Free Radical Biology and Medicine

Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme important for the reduction of toxic lipid peroxide products. Previous studies revealed the importance of mouse Gpx4 in protecting cardiomyocytes from ferroptosis and, subsequently, the development of cardiovascular disease. In this paper, we investigate the transcriptional consequences of cardiac-specific deletion of Gpx4 in mice and compare this response with that observed in human cardiomyopathy. The findings in this study highlight the importance of GPX4 in maintaining both structural and functional stability of the heart and identify key pathway changes resulting from excessive ferroptosis in cardiac tissue. By overlapping common transcriptional programs perturbed in this animal model and human cardiomyopathy, our findings identify putative mechanisms through which ferroptosis contributes to the development and progression of heart disease. These studies may help guide future cardiovascular therapeutics targeting ferroptosis-dependent pathways.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSTransthyretin amyloidosis (ATTR) is a progressive, degenerative disease affecting the heart and other organ systems, as well as the peripheral, autonomic, and central nervous systems. Although pharmacological and genetic evidence establishes aggregation as a driver of ATTR pathology, the mechanism by which aggregation compromises post-mitotic tissue function is poorly understood. We utilized bottom-up proteomics on wild-type (WT) human cardiac (WT/WT genotype) and V122I human cardiac (V122I/WT genotype) tissue, combined with tissue clearing technology to create an optically transparent tissue architecture to visualize three-dimensional relationships, to better understand TTR cardiomyopathy (CM). MethodsFlash-frozen 0.5 mm cardiac tissue slices from human subjects with end-stage WT-TTR CM, end-stage V122I CM, and slices from an age-matched human control were used for these experiments. Fibril extraction from diseased tissue followed published protocols. Strong denaturant-mediated proteome tissue extraction on samples from each subject facilitated bottom-up proteomics by using liquid chromatography (LC)-mass spectrometry (MS)/MS. Tissue clearing was performed on 0.5 mm cardiac slices utilizing a lauryl sulfate-based lipid removal strategy. Slices were stained using indirect immunofluorescence with antibodies to protein targets identified by proteomics. We used an antibody to non-native TTR and AmyTracker 480 (an oligothiophene dye that binds to amyloid fibrils) to image TTR deposits. ATTR fibrils were characterized structurally using cryogenic electron microscopy (cryo-EM) followed by helical reconstruction. ResultsProteomic cardiac analysis afforded high spectral counts for transthyretin (TTR) and proteins typically associated with amyloid fibrils, e.g. serum amyloid P (APCS). Fibril and cardiac homogenate proteomics revealed high levels of angiogenic and hemostatic proteins, including those composing the complement and coagulation cascades. 3D imaging revealed loss of normal microvascular architecture in CM samples with regions of hyper- and hypovascularization. Microvascular obstruction by capillary thrombosis was also observed in CM. ATTR fibrils adopted the common spearhead fold and were decorated with collagen VI (COLVI), an extracellular matrix component. ConclusionsWe hypothesize that ATTR CM is a microangiopathy driven by capillary bed thrombo-inflammation and dysregulated angiogenic revascularization. Phenotypic convergence of WT ATTR CM and V122I ATTR CM was observed via proteomics, 3D imaging, and ex vivo fibril characterization by cryo-EM. We provide evidence of capillary thrombosis in ex vivo ATTR CM tissue. Vasodilation and increased capillary permeability expose components of the vascular basement membrane (VBM) to misfolded TTR. These components are known to promote TTR aggregation and stabilize amyloid fibrils in the extracellular space. Congestion of the VBM prevents appropriate revascularization, reducing cardiac exertional capacity over time, leading to heart failure. Our ATTR CM heart tissue proteomics data shows significant overlap with the proteomic profiles of human AD brain tissues, revealing key amyloid, coagulation, complement, and angiogenesis proteins being changed in amyloidoses.

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.

