Search Results (1,315)

Mitochondrial reactive oxygen species (ROS) play a central role in cardiac ischemia/reperfusion injury, heart failure, and arrhythmogenesis, while also serving essential signaling functions under physiological conditions. Among the eleven identified mitochondrial ROS-producing sites, complexes I and III are considered the major contributors, particularly under conditions of impaired electron flow. However, much of the existing knowledge comes from rodent models or cultured cells and is often assumed to apply to humans. Here, ROS production from complexes I and III was measured directly in human myocardial and skeletal muscle biopsies and compared with corresponding rat tissues under identical experimental conditions. Hydrogen peroxide generation was quantified using Amplex UltraRed, with simultaneous monitoring of mitochondrial respiration using a Clark-type oxygen electrode. Across all examined mechanisms—reverse and forward electron transport at complex I and the ubiquinol oxidation site of complex III, rat tissues produced more ROS than human tissues, consistent with their higher respiratory rates. However, the dominant ROS-producing sites differed: in rats, complex III was the primary source, whereas in human tissues the highest ROS production occurred during reverse electron transport at complex I. When normalized to respiration, human tissues showed relatively greater ROS generation at complex I but markedly lower production at complex III. These direct measurements of mitochondrial ROS production in human myocardium provide new insight into cardiac redox physiology and may explain the limited clinical translation of cardioprotective strategies targeting mitochondrial ROS production, such as interventions aimed at modulating reperfusion injury or preconditioning. Full article

Sugarcane stores sucrose in a living culm for extended periods, yet the respiratory cost of maintaining this storage tissue remains poorly quantified. We quantified growth and maintenance respiration along the culm (internodes 1 to 12) in three genotypes at mid-season (rapid growth) and end-season (maturation) using a composition-based carbon accounting framework derived from measurements of biomass accumulation and composition. Growth respiration was highest in elongating internodes (3 to 6) and declined with maturation, whereas maintenance respiration increased progressively and dominated in mature storage internodes (10 to 12). Consequently, total sink demand remained substantial even after structural growth slowed, indicating that mature internodes continue to require significant metabolic input despite limited biomass production. To evaluate the potential impact of energetic constraints, we simulated reduced mitochondrial energy contribution to assess the sensitivity of respiratory carbon demand to decreased energetic efficiency. These simulations predicted an increase in glucose requirement for respiration across all internodes, with the largest proportional effect in mature tissue where maintenance costs dominated. Despite this predicted increase in respiratory demand, sucrose accumulation was maintained in mature culms, indicating that respiratory carbon loss remains constrained during storage. This suggests that storage tissue operates with relatively high carbon-use efficiency during maintenance-dominated metabolism. We interpret this pattern as consistent with metabolic configurations that reduce ATP demand, potentially involving partial substitution of ATP-dependent reactions by pyrophosphate (PPi)-dependent pathways, although this mechanism was not directly measured. These findings highlight the importance of maintenance respiration and energetic efficiency in determining sink strength and sucrose yield, and they provide a physiological framework for understanding carbon conservation in long-lived storage organs. Full article

The emerging problem that microplastics pose to our society is reflected in the exponential growth in investigations devoted to uncovering their toxicological potential in humans. However, these studies present several limitations, one of the most significant being the use of microplastics that do not represent their environmental counterparts. In this study, we evaluated the impact of two types of polyethylene microplastics (27–32 µm)—non-fluorescent and fluorescent—on the liver and intestine, targeting mitochondria. FVB/n mice were subjected to a subacute exposure to two concentrations representative of human exposure (0.002% (w/w) and 0.006% (w/w)). Both types of microplastics impaired mitochondrial respiration through disruption of NADH-linked pathways, with more pronounced effects at the highest concentration of fluorescent MPs. Electron transport chain complexes, particularly CIII and CIV, were affected, partially explaining the observed alterations in mitochondrial respiratory capacity. An increased SOD and GPx activity supported the link between mitochondrial dysfunction and increased reactive oxygen species overproduction under MPs exposure. Hepatic mitochondrial lipid remodelling was detected following exposure to fluorescent microplastics, while intestinal epithelial cells displayed impaired mitochondrial activity together with compromised cellular integrity, indicative of stress response. In parallel, shifts in gut composition suggest that PE MPs may contribute to intestinal barrier dysfunction. Overall, fluorescent MPs induced more severe mitochondrial and biochemical disturbances in both the liver and the intestine than their non-fluorescent counterparts. Our findings highlight mitochondria as central targets for microplastic-induced toxicity and underscore the need for improved MPs models in toxicological research. Full article

