Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA dam age response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mech anistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investi gate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the mor phological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined

Assessment of mRNA and microRNA Stabilization in Peripheral Human Blood for Multicenter Studies and Biobanks

In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis laboratory. Both examined systems were clearly appropriate for RNA stabilization in human blood independently of the shipping conditions. The isolated RNA is characterized by good quantity and quality and well suited for downstream applications like quantitative RT-PCR analysis of mRNA and microRNA. Superior yield and integrity values were received using RNAlater. It would be reasonable to consider the production and approval of blood collection tubes prefilled with RNAlater to facilitate the use of this excellent RNA stabilization system in large studies

Differences in DNA repair capacity, cell death and transcriptional response after irradiation between a radiosensitive and a radioresistant cell line

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols

Genetic modifiers of radon-induced lung cancer risk: a genome-wide interaction study in former uranium miners

PURPOSE: Radon is a risk factor for lung cancer and uranium miners are more exposed than the general population. A genome-wide interaction analysis was carried out to identify genomic loci, genes or gene sets that modify the susceptibility to lung cancer given occupational exposure to the radioactive gas radon. METHODS: Samples from 28 studies provided by the International Lung Cancer Consortium were pooled with samples of former uranium miners collected by the German Federal Office of Radiation Protection. In total, 15,077 cases and 13,522 controls, all of European ancestries, comprising 463 uranium miners were compared. The DNA of all participants was genotyped with the OncoArray. We fitted single-marker and in multi-marker models and performed an exploratory gene-set analysis to detect cumulative enrichment of significance in sets of genes. RESULTS: We discovered a genome-wide significant interaction of the marker rs12440014 within the gene CHRNB4 (OR = 0.26, 95% CI 0.11-0.60, p = 0.0386 corrected for multiple testing). At least suggestive significant interaction of linkage disequilibrium blocks was observed at the chromosomal regions 18q21.23 (p = 1.2 × 10-6), 5q23.2 (p = 2.5 × 10-6), 1q21.3 (p = 3.2 × 10-6), 10p13 (p = 1.3 × 10-5) and 12p12.1 (p = 7.1 × 10-5). Genes belonging to the Gene Ontology term "DNA dealkylation involved in DNA repair" (GO:0006307; p = 0.0139) or the gene family HGNC:476 "microRNAs" (p = 0.0159) were enriched with LD-blockwise significance. CONCLUSION: The well-established association of the genomic region 15q25 to lung cancer might be influenced by exposure to radon among uranium miners. Furthermore, lung cancer susceptibility is related to the functional capability of DNA damage signaling via ubiquitination processes and repair of radiation-induced double-strand breaks by the single-strand annealing mechanism

The German Uranium Miners’ Biobank—A Biobank for OMICs Radiation Research

Systematic bio- and databanks are key prerequisites for modern radiation research to investigate radiation response mechanisms in the context of genetic, environmental and lifestyle-associated factors. This report presents the current status of the German Uranium Miners’ Biobank. In 2008, the bio- and databank was established at the Federal Office for Radiation Protection, and the sampling of biological materials from former uranium miners with and without lung cancer was initiated. For this purpose, various biological specimens, such as DNA and RNA, were isolated from blood samples as well as from formalin-fixed paraffin-embedded lung tissue. High-quality biomaterials suitable for OMICs research and the associated data on occupational radiation and dust exposure, and medical and lifestyle data from over 1000 individuals have been stored so far. Various experimental data, e.g., genome-wide SNPs, whole genome transcriptomic and miRNA data, as well as individual chromosomal aberration data from subgroups of biobank samples, are already available upon request for in-depth research on radiation-induced long-term effects, individual radiation susceptibility to lung cancer and radon-induced fingerprints in lung cancer. This biobank is the first systematic uranium miners´ biobank worldwide that is suitable for OMICs research on radiation-exposed workers. It offers the opportunity to link radiation-induced perturbations of biological pathways or processes and putative adverse outcome(s) by OMICs profiling at different biological organization levels

Ionizing Radiation Protein Biomarkers in Normal Tissue and Their Correlation to Radiosensitivity: A Systematic Review

