This study investigates the effects of caloric restriction (CR) on renal injury and fibrosis following ischemia-reperfusion injury (IRI), with a focus on the roles of the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling and autophagy.

Metabolic pathways fuel tumor progression and resistance to stress conditions including chemotherapeutic drugs, such as DNA damage response (DDR) inhibitors. Yet, significant gaps persist in how metabolic pathways confer resistance to DDR inhibition in cancer cells. Here, we employed a metabolism-focused CRISPR knockout screen and identified genetic vulnerabilities to DDR inhibitors. We unveiled Peroxiredoxin 1 (PRDX1) as a synthetic lethality partner with Ataxia Telangiectasia Mutated (ATM) kinase. Tumor cells depleted of PRDX1 displayed heightened sensitivity to ATM inhibition in vitro and in mice in a manner dependent on p53 status. Mechanistically, we discovered that the ribosomal protein RPL32 undergoes redox modification on active cysteine residues 91 and 96 upon ATM inhibition, promoting p53 stability and altered cell fitness. Our findings reveal a new pathway whereby RPL32 senses stress and induces p53 activation impairing tumor cell survival.

Caffeic acid phenethyl ester (CAPE) is a hydrophobic phytochemical typically found in propolis that acts as an antioxidant, anti-inflammatory and cardiovascular protector, among several other properties. However, the molecular entity responsible for recognising CAPE is unknown, and whether that molecular interaction is involved in developing an antioxidant response in the target cells remains an unanswered question. Herein, we hypothesized that a subfamily of TRP ion channels works as the molecular entity that recognizes CAPE at the plasma membrane and allows a fast shift in the antioxidant capacity of intact endothelial cells (EC). By monitoring cytoplasmic Ca in a microvascular EC model, we compared the calcium responses evoked by three structurally related compounds: caffeic acid phenethyl ester, neochlorogenic acid and caffeic acid. Only CAPE induced rapid and transient calcium responses at nanomolar concentrations together with a gradual increase in cytoplasmic sodium levels, suggesting the activation of a non-selective cationic permeation at the plasma membrane. Electrophysiological as well as pharmacological, and RNA silencing assays confirmed the involvement of TRPV1 in the recognition of CAPE by ECs. Finally, we demonstrated that Ca influx by TRPV1 was necessary for recording CAPE-induced cytoplasmic redox changes, a phenomenon captured in real-time in ECs expressing the HyPer biosensor. Our data depict a molecular mechanism behind the antioxidant effect of CAPE in endothelial cells, connecting the activation of TRPV1 ion channels, cytoplasmic calcium increase, and a reduction of disulfide bonds on a redox biosensor. This phenomenon occurs within seconds to minutes and contributes to a better understanding of the mechanisms underlying the vasodilatory effect of CAPE and other compounds that interact with TRPV1 in the vascular bed.

Myopia is an evolving global health challenge, with estimates suggesting that by 2050 it will affect half of the world's population, becoming the leading cause of irreversible vision loss. Moreover, myopia can lead to various complications, including the earlier onset of cataracts. Given the progressive aging of the population and the increase in life expectancy, this will contribute to a rising demand for cataract surgery, posing an additional challenge for healthcare systems. The pathogenesis of nuclear and posterior subcapsular cataract (PSC) development in axial myopia is complex and primarily involves intensified liquefaction of the vitreous body, excessive production of reactive oxygen species, impaired antioxidant defense, and chronic inflammation in the eyeball. These factors contribute to disruptions in mitochondrial homeostasis, abnormal cell signaling, lipid peroxidation, protein and nucleic acid damage, as well as the induction of adverse epigenetic modifications. Age-related and oxidative processes can cause destabilization of crystallins with subsequent protein accumulation, which finally drives to a lens opacification. Moreover, an altered redox status is one of the major contributors to the pathogenesis of PSC. This review aims to summarize the mechanisms known to be responsible for the accelerated development of cataracts in axial myopia and to enhance understanding of these relationships.

