. Although numerous studies implicate OS in cardiomyopathy progression, the precise molecular mechanisms remain incompletely defined. This review provides an updated synthesis of current findings on OS-related signaling pathways across cardiomyopathy subtypes, emphasizing emerging therapeutic targets within redox-regulatory networks. A deeper understanding of these mechanisms may guide the development of targeted antioxidant strategies to improve clinical outcomes in affected patients.

Calcium oxalate (CaOx) nephrolithiasis, as one of the most common types of kidney stones, poses a major threat to human health. This study aimed to investigate the role of 3-hydroxyisobutyrate dehydrogenase (HIBADH) in the pathogenesis of CaOx nephrolithiasis. CaOx nephrolithiasis models were established in rats via 1 % ethylene glycol and 2 % ammonium chloride induction and in HK-2 cells using calcium oxalate monohydrate (COM, 100 μg/mL). HIBADH expression was modulated through plasmid transfection and siRNA knockdown in vitro, and AAV2/9-mediated gene transfer in vivo. Multiple parameters were assessed, including cell crystal adhesion, apoptosis, cell cycle distribution, oxidative stress markers (SOD, MDA, MitoSOX fluorescence), and mitochondrial function (ATP level, mitochondrial membrane potential), using various techniques such as crystal adhesion assay, flow cytometry, western blot, qRT-PCR, and fluorescence microscopy. Kidney tissues were analyzed through H&E, Von Kossa, and PAS staining. Results demonstrated that HIBADH expression was significantly downregulated in CaOx nephrolithiasis rats and COM-treated HK-2 cells. In vitro, HIBADH overexpression reduced cell crystal adhesion and apoptosis, promoted cell cycle progression, mitigated mitochondria-involved cellular oxidative stress, and enhanced mitochondrial function in COM-induced HK-2 cells. In vivo, AAV2/9-mediated HIBADH overexpression attenuated crystal deposits and tubular injury, reduced apoptosis, and mitigated mitochondria-involved cellular oxidative stress in kidney tissues. The mitochondria-targeted antioxidant Mito-TEMPO counteracted the effects of HIBADH silencing, highlighting the role of mitochondrial function in HIBADH's protective mechanism. This study identifies HIBADH as a critical regulator in CaOx nephrolithiasis, exerting its protective effects through modulation of mitochondrial function and mitochondria-involved cellular oxidative stress, cell crystal adhesion, and apoptosis. Our findings elucidate the link between mitochondrial metabolism and kidney stone formation, positioning HIBADH as a key protective factor and a promising candidate with therapeutic potential for CaOx nephrolithiasis.

Cardiovascular diseases (CVDs) represent a profound challenge with inflammation playing a significant role in their pathophysiology. Extracellular vesicles (EVs), which are membranous structures encapsulated by a lipid bilayer, are essential for intercellular communication by facilitating the transport of specific bioactive molecules, including microRNAs, proteins, and lipids. Emerging evidence suggests that the regulatory mechanisms governing cardiac resident cells are influenced by EVs, which function as messengers in intercellular communication and thereby contribute to the advancement of CVDs. In this review, we discuss the multifaceted biological functions of EVs and their involvement in the pathogenesis of various CVDs, encompassing myocardial infarction, ischemia-reperfusion injury, heart failure, atherosclerosis, myocarditis, cardiomyopathy, and aneurysm. Furthermore, we summarize the recent advancements in utilizing EVs as non-invasive biomarkers and in cell-free therapy based on EVs for the diagnosis and treatment of CVDs. Future research should investigate effective techniques for the isolation and purification of EVs from body fluids, while also exploring the pathways for the clinical translation of therapy based on EVs. Additionally, it is imperative to identify appropriate EV-miRNA profiles or combinations present in the circulation of patients, which could serve as biomarkers to improve the diagnostic accuracy of CVDs. By synthesizing and integrating recent research findings, this review aims to provide innovative perspectives for the pathogenesis of CVDs and potential therapeutic strategies.