Background and objectives: Exposure to ionizing radiation (IR) has increased immensely over the past years, owing to diagnostic and therapeutic reasons. However, certain radiosensitive individuals show toxic enhanced reaction to IR, and it is necessary to specifically protect them from unwanted exposure. Although predicting radiosensitivity is the way forward in the field of personalised medicine, there is limited information on the potential biomarkers. The aim of this systematic review is to identify evidence from a range of literature in order to present the status quo of our knowledge of IR-induced changes in protein expression in normal tissues, which can be correlated to radiosensitivity. Methods: Studies were searched in NCBI Pubmed and in ISI Web of Science databases and field experts were consulted for relevant studies. Primary peer-reviewed studies in English language within the time-frame of 2011 to 2020 were considered. Human non-tumour tissues and human-derived non-tumour model systems that have been exposed to IR were considered if they reported changes in protein levels, which could be correlated to radiosensitivity. At least two reviewers screened the titles, keywords, and abstracts of the studies against the eligibility criteria at the first phase and full texts of potential studies at the second phase. Similarly, at least two reviewers manually extracted the data and accessed the risk of bias (National Toxicology Program/Office for Health Assessment and Translation—NTP/OHAT) for the included studies. Finally, the data were synthesised narratively in accordance to synthesis without meta analyses (SWiM) method. Results: In total, 28 studies were included in this review. Most of the records (16) demonstrated increased residual DNA damage in radiosensitive individuals compared to normo-sensitive individuals based on γH2AX and TP53BP1. Overall, 15 studies included proteins other than DNA repair foci, of which five proteins were selected, Vascular endothelial growth factor (VEGF), Caspase 3, p16INK4A (Cyclin-dependent kinase inhibitor 2A, CDKN2A), Interleukin-6, and Interleukin-1β, that were connected to radiosensitivity in normal tissue and were reported at least in two independent studies. Conclusions and implication of key findings: A majority of studies used repair foci as a tool to predict radiosensitivity. However, its correlation to outcome parameters such as repair deficient cell lines and patients, as well as an association to moderate and severe clinical radiation reactions, still remain contradictory. When IR-induced proteins reported in at least two studies were considered, a protein network was discovered, which provides a direction for further studies to elucidate the mechanisms of radiosensitivity. Although the identification of only a few of the commonly reported proteins might raise a concern, this could be because (i) our eligibility criteria were strict and (ii) radiosensitivity is influenced by multiple factors. Registration: PROSPERO (CRD42020220064)

Advanced Omics and Radiobiological Tissue Archives: The Future in the Past

Archival formalin-fixed, paraffin-embedded (FFPE) tissues and their related diagnostic records are an invaluable source of biological information. The archival samples can be used for retrospective investigation of molecular fingerprints and biomarkers of diseases and susceptibility. Radiobiological archives were set up not only following clinical performance such as cancer diagnosis and therapy but also after accidental and occupational radiation exposure events where autopsies or cancer biopsies were sampled. These biobanks provide unique and often irreplaceable materials for the understanding of molecular mechanisms underlying radiation-related biological effects. In recent years, the application of rapidly evolving “omics” platforms, including transcriptomics, genomics, proteomics, metabolomics and sequencing, to FFPE tissues has gained increasing interest as an alternative to fresh/frozen tissue. However, omics profiling of FFPE samples remains a challenge mainly due to the condition and duration of tissue fixation and storage, and the extraction methods of biomolecules. Although biobanking has a long history in radiation research, the application of omics to profile FFPE samples available in radiobiological archives is still young. Application of the advanced omics technologies on archival materials provides a new opportunity to understand and quantify the biological effects of radiation exposure. These newly generated omics data can be well integrated into results obtained from earlier experimental and epidemiological analyses to shape a powerful strategy for modelling and evaluating radiation effects on health outcomes. This review aims to give an overview of the unique properties of radiation biobanks and their potential impact on radiation biology studies. Studies recently performed on FFPE samples from radiobiology archives using advanced omics are summarized. Furthermore, the compatibility of archived FFPE tissues for omics analysis and the major challenges that lie ahead are discussed

Having a real prototype of your own product design – small effort but big eureka moment for students

[EN] This paper presents a project-based product design course, which is the only course on product design in our curriculum. We noticed that the building of a prototype is often omitted in academic teaching. However, students benefit from building a prototype of their design. Hence, we reduced the theoretical content to give students time to design and build a prototype. To illustrate the course two student projects are shown. We want to motivate lecturers to introduce prototyping to courses, since our experience is that the benefits of building a prototype outweigh the decrease in theoretical knowledge.Riess, C.; Walter, M.; Tyroller, M.; Gomolka, L.; Augustin, J.; Altieri, M. (2023). Having a real prototype of your own product design – small effort but big eureka moment for students. Editorial Universitat Politècnica de València. 266-279. https://doi.org/10.4995/BMT2022.2022.1562426627