Methamphetamine is a widely abused drug associated with significant neuroinflammation and neurodegeneration, mainly through the activation of glial cells and neurons in the central nervous system. This study investigates the role of the astrocyte-specific NOD-like receptor family pyrin domain-containing protein 6 (NLRP6) inflammasome in methamphetamine-induced astrocytic pyroptosis and neuroinflammation. Our findings demonstrate that methamphetamine exposure induces NLRP6-dependent pyroptosis, astrocyte activation, and the release of proinflammatory cytokines in mouse primary astrocytes. Gene silencing of NLRP6 reduces methamphetamine-induced pyroptosis and proinflammatory cytokines release. We also identified miR-152 as a critical upstream regulator of NLRP6, which is downregulated in methamphetamine-exposed astrocytes. Overexpression of miR-152 decreases NLRP6 expression, mitigating methamphetamine-induced pyroptosis and inflammation. In vivo and ex vivo studies in methamphetamine-exposed mice confirmed these results and showed that methamphetamine induces anxiety-like, cognitive impairment, and depression-like behavior, further linking astrocyte-specific NLRP6 signaling to methamphetamine-induced neuroinflammation. This study highlights the potential of targeting the NLRP6 inflammasome in astrocytes as a therapeutic approach to alleviate methamphetamine-induced central nervous system pathology. Further research is warranted to explore clinical applications and identify therapeutic targets for methamphetamine-related neurological disorders.

Available evidence indicates that neuregulin-1 (NRG-1) can provide a protection against myocardial ischemia/reperfusion (I/R) injury and is involved in various cardioprotective interventions by potential regulation of mitophagy. However, the molecular mechanisms linking NRG-1 and mitophagy remain to be clarified. In this study, both an in vivo myocardial I/R injury model of rats and an in vitro hypoxia/reoxygenation (H/R) model of H9C2 cardiomyocytes were applied to determine whether NRG-1 postconditioning attenuated myocardial I/R injury through the regulation of mitophagy and to explore the underlying mechanisms. In the in vivo experiment, cardioprotective effects of NRG-1 were determined by infarct size, cardiac enzyme and histopathologic examinations. The potential downstream signaling pathways and molecular targets of NRG-1 were screened by the RNA sequencing and the Protein-Protein Interaction Networks. The expression levels of mitochondrial uncoupling protein 2 (UCP2) and mitophagy-related proteins in both the I/R myocardium and H/R cardiomyocytes were measured by immunofluorescence staining and Western blots. The activation of mitophagy was observed with transmission electron microscopy and JC-1 staining. The KEGG and GSEA analyses showed that the mitophagy-related signaling pathways were enriched in the I/R myocardium treated with NRG-1, and UCP2 exhibited a significant correlation between mitophagy and interaction with PINK1. Meanwhile, the treatment with mitophagy inhibitor Mdivi-1 significant eliminated the cardioprotective effects of NRG-1 postconditioning in vivo, and the challenge with UCP2 inhibitor genipin could also attenuate the activating effect of NRG-1 postconditioning on mitophagy. Consistently, the in vitro experiment using H9C2 cardiomyocytes showd that NRG-1 treatment significantly up-regulated the expression levels of UCP2 and mitophagy-related proteins, and activated the mitophagy, whereas the challenge with small interfering RNA-mediated UCP2 knockdown abolished the effects of NRG-1. Thus, it is conclused that NRG-1 postconditioning can produce a protection against the myocardial I/R injury by activating mitophagy through the UCP2/PINK1/LC3B signaling pathway.