Oxidative stress results from excessive accumulation of reactive oxygen species (ROS) and plays a central role in numerous physiological and pathological processes. Accurate quantification of antioxidant enzyme activities is therefore essential in redox biology research. However, data analysis for commonly used assays, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), is frequently performed using spreadsheets or manual calculations, which are time-consuming and prone to error. Here, we present Redoxyme, a free, open-source, Python-based graphical user interface designed to standardize and automate the calculation of antioxidant enzyme activities. The software integrates protein normalization, enzyme-specific calculation routines, data visualization, and Excel export within an intuitive interface that does not require programming expertise. Redoxyme was validated using experimental data obtained from animal tissues (rats and mice), demonstrating excellent agreement with manual calculations and established analytical methods. Redoxyme provides a practical solution for improving reproducibility and efficiency in antioxidant enzyme activity analysis. The software is currently distributed as a standalone executable for Windows (locally installed), and an interactive web-based calculator implemented in Streamlit, enabling direct use without local installation. The source code and version-controlled development history are openly accessible via GitHub, promoting transparency, reproducibility, community-driven improvements, and can, in principle, be adapted for other operating systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/703993v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@120cc68org.highwire.dtl.DTLVardef@4be246org.highwire.dtl.DTLVardef@1f47134org.highwire.dtl.DTLVardef@1341100_HPS_FORMAT_FIGEXP M_FIG C_FIG

A method to measure telomere length in S. cerevisiae was developed based on bioluminescence resonance energy transfer (BRET). The system uses energy transfer between a luciferase-Rif2 fusion protein and fluorescently tagged Rap1. The study demonstrates that the BRET ratio correlates with the Rap1/Rif2 complex at the telomeres and thus the availability of telomeric Rap1 binding sites. This enables the measurement of telomere length in living cells. The system was able to reproduce reported deviations in telomere length in mutants lacking telomere length regulators, cells treated with telomere length modifying compounds and strains expressing inducible telomerase. The BRET ratio linearly correlated with the average number of telomeric nucleotides derived from long-read sequencing data using a novel algorithm for telomere length calculation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711003v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1850c4dorg.highwire.dtl.DTLVardef@1ead295org.highwire.dtl.DTLVardef@1a76358org.highwire.dtl.DTLVardef@6b3183_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reactive oxygen species (ROS) play a dual role in cellular homeostasis, but excessive levels of ROS lead to oxidative stress, accelerating skin aging. Environmental stressors like UV radiation induce ROS overproduction, overwhelming endogenous antioxidant defenses and causing cellular damage. While the skin possesses an intrinsic antioxidant network that provides moderate protection, excessive oxidative stress can trigger inflammatory responses, thereby necessitating exogenous antioxidant intervention. Microbe-derived antioxidants (MA), produced via probiotic fermentation of sea buckthorn and chestnut rose, have shown promise in mitigating ROS-induced damage. In this study, we evaluated two MA formulations, MA1 and MA2, for their ability to scavenge free radicals and alleviate hydrogen peroxide (H2O2)-induced oxidative stress in human dermal fibroblasts (HDF) and dermal papilla cells (HDP). Both formulations displayed dose-dependent DPPH radical scavenging activity and enhanced cell viability at low concentrations. Under H2O2-induced oxidative stress, MA1 and MA2 effectively restored intracellular ROS to baseline levels, demonstrating significant cytoprotective effects. UHPLC-MS/MS profiling identified 12 compounds shared by both formulations, and Gene Ontology Biological Process enrichment analysis revealed that their associated target genes were significantly enriched in antioxidant-related pathways. Five compounds--adenosine, citric acid, 5-hydroxymethylfurfural, myricetin, and phenylalanine--emerged as key contributors to the observed antioxidative effects. Together, these findings highlight the potential of fermented microbial antioxidants to re-establish redox homeostasis in human skin cells and support their further development as therapeutic or cosmetic interventions targeting oxidative stress and skin aging. Given the heightened oxidative sensitivity of aged fibroblasts, MAs ability to alleviate ROS may offer novel therapeutic strategies against skin aging and related pathologies.

