Search Results (139)

Colorectal tumors consist of diverse cell populations, including cancer cells and immune cells. Sorafenib (Nexavar), an oral multikinase inhibitor, targets tumor growth and angiogenesis while inducing apoptosis. However, its clinical use is hindered by poor solubility, rapid metabolism, and low bioavailability. This study explores a nanotechnology-based approach to enhance Sorafenib’s efficacy against colon cancer. Nexavar was encapsulated into nanoparticles using an oil phase and Span 80 as a stabilizer to produce sub-100 nm droplets. The resulting Nano-Nexavar was evaluated for cytotoxicity on Caco-2 colorectal cancer cells and compared with free Nexavar on both Caco-2 and normal NCM-460 colon cells. Nano-Nexavar significantly reduced cancer cell viability at lower concentrations, with no observed toxicity to normal cells. Both formulations induced lactate dehydrogenase release and cell reduction at 2.5 µM, but Nano-Nexavar triggered nearly 60% apoptosis in Caco-2 cells. It inhibited Raf-1, NFκB, and ERK signaling, and reduced epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) levels over time. Notably, unlike Nexavar, the Nano-Nexavar suppressed aspartate β-hydroxylase (ASPH) and enhanced mitochondrial-mediated apoptosis by increasing Bax expression, mitochondrial accumulation, and mtDNA levels indicated by immunofluorescence, immunoblotting, flow cytometry, and qRT-PCR. These data demonstrate that Nano-Nexavar potentiates Sorafenib’s anticancer activity by targeting ASPH, thereby amplifying mitochondrial signaling–induced cell death. Full article

Background: The senescence of testicular Leydig cells (LCs) is a key cause of age-related testosterone deficiency, in which oxidative stress (OS) and mitochondrial dysfunction are critical driving mechanisms. We explore whether the bioactive peptide C248 of PRDX4, an intracellular antioxidant, exerts mitochondrial protection to ameliorate LCs’ function. Methods: Based on the antioxidant domains of the PRDX4 protein, small molecular peptides were designed, and bioactive peptide C248 stood out from the crowd. An OS-induced senescence model of LCs was constructed by treating the MLTC-1 cell line with hydrogen peroxide (H₂O₂). C248 peptide or nicotinamide mononucleotide (NMN), as the positive control, was administered in the culture medium. The cellular function-related indicators, including DPPH free radical scavenging rate, cell viability, testosterone level, hydrogen peroxide (H₂O₂) content, senescence-associated β-galactosidase (SA-β-gal) activity, 8-hydroxy-2′-deoxyguanosine (8-OHDG) level, and 4-hydroxynonenal (4-HNE) level, were evaluated. The mitochondrial function and structural indicators, such as mitochondrial membrane potential, ATP production, mitochondrial morphology, and mitochondrial DNA (mtDNA) copy number, were subsequently tested. Results: In vitro experiments confirmed that C248 could scavenge DPPH free radicals in a dose-dependent manner, reduce the levels of reactive oxygen species, and increase antioxidant enzyme activity in LCs (p < 0.01). Both C248 and NMN increased testosterone secretion and improved cell viability (p < 0.01). Both C248 and NMN increased mitochondrial morphology and quantity, mitochondrial membrane potential (p < 0.01), ATP production (p < 0.01), and mitochondrial DNA (mtDNA) copy number (p < 0.01). Conclusion: This study reveals that the small molecular C248, a bioactive peptide of PRDX4, is a new candidate molecule for intervening in LC senescence and confirms that mitochondrial protection is a key strategy for improving age-related testicular dysfunction. Full article