The C-terminal binding protein 1 (CTBP1) is a transcriptional corepressor with a major role in nervous system growth and development. There are only 20 published cases with CTBP1 mutations, displaying a phenotype of Hypotonia, Ataxia, Developmental Delay and Tooth enamel defect Syndrome (HADDTS). Histochemical evidence of decreased mitochondrial respiratory chain activity has been previously reported, but comprehensive data on the metabolic phenotype assessed by various cellular respiration parameters are still missing. We present a 10-year-old female with typical HADDTS features, harboring the most reported de novo heterozygous CTBP1 mutation c.991C>T. To elucidate her metabolic phenotype, we quantified mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) utilizing an analyzer for assessing mitochondrial function (Seahorse XFp). Real-time metabolic assays revealed profound mitochondrial dysfunction with significantly attenuated maximal respiration and spare respiratory capacity compared to neurotypical controls. Following mitochondria-targeted nutritional support for one-year measurable bioenergetic improvements and reduced number of respiratory infections were registered. However, neurological recovery and new skill acquisition were not observed. We present a novel case of CTBP1-related neurodevelopmental disorder and demonstrate, for the first time, the application of non-invasive, real-time mitochondrial functional assessment in this setting, providing additional evidence for mitochondrial dysfunction in HADDTS. Full article

The ATP-dependent inhibition of cytochrome c oxidase (CytOx, complex IV of the electron transport chain) is the second mechanism of respiratory control adjusting mitochondrial respiration in order to prevent excessive electron flow and reactive oxygen species (ROS) production. Here, we investigate how tricarboxylic acid (TCA) cycle metabolites and the subsequent complex I or complex II activities influence this regulatory mechanism. Therefore, CytOx activity was assessed by the oxygen consumption rate after cytochrome c (Cyt c) titration to stimulate complex IV activity in isolated rat heart mitochondria (RHM) and permeabilized AC16 cells. Mitochondrial membrane potential (Δψm) and ROS formation were analysed by flow cytometry. Our results show that TCA cycle intermediates differed in their impact on CytOx activity and subsequent ROS formation. NADH-linked substrates such as α-ketoglutarate, glutamate and malate increased respiratory capacity, but preserved ATP-dependent control of CytOx, indicating that elevated electron supply alone does not necessarily abolish ATP sensitivity. In contrast, succinate, which feeds electrons directly into complex II, strongly increased respiration causing the loss of ATP-dependent respiratory control in both model systems. Despite this strong respiratory effect, succinate induced only modest changes in mitochondrial membrane potential in isolated mitochondria, whereas permeabilized cardiomyocytes exhibited reduced polarization accompanied by increased superoxide formation. Together, these findings demonstrate that the effectiveness of ATP-dependent CytOx inhibition is influenced by TCA cycle activity and depends on the site of electron entry into the respiratory chain. Thus, substrate-dependent modulation of respiratory control links metabolite availability to mitochondrial redox regulation in cardiac cells. Full article

Mitochondrial dysfunction is a hallmark of aging and age-related physical decline in people living with HIV (PLWH) who experience accelerated aging. This pilot study investigated the relationships between platelet mitochondrial function, physical performance, and body composition in older, sedentary PLWH compared with older, sedentary HIV-negative controls. Platelets have the potential to act as minimally invasive and easily accessible biomarkers for systemic mitochondrial bioenergetics and may serve as a practical biomarker in aging-related research. We analyzed correlations between mitochondrial parameters, protein levels, and measures of physical performance and body composition in a cohort of predominantly African American men (n = 7 PLWH, n = 7 controls). Body composition was assessed using dual-energy X-ray absorptiometry (DXA), and exercise capacity through VO₂ peak and strength tests. Platelet mitochondrial bioenergetic parameters were measured by oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Key mitochondrial proteins SIRT3, COXII, DRP1, and OPA1 were evaluated by Western blotting. The PLWH and HIV-negative control groups were similar in age and cardiorespiratory fitness. In PLWH, basal OCR and ATP-linked respiration showed strong positive correlations with VO₂ peak (r = 0.874, p < 0.05 and r = 0.862, p < 0.05, respectively) and negative correlations with BMI (r = −0.856, p < 0.05 and r = −0.849, p < 0.05, respectively). SIRT3 emerged as a potential key player, demonstrating strong positive correlations with basal OCR (r = 0.804, p < 0.05), ATP-linked respiration (r = 0.787, p < 0.05), and VO₂ peak (r = 0.970, p < 0.001), and negative correlations with BMI (r = −0.830, p < 0.05) and fat mass (r = −0.827, p < 0.05) in PLWH. Analyses focused on within-group associations in PLWH because bioenergetic measures were obtained using different Seahorse platforms in PLWH and controls, precluding valid direct quantitative comparisons between groups. Our findings provide evidence for significant associations between platelet mitochondrial bioenergetics, specific mitochondrial proteins (particularly SIRT3), and key physical attributes in older, sedentary PLWH. These preliminary findings suggest that platelets may serve as minimally invasive biomarkers of systemic mitochondrial health, contribute to our understanding of mitochondrial function in HIV-associated accelerated aging, and inform future interventions to enhance mitochondrial function and improve health outcomes in this vulnerable population. However, results should be interpreted cautiously given the small sample size and exploratory design and should be considered hypothesis-generating rather than definitive. Larger, demographically more diverse studies that include HIV-negative controls are needed to validate these associations and determine their clinical relevance. Full article

