Search Results (595)

Histone post-translational modifications (PTMs) have emerged as promising epigenetic biomarkers with increasing forensic relevance. Unlike conventional genetic markers such as short tandem repeats (STRs), histone modifications can offer additional layers of biological information, capturing individual-specific regulatory states and remaining detectable even in degraded forensic samples. This review highlights recent advances in understanding histone PTMs in forensic contexts, focusing on three key domains: analysis of degraded biological evidence, differentiation of monozygotic (MZ) twins, and postmortem interval (PMI) estimation. We summarize experimental findings from human cadavers, animal models, and typical forensic samples including bone, blood, and muscle, illustrating the stability and diagnostic potential of marks such as H3K4me3, H3K27me3, and γ-H2AX. Emerging technologies including CUT&Tag, MALDI imaging, and nanopore-based sequencing offer novel opportunities to profile histone modifications at high resolution and low input. Despite technical challenges, these findings support the feasibility of histone-based biomarkers as complementary tools for forensic identification and temporal analysis. Future work should prioritize methodological standardization, inter-laboratory validation, and integration into forensic workflows. However, the forensic applicability of these modifications remains largely unvalidated, and further studies are required to assess their reliability in casework contexts. Full article

Accurately estimating the radiation dose received by an individual is essential for evaluating potential damage caused by exposure to ionizing radiation. Most retrospective dosimetry methods require the time since exposure to be known and rely on calibration curves specific to that time point. In this work, we introduce a novel method tailored to the γ-H2AX assay, which is a protein-based biomarker for radiation exposure, that enables the estimation of both the radiation dose and the time of exposure within a plausible post-exposure interval. Specifically, we extend calibration curves available at two distinct time points by incorporating the biological decay of foci, resulting in a model that captures the joint dependence of foci count on both dose and time. We demonstrate the applicability of this approach using both real-world and simulated data. Full article

Egg yolk immunoglobulin Y (IgY) possesses advantages such as low cost, easy availability, simple preparation, high antigen specificity, absence of drug residues, and compliance with animal welfare standards, making it an environmentally friendly and safe alternative to antibiotics. This research utilizes IgY antibody technology to develop a multivalent passive immune vaccine for major pathogenic bacteria in aquaculture. In this study, IgY antibodies against live Shewanella xiamenensis (LSX-IgY) and inactivated S. xiamenensis (ISX-IgY) were prepared by immunizing laying hens, and passive immunization protection experiments were conducted in Carassius auratus infected with S. xiamenensis and Aeromonas hydrophila. The passive immunization protection rates of LSX-IgY and ISX-IgY against S. xiamenensis were 63.64% and 72.73%, respectively, and the passive cross-protection rates against A. hydrophila were 50% and 71.43%, respectively. Further, C. auratus sera could specifically bind to S. xiamenensis or A. hydrophila in vitro, and the phagocytic activity of leukocytes was increased. LSX-IgY and ISX-IgY could reduce the bacterial load in the C. auratus kidneys. Meanwhile, they could significantly reduce the levels of antioxidant factors in serum and inhibit the mRNA expression of inflammation-related factors in the kidneys and spleens. Additionally, histopathology and immunofluorescence analysis showed that both IgY preparations preserved tissue integrity and reduced the expression of apoptosis factor (p53) and DNA damage factor (γH2A.X) of visceral organs, respectively. In summary, LSX-IgY and ISX-IgY can combat various bacterial infections, with no significant difference between the two. Additionally, inactivated bacterial immunization is more aligned with animal welfare standards for laying hens. Therefore, ISX-IgY is expected to serve as a multivalent vaccine against major aquaculture pathogens. Full article

