Search Results (85)

Sepsis is a clinical syndrome characterized by a dysregulated host response to infection, frequently resulting in septic shock and multi-organ failure. Emerging evidence highlights the critical role of neutrophil extracellular traps (NETs) in the pathophysiology of sepsis. NETs are extracellular structures composed of chromatin DNA, histones, and granular proteins released by neutrophils through a specialized form of cell death known as NETosis. While NETs contribute to the containment of pathogens, their excessive or dysregulated production in sepsis is associated with endothelial damage, immunothrombosis, and organ dysfunction. Several NET-associated biomarkers have been identified, including circulating cell-free DNA (cfDNA), histones, MPO-DNA complexes, and neutrophil elastase–DNA complexes, which correlate with the disease severity and prognosis. Therapeutic strategies targeting NETs are currently under investigation. Inhibition of NET formation using PAD4 inhibitors or ROS scavengers has shown protective effects in preclinical models. Conversely, DNase I therapy facilitates the degradation of extracellular DNA, reducing the NET-related cytotoxicity and thrombotic potential. Additionally, heparin and its derivatives have demonstrated the ability to neutralize NET-associated histones and mitigate coagulopathy. Novel approaches include targeting upstream signaling pathways, such as TLR9 and IL-8/CXCR2, offering further therapeutic promise. Full article

Background/Objectives: Hodgkin’s lymphoma (HL) affects 2–4 individuals per 100,000 annually. Standard treatment includes radiotherapy and ABVD chemotherapy, achieving a 95% survival rate. However, HL survivors face an elevated risk of treatment-related morbidity, particularly the development of secondary malignancies. Previous studies have demonstrated that ABVD treatment induces a high frequency of chromosomal aberrations (CAs) in lymphocytes from HL patients, with higher frequencies one year after treatment than during treatment. This study aimed to determine whether HL treatment also induces unclassified chromosomal/nuclear aberrations (UnCAs) in the lymphocytes of HL patients, and whether these alterations may serve as complementary indicators of genomic instability. Methods: Peripheral blood lymphocytes from HL patients were collected at three time points: before treatment (BT), during treatment (DT), and one year after treatment (1yAT) with ABVD chemotherapy and radiotherapy. A minimum of 3000 nuclei were analyzed per patient to identify and quantify UnCAs. These results were compared to UnCA frequencies in healthy individuals. Results: The percentage of cells presenting UnCAs per 3000 nuclei was 23.92% BT, 18.58% DT, and 30.62% 1yAT. All values were significantly higher (p < 0.016) than the 8.16% observed in healthy controls. The increase was primarily driven by free chromatin and micronuclei clusters. UnCA frequency was lower during treatment than one year after, likely due to the elimination of highly damaged cells through apoptosis or lack of proliferative capacity. Over time, however, persistent genomic damage appears to accumulate in surviving cells, becoming more evident post-treatment. A parallel trend was observed between the frequencies of UnCAs free chromatin, micronucleus and micronuclei clusters, and classical CAs, showing a similar pattern of genomic damage induced by therapy. Conclusions: The post-treatment increase in UnCAs indicates ongoing genomic instability, possibly driven by the selective survival of hematopoietic stem cells with higher genomic fitness. Given their persistence and association with therapy-induced damage, free chromatin and micronuclei clusters may serve as early biomarkers for secondary cancer risk in HL survivors. Full article

Background: Breast cancer (BrCa) patients with tumors expressing high interleukin-6 (IL6) levels have poor clinical outcomes. In BrCa, altered occupancy of CCCTC-binding factor (CTCF) within its DNA binding sites deregulates the expression of its targeted genes. In this study, we investigated whether CTCF contributes to the altered IL6 expression in BrCa. Methods/Results: We performed CTCF gain- and loss-of-function assays in BrCa cell lines and observed an inverse correlation between CTCF and IL6 expression levels. To understand how CTCF negatively regulates IL6 gene expression, we performed luciferase gene reporter assays, site-directed mutagenesis assays, and chromatin immunoprecipitation assays. Our findings revealed that CTCF interacted with the IL6 promoter, a form of regulation disrupted in a CpG methylation-independent fashion in MDA-MB-231 and Tamoxifen-resistant MCF7 cells. Data from TCGA and GEO databases allowed us to explore the clinical implications of our results. An inverse correlation between CTCF and IL6 expression levels was seen in disease-free survival BrCa patients but not in patients who experienced cancer recurrence. Conclusions: Our findings provide evidence that the CTCF-mediated negative regulation of the IL6 gene is lost in highly tumorigenic BrCa cells. Full article