The invasive fungal pathogen Pseudogymnoascus destructans is responsible for the collapse of several North American bat species through an infectious fungal skin disease known as White-Nose Syndrome (WNS). Recent transcriptomic studies have suggested that trace copper ion acquisition is essential for P. destructans propagation on its animal hosts. However, little is known about the mechanistic details of P. destructans adaptation occurring at the protein level. In this study, we report the global proteomic adaptation of P. destructans under chronic Cu-stress growth conditions employing chemically defined media. We identify 4340 P. destructans proteins, or approximately 47.8% of the predicted proteome, spanning a dynamic intensity range of six orders of magnitude. Chronic Cu-withholding stress leads to substantial alterations in the proteome, with 1398 differentially abundant proteins (DAPs) exhibiting statistically significant (p < 0.05) changes in protein levels compared to control growth conditions. We find that Cu-withholding stress induces increased levels of proteins associated with high-affinity Cu-acquisition, changes in intracellular superoxide dismutase (SOD) levels, and alterations in mitochondrial proteins related to aerobic respiration. In contrast, chronic Cu-overload stress leads to 390 DAPs (p < 0.05), which are more widely distributed across the proteome, with several DAPs associated with genomic stability and basic metabolism. Additionally, in this report, we present assessment of antisera products against intracellular and cell-surface protein targets of P. destructans that are effective for indicating Cu-withholding stress by western blotting. Together this report, provides insight into P. destructans adaptability to copper stress and identifies fungal proteins that may alleviate copper stress in the WNS infection niche. Full article

Garrya flavescens S. Wats. (GF) has been traditionally used to treat gastrointestinal spasms, yet its bioactivity within the central nervous system remains unexplored. This study aimed to characterize the bioactive constituents of GF and evaluate its anti-inflammatory and metabolic regulatory effects in lipopolysaccharide-activated microglia. Phytochemical profiling using LC-HRMS and HPLC identified rutin as a primary bioactive component, present at an exceptionally high concentration (9309 μg/g). In BV-2 microglial and RAW 264.7 cells, GF treatment significantly suppressed the expression of pro-inflammatory cytokines and mediators in a dose-dependent manner. Mechanistic studies revealed that GF specifically modulated the ERK signaling pathway. Furthermore, Seahorse XF analysis demonstrated that GF restored mitochondrial homeostasis by reducing basal respiration and proton leak while significantly enhancing spare respiratory capacity. Finally, conditioned medium from GF-treated microglia improved the viability of N2A neuronal cells. These findings highlight GF as a potent botanical source with significant neuroprotective potential, offering a promising candidate for functional food or nutraceutical applications targeting neuroinflammatory disorders. Full article

Cytochrome c (Cytc) tyrosine 48 (Y48) has been previously shown to be phosphorylated in bovine liver, and phosphomimetic substitution (Y48E) inhibits key functions of Cytc in vitro, including respiration and apoptosis. In this study, we investigated the effect of Y48 modification in a double-knockout cell culture model that stably expressed either unphosphorylated wild-type (WT) Cytc, control Y48F Cytc, or phosphomimetic Y48E Cytc. Our findings revealed that Y48E Cytc caused partial inhibition of mitochondrial respiration in intact cells, which corresponded with lower mitochondrial membrane potentials (ΔΨ_m) and reduced reactive oxygen species (ROS) production. When subjected to an oxygen–glucose deprivation/reoxygenation (OGD/R) model, which simulates ischemia/reperfusion injury, the Y48E phosphomimetic cell line showed lower ROS production compared to the unphosphorylated WT and Y48F Cytc cell lines, the latter of which generated higher levels of ROS upon reoxygenation. As a result, the Y48E Cytc cell line had significantly lower cell death rates when exposed to OGD/R, confirming the cytoprotective role of Y48 phosphorylation of Cytc. In summary, our research indicates that the loss of Y48 phosphorylation in Cytc during ischemia leads to reperfusion injury by driving maximum electron transport chain flow, hyperpolarization of ΔΨ_m, bursts of ROS, and death of cells through apoptosis. Full article

