Minimum reporting standards about dosimetry of radiation sources used in radiation research studies.

The necessity of precise dosimetry and its documentation in research is less obvious than in medicine and in radiological protection. However, in radiation research, results can only be validated if experiments were carried out with sufficient precision and described with sufficient details, especially information regarding dosimetry. In order to ensure this, an initiative was launched to establish reproducible dosimetry reporting parameters in published studies. Minimum standards for reporting radiation dosimetry information were developed and published in parallel in the International Journal of Radiation Biology and Radiation Research. As editors of Radiation and Environmental Biophysics, we support this initiative and reproduce the agreed minimum irradiation parameters that should be reported in publications on radiation biology submitted to our journal.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus.

Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Dose limits for occupational exposure to ionising radiation and genotoxic carcinogens: a German perspective.

This paper summarises the view of the German Commission on Radiological Protection ("Strahlenschutzkommission", SSK) on the rationale behind the currently valid dose limits and dose constraints for workers recommended by the International Commission on Radiological Protection (ICRP). The paper includes a discussion of the reasoning behind current dose limits followed by a discussion of the detriment used by ICRP as a measure for stochastic health effects. Studies on radiation-induced cancer are reviewed because this endpoint represents the most important contribution to detriment. Recent findings on radiation-induced circulatory disease that are currently not included in detriment calculation are also reviewed. It appeared that for detriment calculations the contribution of circulatory diseases plays only a secondary role, although the uncertainties involved in their risk estimates are considerable. These discussions are complemented by a review of the procedures currently in use in Germany, or in discussion elsewhere, to define limits for genotoxic carcinogens. To put these concepts in perspective, actual occupational radiation exposures are exemplified with data from Germany, for the year 2012, and regulations in Germany are compared to the recommendations issued by ICRP. Conclusions include, among others, considerations on radiation protection concepts currently in use and recommendations of the SSK on the limitation of annual effective dose and effective dose cumulated over a whole working life.

Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells.

Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

Current concepts in clinical radiation oncology.

Based on its potent capacity to induce tumor cell death and to abrogate clonogenic survival, radiotherapy is a key part of multimodal cancer treatment approaches. Numerous clinical trials have documented the clear correlation between improved local control and increased overall survival. However, despite all progress, the efficacy of radiation-based treatment approaches is still limited by different technological, biological, and clinical constraints. In principle, the following major issues can be distinguished: (1) The intrinsic radiation resistance of several tumors is higher than that of the surrounding normal tissue, (2) the true patho-anatomical borders of tumors or areas at risk are not perfectly identifiable, (3) the treatment volume cannot be adjusted properly during a given treatment series, and (4) the individual heterogeneity in terms of tumor and normal tissue responses toward irradiation is immense. At present, research efforts in radiation oncology follow three major tracks, in order to address these limitations: (1) implementation of molecularly targeted agents and 'omics'-based screening and stratification procedures, (2) improvement of treatment planning, imaging, and accuracy of dose application, and (3) clinical implementation of other types of radiation, including protons and heavy ions. Several of these strategies have already revealed promising improvements with regard to clinical outcome. Nevertheless, many open questions remain with individualization of treatment approaches being a key problem. In the present review, the current status of radiation-based cancer treatment with particular focus on novel aspects and developments that will influence the field of radiation oncology in the near future is summarized and discussed.

Stain-Free technology as a normalization tool in Western blot analysis.

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.