NETosis is assumed to be involved in the pathogenesis of common rheumatic diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS). However, the levels of circulating NETosis biomarkers and the extent of changes in these markers in specific rheumatic diseases are not fully understood. In this study, cell-free DNA (cfDNA) concentration as a non-specific marker, as well as myeloperoxidase (MPO) and citrullinated histone H3 (H3cit) as specific markers of NETosis, were investigated in SLE, RA, PsA, and AS. Analysis of covariance, accounting for sex, age and disease duration, showed that total cfDNA was elevated in SLE and AS compared with healthy subjects. Nuclear and mitochondrial cfDNA were elevated in four diseases. However, nuclear cfDNA was increased to a greater extent in SLE but mitochondrial cfDNA was higher in RA. MPO and H3cit were significantly elevated in SLE compared with other diseases, although MPO was also higher in RA. Elevated concentrations of MPO and H3cit in SLE were associated with the presence of concomitant cardiovascular diseases. The effect of biological therapy on mitochondrial cfDNA, MPO, and H3cit was also shown. The proinflammatory cytokine IL-18, implicated in the induction of NETosis, was similarly elevated in the four rheumatic diseases. Thus, the most striking signs of NETosis are found in SLE, although they are also present in RA. PsA and AS were mainly characterized by an increase in cfDNA. These data highlight characteristic changes in NETosis markers in four rheumatic diseases. Full article

This article aims to point out new perspectives opened by genomics and epigenomics in skin rejuvenation strategies which target the main hallmarks of the ageing. In this respect, this article presents a concise overview on: the clinical relevance of the most important clocks and biomarkers used in skin anti-ageing strategy evaluation, the fundamentals, the main illustrating examples preclinically and clinically tested, the critical insights on knowledge gaps and future research perspectives concerning the most relevant skin anti-ageing and rejuvenation strategies based on novel epigenomic and genomic acquisitions. Thus the review dedicates distinct sections to: senolytics and senomorphics targeting senescent skin cells and their senescent-associated phenotype; strategies targeting genomic instability and telomere attrition by stimulation of the deoxyribonucleic acid (DNA) repair enzymes and proteins essential for telomeres’ recovery and stability; regenerative medicine based on mesenchymal stem cells or cell-free products in order to restore skin-resided stem cells; genetically and chemically induced skin epigenetic partial reprogramming by using transcription factors or epigenetic small molecule agents, respectively; small molecule modulators of DNA methylases, histone deacetylases, telomerases, DNA repair enzymes or of sirtuins; modulators of micro ribonucleic acid (miRNA) and long-non-coding ribonucleic acid (HOTAIR’s modulators) assisted or not by CRISPR-gene editing technology (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats); modulators of the most relevant altered nutrient-sensing pathways in skin ageing; as well as antioxidants and nanozymes to address mitochondrial dysfunctions and oxidative stress. In addition, some approaches targeting skin inflammageing, altered skin proteostasis, (macro)autophagy and intercellular connections, or skin microbiome, are very briefly discussed. The review also offers a comparative analysis among the newer genomic/epigenomic-based skin anti-ageing strategies vs. classical skin rejuvenation treatments from various perspectives: efficacy, safety, mechanism of action, evidence level in preclinical and clinical data and regulatory status, price range, current limitations. In these regards, a concise overview on senolytic/senomorphic agents, topical nutrigenomic pathways’ modulators and DNA repair enzymes, epigenetic small molecules agents, microRNAs and HOTAIRS’s modulators, is illustrated in comparison to classical approaches such as tretinoin and peptide-based cosmeceuticals, topical serum with growth factors, intense pulsed light, laser and microneedling combinations, chemical peels, botulinum toxin injections, dermal fillers. Finally, the review emphasizes the future research directions in order to accelerate the clinical translation of the (epi)genomic-advanced knowledge towards personalization of the skin anti-ageing strategies by integration of individual genomic and epigenomic profiles to customize/tailor skin rejuvenation therapies. Full article