Cutaneous melanoma is the most lethal form of skin cancer because of its aggressiveness, rapid metastasis, and high therapeutic resistance. The 2018 World Health Organization (WHO) classification emphasized that melanoma comprises distinct subtypes defined by cumulative sun damage, site of origin, and molecular characteristics, which explain differences in mutational burden, immunogenicity, and treatment response. Immunotherapy with anti-PD-1 therapy such as nivolumab and pembrolizumab changed the therapeutic landscape by restoring CD8+ T-cell activity and improving survival. Still, many patients show primary or acquired resistance influenced by low PD-L1 expression, loss of antigen presentation, tumor metabolic plasticity, and an immunosuppressive microenvironment. Mitochondria are central to this process. They regulate ATP generation through oxidative phosphorylation (OXPHOS), redox control, apoptosis, and the metabolic programming needed for T-cell activation. In the tumor microenvironment (TME), hypoxia, nutrient restriction, and PD-1 signaling reduce mitochondrial biogenesis, increase fission and reactive oxygen species (ROS) accumulation, and lead to exhaustion and impaired effector function. Moreover, tumor cells outcompete immune cells for key nutrients such as glucose and glutamine, while increased lactate production and extracellular acidosis further suppress mitochondrial respiration in T cells. Strategies to overcome resistance include restoring oxidative metabolism, activating PGC-1α, supplying metabolic substrates, and combining checkpoint blockade with inhibitors of glycolysis or glutaminolysis to enhance the immune response. Full article

Neuroblastoma remains a formidable pediatric malignancy characterized by profound metabolic plasticity and limited therapeutic responsiveness in high-risk disease. Emerging evidence positions the interplay between Reactive Oxygen Species (ROS) and the metabolic sentinel AMP-activated protein kinase (AMPK) as a critical regulator of tumor metabolic stress and apoptotic susceptibility, with additional implications in the systemic pathology of Cancer Cachexia. Building on our previous work demonstrating that 1,1-Diethoxyethane (1,1-DEE; Biosacetalin), a volatile aroma compound inhibits mitochondrial complex I, induces ROS production, and activates AMPK-PGC1α-mediated mitochondrial biogenesis accompanying enhancement of aerobic respiration, leading to anti-Warburg effect. We identify 1,1-DEE as a previously unrecognized metabolic modulator with potent antitumor activity. 1,1-DEE triggers ROS-induced AMPK activation, leading to apoptotic elimination of neuroblastoma cells (SH-SY5Y), robust suppression of tumor growth, and significant prolongation of survival (median survival 77 days) in tumor-bearing NSG mice. Strikingly, 1,1-DEE simultaneously alleviates cancer-associated cachexia by preserving body weight. Mechanistically, our findings reveal a ROS–AMPK–centered signaling axis through which 1,1-DEE integrates tumor-selective cytotoxicity with systemic metabolic protection, highlighting a unified therapeutic strategy for targeting both tumor progression and cachexia in neuroblastoma. Full article

Copper is a crucial cofactor that sustains multiple cellular electron-transfer reactions, making it an essential element for life. However, cytotoxic levels of copper can cause structural damage and cell death through the production of reactive oxygen species (ROS) and nonspecific attacks on proteins. Moreover, immune cells, including neutrophils and macrophages, accumulate copper to induce oxidative bursts that kill engulfed pathogens. Therefore, a well-regulated copper homeostasis system is required for the human commensal fungus Candida albicans to thrive in extreme host environments. Remarkably, C. albicans exhibits higher copper tolerance than the nonpathogenic model yeast Saccharomyces cerevisiae, suggesting the presence of a specific copper tolerance mechanism that supports its adaptability to copper stress. Ndt80 is a versatile transcription factor that regulates several biological processes in C. albicans, ranging from morphological control to drug resistance. This study further reveals that Ndt80 may contribute to copper tolerance by regulating copper transporters and copper-dependent superoxide dismutases (Sods). Additionally, RNA sequencing and complementary approaches uncovered the involvement of Ndt80 in plasma membrane integrity and mitochondrial respiration under copper stress, further linking Ndt80 to copper tolerance. Together, these results broaden our understanding of Ndt80 functions and provide new insights into copper tolerance in C. albicans. Full article

