This roadmap reviews the new, highly interdisciplinary research field studying the behavior of condensed matter systems exposed to radiation. The Review highlights several recent advances in the field and provides a roadmap for the development of the field over the next decade. Condensed matter systems exposed to radiation can be inorganic, organic, or biological, finite or infinite, composed of different molecular species or materials, exist in different phases, and operate under different thermodynamic conditions. Many of the key phenomena related to the behavior of irradiated systems are very similar and can be understood based on the same fundamental theoretical principles and computational approaches. The multiscale nature of such phenomena requires the quantitative description of the radiation-induced effects occurring at different spatial and temporal scales, ranging from the atomic to the macroscopic, and the interlinks between such descriptions. The multiscale nature of the effects and the similarity of their manifestation in systems of different origins necessarily bring together different disciplines, such as physics, chemistry, biology, materials science, nanoscience, and biomedical research, demonstrating the numerous interlinks and commonalities between them. This research field is highly relevant to many novel and emerging technologies and medical applications.

• Publication type
• Journal Article MeSH
• Review MeSH

The cell as a system of many components, governed by the laws of physics and chemistry drives molecular functions having an impact on the spatial organization of these systems and vice versa. Since the relationship between structure and function is an almost universal rule not only in biology, appropriate methods are required to parameterize the relationship between the structure and function of biomolecules and their networks, the mechanisms of the processes in which they are involved, and the mechanisms of regulation of these processes. Single molecule localization microscopy (SMLM), which we focus on here, offers a significant advantage for the quantitative parametrization of molecular organization: it provides matrices of coordinates of fluorescently labeled biomolecules that can be directly subjected to advanced mathematical analytical procedures without the need for laborious and sometimes misleading image processing. Here, we propose mathematical tools for comprehensive quantitative computer data analysis of SMLM point patterns that include Ripley distance frequency analysis, persistent homology analysis, persistent 'imaging', principal component analysis and co-localization analysis. The application of these methods is explained using artificial datasets simulating different, potentially possible and interpretatively important situations. Illustrative analyses of real complex biological SMLM data are presented to emphasize the applicability of the proposed algorithms. This manuscript demonstrated the extraction of features and parameters quantifying the influence of chromatin (re)organization on genome function, offering a novel approach to study chromatin architecture at the nanoscale. However, the ability to adapt the proposed algorithms to analyze essentially any molecular organizations, e.g., membrane receptors or protein trafficking in the cytosol, offers broad flexibility of use.

• Keywords
• Application of mathematical analysis tools to chromatin organization and DNA repair processes, DSB, DNA double-strand break, HR, homologous recombination, IRIF, ionizing radiation induced foci, LET, linear energy transfer, NHEJ, non-homologous end joining, NN, nearest neighbor, PCA, principal component analysis, Persistent homology, Persistent image, Principal component analysis, Ripley distance frequency histograms, SMLM, single molecule localization microscopy, Single molecule localization microscopy (SMLM),
• Publication type
• Journal Article MeSH

Complex functioning of the genome in the cell nucleus is controlled at different levels: (a) the DNA base sequence containing all relevant inherited information; (b) epigenetic pathways consisting of protein interactions and feedback loops; (c) the genome architecture and organization activating or suppressing genetic interactions between different parts of the genome. Most research so far has shed light on the puzzle pieces at these levels. This article, however, attempts an integrative approach to genome expression regulation incorporating these different layers. Under environmental stress or during cell development, differentiation towards specialized cell types, or to dysfunctional tumor, the cell nucleus seems to react as a whole through coordinated changes at all levels of control. This implies the need for a framework in which biological, chemical, and physical manifestations can serve as a basis for a coherent theory of gene self-organization. An international symposium held at the Biomedical Research and Study Center in Riga, Latvia, on 25 July 2022 addressed novel aspects of the abovementioned topic. The present article reviews the most recent results and conclusions of the state-of-the-art research in this multidisciplinary field of science, which were delivered and discussed by scholars at the Riga symposium.

• Keywords
• database pattern analysis, dynamic genome organization, epigenetic interactions, fluorescence microscopy, gene activity oscillations, heterochromatin and self-organization, nucleotide k-mers, organizational and functional networks, topological genome analysis, transposon-effected regulation,
• MeSH
• Cell Differentiation genetics   MeSH
• Cell Nucleus * metabolism   MeSH
• Genome * MeSH
• Publication type
• Congress MeSH
• Review MeSH

(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley's distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).

• Keywords
• DNA double strand breaks (DSBs), DNA repair, DNA repair nano-clusters, chromatin nano-architecture rearrangements, gold nanoparticles (GNP), ionizing radiation-induced (repair) foci (IRIF), nanoparticle-enhanced cancer radiotherapy, single-molecule localization microscopy (SMLM),
• Publication type
• Journal Article MeSH

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.