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a common form of endocrine hypertension. Here, we demonstrate that Early Growth Response 1 (EGR1) plays a dual role in adrenal cell biology, regulating both oxidative stress and aldosterone production. Using RNA sequencing of RSL3-treated human adrenal cells and spatial transcriptomics of adrenal glands from patients with primary aldosteronism, we identify EGR1 as a key gene associated with RSL3-related oxidative stress and APAs. We show that EGR1 silencing decreases oxidative stress and increases CYP11B2 gene expression and aldosterone production in adrenal cells, while its overexpression has the opposite effects. Notably, EGR1 expression is downregulated in APAs and aldosterone-producing micronodules compared to the adjacent adrenal cortex, which correlates in part with decreased levels of oxidative stress markers. The adrenal cortex of pigs with secondary hyperaldosteronism shows decreased immunostaining of EGR1 and a marker of oxidative stress, suggesting a potential link between EGR1 expression, oxidative stress levels, and adrenocortical function. These findings reveal a novel mechanism linking EGR1 to oxidative stress regulation and aldosterone production in adrenal cells, with potential implications for the pathogenesis of APAs and other adrenocortical tumors.

Repeated use of nitroglycerin results in a loss of its vasodilatory efficacy which limits its clinical use for the treatment of angina pectoris. This tolerance phenomenon is a defining characteristic of all compounds classified as nitrodilators, which includes NTG as well as S-nitrosothiols and dinitrosyl iron complexes. These compounds vasodilate via activation of soluble guanylate cyclase, although they do not release requisite amounts of free nitric oxide (NO) and some do not even cross the plasma membrane. Here we demonstrate that nitrodilators cause vasodilation via mobilization of NO moiety from a nitrodilator-activated NO store (NANOS) pre-formed in the vascular smooth muscle cell, similar to the mechanism by which UV light is also known to cause vasodilation and tolerance. Intraperitoneal nitrite prevented NTG tolerance in coronary arteries of rats that received NTG transdermal patches for 4 days, and potentiated NTG- and GSNO- mediated mesenteric vasodilation in intact rats. Consistent with the incorporation of nitrite into the depletable NANOS, incubation of arteries with N-nitrite resulted in the accumulation of high molecular weight N-NO-containing compounds in arteries, and subsequent exposure to NTG, GSNO, or UV light resulted in efflux of N-NO species. In addition, HO and metal/metalloproteins synergistically facilitated NO release from nitrite, while the oxidative stress associated with inflammation and nitrite synergistically potentiated the nitrodilator-mediated vasodilation. In conclusion, NTG mediates vasodilation via activation of a depletable intracellular store of NO that can be replenished by nitrite, thereby preventing tolerance.

Sarcopenia, the age-related decline in muscle mass and function, is a significant contributor to increased frailty and mortality in the elderly. Currently, no FDA-approved treatment exists for sarcopenia. Here, we identified norharmane (NR), a β-carboline alkaloid, as a potential therapeutic agent for mitigating muscle aging. We aimed to determine the ability of NR to delay muscle aging in Caenorhabditis elegans (C. elegans), mouse, and muscle cells in mice and humans. NR treatment improved swimming ability and increased the maximum velocity in aged C. elegans. Transcriptomic analysis revealed that NR upregulated detoxification genes in C. elegans, including cytochrome P450, UGT, and GST enzymes. NR-induced benefits were dependent on the SKN-1/Nrf2 stress response pathway. In mammalian models, NR delayed cellular senescence in human skeletal muscle myoblasts and enhanced myogenesis in C2C12 cells and primary aged myoblasts. NR supplementation in aged mice prevented muscle loss, improved muscle function, and reduced markers of cellular senescence. We found that the p38 MAPK pathway mediated NR activation of Nrf2 by disrupting the Nrf2-Keap1 interaction. NR also improved oxygen consumption rates and promoted mitochondrial biogenesis. These findings suggest that NR is a promising candidate for preventing sarcopenia and improving muscle health.