Background/Objectives: Radiotherapy (RT) is a mainstay of clinical cancer therapy that causes broad immune responses. The complement system is a pivotal effector mechanism in the innate immune response, but the impact of RT is less well understood. This study investigates the interaction between RT and the complement system as a possible approach to improve immune responses in cancer treatment. Methods: Human solid cancer (lung, prostate, liver, breast cancer), lymphoma, and leukemia cells were irradiated using X-rays and treated with polyclonal antibodies or anti-CD20 monoclonal antibodies, respectively. Chromium release assay was applied to measure cell lysis after radiation with or without complement-activating antibody treatment. The expression of membrane-bound complement regulatory proteins (mCRPs; CD46, CD55, CD59), which confer resistance against complement activation, CD20 expression, apoptosis, and radiation-induced DNA double-strand breaks (γH2AX), was measured by flow cytometry. The radiosensitivity of tumor cells was assessed by colony-forming assay. Results: We demonstrate that RT profoundly impacts complement function by upregulating the expression of membrane-bound complement regulatory proteins (mCRPs) on tumor cells in a dose- and time-dependent manner. Impaired complement-mediated tumor cell lysis could thus potentially contribute to radiotherapeutic resistance. We also observed RT-induced upregulation of CD20 expression on lymphoma and leukemic cells. Notably, complement activation prior to RT proved more effective in inducing RT-dependent early apoptosis compared to post-irradiation treatment. While complement modulation does not significantly alter RT-induced DNA-damage repair mechanisms or intrinsic radiosensitivity in cancer cells, our results suggest that combining RT with complement-based anti-cancer therapy may enhance complement-dependent cytotoxicity (CDC) and apoptosis in tumor cells. Conclusions: This study sheds light on the complex interplay between RT and the complement system, offering insights into potential novel combinatorial therapeutic strategies and a potential sequential structure for certain tumor types. Full article

Integrating spatial transcriptomic data with immunofluorescence image data is challenging using existing tools due to their differences in spatial resolution. Immunofluorescence provides information about protein expression at the cellular or subcellular level, whereas spatial transcriptomic platforms typically rely on multicellular “spots” for RNA profiling. Our study coupled spatial transcriptomics of irradiated glioblastoma tissues with immunofluorescence for γH2AX, a marker of DNA damage within the nuclei of cells. We then compared gene expression in γH2AX-positive and negative regions within the tissue. There was significant interobserver variability in manual annotation of γH2AX positivity in multicellular spots by three different researchers (Kappa statistic = 0.345), despite all of them being familiar with γH2AX immunofluorescence and having predefined imaging parameters for annotation. This variability led to different researchers nominating different genes as being associated with DNA repair. To overcome this problem, we have developed a new tool using MATLAB. This tool performs “spot”-wise image analysis and uses researcher-defined parameters such as immunofluorescent marker intensity threshold and number of positive cells to annotate the “spots” as γH2AX positive or negative. The tissue with the most variability in manual annotation was annotated reproducibly by our MATLAB tool, leading to reproducible downstream analysis. Full article

Ovarian cancer cells overexpress HDAC6, and selective HDAC6 inhibitors have been considered potential new drugs for ovarian cancer either alone or in combination with other anticancer agents. We screened 46 potential novel HDAC6 inhibitors in ES-2 ovarian cancer cells and showed that compound 25253 demonstrated the most potent anti-proliferative activity and effective synergy with paclitaxel, which was also validated in TOV21G ovarian cancer cells. The combination of 25253 and paclitaxel significantly induced subG1 and apoptotic cells, revealed by PI staining assay and Annexin V-FITC/PI double staining assay, respectively. Western blot analysis showed downregulation of Bcl-2 and Bcl-XL, and upregulation of Bax and Bak, indicating that apoptosis was mediated through the intrinsic pathway. The combination increased γ-H2AX and p-p53 protein levels, suggesting the induction of DNA damage. Furthermore, HDAC6 was downregulated and acetylated α-tubulin was profoundly increased. Compound 25253 enhanced the inhibitory effect of paclitaxel on cell migration and invasion, possibly due to the extensive accumulation of acetylated α-tubulin, which affected microtubule dynamics. Taken together, the combination of 25253 and paclitaxel synergistically inhibited the growth, migration, and invasion of ovarian cancer cells and induced apoptosis, providing supporting evidence that the combination of HDAC6 inhibitors and paclitaxel may be a promising treatment strategy for ovarian cancer. Full article