Genetic inhibition of cyclophilin D (CypD) delays the opening of the mitochondrial permeability transition pore (MPTP) and therefore reduces necrotic cell death. Elucidation of factors that impact CypD activity is therefore key to understanding the regulation of MPTP opening. Glycogen synthase kinase-3{beta} (GSK3{beta}) is a serine/threonine kinase that has been shown to modulate MPTP and cell death, potentially through phosphorylation of CypD. Therefore, we hypothesized that the mitochondrial fraction of GSK3{beta} directly phosphorylates CypD and promotes opening of MPTP. Overexpression of full length GSK3{beta} in mouse embryonic fibroblasts sensitized the MPTP and exacerbated oxidative stress-induced necrosis. In contrast, genetic inhibition of GSK3{beta} protected against oxidant-induced cytotoxicity but did not affect the MPTP. Recombinant GSK3{beta} could directly bind to and phosphorylate recombinant CypD. Mass spectrometry revealed several putative GSK3{beta} phosphorylation sites on CypD. However, mutation of these sites did not affect the peptidyl prolyl isomerase activity of CypD and reconstitution of these phosphomutants in CypD-deficient cells increased MPTP sensitivity and oxidative-induced cell death to the same extent as wild-type CypD. Further, targeted overexpression of either wild-type or kinase-inactive GSK3{beta} in the mitochondrial matrix did not impact MPTP or cell death. Moreover, while proteinase-K digestion of cardiac mitochondria showed a significant amount of GSK3{beta} in the mitochondria, it was not localized to the matrix. Finally, overexpression of GSK3{beta} was still able to increase MPTP sensitivity and oxidative stress-induced death in CypD-null cells. Taken together, these data indicate that, while GSK3{beta} can modulate MPTP, this appears to be independent of GSK3{beta}s interaction with, or phosphorylation of CypD.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in ageing populations, with oxidative stress recognised as a key pathogenic driver. The dietary antioxidant and cytoprotectant, L-ergothioneine (ET), is avidly accumulated in many tissues, especially the eye. However its relationship to AMD has not been investigated. Here, we examined ETs distribution in ocular tissue and assessed circulating and intraocular ET levels in patients with neovascular AMD. Compared with ocularly-normal age-matched individuals, AMD patients exhibited significantly lower serum ET; elevated levels of ET metabolites, hercynine and ETSO, which may be generated by oxidative stress; and elevated levels of serum allantoin, a product of oxidative damage to urate in humans. Levels of ET in aqueous humour in AMD patients were marginally lower than cataractous patients who are already known to have significantly lower ET levels than healthy eyes. High ET levels were seen in human ocular tissues concentrating in regions vulnerable to oxidative injury, including the lens, retina, retinal pigment epithelium, and choroid, supporting a physiological protective role of ET in the eye. These findings identify the strong association between low ET levels and AMD, warranting further studies to determine whether ET supplementation can modify AMD risk or progression.