Hypertension, a chronic condition marked by elevated blood pressure, poses a significant health risk globally. This review explores the potential of sodium nitrate supplementation to enhance nitric oxide (NO) bioavailability and reduce oxidative stress in patients with hypertension. NO, known for its vasodilatory properties, plays a crucial role in maintaining endothelial function and cardiovascular health. Additionally, this study provides a comprehensive analysis of current research on the mechanisms through which sodium nitrate enhances nitric oxide (NO) levels, thereby improving endothelial function, reducing oxidative stress, and lowering blood pressure. The findings underline sodium nitrate's promising capacity to reduce dependence on conventional antihypertensive therapies, offering a cost-effective strategy for enhancing cardiovascular outcomes. Effective dosage ranges, such as 6-12 mmol/day (approximately 510-1020 mg nitrate), derived from dietary sources like leafy greens and beetroot juice, are proposed as practical solutions. Future studies are warranted to substantiate these benefits, refine dosing protocols, and establish guidelines for safe and effective clinical application. Integrating sodium nitrate into treatment frameworks could significantly advance hypertension management, improve patient quality of life, and reduce healthcare expenditures.

A simple and reliable total antioxidant capacity (TAC) assay is essential for food safety evaluation and human health monitoring. Herein, a trimetallic nanozyme (Fe3O4@Ag@Pt) was synthesized and exhibited OXD-, POD- and SOD-like activities, which could generate a synergistic catalytic system. Fe3O4@Ag@Pt can catalyze oxygen to produce various reactive oxygen intermediates, and the endogenous product H2O2 could be captured and further dissociated efficiently into •OH, due to its strong substrate binding affinity. Since antioxidants can compete with TMB and lead to an antioxidant concentration-dependent color change, a colormetric sensing platform was constructed with a detection limit of 1.97 μM, 5.06 μM, and 8.99 μM for GSH, AA and Trolox, respectively. The proposed Fe3O4@Ag@Pt based assay was suitably employed to quantify TAC in fruit samples, beverages and cells with the aid of spike recovery and reference method validation, which hold vast promise as an analytical platform for food safety and biomedical diagnosis.

We conducted the current systematic review to investigate the association between dietary inflammatory index (DII) and severe headaches or migraine among adults via synthesizing observational evidence.

We conducted a systematic literature search of observational studies through PubMed, Scopus, and Web of Science databases from inception until July 2024. The PECO framework was implemented to select eligible studies as follows: Population (adults with severe headache or migraine), Exposure (individuals with the highest adherence to a pro-inflammatory diet), Comparison (individuals with the lowest adherence to a pro-inflammatory diet), Outcome (risk of developing severe headache or migraine, headaches frequency, duration, severity, and migraine-related disability).

After reviewing six studies involving 31,958 individuals, we found that following an anti-inflammatory diet is associated with a lower frequency and severity of migraine headaches. Additionally, our research revealed that individuals with migraines tend to have lower adherence to an anti-inflammatory diet when compared to people without migraines. Surprisingly, adherence to a pro-inflammatory diet was linked to a reduced risk of chronic daily headaches.

Present findings imply a negative link between an inflammatory diet and severe headaches or migraine. However, further well-designed longitudinal studies are needed to interpret the causality and shed light on the underlying mechanisms.

Psoriasis is a chronic, systemic immune-mediated skin disease. Although many new strategies for psoriasis treatment have been developed, there is great need in clinic for treating psoriasis. Gentiopicroside (GPS), derived from Gentiana manshurica Kitagawa, has multiple pharmacological activities including anti-inflammatory, anti-oxidative and antiviral activities. In this study, we investigated the potential effects of GPS in imiquimod (IMQ)-induced psoriasis mouse model and the underlying mechanisms. The mice were sensitized on their shaved back with IMQ cream for 7 days with or without topical application of 1% or 2% GPS cream. We showed that the application of GPS cream significantly ameliorated psoriasis-like skin lesions; GPS effect was better than that of calcipotriol. GPS rectified the immune cells infiltration and keratinocytes activation in the skin lesions, and significantly inhibited TNF-α/IFN-γ stimulated human keratinocyte (HaCaT) activation in vitro. Proteomic analysis from keratinocytes with and without GPS treatment prompted that GPS regulated the Keap1-Nrf2 pathway, which was the most important pathway in regulating oxidative stress and inflammation. We demonstrated that GPS regulated the protein expression of p62 and Keap1, induced Nrf2 nuclear translocation followed by transcription of Nrf2 downstream antioxidant genes in HaCaT cells. Furthermore, the antioxidant effects of GPS were abolished in Nrf2-/- keratinocytes. Simultaneously, Nrf2-/- mice showed increased psoriasiform symptoms with a diminished protective effect in response to GPS treatment. Collectively, the study discloses that GPS inhibits keratinocyte activation and ameliorates psoriasis-like skin lesions in an Nrf2-dependent manner.