Objectives: Beta-emitting radionuclide therapy, exemplified by ¹⁷⁷Lu-Oxodotreotide (Lutathera^®), enables targeted treatment of neuroendocrine tumors by delivering β-radiation to tumor cells. However, the dose-dependent molecular mechanisms underlying cellular damage remain insufficiently characterized. This study aimed to investigate the phenotypic changes in IMR-32 human neuroblastoma cells following Lutathera exposure, with a focus on the dose-dependent relationship between radiation and cellular damage. Methods: IMR-32 cells were allocated to control, low- (0.05 MBq/mL), medium- (0.5 MBq/mL), and high-dose (5 MBq/mL) groups and treated with ¹⁷⁷Lu-Oxodotreotide for 24 h. Flow cytometry was employed to assess cell viability, apoptosis, mitochondrial membrane potential, γ-H2AX expression (a marker of DNA damage), and proliferation. Results: Lutathera induced dose-dependent cytotoxic effects. Cell viability declined linearly with increasing dose (control: 100% vs. high-dose: 13.48%; r = −0.955, p < 0.001). Apoptosis was significantly elevated (control: 35.34% vs. high-dose: 88.12%; r = 0.999), accompanied by increased γ-H2AX levels (control: 5.26 × 10⁴ vs. high-dose: 13.13 × 10⁴; r = 0.930), indicating DNA double-strand breaks. Mitochondrial membrane potential decreased (control: 6.06 × 10⁴ vs. high-dose: 46.27 × 10⁴; r = 0.999), and proliferation was suppressed (control: 91.10 × 10⁴ vs. high-dose: 103.84 × 10⁴; r = 0.954), both showing strong dose correlations (p < 0.001). Conclusions: ¹⁷⁷Lu-Oxodotreotide exerts dose-dependent cytotoxicity in IMR-32 cells via DNA damage, mitochondrial dysfunction, and apoptosis induction. These findings underscore the necessity of optimizing dosing regimens to balance therapeutic efficacy and safety in clinical settings, providing a foundation for personalized β-emitter therapies. Full article

Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by the degeneration of dopaminergic neurons in the substantia nigra, leading to decreased dopamine levels in the striatum and causing a range of motor and non-motor impairments. Although the molecular mechanisms driving PD progression remain incompletely understood, emerging evidence suggests that the buildup of nuclear DNA damage, especially DNA double-strand breaks (DDSBs), plays a key role in contributing neurodegeneration, promoting senescence and neuroinflammation. Despite the pathogenic role for DDSB in neurodegenerative disease, targeting DNA repair mechanisms in PD is largely unexplored as a therapeutic approach. Ataxia telangiectasia mutated (ATM), a key kinase in the DNA damage response (DDR), plays a crucial role in neurodegeneration. In this study, we evaluated the therapeutic potential of AZD1390, a highly selective and brain-penetrant ATM inhibitor, in reducing neuroinflammation and improving behavioral outcomes in a mouse model of α-synucleinopathy. Four-month-old C57BL/6J mice were unilaterally injected with either an empty AAV1/2 vector (control) or AAV1/2 expressing human A53T α-synuclein to the substantia nigra, followed by daily AZD1390 treatment for six weeks. In AZD1390-treated α-synuclein mice, we observed a significant reduction in the protein level of γ-H2AX, a DDSB marker, along with downregulation of senescence-associated markers, such as p53, Cdkn1a, and NF-κB, suggesting improved genomic integrity and attenuation of cellular senescence, indicating enhanced genomic stability and reduced cellular aging. AZD1390 also significantly dampened neuroinflammatory responses, evidenced by decreased expression of key pro-inflammatory cytokines and chemokines. Interestingly, mice treated with AZD1390 showed significant improvements in behavioral asymmetry and motor deficits, indicating functional recovery. Overall, these results suggest that targeting the DDR via ATM inhibition reduces genotoxic stress, suppresses neuroinflammation, and improves behavioral outcomes in a mouse model of α-synucleinopathy. These findings underscore the therapeutic potential of DDR modulation in PD and related synucleinopathy. Full article

Objectives: Mesothelin (MSLN) is overexpressed in pancreatic ductal adenocarcinoma (PDAC), promoting cell proliferation, migration, and inhibiting apoptosis. While its oncogenic properties have been documented, the role of MSLN in regulating cellular senescence—a tumor-suppressive mechanism—has remained unexplored. This study is the first to identify and characterize a novel mesothelin-associated anti-senescence (MAAS) effect in PDAC. Methods: A proteogenomic analysis of PDAC tissue samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) was performed to evaluate MSLN-associated senescence pathways using WebGestalt. Human and murine PDAC cell lines with modified MSLN expression were analyzed for senescence phenotypes via SA-β-gal staining, Western blotting of key regulators (P53, P21^waf1, and P16^ink4a), γH2AX immunoblotting, and IL-8 quantification using ELISA. Results: The CPTAC analysis revealed an inverse correlation between MSLN expression and DNA damage/repair pathways. MSLN-deficient cells exhibited classic senescence features—growth arrest, an enlarged morphology, and elevated SA-β-gal activity. The expression of P53, P21^waf1, and P16^ink4a was upregulated, alongside increased γH2AX levels, indicating the activation of the DNA damage response. IL-8 secretion was significantly higher in the MSLN knockdown cells and reduced in the MSLN-overexpressing cells, consistent with the modulation of the SASP. Notably, MSLN deficiency impaired cell viability without inducing overt cytotoxicity, supporting a shift toward senescence. Conclusions: Our findings uncover a previously unrecognized mechanism through which MSLN promotes tumor progression by suppressing senescence via P53-associated pathways. Targeting the MAAS pathway may offer a novel therapeutic strategy to restore tumor-suppressive senescence and enhance treatment efficacy in PDAC. Full article

