-
International Journal of Molecular... Jun 2024Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be...
Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.
Topics: Humans; K562 Cells; Leukocytes; Electrons; Telomere; Leukemia; Telomere Homeostasis; In Situ Hybridization, Fluorescence; Telomere Shortening; DNA Damage; Dose-Response Relationship, Radiation
PubMed: 38928414
DOI: 10.3390/ijms25126709 -
International Journal of Molecular... Jun 2024Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It...
Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.
Topics: Humans; Virus Replication; Oncolytic Viruses; RNA, Viral; Adenoviridae; Animals; Oncolytic Virotherapy; Mice; Cell Line, Tumor; Neoplasms; Female
PubMed: 38928259
DOI: 10.3390/ijms25126551 -
Genes May 2024Protein-DNA complex interactivity plays a crucial role in biological activities such as gene expression, modification, replication and transcription. Understanding the...
Protein-DNA complex interactivity plays a crucial role in biological activities such as gene expression, modification, replication and transcription. Understanding the physiological significance of protein-DNA binding interfacial hot spots, as well as the development of computational biology, depends on the precise identification of these regions. In this paper, a hot spot prediction method called EC-PDH is proposed. First, we extracted features of these hot spots' solid solvent-accessible surface area (ASA) and secondary structure, and then the mean, variance, energy and autocorrelation function values of the first three intrinsic modal components (IMFs) of these conventional features were extracted as new features via the empirical modal decomposition algorithm (EMD). A total of 218 dimensional features were obtained. For feature selection, we used the maximum correlation minimum redundancy sequence forward selection method (mRMR-SFS) to obtain an optimal 11-dimensional-feature subset. To address the issue of data imbalance, we used the SMOTE-Tomek algorithm to balance positive and negative samples and finally used cat gradient boosting (CatBoost) to construct our hot spot prediction model for protein-DNA binding interfaces. Our method performs well on the test set, with AUC, MCC and F1 score values of 0.847, 0.543 and 0.772, respectively. After a comparative evaluation, EC-PDH outperforms the existing state-of-the-art methods in identifying hot spots.
Topics: Machine Learning; DNA; Algorithms; Protein Binding; DNA-Binding Proteins; Computational Biology; Binding Sites
PubMed: 38927611
DOI: 10.3390/genes15060676 -
Antibiotics (Basel, Switzerland) Jun 2024The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an...
The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against was 32-64 µg/mL, including strains carrying , which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene increased the MIC of 5-FU against by 32-fold, indicating the role of the gene in 5-FU resistance. Additionally, transcriptomic analysis of treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.
PubMed: 38927194
DOI: 10.3390/antibiotics13060528 -
Biomolecules Jun 2024Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for...
Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.
Topics: DNA Repair; Humans; Pyrimidine Nucleotides; Click Chemistry; DNA-Directed DNA Polymerase; Deoxyuridine; DNA; DNA Replication; Uracil-DNA Glycosidase
PubMed: 38927084
DOI: 10.3390/biom14060681 -
Nature Communications Jun 2024Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR),...
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RAS oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RAS. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RAS induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
Topics: Humans; Exodeoxyribonucleases; Phosphoproteins; MRE11 Homologue Protein; Signal Transduction; Cellular Senescence; DNA Replication; DNA Damage; Fibroblasts; Interferon-beta
PubMed: 38926338
DOI: 10.1038/s41467-024-49740-w -
Journal of Fungi (Basel, Switzerland) Jun 2024In budding yeast, Rad5 and Rad7-Rad16 play respective roles in the error-free post-replication repair and nucleotide excision repair of ultraviolet-induced DNA damage;...