(1) Background: Mild traumatic brain injury (mTBI), the most prevalent form of traumatic brain injury, often results from repetitive impacts to the head and is associated with long-term neurological impairment. The pathophysiology of mTBI is multifactorial and involves alterations in mitochondrial bioenergetics, a key determinant of neuronal function and survival. Although mitochondrial dysfunction is recognized as a hallmark of mTBI, its long-term effects on bioenergetics and the roles of regulatory cytosolic and mitochondrial proteins remain poorly understood. We hypothesized that repeated mTBI (rmTBI) induces sustained deficits in mitochondrial bioenergetics that are associated with long-term changes in key bioenergetic and other regulatory proteins. (2) Methods: Using the repeated CHIMERA injury model in adult male rats, randomly assigned to sham or rmTBI groups, we assessed mitochondrial respiration in isolated mitochondria and whole cerebral cortex homogenates using a Clark O₂ electrode and an Oroboros O₂k respirometer at time points ranging from 1 day to 2 months post-injury. Western blotting was performed for expression of regulatory proteins HKI, DRP1, MFN2, VDAC1, and ANT2. (3) Results: At 2 months post-rmTBI, respiration was faster and uncoupled, while ATP synthesis was significantly slowed compared with sham rats. This was accompanied by decreased expression of mitochondrial MFN2 and ANT2, by increased mitochondrial expression of DRP1, and by decreased translocation of HKI to mitochondria. There was no significant difference in VDAC1 expression. Earlier time points showed no significant differences in bioenergetics or protein expression, but neuro-inflammatory markers (GFAP and Iba1) were significantly elevated at these earlier time points of post-injury. (4) Conclusions: These findings indicate that rmTBI leads to a delayed long-term impairment of mitochondrial bioenergetics associated with alterations in proteins critical for bioenergetic regulation and mitochondrial control. This suggests a pathophysiologic mechanism for the persistent cognitive and behavioral deficits observed following rmTBI. Full article

Oxidative stress occurs when there is an excess of reactive oxygen species (ROS) in the cell, primarily produced by mitochondria. Excess ROS trigger membrane lipid peroxidation (LPO), cause mitochondrial swelling, and release proapoptotic proteins into the cytoplasm, which can lead to apoptosis. It is assumed that antioxidants that reduce excessive ROS formation by mitochondria can increase the body’s resistance to stress factors. We investigated the effects of hypoxia and the antioxidant Ambiol (2-methyl-4-dimethylaminomethylbenzimidazole-5-ol dihydrochloride) on the functional characteristics of mitochondria, which were assessed by measuring lipid peroxidation intensity using spectrofluorimetry, mitochondrial membranes fatty acid composition using chromatography, mitochondrial morphology using atomic force microscopy, and respiration rate using polarography. Injecting mice with Ambiol at a dose of 10⁻⁶ mol/kg for 5 days prevented the stress-induced activation of lipid peroxidation, a decrease in the unsaturation index of C₁₈ and C₂₀ fatty acids in mitochondrial membranes, and swelling of these organelles. The drug also increased the efficiency of oxidative phosphorylation during the oxidation of NAD-dependent substrates. Furthermore, Ambiol increased the lifespan of mice by 3.0–4.0 times under various types of hypoxia. Ambiol’s ability to maintain initial (control) levels of C₁₈ and C₂₀ unsaturated fatty acids appears to protect against stress-induced mitochondrial dysfunction. Full article

A549 cells are widely used as an in vitro model of alveolar type II (ATII) epithelial cells; however, their phenotype and metabolic state are highly sensitive to culture conditions, cell density, and the duration of static, non-passaged cultivation. Here, we examined how prolonged static culture affects lipid metabolism, mitochondrial bioenergetics, and viability in A549 cells. A549 cultures were maintained without passaging for up to 25 days in DMEM or Ham’s F-12 and analyzed using lipid secretion assays, targeted lipidomics, [¹⁴C]-acetate incorporation, Seahorse bioenergetic profiling, and transcriptional analysis of stress-associated markers. Several surfactant-associated readouts were highest during early culture, peaking on day 7, as evidenced by elevated expression of ABCA3 and SP-A and maximal secretion of surfactant-associated phospholipids. With prolonged cultivation and increasing culture density, cellular phosphatidylglycerol levels declined progressively and became nearly undetectable by day 25, accompanied by reduced anabolic lipid metabolism, lower oxygen consumption, and impaired glycolytic activity. These changes coincided with increased reactive oxygen species, elevated intracellular Ca²⁺ levels, and increased expression of stress-associated transcripts, including CASP1, IL1B, and C3. Later stages were also associated with reduced mitochondrial respiration and decreased viability. Collectively, our findings show that prolonged static culture is associated with metabolic remodeling and reduced bioenergetic capacity in A549 cells. Full article