Purpose: The development of acquired resistance to arginine deprivation therapy (ADT) is a major barrier to its efficacy. This study aimed to elucidate the possible mechanisms underlying the resistance to ADT. Methods: We applied repeated ADT and established a subline SAS-R9 of the human head and neck squamous cell carcinoma (HNSCC) cells semi-resistant to arginine (Arg) deprivation in vitro. This subline was compared to the parental SAS cell lines for its relative clonogenic proliferation, aggregation, adhesion, and migration capacities. The transcriptomic changes were assessed by RNA sequencing. Signaling pathway alterations were confirmed by RT-PCR and Western blotting. Relative cell radioresistance was analyzed by radiobiological clonogenic survival assay. DNA double-strand break (DSB) repair was assessed by γH2A.X foci analysis. Results: SAS-R9 cells showed higher survival in response to ADT and radiotherapy, elevated clonogenic proliferation rate, cell–cell aggregation, and cell–matrix adhesion, along with increased epithelial–mesenchymal transition (EMT) markers and enhanced DNA DSB repair, potentially related to a more aggressive and therapy-resistant phenotype. Conclusions: While acute ADT has radiosensitizing potential, this new study suggests that long-term, repeated ADT is associated with cell selection and reprogramming, resulting in resistance to radiotherapy-induced DNA damage and higher tumor cell aggressiveness. Full article

In this study, the chemopreventive effects of rosmarinic acid (RA), a major phenolic acid of the plant Rosmarinus officinalis L., against the carcinogenic naturally occurring mycotoxin aflatoxin B1 (AFB1) were investigated using both in silico and in vitro approaches. The in silico investigation of the chemical reactions between rosmarinic acid and the carcinogenic metabolite of AFB1, aflatoxin B1 exo-8,9-epoxide (AFBO), was conducted by activation free energies calculations with DFT functionals M11-L and MN12-L, in conjunction with the 6-311++G(d,p) flexible basis set and implicit solvation model density (SMD), according to a newly developed quantum mechanics-based protocol for the evaluation of carcinogen scavenging activity (QM-CSA). Following the computational analyses, the chemoprotective effects of RA were further studied in vitro in human hepatocellular carcinoma HepG2 cells by analyzing its influence on AFB1-induced genotoxicity using a comet assay, γH2AX, and p-H3, while its impact on cell proliferation and cell cycle modulation was assessed using flow cytometry. Our computational results revealed that the activation free energy required for the reaction of RA with AFBO (14.86 kcal/mol) is significantly lower than the activation free energy for the competing reaction of AFBO with guanine (16.88 kcal/mol), which indicates that RA acts as an efficient natural scavenger of AFBO, potentially preventing AFB1-specific DNA adduct formation. The chemoprotective activity of RA was confirmed through in vitro experiments, which demonstrated a statistically significant (p < 0.05) reduction in AFB1-induced single- and double-strand breaks in HepG2 cells exposed to a mixture of AFB1 and RA at non-cytotoxic concentrations. In addition, RA reversed the AFB1-induced reduction in cell proliferation. Full article

Background/Objectives: Uric acid can act as a prooxidant or an antioxidant; therefore, its effects on human umbilical vein endothelial cells (HUVECs) were investigated to better understand its role in promoting cellular senescence and vascular dysfunction. Methods: HUVECs were exposed to different concentrations of exogenous uric acid levels typically found in patients with cardiovascular conditions (5 mg/dL, 7.5 mg/dL, and 10 mg/dL) to assess cell viability, proliferation, and senescence markers including SA-β-Gal activity, γ-H2A.X and 53BP1 expression, as well as mitochondrial dysfunction parameters such as reactive oxygen species (ROS) production, mitochondrial mass, and mitochondrial membrane potential (ΔΨm). Additionally, the secretion of factors related to the senescence-associated secretory phenotype (SASP) was quantified. Results: Uric acid concentrations of 7.5 mg/dL and above significantly reduced HUVEC viability, enhanced proliferation, and increased markers of cellular senescence, including SA-β-Gal activity and γ-H2A.X/53BP1 expression. Higher uric acid levels also led to increased ROS production, increased mitochondrial mass, and reduced membrane potential. Uric acid also dose-dependently increased IL-6, IL-8, HGF, GRO-1, and TGF-β1 levels. Conclusions: High uric acid concentrations (≥7.5 mg/dL) promote HUVEC senescence, possibly due to ROS-induced DNA damage. In addition, uric acid triggers the production of pro-inflammatory cytokines and growth factors. Full article