African swine fever virus (ASFV) is the cause of a severe hemorrhagic disease in domestic pigs and wild boar. Currently, a highly virulent genotype II ASFV is causing massive pig mortality worldwide. In its acute form, the disease is characterized by high fever, a range of non-specific clinical signs and cell death. In this study, we demonstrate a greatly elevated level (>1000-fold) of cell-free DNA (cfDNA), more specifically, fragmented host genomic DNA (gDNA), in serum from both wild boar and domestic pigs infected with a highly virulent genotype II ASFV. Increases were also observed, to a lesser extent, in the serum levels of mitochondrial DNA (between 4- to >500-fold). For comparison, release of the cytoplasmic enzyme, lactate dehydrogenase, which is a commonly used marker for cellular damage, was also found to be elevated in some animals, but with less consistency. These results indicate that gDNA in serum (i.e., cfDNA) can be a useful marker for cell death during infection with highly virulent variants of the virus, and could be a promising biomarker to elucidate the pathogenesis of ASFV infection in both domestic pigs and wild boar in future studies. Full article

Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by the gradual and irreversible loss of neurons, especially within the substantia nigra region of the midbrain. Early and accurate diagnosis remains a significant challenge in both research and clinical practice. This difficulty is further compounded by the substantial clinical and molecular heterogeneity of PD, emphasizing the urgent need for reliable biomarkers to enhance diagnostic precision and guide therapeutic strategies. One promising candidate biomarker is cell-free DNA (cfDNA), comprising short DNA fragments composed of mitochondrial (cf-mtDNA) and nucleus-derived (cf-ntDNA) DNA. cfDNA is released into body fluids through physiological or pathological processes such as apoptosis, necrosis, NETosis, or active secretion. The presence of cfDNA in human biological fluids has been utilized for years in oncology and prenatal medicine and, more recently, it has gained attention as a non-invasive diagnostic tool in the context of neurodegenerative diseases such as PD. This review article aims to provide a comprehensive overview of the current knowledge on the origin of cfDNA, highlighting the roles of the mitochondria and cf-mtDNA in PD, mitochondria quality control, and neuroinflammation in cfDNA biogenesis. The review collates available research on cfDNA types in human serum, plasma, and CSF, sequence analysis, and its potential application as a biomarker in the diagnosis and monitoring of PD, contributing to the ongoing search for non-invasive biomarkers of neurodegenerative diseases. Full article

Ischemic stroke remains one of the most catastrophic diseases in neurology, in which, due to a disturbance in the cerebral blood flow, the brain is acutely deprived of its oxygen and glucose oligomer, which in turn rapidly leads to energetic collapse and progressive cellular death. There is now increasing evidence that this type of stroke is not simply a type of ‘oxidative stress’ but rather a programmable loss-of-redox homeostasis, within which electron flow and the balance of oxidants/reductants are cumulatively displaced at the level of the single molecule and at the level of the cellular area. The advances being made in cryo-electron microscopy, lipidomics, and spatial omics are coupled with the introduction of a redox code produced by the interaction of the couples NADH/NAD⁺, NADPH/NADP⁺, GSH/GSSG, BH₄/BH₂, and NO/SNO, which determine the end results of the fates of the neurons, glia, endothelium, and pericytes. Within the mitochondria, pathophysiological events, including reverse electron transport, succinate overflow, and permeability transition, are found to be the first events after reperfusion, while signals intercommunicating via ER–mitochondria contact, peroxisomes, and nanotunnels control injury propagation. At the level of the tissue, events such as the constriction of the pericytes, the degradation of the glycocalyx, and the formation of neutrophil extracellular traps underlie microvascular failure (at least), despite the effective recanalization of the vessels. Systemic influences such as microbiome products, oxidized lipids, and free mitochondrial DNA in cells determine the redox imbalance, but this generally occurs outside the brain. We aim to synthesize how the progressive stages of ischemic injury evolve from the cessation of flow to the collapse of the cell structure. Within seconds of injury, there is reverse electron transport (RET) through mitochondrial complex I, with bursts of superoxide (O₂•⁻) and hydrogen peroxide (H₂O₂) being produced, which depletes the stores of superoxide dismutase, catalase, and glutathione peroxidase. Accumulated succinate and iron-induced lipid peroxidation trigger ferroptosis, while xanthine oxidase and NOX2/NOX4, as well as uncoupled eNOS/nNOS, lead to oxidative and nitrosative stress. These cascades compromise the function of neuronal mitochondria, the glial antioxidant capacity, and endothelial–pericyte integrity, leading to the degradation of the glycocalyx with microvascular constriction. Stroke, therefore, represents a continuum of redox disequilibrium, a coordinated biochemical failure linking the mitochondrial metabolism with membrane integrity and vascular homeostasis. Full article