Cellular nucleosomes—the structural and functional units of chromatin—are inherently present in cells. During cellular damage or cell death, nucleosomes are released into circulation, either actively or passively. Once released, nucleosomes can become immunogenic entities through various mechanisms. The nucleosomal proteins in nucleosomes, called histones, play a pivotal role in inducing immunogenicity. However, intact nucleosomes are more immunogenic than the histones alone, as nucleosomal double-stranded deoxyribonucleic acid (dsDNA) enhances its immunogenic potential. Our recent study has shown that circulating histones are predominantly nucleosomal histones rather than free histones. Consequently, circulating histones primarily function as integral parts of circulating nucleosomes rather than acting independently. Circulating nucleosomes and their associated histones are implicated in the pathogenesis of a wide array of diseases. Notably, they are critical in the pathogenesis of lung injury and sepsis. These diseases have high morbidity and mortality rates and lack early diagnostic biomarkers. Further investigation is required to fully elucidate the role of circulating nucleosomes and their associated histones in disease processes. This review aims to discuss the current understanding of circulating nucleosomes and histones in the pathogenesis of lung injury and sepsis, with a focus on the underlying mechanisms. Full article

Resveratrol, a bioactive phytoalexin, has been extensively studied as a pharmaceutical and nutraceutical candidate for the treatment of various diseases. Although its therapeutic effects have been largely attributed to its anti-oxidant properties, its underlying mechanisms and dose dependency are not well understood. Recent studies have shown that cell-free chromatin particles (cfChPs), which are released daily from billions of dying cells, can enter circulation and be internalized by healthy cells, wherein they trigger various damaging effects, including double-strand DNA breaks. Notably, deactivating cfChPs using a mixture of resveratrol and copper can neutralize their harmful effects. The addition of copper imparts a novel therapeutic property to resveratrol viz. the generation of reactive oxygen species (ROS), which are capable of deactivating cfChPs without damaging the genomic DNA. This perspective article discusses how the deactivation of cfChPs via the ROS generated by combining resveratrol with copper can have multiple therapeutic effects. Exploiting the damaging effects of ROS to deactivate cfChPs and ameliorate disease conditions may be a viable therapeutic approach. Full article

Retinoic acid (RA) plays a critical role in initiating meiosis in primordial germ cells (PGC), yet the specific mechanisms of its interaction with PGC remain unclear. In this study, we used an in vitro feeder-free culture system with chicken PGC as a model to explore the mechanisms by which RA induces the entry of PGC into meiosis. Results demonstrated that exogenous RA treatment altered the cell cycle distribution of PGC, significantly increasing the proportion of cells in the G1 phase and decreasing those in the G2 phase, suggesting that RA may promote the transition of PGC from proliferation to differentiation. Giemsa staining further revealed that chromosomes in a subset of RA-treated PGC exhibited meiotic characteristics. Through combined RNA-seq and ATAC-seq analyses, we identified that CYP26A1, a gene involved in RA degradation, was significantly upregulated in the RA-treated group, with enhanced accessibility in its chromatin regions. This finding suggests a robust mechanism for self-regulation of RA levels within PGC, indicating that CYP26A1 may play a pivotal role in the degradation of exogenous RA in chicken PGC. This study elucidated the effects of RA on chicken PGC and provided new insights into the role of RA in germ cell differentiation. Full article

