Search Results (1,445)

Precise manipulation of mitochondrial DNA (mtDNA) by CRISPR-Cas systems remains challenging, largely due to inefficient import of guide RNAs, motivating the exploration of alternative programmable nucleases. Here, we show that prokaryotic Argonaute nucleases (pAgos) of various classes can be efficiently targeted to human mitochondria. Using the Su9 mitochondrial targeting sequence from Neurospora crassa, we achieved robust mitochondrial import of four pAgos—DecAgo, CbuAgo, KmaAgo and RslAgo. As a functional readout of their activity in cells, we targeted the single-stranded D-loop region, which plays a central role in mtDNA replication and maintenance, reasoning that cleavage at this site was expected to potentially result in a reduction in mtDNA copy number. Of the four enzymes, only RNA-guided DecAgo induced a pronounced reduction in mtDNA levels, decreasing copy number approximately fivefold within 48 h. Unexpectedly, this effect occurred independently of exogenous guides, suggesting that DecAgo may utilize endogenous mitochondrial guide RNAs. These findings identify DecAgo as an active nuclease in human mitochondria and reveal a previously unrecognized mode of targeting, highlighting the need to further investigate the underlying mechanism and the potential role of endogenous guide molecules, as well as improving targeting specificity. Full article

P. massoniana is an important native economic and ecological tree species in southern China, where seasonal drought has emerged as a critical factor limiting its productivity. The SAUR gene family, recognized as core early auxin-responsive genes, plays a crucial role in balancing plant growth, development, and stress adaptation; however, research related to this family in conifers remains limited. Utilizing the chromosome-level genome of P. massoniana, this study identified 73 SAUR genes (PmSAUR1~73) through bioinformatics methods, systematically analyzing the physicochemical properties of the encoded proteins, chromosomal localization, phylogenetic relationships, gene structures, and cis-acting elements. Combined with transcriptome sequencing and molecular experiments, the drought stress response patterns of these genes were further elucidated. The results indicated that PmSAUR genes predominantly encode alkaline proteins, primarily localized in mitochondria and nuclei, with an uneven distribution across nine chromosomes, where tandem duplication serves as the primary mechanism driving family expansion. Phylogenetic analysis classified these genes into seven subfamilies, which include both conserved clades homologous to angiosperms and branches specific to P. massoniana. All members contain the Auxin_inducible conserved domain, with motif1 identified as the core essential motif. Promoter regions were enriched with MeJA (methyl jasmonate)-responsive (56%), ABA-responsive, and drought stress-related cis-elements. Under drought stress, 38 PmSAUR genes exhibited diverse temporal expression patterns. Four key genes (PmSAUR14, PmSAUR28, PmSAUR54, and PmSAUR73), which are localized in the nucleus and exhibit high expression specifically in male cones or roots, were identified. These genes exhibit an expression pattern consistent with an auxin-negative response (i.e., repressed by IAA and induced by drought) and display a distinctive response pattern characterized by drought-induced upregulation coupled with IAA-mediated downregulation. This mechanism may contribute to the drought adaptation strategies of P. massoniana, involving regulatory processes for aboveground reproduction and adaptation of the underground root system. This study represents the first effort to elucidate the evolutionary characteristics and drought response patterns of the SAUR gene family in P. massoniana, thereby addressing the existing research gap regarding the functions of SAUR genes in coniferous trees. Furthermore, it offers candidate gene resources and theoretical support for the molecular breeding of stress resistance in P. massoniana. In addition, two auxin-induced SAUR genes (PmSAUR22 and PmSAUR37) were identified as contrasting examples, but the main focus of this study is on the four auxin-repressed genes. Full article

Mitochondrial DNA (mtDNA) mutations are associated with a diverse spectrum of diseases and pose a significant threat to human health. Despite their importance as therapeutic targets, the unique structural and electrochemical properties of mitochondria—most notably the impermeable inner mitochondrial membrane and the high membrane potential—present formidable challenges for the targeted delivery of therapeutic agents. Currently, there are no approved curative treatments for patients harboring pathogenic mtDNA mutations. In this review, we discuss recent advancements in gene therapy for mitochondrial genome-related disorders, with a particular focus on allotopic expression of mtDNA-encoded genes and mitochondrial genome editing technologies. We conclude that allotopic expression currently stands as the most promising approach for near-term clinical implementation. But we also pay great attention to programmable nucleases and base editors utilizing RNA-independent DNA recognition which are evolving with remarkable speed. Full article