• Keywords
• 53BP1, P53-binding protein 1, Biodosimetry, CNN, convolutional neural network, Confocal Microscopy, Convolutional Neural Network, DNA Damage and Repair, DSB, DNA double-strand break, Deep Learning, FOV, field of view, GUI, graphical user interface, IRIF, ionizing radiation-induced (repair) foci, Image Analysis, Ionizing Radiation-Induced Foci (IRIFs), MSER, maximally stable extremal region (algorithm), Morphometry, NHDFs, normal human dermal fibroblasts, RAD51, DNA repair protein RAD51 homolog 1, U-87, U-87 glioblastoma cell line, γH2AX, histone H2AX phosphorylated at serine 139,
• Publication type
• Journal Article MeSH

DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

• Keywords
• DNA double-strand breaks, ionizing photon irradiation, persistent homology, single-molecule localization microscopy, topology of MRE11 clusters, topology of γH2AX clusters,
• Publication type
• Journal Article MeSH

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a "double-edged sword"-it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

• Keywords
• chromatin rearrangements after irradiation, ionizing radiation-induced foci (IRIF), nano-architecture, single molecule localization microscopy (SMLM), topology of DNA double strand breaks,
• MeSH
• Chromatin genetics   ultrastructure   MeSH
• DNA Breaks, Double-Stranded radiation effects   MeSH
• HeLa Cells MeSH
• Radiation, Ionizing MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Cell Line, Tumor MeSH
• Neoplasms genetics   MeSH
• DNA Repair genetics   radiation effects   MeSH
• DNA Damage genetics   radiation effects   MeSH
• Single Molecule Imaging methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH

DNA double-strand breaks (DSBs) have been recognized as the most serious lesions in irradiated cells. While several biochemical pathways capable of repairing these lesions have been identified, the mechanisms by which cells select a specific pathway for activation at a given DSB site remain poorly understood. Our knowledge of DSB induction and repair has increased dramatically since the discovery of ionizing radiation-induced foci (IRIFs), initiating the possibility of spatiotemporally monitoring the assembly and disassembly of repair complexes in single cells. IRIF exploration revealed that all post-irradiation processes-DSB formation, repair and misrepair-are strongly dependent on the characteristics of DSB damage and the microarchitecture of the whole affected chromatin domain in addition to the cell status. The microscale features of IRIFs, such as their morphology, mobility, spatiotemporal distribution, and persistence kinetics, have been linked to repair mechanisms. However, the influence of various biochemical and structural factors and their specific combinations on IRIF architecture remains unknown, as does the hierarchy of these factors in the decision-making process for a particular repair mechanism at each individual DSB site. New insights into the relationship between the physical properties of the incident radiation, chromatin architecture, IRIF architecture, and DSB repair mechanisms and repair efficiency are expected from recent developments in optical superresolution microscopy (nanoscopy) techniques that have shifted our ability to analyze chromatin and IRIF architectures towards the nanoscale. In the present review, we discuss this relationship, attempt to correlate still rather isolated nanoscale studies with already better-understood aspects of DSB repair at the microscale, and consider whether newly emerging "correlated multiscale structuromics" can revolutionarily enhance our knowledge in this field.

• Keywords
• DNA damage and repair, DNA double-strand breaks (DSBs), DSB repair pathway choice and hierarchy, chromatin architecture, ionizing radiation, ionizing radiation-induced foci (IRIFs), linear energy transfer (LET), single-molecule localization microscopy (SMLM), superresolution microscopy,
• Publication type
• Journal Article MeSH
• Review MeSH

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

• MeSH
• Chromatin drug effects   genetics   MeSH
• Dimethyl Sulfoxide pharmacology   MeSH
• DNA Breaks, Double-Stranded drug effects   MeSH
• Fibroblasts MeSH
• Cryopreservation methods   MeSH
• Cryoprotective Agents pharmacology   MeSH
• Skin cytology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• S Phase drug effects   MeSH
• Cell Survival drug effects   genetics   MeSH
• Freezing adverse effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Chromatin MeSH
• Dimethyl Sulfoxide MeSH
• Cryoprotective Agents MeSH

BACKGROUND: Tumor targeting of radiotherapy represents a great challenge. The addition of multimodal nanoparticles, such as 3 nm gadolinium-based nanoparticles (GdBNs), has been proposed as a promising strategy to amplify the effects of radiation in tumors and improve diagnostics using the same agents. This singular property named theranostic is a unique advantage of GdBNs. It has been established that the amplification of radiation effects by GdBNs appears due to fast electronic processes. However, the influence of these nanoparticles on cells is not yet understood. In particular, it remains dubious how nanoparticles activated by ionizing radiation interact with cells and their constituents. A crucial question remains open of whether damage to the nucleus is necessary for the radiosensitization exerted by GdBNs (and other nanoparticles). METHODS: We studied the effect of GdBNs on the induction and repair of DNA double-strand breaks (DSBs) in the nuclear DNA of U87 tumor cells irradiated with γ-rays. For this purpose, we used currently the most sensitive method of DSBs detection based on high-resolution confocal fluorescence microscopy coupled with immunodetection of two independent DSBs markers. RESULTS: We show that, in the conditions where GdBNs amplify radiation effects, they remain localized in the cytoplasm, i.e. do not penetrate into the nucleus. In addition, the presence of GdBNs in the cytoplasm neither increases induction of DSBs by γ-rays in the nuclear DNA nor affects their consequent repair. CONCLUSIONS: Our results suggest that the radiosensitization mediated by GdBNs is a cytoplasmic event that is independent of the nuclear DNA breakage, a phenomenon commonly accepted as the explanation of biological radiation effects. Considering our earlier recognized colocalization of GdBNs with the lysosomes and endosomes, we revolutionary hypothesize here about these organelles as potential targets for (some) nanoparticles. If confirmed, this finding of cytoplasmically determined radiosensitization opens new perspectives of using nano-radioenhancers to improve radiotherapy without escalating the risk of pathologies related to genetic damage.

• Keywords
• DNA double-strand breaks, DNA repair, Gadolinium, Nanomedicine, Nanoparticles, Radiosensitization, Radiotherapy, Theranostic,
• MeSH
• DNA Breaks, Double-Stranded drug effects   MeSH
• Gadolinium toxicity   MeSH
• Glioblastoma metabolism   MeSH
• Metal Nanoparticles toxicity   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• DNA Repair drug effects   MeSH
• DNA Damage drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Gadolinium MeSH