Background/Objectives: Estrogen receptor-α coactivator MED1 is overexpressed in 40–60% of human breast cancers, and its high expression correlates with poor disease-free survival of patients undergoing anti-estrogen therapy. However, the molecular mechanism underlying MED1 upregulation and activation in breast cancer treatment resistance remains elusive. Methods: miRNA and mRNA expression analysis was performed using the NCBI GEO database. MED1 targeting and its impact on therapy resistance was evaluated in control and tamoxifen-resistant breast cancer cell lines by miR-205 overexpression and inhibition. Immunoblotting, chromatin immunoprecipitation, and luciferase reporter assays were used to understand the molecular mechanism of MED1-mediated tamoxifen resistance. Mice xenograft models were used to validate treatment efficacy and molecular mechanisms in vivo. Results: miR-205 was found to directly target and suppress the expression of MED1 through bioinformatic analyses and experimental validations. An inverse correlation of miR-205 and MED1 was observed in breast cancer patients with high MED1/low miR-205, indicative of poor prognosis in long-term anti-estrogen treatment. Furthermore, the depletion of miR-205 was observed in tamoxifen-resistant breast cancer cells overexpressing MED1. The restoration of miR-205 expression attenuated MED1 expression and re-sensitized cells to tamoxifen both in vitro and in vivo. Interestingly, miR205 was also found to target another key regulatory gene, HER3, which drives PI3K/Akt signaling and MED1 activation by phosphorylation. Importantly, we found ER target gene transcription and promoter cofactor recruitment by tamoxifen can be reversed by induced miR205 expression. Conclusions: Altogether, miR-205 functions as a negative regulator of MED1 and HER3, affecting the regulation of the HER3-PI3K/Akt-MED1 axis in anti-estrogen resistance, and could serve as a potential therapeutic regime to overcome treatment resistance. Full article

Background: Neuronal loss is a major pathological feature of neurodegenerative diseases. The analysis of plasma cell-free DNA (cfDNA) is an emerging approach to track cell death events in a minimally invasive way and from inaccessible areas of the body, such as the brain. Previous studies showed that DNA methylation (DNAm) profiles can be used to map the tissue of origin of cfDNA and to identify molecules released from the brain upon cell death. The aim of the present study is to contribute to this research field, presenting the development and validation of an assay for the detection of brain-derived cfDNA (bcfDNA). Methods: To identify CpG sites with brain-specific DNAm, we compared brain and non-brain tissues for their chromatin state profiles and genome-wide DNAm data, available in public datasets. The selected target genomic regions were experimentally validated by bisulfite sequencing on DNA extracted from 44 different autoptic tissues, including multiple brain regions. Sequencing data were analysed to identify brain-specific epihaplotypes. The developed assay was tested in plasma cfDNA from patients with immune effector cell-associated neurotoxicity syndrome (ICANS) following chimeric antigen receptor T (CAR-T) therapy. Results: We validated five genomic regions with brain-specific DNAm (four hypomethylated and one hypermethylated in the brain). DNAm analysis of the selected genomic regions in plasma samples from CAR-T patients revealed higher levels of bcfDNA in participants with ongoing neurotoxicity syndrome. Conclusions: We developed an assay for the analysis of bcfDNA in plasma. The assay is a promising tool for the early detection of neuronal loss in neurodegenerative diseases. Full article

KMT2A (alias: mixed-lineage leukemia [MLL]) gene mapping on chromosome 11q23 encodes the lysine-specific histone N-methyltransferase 2A and promotes transcription by inducing an open chromatin conformation. Numerous genomic breakpoints within the KMT2A gene have been reported in young children and adults with hematologic disorders and are present in up to 10% of acute leukemias. These rearrangements describe distinct features and worse prognosis depending on the fusion partner, characterized by chemotherapy resistance and high rates of relapse, with a progression-free survival of 30–40% and overall survival below 25%. Less intensive regimens are used in pediatric patients, while new combination therapies and targeted immunotherapeutic agents are being explored in adults. Beneficial therapeutic effects, and even cure, can be reached with hematopoietic stem cell transplantation, mainly in young children with dismal molecular lesions; however, delayed related toxicities represent a concern. Herein, we summarize the translocation partner genes and partial tandem duplications of the KMT2A gene, their molecular impact, clinical aspects, and novel targeted therapies. Full article