The Kelch-repeat superfamily genes played important roles in regulating plant growth and development; however, their functions in foxtail millet (Setaria italica) have not yet been characterized. In this study, SiNL4, a homolog of ZmNL4 controlling leaf width in maize, was knocked out using the CRISPR/Cas9 technology, and two homozygous knockout lines (ko1 and ko2) were obtained. Phenotypic analysis showed that compared with the wild-type Ci846, ko1 and ko2 exhibited reduced leaf width and decreased yield related traits (e.g., panicle weight, grain width, and 1000-grain weight). Cytological analysis showed that changes in leaf width of ko1 and ko2 resulted from a decrease in leaf epidermal cell width and the number of small vascular bundles (SVBs) close to the leaf edge, suggesting that SiNL4 might regulate leaf width by influencing cell expansion and the development of SVB. Spatiotemporal expression analysis indicated that the relative expression level of SiNL4 was high in the stem, leaf, and young panicle. Subcellular localization showed that SiNL4 was mainly localized in the mitochondria and plasma membrane. In addition, the T-DNA insertion mutant (Atnl4) of AT5G18590, the ortholog of SiNL4 in Arabidopsis thaliana, exhibited similar phenotypes with reduced rosette leaf width, seed width, and 1000-seed weight. Moreover, complementary expression of SiNL4 in Atnl4 not only restored the phenotypes, but also significantly increased the 1000-seed weight, indicating that the function of these two genes might be conserved. Meanwhile, we found that SiNL4 knockout caused a decrease in chlorophyll content and net photosynthetic rate (Pn), showing that SiNL4 might be involved in regulating photosynthesis. In summary, this study revealed the function of SiNL4 in regulating leaf width in foxtail millet, providing a potential gene for the genetic improvement of foxtail millet. Full article

Nasopharyngeal carcinoma-associated malignant epithelial models remain useful for exploring integrin-related therapeutic strategies. In this study, we evaluated the antitumor activity and potential mechanisms of rRGD3^mu, a recombinant peptide with a triple-RGD architecture. Using CNE2 cells as the primary experimental model, we evaluated cell viability, colony formation, migration, invasion, adhesion, apoptosis-related marker expression, and EMT-associated molecular changes. In vivo efficacy was assessed using a CNE2 cell-derived BALB/c nude mouse xenograft model. rRGD3^mu inhibited CNE2 cell viability, clonogenic growth, migration, invasion, and adhesion in a dose-dependent manner and suppressed xenograft tumor growth under the tested dosing schedule. Mechanistically, rRGD3^mu promoted mitochondria-associated apoptosis, as indicated by an increased Bax/Bcl-2 ratio and caspase-9/3 activation, and modulated the expression of EMT-associated markers, including E-cadherin, N-cadherin, vimentin, and MMP2. Bioinformatic analysis and experimental validation suggested that ITGB1-containing integrin complexes might serve as important mediators and putative cellular engagement sites of rRGD3^mu. rRGD3^mu treatment reduced ITGB1 protein abundance and attenuated FAK/AKT signaling. ITGB1 knockdown partially mimicked the effects of rRGD3^mu and reduced the additional cellular response to rRGD3^mu treatment, supporting the substantial contribution of ITGB1-associated signaling. These findings provide preliminary mechanistic evidence that rRGD3^mu suppresses malignant phenotypes in CNE2-based models, at least in part through modulation of ITGB1-associated FAK/AKT signaling. Full article

Mitochondria are central regulators of cellular bioenergetics, redox balance, and signaling pathways that integrate metabolic and immune responses. Emerging evidence indicates that biological sex is an important determinant of mitochondrial function, in part through the regulatory effects of sex hormones on mitochondrial biogenesis, oxidative phosphorylation, reactive oxygen species production, and quality control mechanisms. Estrogen, testosterone, and progesterone differentially modulate mitochondrial dynamics, substrate utilization, antioxidant capacity, and immune signaling, resulting in distinct mitochondrial phenotypes that may influence disease susceptibility across the lifespan. In this review, we synthesize current knowledge on the mechanistic basis of sex differences in mitochondrial function and highlight mitochondria as key mediators linking endocrine signaling to immunometabolic regulation. We discuss how mitochondrial-derived signals, including mitochondrial reactive oxygen species, mitochondrial DNA release, and cardiolipin exposure, activate inflammatory pathways such as NF-κB, cGAS–STING, and NLRP3 inflammasome signaling. These pathways may contribute to chronic inflammation, gut barrier dysfunction, and systemic metabolic disruption. We further examine the impact of major endocrine transitions, including pregnancy, the postpartum period, menopause, and androgen imbalance in conditions such as polycystic ovary syndrome, on mitochondrial function and disease risk. Particular emphasis is placed on the gastrointestinal tract as a metabolically active and mitochondria-dependent interface, where mitochondrial dysfunction may contribute to epithelial barrier disruption, microbial dysbiosis, and systemic inflammation. Finally, we discuss emerging therapeutic strategies targeting mitochondrial function, including exercise, hormone-based therapies, mitochondria-targeted antioxidants, and interventions aimed at improving mitochondrial quality control. Understanding sex-specific mitochondrial regulation may provide a framework for improved endocrine stratification, mitochondrial phenotyping, and precision medicine approaches across diverse clinical contexts. Full article