In budding yeast, Rad5 and Rad7-Rad16 play respective roles in the error-free post-replication repair and nucleotide excision repair of ultraviolet-induced DNA damage; however, their homologs have not yet been studied in non-yeast fungi. In the fungus , a deficiency in the Rad7 homolog, Rad5 ortholog and two Rad16 paralogs (Rad16A/B) instituted an ability to help the insect-pathogenic fungus to recover from solar UVB damage through photoreactivation. The fungal lifecycle-related phenotypes were not altered in the absence of , or while severe defects in growth and conidiation were caused by the double deletion of and . Compared with the wild-type and complemented strains, the mutants showed differentially reduced activities regarding the resilience of UVB-impaired conidia at 25 °C through a 12-h incubation in a regime of visible light plus dark (L/D 3:9 h or 5:7 h for photoreactivation) or of full darkness (dark reactivation) mimicking a natural nighttime. The estimates of the median lethal UVB dose LD from the dark and L/D treatments revealed greater activities of Rad5 and Rad16B than of Rad16A and additive activities of Rad16A and Rad16B in either NER-dependent dark reactivation or photorepair-dependent photoreactivation. However, their dark reactivation activities were limited to recovering low UVB dose-impaired conidia but were unable to recover conidia impaired by sublethal and lethal UVB doses as did their photoreactivation activities at L/D 3:9 or 5:7, unless the night/dark time was doubled or further prolonged. Therefore, the anti-UV effects of Rad5, Rad16A and Rad16B in depend primarily on photoreactivation and are mechanistically distinct from those for their yeast homologs.
PubMed: 38921406
DOI: 10.3390/jof10060420 -
Journal of Fungi (Basel, Switzerland) May 2024DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in...
DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen , their targets remain poorly understood. In this study, we explored the impact of deleting on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of only affected a limited number of known DNA damage-responsive genes, including and . Unlike , the downregulation of transcription in under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of . Furthermore, Rad53 was observed to directly interact with the promoter region of , thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in and show species-specific regulation on ; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.
PubMed: 38921373
DOI: 10.3390/jof10060387 -
Frontiers in Genetics 2024Fracture healing is a complex process that involves multiple molecular events, and the regulation mechanism is not fully understood. We acquired miRNA and mRNA...
Fracture healing is a complex process that involves multiple molecular events, and the regulation mechanism is not fully understood. We acquired miRNA and mRNA transcriptomes of mouse fractures from the Gene Expression Omnibus database (GSE76197 and GSE192542) and integrated the miRNAs and genes that were differentially expressed in the control and fracture groups to construct regulatory networks. There were 130 differentially expressed miRNAs and 4,819 differentially expressed genes, including 72 upregulated and 58 downregulated miRNAs, along with 2,855 upregulated and 1964 downregulated genes during early fracture healing. Gene ontology analysis revealed that most of the differentially expressed genes were enriched in the extracellular matrix (ECM) structure and the ECM organization. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested cell cycle, DNA replication, and mismatch repair were involved in the progression of fracture healing. Furthermore, we constructed a molecular network of miRNAs and mRNAs with inverse expression patterns to elucidate the molecular basis of miRNA-mRNA regulation in fractures. The regulatory network highlighted the potential targets, which may help to provide a mechanistic basis for therapies to improve fracture patient outcomes.
PubMed: 38919952
DOI: 10.3389/fgene.2024.1408404 -
Frontiers in Immunology 2024The global impact of the SARS-CoV-2 pandemic has been unprecedented, posing a significant public health challenge. Chronological age has been identified as a key... (Review)
Review
The global impact of the SARS-CoV-2 pandemic has been unprecedented, posing a significant public health challenge. Chronological age has been identified as a key determinant for severe outcomes associated with SARS-CoV-2 infection. Epigenetic age acceleration has previously been observed in various diseases including human immunodeficiency virus (HIV), Cytomegalovirus (CMV), cardiovascular diseases, and cancer. However, a comprehensive review of this topic is still missing in the field. In this review, we explore and summarize the research work focusing on biological aging markers, i.e., epigenetic age and telomere attrition in COVID-19 patients. From the reviewed articles, we identified a consistent pattern of epigenetic age dysregulation and shortened telomere length, revealing the impact of COVID-19 on epigenetic aging and telomere attrition.
Topics: Humans; COVID-19; Aging; SARS-CoV-2; Epigenesis, Genetic; Telomere; Telomere Shortening
PubMed: 38919619
DOI: 10.3389/fimmu.2024.1399676