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Objective: Incorporating iodine into deoxyribonucleic acid (DNA) bases offers a strategy to enhance radiotherapy. The iodine increases the photoabsorption cross-section and can promote DNA disruption and cell death in cancerous tissue. In this study, we investigate the local fragmentation mechanisms of iodinated DNA and the spatial extent of damage propagation following photoactivation.

Approach: Single-stranded DNA oligonucleotides consisting of 2-5 bases, in which the methyl group of thymine is substituted with an iodine atom, were irradiated with synchrotron x-rays above the iodine L-shell ionisation threshold (4900 eV). Fragmentation patterns were extracted by subtracting background spectra obtained below the threshold (4500 eV), and the results were complemented by Born-Oppenheimer molecular dynamics simulations to resolve bond breaking at the atomic level.

Main results: We find that longer oligonucleotide chains predominantly generate larger, high- m/z fragments, while shorter sequences produce a wider variety of small fragments. Backbone cleavage is observed in all sequences, with phosphate- and sugar-based ions dominating the spectra. Bond scission extends up to five bases from the iodination site, with the heaviest stable fragment containing two bases.

Significance: Suppose this effect is extrapolated to genomic DNA, which includes about 29.5% thymine. In that case, the amount of thymine replaced by iodinated uracil can help estimate the extent of DNA damage that might occur during radiation therapy using iodine as a radiosensitiser.

Background: Ascorbate is used for combination therapy with promising results in clinical trials. The proposed main ascorbate cytotoxic effect is DNA damage induction through excessive oxidative stress. However, the involvement of DNA double-strand breakage (DSB) formation and repair in ascorbate-induced DNA damage remains to be revealed.  

Methods: We here used wild-type cells and cells with knock-outs (KO) of the key non-homologous end-joining (NHEJ) repair proteins DNA-PKcs and XRCC4 and tested 2D and 3D cell viability, growth, DSB (DNA fragmentation and DSB surrogate markers 53BP1 and γH2AX), apoptosis, necrosis, and cell cycle in response to ascorbate.  

Results: Ascorbate reduced cell survival and viability in a concentration-dependent manner, with a tendency toward XRCC4 KO cells more sensitivity. Unlike radiation, ascorbate did not produce prompt DSB. However, 24 h after exposure, there was a clear increase in 53BP1 foci in both NHEJproficient and KO cells and evident pan-nuclear γH2AX response, especially in XRCC4 KO cells, which may indicate nuclear degradation leading to DSB formation over time. Further, ascorbate induced G2/M arrest with a more pronounced effect in XRCC4 KO cells. For higher doses (>1 mM) we observed a rapid (24 h) necrotic response without activation of apoptosis. The cell line's different response seems to be related to their cell cycle regulation rather than the NHEJ status. These findings provide novel mechanistic insights into DNA damage formation and cellular response to ascorbate.  

Conclusion: Our data suggest that ascorbate does not generate prompt or direct DSBs. Instead, it induces delayed DSBs ste

Radiotherapy is a standard treatment of pediatric brain tumors. Though the survival rate has improved for many tumor types, most patients suffer long-term cognitive decline and there is currently no way of preventing radiation-induced damage to healthy brain tissue. Here, we used a human forebrain organoid model to investigate if the α₂-adrenoceptor and I₁-imidazoline receptor agonist clonidine could prevent radiotoxicity. We found that treatment of organoids with clonidine significantly reduced radiation-induced loss of neural progenitor cells, neurons, astrocytes, and oligodendrocyte lineage cells. Moreover, clonidine reduced overall DNA damage and signs of reactive gliosis in organoids. Our findings demonstrate that pharmacological rescue of radiation neurotoxicity is possible in a human brain organoid model and provides a rationale for future drug repurposing studies aiming to prevent radiation-induced brain injury in children treated with radiotherapy.

Background: Glioblastoma (GBM) is the deadliest form of brain cancer, impacting both adults and children, marked by exceptionally high morbidity and mortality rates, even with current standard treatments such as surgery, radiation therapy, and chemotherapy. Therefore, there is a pressing need for new therapeutic strategies to improve survival and reduce treatment side effects. In this study, we investigated the effect of HSP90 inhibition in combination with radiotherapy in established and patient-derived glioblastoma cell lines.  

Methods: Potential radiosensitizing effects of the HSP90 inhibitor Onalespib were studied in XTT and clonogenic survival assays as well as in tumor-mimicking multicellular spheroid models. Further, migration capacity and effects on protein expression were studied after exposure to Onalespib and radiation using Proximity Extension Assay analysis.  

Results: HSP90 inhibition with Onalespib synergistically enhanced the radiosensitivity of glioblastoma cells grown in 2D and 3D models, resulting in increased cell death, reduced migration capacity and activation of the apoptotic signaling pathway. The proteomic analysis of glioblastoma cells treated with Onalespib, radiation, and their combination revealed significant alterations in protein expression profiles, involved in growth signaling, immune modulation pathways and angiogenesis. Moreover, the combination treatment indicated potential for enhancing cell cycle arrest and apoptosis, suggesting promising antitumor effects.  

Conclusion: These findings demonstrate that HSP90 inhibition may be a promising strategy to enhance the efficacy of radiotherapy in the treatment of GBM, potent

Background and purpose

This study presents a multi-center comparison of in vitro cell survival measurements and RBE calculations following proton irradiations conducted under harmonized experimental conditions across six European institutions participating in the INSPIRE framework.