The single-electron reduction of redox-active drugs and xenobiotics (quinones, aromatic nitrocompounds, and N-oxides) by flavoenzymes, which initiates redox cycling and oxidative stress, is an important factor in their therapeutic/toxic effects. This review summarizes information on the action of mitochondrial flavoenzymes from various organisms in these processes, emphasizing the kinetic and mechanistic aspects. The flavoenzymes discussed also include those of which only a fraction is localized in mitochondria. According to kinetic data, the most effective generator of free radicals of xenobiotics is respiratory Complex I. However, it is unclear to what extent these reactions can compete with the rapid reduction of ubiquinone in normally functioning mitochondria. In specific cases, a very active free radical generator can be the NADPH:adrenodoxin reductase–adrenodoxin complex. The properties of other dehydrogenases–electrontransferases (succinate:ubiquinone reductase, fatty acid oxidation system) are less well characterized. Due to its high catalytic capacity, a potential but poorly studied source of free radicals of xenobiotics may be NADH:cytochrome b₅ reductase and its complex with cytochrome b₅. Flavoenzyme disulfide reductases, with the possible exception of Plasmodium falciparum thioredoxin reductase, are less active free radical generators. Importantly, in most cases, flavoenzymes perform the mixed single- and two-electron reduction of xenobiotics. According to the available data, the reactivity of redox cyclers depends mostly on their standard single-electron reduction potential and is little influenced by their structure. Therefore, in order to intensify these processes or achieve some structural specificity, it is necessary to focus on the selective accumulation of compounds in mitochondria. Full article

We previously demonstrated that fatty acid-binding protein 3 (FABP3) is significantly upregulated in ischemic neurons, and its inhibition mitigates ischemic brain injury in mice and attenuates mitochondrial damage under rotenone-induced oxidative stress. These findings suggest a potential role for FABP3 in mitochondrial dysfunction in ischemic neurons, although the underlying mechanism remains unclear. In this study, we further investigated the role of FABP3 in mitochondrial injury and apoptosis in ischemic neurons. Our findings indicated that FABP3 deficiency significantly decreased infarct volume following middle cerebral artery occlusion/reperfusion (MCAO/R) in mice, improved cognitive and spontaneous activity deficits, and suppressed BAX activation and mitochondrial translocation, caspase-3 activation, and cytochrome c release. In HT22 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), FABP3 deficiency increased cell viability, reduced apoptosis, and alleviated the loss of mitochondrial membrane potential. Conversely, FABP3 overexpression further exacerbated mitochondrial dysfunction and apoptosis, effects that were partially reversed by the BAX inhibitor BAI1. Furthermore, FABP3 overexpression promoted abnormal mitochondrial lipid accumulation and increased lipid peroxidation. Both the mitochondria-targeted antioxidant MitoQ and the ferroptosis inhibitor Ferrostatin-1 alleviated FABP3 overexpression-induced mitochondrial damage and apoptotic signaling. Collectively, our findings suggest that FABP3 is an important promoter of cerebral ischemia–reperfusion injury. FABP3 may aggravate ischemic neuronal injury by promoting abnormal mitochondrial lipid accumulation and lipid peroxidation, thereby enhancing BAX-dependent mitochondrial apoptotic signaling. Targeting FABP3 may provide a potential therapeutic strategy for neuroprotection in ischemic stroke. Full article