Technological advancements in cell-free DNA (cfDNA) liquid biopsy have triggered exponential growth in numerous clinical applications. While cfDNA-based liquid biopsy has made significant strides in personalizing cancer treatment, the exploration and translation of epigenetics in liquid biopsy to clinical practice is still nascent. This comprehensive review seeks to provide a broad yet in-depth narrative of the present status of epigenetics in cfDNA liquid biopsy and its associated challenges. It highlights the potential of epigenetics in cfDNA liquid biopsy technologies with the hopes of enhancing its clinical translation. The momentum of cfDNA liquid biopsy technologies in recent years has propelled epigenetics to the forefront of molecular biology. We have only begun to reveal the true potential of epigenetics in both our understanding of disease and leveraging epigenetics in the diagnostic and therapeutic domains. Recent clinical applications of epigenetics-based cfDNA liquid biopsy revolve around DNA methylation in screening and early cancer detection, leading to the development of multi-cancer early detection tests and the capability to pinpoint tissues of origin. The clinical application of epigenetics in cfDNA liquid biopsy in minimal residual disease, monitoring, and surveillance are at their initial stages. A notable advancement in fragmentation patterns analysis has created a new avenue for epigenetic biomarkers. However, the widespread application of cfDNA liquid biopsy has many challenges, including biomarker sensitivity, specificity, logistics including infrastructure and personnel, data processing, handling, results interpretation, accessibility, and cost effectiveness. Exploring and translating epigenetics in cfDNA liquid biopsy technology can transform our understanding and perception of cancer prevention and management. cfDNA liquid biopsy has great potential in precision oncology to revolutionize conventional ways of early cancer detection, monitoring residual disease, treatment response, surveillance, and drug development. Adapting the implementation of liquid biopsy workflow to the local policy worldwide and developing point-of-care testing holds great potential to overcome global cancer disparity and improve cancer outcomes. Full article

Malignant rhabdoid tumors (MRTs) are among the most aggressive and treatment-resistant malignancies affecting infants, originating in the kidney, brain, liver, and soft tissues. The 5-year event-free survival rate for these cancers is a mere 20%. In nearly all cases of MRT, the SMARCB1 gene (occasionally SMARCA4)—a pivotal component of the SWI/SNF chromatin remodeling complex—is homozygously deleted, although the precise etiology of these tumors remains unknown. While young patients with localized MRT generally show improved outcomes, especially those who are older and have early-stage disease, the overall prognosis remains poor despite optimal standard treatments. This highlights the urgent need for more effective treatment strategies. We investigated the antitumor activity of a PARP1 inhibitor (talazoparib, TLZ) combined with a DNA alkylating agent (temozolomide, TMZ) in MRT xenograft models. PARP1 is a widely targeted molecule in cancer treatment and, beyond its role in DNA repair, it participates in transcriptional regulation by recruiting chromatin remodeling complexes to modulate DNA accessibility for RNA polymerases. To widen the therapeutic window of the drug combination, we employed PEGylated TLZ (PEG~TLZ), which has been reported to reduce systemic toxicity through slow drug release. Remarkably, our findings indicate that five out of six MRT xenografts exhibited an objective response to PEG~TLZ+TMZ therapy. Significantly, the loss of SMARCB1 was found to confer a protective effect, correlating with higher expression levels of DNA damage and repair proteins in SMARCB1-deficient MRT cells. Additionally, we identified MGMT as a potential biomarker indicative of in vivo MRT response to PEG~TLZ+TMZ therapy. Moreover, our analysis revealed alterations in signaling pathways associated with the observed antitumor efficacy. This study presents a novel and efficacious therapeutic approach for MRT, along with a promising candidate biomarker for predicting tumor response. Full article