BackgroundCardiovascular diseases are often associated with altered protein subcellular localization. As a major cause of inherited cardiomyopathy, LMNA deficiency could trigger nuclear envelope rupture and broadly impair the localization of nuclear and cytoplasmic proteins. Systemic approaches to identify, dissect and manipulate the localization of endogenous proteins are important for mechanistic and therapeutic investigation. MethodProximity proteomics of the nuclear lamina was performed specifically in cardiomyocytes in Lmna-deficient murine models. AAV-mediated Cas9-based gene silencing and subcellular gene upregulation were conducted via the nuclear localization signal (NLS) and nuclear export signal (NES). Cas9-based somatic mutagenesis was supplemented with the single-strand DNA templates of AAV to achieve robust homology-directed repair (HDR) and targeted NLS knock-in, which translocated cytoplasmic proteins into nuclei. ResultIn vivo proximity proteomics detected increased epoxide hydrolase 2 (EPHX2) in cardiomyocyte nuclei in mice carrying germline or cardiac-specific Lmna truncating variants. This phenotype was associated with ruptured nuclear envelope. Cas9-mediated Ephx2 knockout in cardiomyocytes ameliorated cardiac dysfunction in Lmna-deficient mice. Strikingly, overexpression of NLS-EPHX2, but not NES-EPHX2, also mitigated cardiac dysfunction. The cardiac protective EPHX2 substrates, epoxyeicosatrienoic acids (EETs), did not alter upon NLS-EPHX2 overexpression. By contrast, the Lmna-related DNA damage marker {gamma}-H2AX was reduced. The EPHX-D333A mutant lacking hydrolase activity recapitulated the effects of wildtype EPHX2 in nuclei. AAV-Cas9-based HDR achieved efficient NLS knock-in and EPHX2 nuclear translocation in more than 60% cardiomyocytes, which improved cardiac function. ConclusionLmna deficiency leads to the nuclear translocation of EPHX2, which ameliorated cardiac dysfunction in a hydrolase-independent manner. AAV-HDR-mediated somatic gene editing provides an efficient approach to manipulate the subcellular localization of endogenous proteins in cardiomyocytes in vivo. What is Known?O_LICardiovascular diseases are regulated by the changes in protein subcellular localization. In particular, LMNA-related cardiomyopathy is associated with nuclear rupture and impaired separation between nuclear and cytoplasmic proteins. C_LIO_LIEpoxide hydrolase 2 (EPHX2) is a cytoplasmic hydrolase that catalyzes the hydrolysis of cardioprotective epoxyeicosatrienoic acids (EETs) and aggravates an array of heart diseases including myocardial infarction and heart failure. C_LIO_LICRISPR/Cas9-mediated homology-directed repair (HDR) exhibits uniquely high gene editing efficiency in cardiomyocytes with an AAV-based DNA donor, which is suitable for somatic genetic knock-in of small DNA fragments in vivo. C_LI What New Information Does This Article Contribute?O_LILamin-targeted proximity proteomics specifically in cardiomyocytes uncovers novel proteins undergoing subcellular localization changes upon Lmna deficiency. C_LIO_LILmna deficiency leads to EPHX2 nuclear translocation that ameliorates cardiac dysfunction by both mechanisms of cytoplasmic reduction and nuclear induction. C_LIO_LIAAV-HDR-mediated knock-in provides a robust platform to manipulate subcellular localization of endogenous proteins specifically in cardiomyocytes in vivo. C_LI Cardiovascular diseases are often associated with altered protein subcellular localization, but systemic approaches to identify, study and manipulate subcellular localization remain incomplete. This study established an in vivo proximity proteomics approach to identify novel proteins undergoing localization changes relative to the nuclear lamina. In murine models of LMNA-related cardiomyopathy, this approach uncovered EPHX2, a classic cytoplasmic hydrolase that aggravates cardiovascular diseases, as a new protein that translocated into cell nucleus and exerted a cardiac protective effect. CRISPR/Cas9-based cardiomyocyte gene editing with an AAV-based DNA donor efficiently achieved NLS knock-in into the Ephx2 gene, promoted EPHX2 protein nuclear translocation and mitigated cardiac dysfunction with Lmna deficiency. These findings indicated a novel avenue to identify and manipulate the subcellular localization changes of endogenous proteins for basic and translational cardiology.

Cardiac transthyretin amyloidosis (ATTR-CA) is caused by myocardial deposition of misfolded transthyretin, leading to progressive heart failure. Disease pathology, however, extends beyond passive amyloid deposition and also involves active processes such as extracellular matrix (ECM) remodeling and immune activation. Mass spectrometry (MS) is the gold standard for amyloid typing in diagnostics. Here, we applied quantitative MS-driven proteomics on formalin-fixed paraffin-embedded whole cardiac tissue sections from six ATTR-CA cases, ten unaffected controls and four AL-CA controls to investigate protein expression changes. In addition to transthyretin, over 500 proteins were upregulated in ATTR-CA biopsies, including complement and coagulation factors as well as extracellular matrix (ECM) remodeling proteins. Among these, members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family, metalloproteinases (MMPs), and Tissue Inhibitor of Metalloproteinases (TIMP3) showed significant upregulation. These proteins are key regulators of ECM turnover and structural integrity. Immunohistochemistry confirmed ADAMTS4 enrichment in amyloid deposits, while TIMP3 showed strong expression in cardiomyocytes and weaker staining within amyloid deposits. Together, these findings indicate that ECM remodeling, alongside complement and coagulation activation, represents a reproducible feature of cardiac ATTR amyloidosis. Whole-tissue proteomics provides biological insights that extend beyond amyloid typing, with potential implications for biomarker discovery and therapeutic targeting in ATTR-CA.