Alzheimer’s disease (AD) is a neurodegenerative disorder involving free radical overload, neuroinflammation, and a deranged cell stress response. In particular, the modulation of the heme oxygenase/biliverdin reductase (HO/BVR) system, a key component of the brain stress response, is currently regarded as a promising therapeutic approach for AD. Cellular senescence, defined as a process of cell cycle arrest due to oxidative stress, DNA damage, mitochondrial dysfunction, and oncogene activation, has been identified as a pivotal factor in the development of AD. A mounting body of research has demonstrated that the accumulation of senescent cells in the brain can lead to a variety of neurotoxic effects, including synaptic dysfunction, the destruction of the blood–brain barrier, and impaired remyelination. Finally, the release of proinflammatory molecules by senescent cells further exacerbates neurodegeneration. A considerable number of xenobiotics, with well-documented neuroprotective effects through the activation of the HO/BVR system, have been shown to modulate pathways involved in cellular senescence outside the brain. Unfortunately, a direct link between HO/BVR and cellular senescence in AD is yet to be established. This compelling evidence should motivate basic and clinical researchers to address such a significant gap in knowledge and conduct novel studies in this field. Full article

Obesity contributes to the development of metabolic disorders such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD) through sustained low-grade inflammation and mitochondrial dysfunction. In obesity, hypertrophied adipose tissue release high levels of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, and elevates circulating free fatty acids. These changes promote systemic insulin resistance and ectopic lipid deposition. Mitochondrial dysfunction, including reduced oxidative phosphorylation, excess reactive oxygen species (ROS) production, and mitochondrial DNA damage, further stimulate inflammatory pathways such as the NLRP3 inflammasome, creating a feedback loop that worsens metabolic stress. Ultimately, this interaction disrupts energy balance, weakens insulin signaling, and accelerates β-cell dysfunction and hepatic steatosis. In both T2DM and MASLD, oxidative stress, defective mitochondrial quality control, and dysregulated immunometabolic responses are consistently observed pathophysiological features. Interventions aimed at reducing inflammation and restoring mitochondrial function—including lifestyle modification, mitochondria-targeted therapies, inflammasome regulation, and enhancement of mitochondrial biogenesis or mitophagy—may retard disease progression. Full article

Background: Mitochondrial DNA (mtDNA), normally enclosed within mitochondria, can be released into circulation in response to cellular stress, hypoxia, or inflammation. Its detection in plasma has been proposed as a marker of cellular injury, particularly in the context of mechanical ventilation. High-frequency jet ventilation is a specialized approach of open-airway ventilation, delivering small tidal volumes through jet gas streams, applied with high pressure and oxygen fraction. It remains unclear whether this mode of ventilation contributes to mitochondrial stress. We therefore hypothesized that circulating mtDNA levels would increase after jet ventilation due to the combined effects of high oxygen exposure and mechanical strain. Furthermore, we explored whether the magnitude of mtDNA change correlates with the duration of ventilation and arterial oxygenation levels. Methods: Plasma levels of cell-free circulating mitochondrial DNA were measured in 30 patients before and following jet ventilation in laryngotracheal surgery. Post hoc analysis of a primary monocentric, randomized cross-over study was conducted to investigate ventilation distribution in high-frequency jet ventilation techniques. Results: Mitochondrial DNA levels significantly decreased after jet ventilation (median T0: 13.57; T1: 6.78; p = 0.0087). No significant associations were found between mtDNA change and jet ventilation duration, type of surgery, or ASA classification. Despite variable air entrainment in the open-jet ventilation system, the arterial partial pressure of oxygen increased significantly during the procedure. Conclusions: Jet ventilation was associated with a significant decrease in circulating mtDNA levels. This contrasts with our initial hypothesis of mtDNA elevation under ventilation-induced stress. These findings suggest that jet ventilation may exert less mitochondrial damage than previously expected. Full article