DNA Damage Tolerance (DDT) mechanisms allow cells to bypass lesions in the DNA during replication. This allows the cells to progress normally through the cell cycle in the face of abnormalities in their DNA. PCNA, a homotrimeric sliding clamp complex, plays a central role in the coordination of various processes during DNA replication, including the choice of mechanism used during DNA damage bypass. Mono-or poly-ubiquitination of PCNA facilitates an error-prone or an error-free bypass mechanism, respectively. In contrast, SUMOylation recruits the Srs2 helicase, which prevents local homologous recombination. The Elg1 RFC-like complex plays an important role in unloading PCNA from the chromatin. We analyze the interaction of mutations that destabilize PCNA with mutations in the Elg1 clamp unloader and the Srs2 helicase. Our results suggest that, in addition to its role as a coordinator of bypass mechanisms, the very presence of PCNA on the chromatin prevents homologous recombination, even in the absence of the Srs2 helicase. Thus, PCNA unloading seems to be a pre-requisite for recombinational repair. Full article

Background: Cytogenotoxic damage caused by the consumption of legal and illegal drugs in drug abusers has been demonstrated, primarily due to alterations in their antioxidant capacity, cellular repair mechanisms, and increased production of free radicals. Folic acid shows antioxidant activity by acting as a reducing agent, neutralizing present free radicals, and reducing genomic damage. Methods: The intervention involved administering 15 mg of folic acid, divided into three doses per day, to a group of 44 drug abusers. The frequency of nuclear abnormalities (NAs) was determined; micronuclei (MNs), nuclear buds (NBUDs), binucleated cells (BNs), abnormally condensed chromatin (CC), karyorrhexis (KX), pyknotic nuclei (PNs), and karyolysis (KL) were determined at different pre-treatment (baseline) and post-treatment time points at 15 and 30 days. Additionally, a group of 44 healthy individuals was used as the control group. Results: We observed a statistically significant decrease in the frequency of NAs in the drug abuser group (28.45 ± 17.74 before supplementation vs. 11.18 ± 7.42 at 15 days and 9.11 ± 10.9 at 30 days of supplementation). Specifically, it decreased the frequency of NBUDs, BNs, CC, KX, and PNs (p < 0.05). Conclusion: Our study demonstrates a clear improvement in cytogenotoxic damage in drug abusers supplemented with folic acid. Full article

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers—chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging. Oxygen content was assessed using phosphorescence lifetime macro-imaging with an oxygen-sensitive probe. For visualization of the perfused microvasculature, an optical coherence tomography-based angiography was used. It was found that PDT induces different alterations in cellular metabolism, depending on the degree of oxygen depletion. Moderate decrease in oxygen in the case of KillerRed was accompanied by an increase in the fraction of free NAD(P)H, an indicator of glycolytic switch, early after the treatment. Severe hypoxia after PDT with Photoditazine resulted from a vascular shutdown yielded in a persistent increase in protein-bound (mitochondrial) fraction of NAD(P)H. These findings improve our understanding of physiological mechanisms of PDT in cellular and vascular modes and can be useful to develop new approaches to monitoring its efficacy. Full article

Multiple studies have shown that cell-free DNA (cfDNA) from cancer patients differ in both fragment length and fragment end motif (FEM) from healthy individuals, yet there is a lack of understanding of how the two factors combined are associated with cancer and gene transcription. In this study, we conducted cfDNA fragmentomics evaluations using plasma from lung cancer patients (n = 12) and healthy individuals (n = 7). A personal gene expression profile was established from plasma using H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). The genes with the highest expression displayed an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94–27.13%, p < 0.0001) compared to the genes with low expression. Furthermore, distinct GC-rich FEMs were enriched after cfChIP. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs resulted in an even further enrichment of the most expressed genes (median = 37.85%, IQR: 30.10–39.49%, p < 0.0001). An in vitro size selection of <150 bp cfDNA could isolate cfDNA representing active genes and the size-selection enrichment correlated with the cfChIP-seq enrichment (Spearman r range: 0.499–0.882, p < 0.0001). This study expands the knowledge regarding cfDNA fragmentomics and sheds new light on how gene activity is associated with both cfDNA fragment lengths and distinct FEMs. Full article