Focal adhesions (FAs), multi-protein complexes that link the extracellular matrix to the intracellular cytoskeleton, are key mediators of cell adhesion, migration, and proliferation. These dynamic structures act as mechanical sensors, transmitting stimuli from the extracellular to intracellular environment activating in this way signaling pathways and enabling cells to adapt to environmental changes. As such, FAs are critical for tissue organization and serve as hubs governing cell spatial arrangement within the organism. The assembly, reactivity, and functional regulation of FAs are tightly controlled by post-translational modifications, including redox modulation by reactive oxygen and nitrogen species. Increasing evidence suggests that redox signaling plays a pivotal role in both the physiological and pathological functions of FAs and their downstream processes. Redox regulation affects various components of the FA complex, including integrins, focal adhesion kinase 1 (FAK1), SRC, adapter proteins, and cytoskeletal elements. In this review, we provide an updated overview of the complex interplay between redox signaling and post-translational modifications in FAs. We explore how redox reactions influence the structure, dynamics, and function of FAs, shedding light on their broader implications in health and disease.

During its catalytic cycle, the homodimeric ATPase topoisomerase II alpha (TOP2A) cleaves double stranded DNA and remains covalently bound to 5' ends via tyrosine phosphodiester bonds. After passing a second, intact duplex through, TOP2A rejoins the break and releases from the DNA. Thereby, TOP2A can relieve strain accumulated during transcription, replication and chromatin remodeling and disentangle sister chromatids for mitosis. Chemotherapy agents such as etoposide are poisons that trap TOP2A mid-cycle, covalently bound to cleaved DNA, leaving behind DNA double strand breaks and activating DNA damage response. While etoposide has been proposed to stabilize the TOP2A-DNA cleavage complex (TOP2Acc) via interfacial inhibition, we have elucidated a complementary mechanism mediated by the ability of etoposide and other TOP2A poisons to induce oxidative stress. Consequently, lipid peroxidation and accumulation of lipid-derived electrophiles such as 4-hydroxynonenal (HNE) results in covalent modification of TOP2A, both blocking ATPase activity and trapping TOP2Acc. HNE modifies multiple sites on human TOP2A in vitro, including alkylating Cys216 in the ATPase domain in a DNA-dependent fashion. Taken together, our data suggest an underappreciated role for TOP2A as a redox sensor in tumor cells, connecting oxidative stress to DNA damage signaling and thereby creating a target for redox-active drugs.

Caffeine (CAFF) is abundant in black coffee. As one of the most widely consumed beverages globally, coffee has been the focus of increasing clinical and basic research, particularly regarding its benefits in alleviating metabolic dysfunction-associated steatotic liver disease (MASLD). However, the therapeutic effects of CAFF on metabolic-associated steatohepatitis (MASH) and the underlying mechanisms remain unclear. In this study, we demonstrated that CAFF potently reduced hepatic steatosis, inflammation, and early-stage liver fibrosis in MASH mice induced by prolonged (36 weeks) high-fat high-carbohydrate (HFHC) diets and high-fat diets combined with carbon tetrachloride (CCl) injections. By using multiple target-identifying strategies, including surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay, we identified dual-specificity phosphatase 9 (Dusp9) as a key therapeutic target, which was diminished by HFHC but restored with CAFF treatment. Dusp9 knockdown in vivo and in vitro exacerbated glycolipid metabolism disorders and stunningly counteracted the systemic therapeutic effects of CAFF in the MASH models. In addition, CAFF inactivated the ASK1-p38/JNK, a downstream signaling pathway of Dusp9, which regulates inflammation and apoptosis. Our study highlights the multifaceted benefits of CAFF in treating MASH by rescuing hepatic Dusp9 expression, thereby reversing glycolipid metabolism disorders, liver inflammation, and fibrosis. These findings provide experimental evidence supporting the clinical and daily use of CAFF and black coffee in managing MASH patients.