Glutathione peroxidases (GPXs) are widely recognized as key antioxidants that mitigate oxidative stress by detoxifying reactive oxygen species (ROS). However, GPXs are largely uncharacterized in citrus. Here, we demonstrated that Citrus sinensis contains four GPX proteins (CsGPX1-4). Unexpectedly, overexpression of CsGPX4, a homolog of AtGPX8 in Arabidopsis, in citrus resulted in typical oxidative stress phenotypes including severe growth inhibition, chlorosis, and elevated intracellular ROS accumulation. Transmission electron microscopy (TEM) analysis further revealed stress responses at cellular level. Whole genome shot gun sequencing analysis showed that T-DNA insertion occurs in the UTR of SWEET2 gene, which is unlikely to be responsible for the oxidative stress phenotypes. Immunoblotting revealed that CsGPX4 accumulates as a truncated protein in citrus, in contrast to the full-length version expressed in Nicotiana benthamiana. MALDI-TOF assays further confirmed the truncation of CsGPX4 in the transgenic line with the predicted cleavage site between L115-K117. This truncation was associated with altered subcellular localization, shifting from cytoplasmic and nuclear distribution in N. benthamiana to membrane association in citrus. Proteomic profiling further indicated extensive reprogramming of pathways involved in detoxification, cytoskeletal stability, hormone signaling, and cell wall modification. Our data suggests that de facto overexpression of truncated CsGPX4 may have dominant-negative effects on proteins interacting with CsGPX4, thus interfering with their normal functions. In conclusion, our study demonstrates CsGPX4 as a critical regulator of redox homeostasis and ROS homeostasis in citrus and reveals selective truncation of CsGPX4 as a unique proteolytic or regulatory strategies in such processes.

BackgroundExercise-induced fatigue is a complex physiological phenomenon involving oxidative stress, inflammation, and metabolic disturbances. Ergothioneine (EGT), a naturally occurring amino acid with potent antioxidant properties, has garnered interest for its potential health benefits. This study aimed to evaluate the anti-fatigue effects of Gene III EGT in a mouse model of exhaustive exercise and to elucidate its underlying mechanisms. MethodsMale C57BL/6 mice were randomly divided into five groups: a control group (CTL), low-dose EGT (EGT-L, 10 mg/kg), medium-dose EGT (EGT-M, 30 mg/kg), high-dose EGT (EGT-H, 50 mg/kg), and a positive control group (Coenzyme Q10, 50 mg/kg). Mice were subjected to a 4-week treadmill training protocol, followed by an exhaustive running test. We measured exercise performance and collected blood and skeletal muscle samples at multiple time points to assess biochemical markers, inflammatory cytokines, antioxidant status, and key signaling proteins. ResultsGene III EGT supplementation, particularly at medium and high doses, significantly extended the time to exhaustion and running distance. Compared to the control group, EGT treatment significantly reduced post-exercise levels of lactic acid (LA), lactate dehydrogenase (LDH), and blood urea nitrogen (BUN). Furthermore, Gene III EGT suppressed the exercise-induced increase in pro-inflammatory cytokines, including IL-1{beta}, IL-6, and TNF-. The anti-fatigue effect of EGT was also associated with a reduction in malondialdehyde (MDA) and an increase in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Mechanistically, EGT promoted the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1) in skeletal muscle, while also increasing the Bcl-2/Bax ratio, suggesting enhanced mitochondrial biogenesis and reduced apoptosis. ConclusionsOur findings demonstrate that Gene III EGT effectively enhances exercise performance and alleviates fatigue. The underlying mechanisms involve the mitigation of oxidative stress and inflammation, as well as the activation of the AMPK/PGC-1 signaling pathway to promote mitochondrial function and cellular protection. These results highlight the potential of Gene III EGT as a nutritional supplement for combating exercise-induced fatigue.