Mitochondrial dysfunction is a key pathological hallmark in amyotrophic lateral sclerosis (ALS), yet the role of circulating cell-free mitochondrial DNA (Cf-mtDNA) as a biomarker remains unclear. This study aimed to investigate serum Cf-mtDNA levels in ALS patients compared to healthy controls and explore its associations with disease biomarkers, clinical progression, and survival. We conducted a case–control study measuring Cf-mtDNA levels in serum samples from 54 ALS patients and 36 age- and sex-matched healthy controls using quantitative droplet digital PCR. Correlations between Cf-mtDNA levels and clinical features, neurofilament concentrations, inflammatory indices, and survival were assessed. The average Cf-mtDNA level in ALS patients was 2,426,315 copies/mL of serum (IQR: 865000–2475000), compared to 1,885,667 copies/mL of serum (IQR: 394250–2492500) in controls (p = 0.308). ROC analysis yielded an AUC of 0.595 (95% CI: 0.468–0.721), indicating very limited discriminant ability. Cf-mtDNA levels were inversely correlated with serum creatinine concentrations (r = –0.335, p = 0.018), but showed no significant associations with ALS phenotype, disease staging, neurofilaments, inflammatory indices, or survival. These findings suggest that, in a predominantly sporadic ALS cohort, serum Cf-mtDNA may not serve as a standalone diagnostic or prognostic biomarker, in contrast to previous reports. Methodological differences, cohort composition, and genetic heterogeneity may account for these discrepancies. Our results underscore the importance of further large-scale, longitudinal studies incorporating genetic stratification and multi-biomarker approaches to better elucidate the role of Cf-mtDNA in ALS pathophysiology. Full article

Background/Objectives: Circulating cell-free DNA (cfDNA) consists of genomic DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) fragments released from cells primarily through apoptosis and necrosis. In healthy individuals, the main source of cfDNA is apoptosis, whereas in cancer patients, necrosis predominates. Considering that in vitro cfDNA models are valuable research tools, this study presents an in vitro characterization of cf-mtDNA patterns released into the culture medium by four human cell lines: normal dermal fibroblasts (Hs27), induced pluripotent stem cells (iPSCs), melanoma cells (BMel), and prostate cancer cells (PC3). Furthermore, as fetal bovine serum (FBS)—a widely used supplement in cell culture media—has been shown to contain bovine cfDNA, species-specific primers were employed to eliminate potential artifacts arising from this contamination in in vitro experiments. Methods: Fragmentation analysis of cf-mtDNA was conducted by amplifying the human MT-CYB gene and the D-loop region in four cell lines using species-specific primers. Two indices, Q and λ, were employed to quantify fragmentation. Results: These indices reveal that cancer cells exhibit the highest degree of fragmentation compared to fibroblasts, whereas stem cells show the lowest degree of fragmentation. This study identified species-specific primers for the human and bovine MT-CYB gene, confirming the presence of bovine cf-mtDNA in cell culture media supplemented with FBS. Conclusions: in vitro cellular models are useful for studying the mechanisms of cfDNA release and fragmentation; designed primers provide a reliable tool for assessing contamination across different growth time points minimizing interference errors and non-specific amplifications. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the most prevalent chronic hepatopathy and a leading precursor of hepatocellular carcinoma (HCC) worldwide. Initially attributed to insulin resistance (IR)-driven metabolic imbalance, recent insights highlight a multifactorial pathogenesis involving oxidative stress (OS), chronic inflammation, and immune dysregulation. The hepatic accumulation of free fatty acids (FFAs) initiates mitochondrial dysfunction and excessive reactive oxygen species (ROS) production, culminating in lipotoxic intermediates and mitochondrial DNA damage. These damage-associated molecular patterns (DAMPs), together with gut-derived pathogen-associated molecular patterns (PAMPs), activate innate immune cells and amplify cytokine-mediated inflammation. Kupffer cell activation further exacerbates OS, while ROS-induced transcriptional pathways perpetuate inflammatory gene expression. Traditional immunity refers to the well-established dichotomy of innate and adaptive immune responses, where innate immunity provides immediate but non-specific defense, and adaptive immunity offers long-lasting, antigen-specific protection. However, a paradigm shift has occurred with the recognition of trained immunity (TI)—an adaptive-like memory response within innate immune cells that enables enhanced responses upon re-exposure to stimuli. Following non-specific antigenic stimulation, TI induces durable epigenetic and metabolic reprogramming, leading to heightened inflammatory responses and altered functional phenotypes. These rewired cells acquire the capacity to produce lipid mediators, cytokines, and matrix-modifying enzymes, reinforcing hepatic inflammation and fibrogenesis. In this context, the concept of immunometabolism has gained prominence, linking metabolic rewiring with immune dysfunction. This literature review provides an up-to-date synthesis of emerging evidence on immunometabolism and trained immunity as pathogenic drivers in MASLD. We discuss their roles in the transition from hepatic steatosis to steatohepatitis, fibrosis, and cirrhosis, and explore their contribution to the initiation and progression of MASLD-related HCC. Understanding these processes may reveal novel immunometabolic targets for therapeutic intervention. Full article