Although leptin-deficient ob/ob mice have been investigated to determine whether hepatic steatosis promotes susceptibility to hepatotoxic insults, carbon tetrachloride (CCl)-induced hepatic fibrosis in ob/ob mice remains largely unknown. In this study, we evaluate the pathogenic mechanisms of hepatic fibrosis in CCl-treated wild-type (WT) and ob/ob mice and analyze some parameters related to lipogenesis, inflammation, fibrosis, oxidative stress, apoptosis, and autophagy. CCl treatment attenuated liver weight and lipogenesis in ob/ob mice. Increased hepatic fibrosis-related proteins were reduced in CCl-treated ob/ob mice compared with CCl-treated WT mice. Specifically, the expression of lipocalin-2 (LCN2) was markedly reduced in CCl-treated ob/ob mice versus CCl-treated WT mice. Compared with CCl-treated WT mice, CCl-treated ob/ob mice had reduced expression of neutrophil-related inflammatory genes and proteins. Hepatic heme oxygenase-1 protein was reduced in CCl-treated ob/ob mice compared with CCl-treated WT mice. However, CCl did not promote hepatic apoptosis in ob/ob mice. Therefore, these findings highlight LCN2 as a key signaling factor in CCl-induced hepatic fibrosis.

Mitochondria are major sites of reactive oxygen species (ROS) production within cells. ROS are important signalling molecules, but excessive production can cause cellular damage and dysfunction. It is therefore crucial to accurately determine when, how and where ROS are produced within mitochondria. Previously, ROS detection involved various chemical probes and fluorescent proteins. These have limitations due to accumulation of the molecules only in the mitochondrial matrix, or the need for a new protein to be expressed for every different species. We report dynamic HO flux changes within all mitochondrial sub-compartments with striking spatial resolution. We combined specific targeting of self-labeling proteins with novel HO-reactive probes. The approach is broad-ranging and flexible, with the same expressed proteins loadable with different dyes and sensors. It provides a framework for concomitant analysis of other chemical species, beyond ROS, whose dynamics within mitochondria are yet unknown, without needing to engineer new proteins.

Accurate and selective techniques for visualizing endogenous peroxynitrite (ONOO) in cerebral ischemia-reperfusion injury (CIRI) models are essential for understanding its complex pathological processes. Here, we introduced a longwave fluorescent probe TJO for detecting ONOO rapidly and sensitively, with a low detection limit of 91 nM. Furthermore, TJO exhibits excellent fluorescence imaging capabilities, enabling detailed visualization of ONOO⁻ in CIRI mice model. This highlights its potential for real-time monitoring of ONOO⁻-related pathological conditions.

Oocyte aging is closely related to a decline in female fertility, accompanied by increased reactive oxygen species levels and changes in protein posttranslational modifications. However, the role of protein palmitoylation in oocyte aging has not been investigated. In the present study, a new association between redox and palmitoylation in aging oocytes was found. We found that the protein level of palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, was increased in maternally aged mice oocytes and follicular fluid of aged (age >35 years) patients with decreased ovarian reserve (DOR). Elevated PPT1 led to decreased S-palmitoylation levels in oocytes, which impaired oocyte maturation and spindle formation. Tubulin was identified as a critical palmitoylated protein regulated by PPT1, whose palmitoylation was also decreased by advanced age, accompanied by abnormalities in membrane localization and microtubule polymerization. Melatonin was found to down-regulate excessive PPT1 and rescue PPT1-induced damage in mouse oocytes, not only by regulating oxidative stress, but also by binding with PPT1 to regulate its lysosomal degradation. In summary, our data demonstrate that PPT1 participates in oocyte aging by regulating tubulin palmitoylation, providing evidence that oxidative stress regulates protein palmitoylation and revealing a novel mechanism of oocyte aging.

The binding of endothelin-1 (ET-1) to endothelin type A receptor (ETAR) performs a critical action in pulmonary arterial smooth muscle cell (PASMC) proliferation leading to pulmonary vascular structural remodeling. More evidence showed that cystathionine γ-lyase (CSE)-catalyzed endogenous hydrogen sulfide (HS) was involved in the pathogenesis of cardiovascular diseases. In this study, we aimed to explore the effect of endogenous HS/CSE pathway on the ET-1/ETAR binding and its underlying mechanisms in the cellular and animal models of PASMC proliferation.