Background & AimsMetabolic disorders such as dyslipidemia, metabolic dysfunction-associated steatotic liver disease (MASLD), and diabetes are promoted by chronic pro-inflammatory and pro-oxidative states. Paraoxonase 1 (PON1), a liver-derived HDL-associated enzyme, plays an important antioxidant role by hydrolyzing oxidized lipids and protecting against oxidative stress- induced damage. Genetic variation in PON1, particularly in promoter and coding regions, modulates enzyme expression and activity, thereby influencing susceptibility to metabolic and cardiovascular diseases. This study investigated the genetic determinants of serum paraoxonase (PONase) activity and their relationship with dysmetabolic phenotypes. MethodsA genome-wide association study was conducted in 922 Portuguese individuals from the PREVADIAB2 cohort. Genetic variants and haplotypes related to PONase activity were analyzed, and associations with dysglycemia and liver fibrosis were evaluated in individuals aged over 55 years. ResultsWe identified two key PON1 variants as determinants of PONase activity: rs2057681 (in strong linkage disequilibrium with the non-synonymous Q192R variant) and rs854572 (located in the promoter region). Analysis of rs854572-rs2057681 haplotypes revealed that specific combinations differentially modulate PONase activity and confer risk or protection for dysglycemia and liver fibrosis, depending on the rs2057681 genotype context. Notably, although PONase activity was strongly associated with PON1 variants, it did not directly correlate with dysmetabolic phenotypes, suggesting that genetic context and haplotype structure, rather than enzyme activity alone, shape disease susceptibility. ConclusionsThese findings highlight the complex genetic architecture of PON1 and its role in metabolic disease risk, supporting the use of PON1 genetic information to uncover predisposition to dysmetabolic conditions. Our results provide insights into the interplay between PON1 genetics, enzyme function, and dysmetabolism, with implications for risk stratification in metabolic liver disease. Lay SummaryPON1 is a liver-derived gene that encodes an enzyme involved in protection against oxidative stress, a key contributor to metabolic liver disease and diabetes. In this study, we found that specific combinations of PON1 genetic variants are associated with abnormalities in blood glucose regulation and with markers of liver fibrosis. These associations were dependent on genetic configuration rather than enzyme activity alone, suggesting that PON1 genetic information may help identify individuals at higher risk of metabolic liver disease.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite that accumulates in diabetes and aging, causes tissue dyshomeostasis, for which therapeutic interventions are limited. Herein, we investigate the potential of tannic acid (TA) in fortifying organ and organismal health against MGO. Anatomical disruption in vivo of hydra bodies and ex vivo decellularization of murine mesenteries with MGO suggested an impaired interaction between cells and their extracellular matrix (ECM); however, pretreatment of these systems with TA reversed this effect. We confirmed this through subsequent exposure of control and TA-pretreated mammalian cell-secreted endogenous matrix, Collagen I, and basement membrane matrix to MGO. TA prevented loss of ECM biochemical characteristics and restored perturbed cell adhesion and spreading on these substrata induced by MGO. NMR titrations confirmed TA-bound MGO in a 1:5 stoichiometry, potentially quenching its electrophilic properties. Our study posits TA as a novel candidate for protecting organ and organismal architectures against the histopathological effects of dicarbonyl stress.

BackgroundAlcoholic fatty liver disease (AFLD) is a progressive hepatic pathology triggered by chronic ethanol consumption, serving as the initial stage of severe liver injury. Currently, there are no FDA-approved pharmacological interventions that specifically target alcohol-induced hepatic steatosis or prevent disease progression, highlighting an urgent need for effective preventive strategies. This study evaluated the preventive efficacy and underlying mechanisms of Gene III Ergothioneine (EGT) in a clinically relevant preclinical model. MethodsC57BL/6 mice were randomized into five groups: a Control group, an alcoholic fatty liver Model group, a Positive control group treated with Silybin (100 mg/kg), and three EGT treatment groups (10, 30, and 50 mg/kg). The NIAAA mouse model was utilized to induce alcoholic fatty liver. Various biochemical, histological, and molecular markers were assessed to evaluate liver damage, alcohol metabolism, lipid profiles, oxidative stress, and inflammation. ResultsGene III EGT treatment significantly ameliorated hepatic steatosis and necrosis, as confirmed by H&E and Oil Red O staining. Notably, EGT accelerated alcohol clearance, reducing serum ethanol levels by up to 54.4% in a dose-dependent manner. Furthermore, EGT restored liver function markers (ALT, AST, GGT) and corrected dyslipidemia by lowering TG, TC, and LDL-C while elevating HDL-C. Mechanistically, EGT suppressed pro-inflammatory cytokines (IL-6, IL-1 {beta}) and mitigated oxidative stress by reducing malondialdehyde (MDA) accumulation and restoring superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. ConclusionGene III Ergothioneine prevents alcoholic liver injury through a dual mechanism: accelerating ethanol metabolism and enhancing hepatocyte antioxidative and anti-inflammatory defenses. These findings position EGT as a promising therapeutic candidate for AFLD management.

BackgroundOxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. MethodsA wide battery of complementary tests from RNA level to high-resolution respirometry on permeabilized muscle fibers was performed. The relationship between respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine the clinical significance of the findings. ResultsThe study included a total of 67 patients with IBM and 45 controls. IBM muscle tissue exhibited reduced maximal respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, males had decreased State 3 intrinsic respiration, whereas females had greater intrinsic respiration in leak states. Complex II control ratio strongly correlated with disease duration and severity only in females. IBM was associated with decreased RNA and protein expression of OXPHOS complexes. Complex I activity was decreased mainly in females. IBM samples exhibited lower maximal H2O2 emission, accompanied by a higher total antioxidant capacity that correlated with disease duration in females. In IBM, there was decreased mtDNA content, and impaired mitophagy, both of which strongly correlated with respirometry measures and markers of disease severity, indicating these pathways are likely interconnected and of clinical significance. ConclusionIBM is characterized by multilevel impairments in mitochondrial coupling efficiency, revealing several potential therapeutic targets to improve mitochondrial fitness, while accounting for sex-specific differences.

Pro-death Bax isoform Bax{Delta}2 forms protein aggregates in Alzheimers neurons, triggering stress granule formation and neuronal cell death. In seeking chemical ligands to prevent Bax{Delta}2 monomer aggregation, we performed in silico screening of FDA-approved drugs using computational docking. This screening identified a group of compounds that bind to the hydrophobic pocket of Bax{Delta}2. Subsequent wet-lab testing revealed that digoxin could block neuronal cell death at nanomolar concentrations (50 to 100 nM). Importantly, digoxins protective role is specific to Bax{Delta}2-induced cell death and is independent of its primary cardio-action on Na/K-ATPase. Further investigation suggests that digoxin does not significantly affect the formation of Bax{Delta}2 aggregates but may instead modulate Bax{Delta}2 protein levels. Although the therapeutic use of digoxin for Alzheimers disease is not feasible due to its narrow therapeutic window and toxicity, these findings open the door for chemical modification of digoxin, or development of similar compounds, to prevent Bax{Delta}2-mediated neuronal cell death in Alzheimers disease.

Exposure to cigarette smoke is one of the major risk factors for developing various diseases such as chronic obstructive pulmonary disease (COPD), cardiovascular disorders, and cancer mediated via cellular oxidative stress and organelle dysfunction. To this end, the current study investigated how cigarette smoke extract (CSE) affects vacuole structure and function in Saccharomyces cerevisiae, as vacuole plays a crucial role in handling oxidative stress-induced misfolded proteins. Our results showed that CSE exposure causes transient vacuolar fragmentation up to 1 h to increase its surface area to facilitate microautophagy in clearing CSE-mediated misfolded protein and promoting cell survival. However, excessive fragmentation or vacuolar fusion sensitizes cells towards CSE-mediated cellular toxicity. Towards understanding the underlying mechanism, the current study demonstrated the involvement of PI3P and PI (3,5) P2-mediated signaling and phospholipase-driven remodeling of lipid moieties. Moreover, the current study also showed the importance of mitochondrial activity in CSE-mediated vacuolar fragmentation. Prolonged exposure to CSE impairs mitochondrial function and thus disrupts fragmentation, the adaptive survival strategy against CS. It results in proteostasis collapse, which is a characteristic shared by many inflammatory and degenerative disorders. Taken together, the current study reveals a previously unrecognized cellular protection mechanism induced by cigarette smoke and highlights potential therapeutic targets for mitigating CS-mediated diseases