Mitochondrial oxidative stress and neuroinflammation are involved in the onset and progression of Alzheimer’s disease (AD). Novel reliable, circulating biomarkers related to these processes were searched in cerebrospinal fluid (CSF) and plasma samples. Paired CSF and plasma samples from 20 subjective memory complaints (SMC) subjects, 20 mild cognitive impairment (MCI) due to AD subjects, and 20 Alzheimer’s dementia (ADd) patients were analyzed. Protein amounts of manganese-containing superoxide dismutase 2 (SOD2), cell-free mitochondrial DNA (cf-mtDNA) level, DNase I, and matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) activities were determined. As for SOD2, an MCI male-specific significant increase in both biofluids and an ADd male-specific significant decrease in plasma were found. No significant differences were demonstrated in cf-mtDNA level. An ADd-specific significant increase in plasma DNase I and MMP-2 activities was found. A SMC female-specific significant higher value in CSF MMP-9 activity in comparison to male counterparts was demonstrated. The present results suggest a male patient-specific (MCI and ADd) regulation of SOD2 expression in plasma and support an ADd-specific increase in plasma DNase I and MMP-2 activities. Therefore, the potential of SOD2 amount, DNase I, and MMP-2 activities in plasma as new markers of ADd should be explored. The SMC female-specific high activity of MMP-9 might contribute to AD female-sex bias. Full article

Due to their clinical heterogeneity, nonspecific symptoms, and the limitations of existing biomarkers and imaging modalities, metabolic brain diseases (MBDs), such as mitochondrial encephalopathies, lysosomal storage disorders, and glucose metabolism syndromes, pose significant diagnostic challenges. This review examines the growing potential of cell-free DNA (cfDNA) derived from cerebrospinal fluid (CSF) epigenetic profiling as a dynamic, cell-type-specific, minimally invasive biomarker approach for MBD diagnosis and monitoring. We review important technological platforms and their use in identifying CNS-specific DNA methylation patterns indicative of neuronal injury, neuroinflammation, and metabolic reprogramming, including cfMeDIP-seq, enzymatic methyl sequencing (EM-seq), and targeted bisulfite sequencing. By synthesizing current findings across disorders such as MELAS, Niemann–Pick disease, Gaucher disease, GLUT1 deficiency syndrome, and diabetes-associated cognitive decline, we highlight the superior diagnostic and prognostic resolution offered by CSF cfDNA methylation signatures relative to conventional CSF markers or neuroimaging. We also address technical limitations, interpretive challenges, and translational barriers to clinical implementation. Ultimately, this review explores CSF cfDNA epigenetic analysis as a liquid biopsy modality. The central objective is to assess whether epigenetic profiling of CSF-derived cfDNA can serve as a reliable and clinically actionable biomarker for improving the diagnosis and longitudinal monitoring of metabolic brain diseases. Full article