Enhancement of glycolysis has been reported in tumor cells, and it is believed that this enhancement is important for maintaining the stemness of tumor cells and contributes to malignant phenotypes. Here, we investigated the effects of Oxamate, which inhibits glycolysis by blocking the conversion of pyruvate to lactate, on radiosensitivity and its molecular mechanisms in T98G glioblastoma cells. Oxamate significantly enhanced radiosensitivity by delaying DNA repair, as indicated by the persistence of γ-H2AX foci up to four days post-irradiation. Mechanistically, Oxamate suppressed the expression and phosphorylation of key DNA repair factors. Furthermore, Oxamate induced apoptosis and promoted cellular senescence, as evidenced by the accumulation of SA-β-gal and the upregulation of pS15-p53 and p21. In addition, Oxamate downregulated EGFR expression, reduced the levels of stem cell markers, and modulated epithelial–mesenchymal transition (EMT) markers, suggesting a potential suppression of EMT-related pathways. Together, these results demonstrate that Oxamate enhances radiosensitivity in glioblastoma cells through multiple mechanisms, including the inhibition of DNA repair, induction of apoptosis and senescence, and suppression of cancer stem cell properties and EMT. Our findings provide new insights into the potential use of Oxamate as a radiosensitizer and warrant further investigation of its clinical application in glioblastoma therapy. Full article

Colibactin toxin-producing Escherichia coli (pks+ E. coli) strains are associated with the occurrence of colorectal cancer in humans. These strains induce DNA damage when in close contact with the cells of the intestinal epithelium. Therefore, maintaining the integrity of the mucus layer that covers the intestinal epithelial mucosa is crucial for counteracting the effects of colibactin. The Vat protein is a mucin protease capable of degrading MUC2 mucus proteins that was previously described in adherent and invasive Escherichia coli strains. Our work shows that the vat gene is found in the genome of all pks+ E. coli strains isolated from patients with colon cancer. In mucus-producing HT29-16E cells, we demonstrated that the Vat protein of E. coli pks+ allows bacteria to penetrate mucus and to reach the epithelial cells. Cells infected with the E. coli pks + vat- strain show a reduction in γ-H2AX staining, a marker of DNA damage. Infection of Apc^Min/+ mice with the E. coli pks + vat+ strain or the E. coli pks + vat- mutant revealed that Vat enhances the ability of pks+ E. coli strains to colonize the intestinal mucosa and, in turn, their pro-carcinogenic effects. This study reveals that Vat promotes crossing of the intestinal mucus layer, gut colonization, and the carcinogenicity of pks+ E. coli. Full article

As the primary active component of Astragalus membranaceus, Astragaloside IV (AS-IV) is widely recognized in pharmacological research for its multifaceted therapeutic potential, particularly its antioxidative, immunostimulatory, and cardioprotective properties. Oxidative stress is an important mechanism in the induction of many diseases. The present study investigates the antioxidative mechanism of Astragaloside IV in zebrafish, using menaquinone exposure to induce oxidative stress conditions. The findings revealed that AS-IV effectively attenuated oxidative stress-induced mortality and morphological abnormalities in zebrafish. AS-IV exhibited a concentration-dependent protective effect against developmental abnormalities, with progressive reduction in pericardial effusion, body curvature, and growth retardation observed at higher doses. Moreover, AS-IV treatment not only effectively reduced reactive oxygen species (ROS) accumulation and attenuated oxidative DNA damage but also significantly decreased apoptosis in the cardiac region of zebrafish embryos under oxidative stress conditions. Western blot analysis revealed that AS-IV treatment significantly reduced the protein levels of both Cleaved Caspase-3 and γ-H2AX, indicating its ability to inhibit DNA damage-induced apoptosis. AS-IV mediates its antioxidant defense mechanisms through the activation of the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, inducing the significant upregulation of cytoprotective enzymes. This molecular mechanism underlies the observed phenotypic improvements in oxidative stress-related damage. Upstream analysis demonstrated that AS-IV activates NRF2 primarily through protein kinase B (AKT/PKB) pathway modulation, independent of KEAP1 regulation. Comprehensive mechanistic analysis reveals that Astragaloside IV mitigates oxidative stress-induced apoptosis in zebrafish through coordinated regulation of the AKT/NRF2/HO-1/Caspase-3 signaling axis. Full article