Synthetic 6-Br-Q0C10 has been shown to exhibit a partial electron transfer activity of native coenzyme Q in the isolated mitochondria. It reduces energy coupling efficiency by approximately 30%, suggesting that it may be useful in modulating cell growth in tissue culture. Whether or not it behaves in the same way in the whole cells, or animal, however, has not yet been fully examined. Recently we have investigated the effect of 6-Br-Q0C10 across multiple cell lines using three detection methods. Treatment with 6-Br-Q0C10 reduces cell proliferation in all cell lines tested, with different effectiveness. Obesity-related cell lines were the most susceptible, and a pronounced inhibitory effect was also observed in cancer cell lines. These results strengthen the idea of using 6-Br-Q0C10 to manage obesity or to retard the growth of rate cancer cells and thus prolonging life.

BACKGROUNDMyocardial ischemia/reperfusion injury (I/RI) represents a serious clinical complication in patients after acute myocardial infarction. Ubiquitin-activating enzyme 1 (UBA1) catalyzes the initial step of ubiquitination and plays a fundamental role in regulating protein homeostasis and related diseases. This study aims to elucidate the functional contribution of UBA1 to the pathogenesis of myocardial I/RI and to uncover its underlying mechanisms. METHODSSingle-cell RNA sequencing was employed to characterize UBA1 expression in human ischemic heart tissues. Myocardial I/R injury was examined in myocardial-specific UBA1 knockout (UBA1cko) mice, UBA1-overexpressing mice (rAAV9-UBA1), and corresponding controls. Neonatal rat cardiomyocytes underwent hypoxia/reoxygenation in vitro. Cardiac function and infarction were evaluated by echocardiography and pathological staining. Protein-protein interactions were analyzed via immunoprecipitation combined with mass spectrometry. The endoplasmic reticulum-mitochondrial contact sites (ERMCSs) and mitochondrial ultrastructure were evaluated through transmission electron microscopy and confocal imaging. RESULTSUBA1 expression was significantly downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. UBA1cko mice exhibited aggravated I/RI with greater infarct size, impaired function, apoptosis, elevated intracellular Ca2+ levels, mitochondrial dysfunction, and ER stress, whereas UBA1 overexpression conferred cardioprotective effects. Mechanistically, UBA1 directly bound to and ubiquitinated Pdzd8, a key ERMCS-tethering protein, thereby promoting its degradation, which inhibited ERMCS formation and improved mitochondrial dysfunction and ER stress. Moreover, knockdown of Pdzd8 via rAAV9-siRNA effectively mitigated UBA1 knockout-induced myocardial damage. Additionally, administration of auranofin (AF), a U.S. Food and Drug Administration-approved drug for treating rheumatoid arthritis, markedly alleviated myocardial I/RI via activating UBA1 in vivo and in vitro. CONCLUSIONSUBA1 confers protection against myocardial I/RI by limiting ERMCS formation through Pdzd8 ubiquitination. Activating UBA1 or targeting Pdzd8 as a potential therapeutic strategy for the treatment of ischemic heart disease. GRAPHIC ABSTRACTA graphic abstract is available for this article. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIUBA1 expression is downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. C_LIO_LICardiac deletion of UBA1 significantly exacerbates myocardial ischemia/reperfusion injury (I/RI), whereas cardiac UBA1 overexpression confers a marked protective effect. C_LIO_LIUBA1 interacts with Pdzd8 (PDZ domain containing 8) and facilitates its ubiquitination and subsequent degradation, which then reduces endoplasmic reticulum-mitochondria contact sites (ERMCSs) and ameliorates mitochondrial dysfunction and ER stress, protecting myocardial I/RI. C_LIO_LIPharmacological activation of UBA1 with the FDA-approved drug auranofin attenuates myocardial I/R injury and improves heart dysfunction. C_LI What Are the Clinical Implications?O_LIUBA1 represents a new therapeutic target for myocardial I/RI. C_LIO_LIActivating UBA1 or targeting Pdzd8 may offer a promising therapeutic strategy for mitigating myocardial I/RI and heart failure, underscoring its potential for clinical translation. C_LI