Renal ischemia-reperfusion (I/R) injury triggers significant oxidative stress and inflammation, leading to tubular epithelial cell (TEC) damage. This study investigates the protective role of Desflurane (DFE) in renal I/R by modulating the ITGB1/CD9 signaling pathway and mitigating oxidative damage.

Adolescent depression is a globally concerned mental health issue, the pathophysiological mechanisms of which remain elusive. Membrane lipids play a crucial role in brain development and function, potentially serving as a crossroad for the abnormalities in neurotransmitters, neuroendocrine, inflammation, oxidative stress, and energy metabolism observed in depressed adolescents. The primary aim of this study was to investigate the erythrocyte membrane lipid profile in adolescent depression. A total of 2838 erythrocyte membrane lipids were detected and quantified in 81 adolescents with depression and 67 matched healthy adolescents using ultra-high performance liquid chromatography-mass spectrometry. Depressed adolescents exhibited significantly different membrane lipid characteristics compared to healthy controls. Specifically, the levels of cholesterol, sphingomyelins, and ceramides were increased, while ether lipids were decreased in patients. Moreover, the patients showed reduced polyunsaturated fatty acids and elevated lipophilic index in membrane, suggesting diminished membrane fluidity. The higher oxidized membrane lipids and plasma malondialdehyde were observed in adolescent depression, indicating the presence of oxidative stress. Importantly, membrane lipid damage was associated with more severe depressive symptoms and worse cognitive function in patients. In addition, reduced polyunsaturated fatty acids and membrane fluidity may be partly responsible for the blunted niacin skin flushing response found in depressed adolescents. In conclusion, our results reveal impaired erythrocyte membrane lipid homeostasis in adolescents with depression, which may implicate membrane dysfunction in the brain. These findings offer new insights into the underlying molecular mechanisms of adolescent depression, highlighting the potential of counteracting membrane damage as a promising avenue for future therapeutic interventions.

Differences in cancer and normal cell oxidative metabolism provide a unique therapeutic opportunity for developing combined modality approaches with redox-active small molecules as radio-chemosensitizers that are well-tolerated by normal tissues. Pentaazamacrocyclic Mn (II)-containing (MnPAM) superoxide dismutase (SOD) mimetics and pharmacological ascorbate given IV to achieve [mM] plasma levels (pharmacological ascorbate: P-AscH‾) have been shown to act individually as cancer cell radio- and chemosensitizers via the generation of HOin vivo. The current study shows that the combination of newly developed MnPAM dismutase mimetic, rucosopasem manganese (RUC) with P-AscH‾ radio-sensitizes non-small cell lung cancer cells (NSCLC) and increases steady state levels of intracellular HO with no additional toxicity to normal human bronchial epithelial cells (HBECs). Conditional over expression of catalase (CAT) in H1299T CATc15 cells demonstrates that the combination of RUC and P-AscH‾ causes radio-sensitization through an HO-dependent mechanism. Interestingly, RUC combined with P-AscH‾ demonstrates more than additive cytotoxicity in both H1299T and A549 NSCLC cells, but conditional over-expression of ferritin heavy chain (FtH) protected only the H1299T, and not the A549, from this toxicity. Most importantly, the combination of RUC + P-AscH‾ was found to sensitize both H1299T and A549 cell types to radio-chemotherapy with cisplatin (CIS) + etoposide (ETOP). Finally, in H1299T NSCLC xenografts the combination of RUC + P-AscH‾ with CIS + ETOP and 12 × 2 Gy radiation significantly inhibits tumor growth and increased median overall over survival. These results support the hypothesis that selective MnPAM dismutase mimetic + P-AscH‾ enhances the efficacy of radio-chemotherapy in NSCLC through a mechanism governed by redox active metals and HO production.