Chalcones, as flavonoid precursors, are known to have biological importance and vast pharmacological effects. These bioactive molecules have been used in traditional medicine for many years for treatment of various diseases, particularly because of their antitumor activity. The aim of this study was to assess antiproliferative activity of selected hydroxychalcones against six cancer cell lines, derived from various human organs and with varying degrees of aggressiveness and resistance to cytostatics (5637, A-431, UM-SCC-17A, SK-MEL-3, MCC13, A172) in comparison to two noncancerous cell lines (MCF-10A and BALB/3T3). The specific goal of the present study was to assess the influence of 2′-hydroxychalcone, 4-hydroxychalcone and 4′-hydroxychalcone, the compounds with the same molar mass but with different positions of the hydroxyl group, on different stages of apoptosis. Additionally, we aimed to determine the involvement of mitochondria in the initiation of the cell death process. The principal cell line used for validation was Merkel cell carcinoma, a rare type of human skin cancer. The aforementioned compounds were supplemented for 24 and 48 h at several concentrations. In this paper we demonstrate the ability of hydroxychalcones to activate an early stage of apoptosis by exposure of phosphatidylserine on the cell surface with simultaneous changes in mitochondrial membrane. Our results acknowledge and strengthen the prospect of using chalcones for development of new therapeutic strategies in various oncological disease models. Full article

Despite current therapies, persistent chronic inflammation remains an important contributor to residual renal risk in diabetic kidney disease (DKD). The NLRP3 inflammasome is a key driver of this inflammatory cascade, with mitochondria serving as a central hub that translates metabolic stress into NLRP3 activation via mitochondrial reactive oxygen species (mtROS) and oxidized mitochondrial DNA (ox-mtDNA). This review delineates the signaling network of the mitochondria–NLRP3 inflammasome axis in DKD and evaluates its potential as a therapeutic target to improve patient outcomes. Full article

Oxidative stress is a fundamental mechanism that impacts reproductive function by altering gamete quality, fertilisation, and the initial development of embryos. Excessive reactive oxygen species lead to the oxidation of polyunsaturated fatty acids in the cell membranes of sperm, oocytes, and adjacent somatic cells. F2-isoprostanes and isofurans are two of the most dependable indicators of oxidative lipid damage among the byproducts generated during free radical-mediated lipid oxidation. Both arise from the non-enzymatic peroxidation of arachidonic acid and provide a chemically stable depiction of in vivo oxidative processes. Reproductive studies indicate that elevated levels of F2-isoprostanes are associated with diminished sperm motility, compromised membrane stability, and an increased risk of DNA fragmentation in various forms of male infertility. Lipid peroxidation products have been detected in follicular fluid inside the female reproductive system, suggesting a relationship between oxidative imbalance, granulosa cell metabolism, and oocyte competency. Isofurans, which are more prevalent in the presence of elevated oxygen levels, may indicate oxidative stress in mitochondria and complications with cellular respiration. The current comprehension of lipid peroxidation indicators in infertility and assisted reproduction remains insufficient. This review aims to synthesise current information on isoprostanes and isofurans as reliable indicators of oxidative lipid damage in reproductive biology, highlighting their effects on gamete quality, mitochondrial dysfunction, and results in assisted reproduction. Our research seeks to clarify the biological importance of current experimental and clinical findings, highlighting their potential as clinically relevant biomarkers in reproductive medicine. Full article

To maintain homeostatic conditions and optimal function during stressors, mitochondria initiate retrograde signaling. The mitochondrial integrated stress response (ISR) and unfolded protein response (UPR^mt) are critical quality control mechanisms activated during instances of mitochondrial perturbations. Restoration of mitochondrial homeostasis is orchestrated by three transcription factors, ATF4, CHOP, and ATF5, which upregulate protective genes to counteract stress. As the health and function of skeletal muscle are heavily dependent on a highly adaptive mitochondrial network, defining how mitochondrial health is maintained across various conditions is essential. Although several studies demonstrate the importance of these responses following instances of stress, the signaling mechanisms required to initiate such pathways remain poorly characterized in skeletal muscle. This review examines how the mitochondrial ISR/UPR^mt and related transcription factors respond to organellar stress by emphasizing the molecular events that occur during exercise, aging and muscle disuse. By consolidating the literature, this work aims to highlight the current understanding of mitochondrial stress response signaling within skeletal muscle and thus emphasize areas for future research and potential therapeutic strategies during divergent metabolic conditions. Full article