Materials and methods

V79-4 cells were irradiated using spread-out Bragg peak (SOBP) proton fields of two configurations delivering 6 and 8 Gy with widths of 6 and 4 cm, respectively. Each center adhered to a standardized protocol, utilizing the same phantom design to minimize uncertainties related to sample positioning. X-ray reference irradiations were also performed to assess cell radiosensitivity across the participating centers.

Results

Despite the consistent protocol, significant inter-institutional variability was observed in the survival measurements. For both treatment plans, the largest variation was detected in the most distal points of the SOBP (coefficients of variation of 43 % and 60 % for the 6 Gy and 8 Gy plans, respectively). Kruskal-Wallis statistical test confirmed the significant differences between the centers for each of the measured position in the proton field for both SOBP configurations. Discrepancies were observed in calculated RBE data as well, albeit preserving the expected trend for the values to slightly increase towards the distal edge of the SOBP (up to 1.5 and 1.3 for the 6 Gy and 8 Gy plans, respectively).

Conclusion

The results of the study highlight the minimal biological variation one could expect performing proton RBE measurements in well-aligned experimental conditions and challenges in conducting large-scale, multi-center radiobiological experiments and inter-comparisons between literature data sets.

The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 x 10-6 with an enrichment in active promoter regions.

The effectiveness of radiation therapy can be enhanced by understanding the fragmentation mechanisms of iodine-doped DNA oligonucleotide under tender X-rays, as explored experimentally and computationally in our study. By primarily targeting iodine atoms above their L-edge ionization energies, we observed a significant increase in the production of fragments critical to DNA backbone breakage, particularly within mass ranges associated with phosphate and sugar groups. The mass spectroscopy experiments demonstrated that iodine-doped DNA oligonucleotides undergo intense fragmentation at long distances from the initial photoactivation site. Born–Oppenheimer based molecular dynamics simulations confirmed the generation of numerous small fragments, including reactive oxygen species, which are pivotal in enhancing the radiation damage. These findings highlight the effectiveness of iodine doping in amplifying DNA damage in radiotherapy via iodine photoactivation, thereby improving the potential for targeted cancer treatment.

  Background

The local anaesthetic lidocaine is widely used in the neonatal intensive unit to treat seizures in premature babies. However, other antiepileptics administered during early development in various animal models have shown negative long-term behavioural effects. Since no long-term behavioural data so far exist regarding lidocaine exposure at an early age, we decided to perform this extended follow-up study using a sensitive behavioural test.

Methods

Neonatal mice received a subcutaneous administration of saline or one dose of lidocaine (0.5, 4, or 12 mg kg⁻¹) on postnatal day 10 (P10; peak of the Brain Growth Spurt). A well-established test to detect long-term behavioural alterations was conducted at 2 and 6 months of age, corresponding to early and late adulthood in humans.

Results

All animal survived to later testing. No signs of acute toxicity were observed. Lidocaine exposure did not result in any negative behavioural effects during habituation to a new home environment at any of the two studied time points, compared to saline placebo.

Conclusions

Lidocaine does not by itself produce any negative long-term behavioural effects in mice exposed in early life (P10) despite long-term follow-up. This is reassuring regarding the current practice of treating seizures in premature babies with intravenous lidocaine.

Background: Proton therapy has garnered attention as an advanced radiation treatment modality for breast cancer due to its ability to deliver highly precise doses to the target area while minimizing exposure to surrounding healthy tissues.

The aim was to detect potential variations in radiobiological response along different parts of the proton depth-dose curve.

Materials and methods: MDA-MB-231 cells were specifically irradiated before, within, and beyond the Bragg peak with a 5 Gy dose, with photons used as a reference. The radiobiological response was evaluated using clonogenic assays, relative γH2AX levels, and quantitative polymerase chain reaction (qPCR) analysis of DNA damage response genes.

Results: A trend of increasing magnitude in radiobiological response was observed with increasing depth of cell irradiation, accompanied by a decrease in survival fraction. Furthermore, differences were noted, particularly in γH2AX levels along the Bragg peak, with higher values of DNA double-strand breaks (DNA DSB) observed at the end of the depth-dose curve.

Conclusions: These findings suggest that despite administering a consistent proton dose to the target area, there can be a range of different biological reactions, which might have significant indications for clinical procedures.

Research on different types of ionizing radiation’s effects has been ongoing for years, revealing its efficacy in damaging cancer cells. Solid tumors comprise diverse cell types, each being able to respond differently to radiation. This study evaluated the radiobiological response of established (MDA-MB-231 (Triple negative breast cancer, TNBC), MCF-7 (Luminal A)) and patient-derived malignant cell lines, cancer-associated fibroblasts, and skin fibroblasts following proton IRR. All cell line types were irradiated with the proton dose of 2, 4, and 6 Gy. The radiobiological response was assessed using clonogenic assay, γH2AX, and p53 staining. It was noticeable that breast cancer lines of different molecular subtypes displayed no significant variations in their response to proton IRR. In terms of cancer-associated fibroblasts extracted from the tumor tissue, the line derived from a TNBC subtype tumor demonstrated higher resistance to ionizing radiation compared to lines isolated from luminal A tumors. Fibroblasts extracted from patients’ skin responded identically to all doses of proton radiation. This study emphasizes that tumor response is not exclusively determined by the elimination of breast cancer cells, but also takes into account tumor microenvironmental variables and skin reactions.