Intercellular mitochondrial trafficking has emerged as an important mechanism influencing tumor progression, metabolic adaptability, and cancer cell plasticity. Beyond their classical bioenergetic functions, mitochondria act as central regulators of redox homeostasis, signaling pathways, and epigenetic remodeling. Increasing evidence suggests that mitochondria can be transferred between tumor, stromal, and immune cells through tunneling nanotubes (TNTs), extracellular vesicles (EVs), gap junctions, and cell fusion within the tumor microenvironment. This dynamic excshange enables metabolically compromised cancer cells to restore oxidative phosphorylation, optimize energy production, and survive under hypoxia and therapeutic stress. Mitochondrial transfer has been increasingly associated with enhanced cellular plasticity and adaptive phenotypic transitions, including the acquisition of stem-like features that contribute to tumor heterogeneity, metastasis, and treatment resistance. In addition to bioenergetic restoration, transferred mitochondrial DNA and metabolites participate in retrograde signaling, linking metabolic state to epigenetic regulation and transcriptional reprogramming. This metabolic epigenetic interplay supports tumor cell adaptation to environmental stress and therapeutic pressure. Although significant progress has been made, the precise mechanisms governing mitochondrial integration and their long-term impact on cellular phenotypes remain incompletely understood. A deeper understanding of these processes may reveal novel therapeutic strategies to disrupt tumor adaptability and progression. Specifically, targeting intercellular mitochondrial trafficking and its associated metabolic and epigenetic effects could help limit tumor plasticity, overcome treatment resistance, reduce disease recurrence, and improve overall clinical outcomes in cancer patients. Full article

Mitochondria–endoplasmic reticulum contact sites (MERCs) are known as the specialized areas that are involved in a large number of intracellular signaling pathways that regulate Ca²⁺ homeostasis, lipid transport, mitochondrial dynamics, cell death, and autophagy. Understanding MERC dynamics has important therapeutic implications in cancer, as these contacts regulate fundamental cellular processes and MERCs represent promising targets for therapeutic interventions aimed at improving cancer treatment outcomes. Despite the accumulated data, the role of MERCs in carcinogenesis still remains unknown; thus, it seems promising to search for new tools facilitating the study of MERCs in tumor cells. The structure of MERCs can be examined in great detail using transmission electron microscopy (TEM). Currently, several hundred TEM images are required to obtain reliable data on these contacts. The speed of data processing can be significantly improved by using fast and accurate image analysis techniques based on deep learning models. In this study, five U-Net models with a ResNet34 encoder network were evaluated, including the basic U-Net-Vanilla architecture as well as models incorporating various attention blocks and blocks capturing multilevel image structure, for the segmentation of mitochondria and the endoplasmic reticulum (ER). The best performance on the test dataset was demonstrated by the U-Net-scSE network, with F1 scores of 0.872 for mitochondria and 0.744 for the ER being achieved. Two models were tested for their ability to leverage pre-training on external datasets (Lucchi++, Kasthuri++, and DeepPi-EM). Additionally, models pre-trained on the CEM500K dataset were evaluated after the parameters had been tuned on the data. It was demonstrated by the results that pre-training or the use of pre-trained networks did not lead to an improvement in the IoU and F1 metrics on the test dataset. Subsequent image analysis was conducted to assess two types of MERCs in the segmented images. Finally, the free and user-friendly UltraNet web server was developed for automated analysis of mitochondria, ER, and MERCs using TEM images. Full article

The discovery of melatonin as a multifunctional free radical scavenger and its possible synthesis in the mitochondrial matrix of peripheral eukaryotic somatic cells highlights a critical new perspective on the importance of this indole. Experimental evidence supporting these findings is substantial, but there are still lingering questions whether melatonin is a direct radical scavenger in vivo and whether it is synthesized in the mitochondrial matrix. We systematically analyze the innovative experimental approaches that support melatonin’s radical scavenging actions and assess the compelling data supporting its production in mitochondria. Melatonin concentrations are reportedly higher in this organelle than in other cellular compartments. Proteins for the enzymes required to convert serotonin to melatonin are present in the mitochondrial matrix and purified mitochondria synthesize melatonin. In the mitochondrial matrix, melatonin is likely located within the “damage radius” of highly reactive oxygen species. We also summarize novel actions of melatonin associated with its regulation of membrane fluidity, determine the molecular composition of membrane lipid rafts, and modulate liquid–liquid phase separation and biomolecular condensates intracellularly. If the findings discussed herein continue to be validated, melatonin would be in an optimal position to function as an antioxidant and may be a key driver in the context of preserving mitochondrial redox homeostasis and disease mitigation. Full article