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In radiation tumor therapy, irradiation, on one hand, should cause cell death to the tumor. On the other hand, the surrounding non-tumor tissue should be maintained unaffected. Therefore, methods of local dose enhancements are highly interesting. Gold nanoparticles, which are preferentially uptaken by very-fast-proliferating tumor cells, may enhance damaging. However, the results in the literature obtained from cell culture and animal tissue experiments are very contradictory, i.e., only some experiments reveal increased cell killing but others do not. Thus, a better understanding of cellular mechanisms is required. Using the breast cancer cell model SkBr3, the effects of gold nanoparticles in combination with ionizing radiation on chromatin network organization were investigated by Single-Molecule Localization Microscopy (SMLM) and applications of mathematical topology calculations (e.g., Persistent Homology, Principal Component Analysis, etc.). The data reveal a dose and nanoparticle dependent re-organization of chromatin, although colony forming assays do not show a significant reduction of cell survival after the application of gold nanoparticles to the cells. In addition, the spatial organization of γH2AX clusters was elucidated, and characteristic changes were obtained depending on dose and gold nanoparticle application. The results indicate a complex response of ALU-related chromatin and heterochromatin organization correlating to ionizing radiation and gold nanoparticle incorporation. Such complex whole chromatin re-organization is usually associated with changes in genome function and supports the hypothesis that, with the application of gold nanoparticles, not only is DNA damage increasing but also the efficiency of DNA repair may be increased. The understanding of complex chromatin responses might help to improve the gold nanoparticle efficiency in radiation treatment. [ABSTRACT FROM AUTHOR]

Background: It is widely accepted that the 3D chromatin organization in human cell nuclei is not random and recent investigations point towards an interactive relation of epigenetic functioning and chromatin (re-)organization. Although chromatin organization seems to be the result of selforganization of the entirety of all molecules available in the cell nucleus, a general question remains open as to what extent chromatin organization might additionally be predetermined by the DNA sequence and, if so, if there are characteristic differences that distinguish typical regions involved in dysfunction-related aberrations from normal ones, since typical DNA breakpoint regions involved in disease-related chromosome aberrations are not randomly distributed along the DNA sequence. Methods: Highly conserved k-mer patterns in intronic and intergenic regions have been reported in eukaryotic genomes. In this article, we search and analyze regions deviating from average spectra (ReDFAS) of k-mer word frequencies in the human genome. This includes all assembled regions, e.g., telomeric, centromeric, genic as well as intergenic regions. Results: A positive correlation between k-mer spectra and 3D contact frequencies, obtained exemplarily from given Hi-C datasets, has been found indicating a relation of ReDFAS to chromatin organization and interactions. We also searched and found correlations of known functional annotations, e.g., genes correlating with ReDFAS. Selected regions known to contain typical breakpoints on chromosomes 9 and 5 that are involved in cancerrelated chromosomal aberrations appear to be enriched in ReDFAS. Since transposable elements like ALUs are often assigned as major players in 3D genome organization, we also studied their impact on our examples but could not find a correlation between ALU regions and breakpoints comparable to ReDFAS. Conclusions: Our findings might show that ReDFAS are associated with instable regions of the genome and regions with many chromatin contacts which is in line with current research indicating that chromatin loop anchor points lead to genomic instability. [ABSTRACT FROM AUTHOR]

The specific characteristics of k-mer words (2 ≤ k ≤ 11) regarding genomic distribution and evolutionary conservation were recently found. Among them are, in high abundance, words with a tandem repeat structure (repeat unit length of 1 bp to 3 bp). Furthermore, there seems to be a class of extremely short tandem repeats (≤12 bp), so far overlooked, that are non-random-distributed and, therefore, may play a crucial role in the functioning of the genome. In the following article, the positional distributions of these motifs we call super-short tandem repeats (SSTRs) were compared to other functional elements, like genes and retrotransposons. We found length- and sequence-dependent correlations between the local SSTR density and G+C content, and also between the density of SSTRs and genes, as well as correlations with retrotransposon density. In addition to many general interesting relations, we found that SINE Alu has a strong influence on the local SSTR density. Moreover, the observed connection of SSTR patterns to pseudogenes and -exons might imply a special role of SSTRs in gene expression. In summary, our findings support the idea of a special role and the functional relevance of SSTRs in the genome. [ABSTRACT FROM AUTHOR]

Dinucleotides are known as determinants for various structural and physiochemical properties of DNA and for binding affinities of proteins to DNA. These properties (e.g., stiffness) and bound proteins (e.g., transcription factors) are known to influence important biological functions, such as transcription regulation and 3D chromatin organization. Accordingly, the question arises of how the considerable variations in dinucleotide contents of eukaryotic chromosomes could still provide consistent DNA properties resulting in similar functions and 3D conformations. In this work, we investigate the hypothesis that coupled dinucleotide contents influence DNA properties in opposite directions to moderate each other's influences. Analyzing all 2478 chromosomes of 155 eukaryotic species, considering bias from coding sequences and enhancers, we found sets of correlated and anti-correlated dinucleotide contents. Using computational models, we estimated changes of DNA properties resulting from this coupling. We found that especially pure A/T dinucleotides (AA, TT, AT, TA), known to influence histone positioning and AC/GT contents, are relevant moderators and that, e.g., the Roll property, which is known to influence histone affinity of DNA, is preferably moderated. We conclude that dinucleotide contents might indirectly influence transcription and chromatin 3D conformation, via regulation of histone occupancy and/or other mechanisms. [ABSTRACT FROM AUTHOR]

We aim to establish a rough first prospect on the potential of certain biorelevant solvents (water, ammonia, and methane) being present in liquid form inside the uppermost few meters of several modeled rocky and icy surfaces of hypothetical bodies orbiting active galactic nuclei (AGNs) and investigate under which constraints this might occur. For this, we adjust and average X-ray spectra from a sample of 20 Type-1 Seyfert galaxies to calculate the mean snowline of the sample used. We then vary the hypothetical body's orbit between 10% and 100% of the snowline radius and calculate a sub-surface attenuation within four different model surface compositions for each. We then use this as a continuous source term for a thermal model. Example bodies are systematically investigated with sizes between 1/30 and 20 earth radii, with further variations also considered (such as possible bound rotation), to end up with a perspective of solvent phases under a wide slew of different conditions. We find that liquid solvents are possible under a multitude of parameters, with temperature being the main constraint to liquid water whereas body size and pressure are the main constraint to liquid methane and ammonia. [ABSTRACT FROM AUTHOR]

Featured Application: The article is presenting simulations for tidal accelerations in multi-body planetary systems under the precondition of having only few physical and orbital parameters experimentally available. Tidal acceleration vectors were calculated under missing most orbital parameters for the solar system as calibration standard and the TRAPPIST-1 system as an extrasolar example. Volcanism powered by tidal forces inside celestial bodies can provide enough energy to keep important solvents for living systems in the liquid phase. A prerequisite to calculate such tidal interactions and consequences is depending on simulations for tidal accelerations in a multi-body system. Unfortunately, from measurements in many extrasolar planetary systems, only few physical and orbital parameters are well-known enough for investigated celestial bodies. For calculating tidal acceleration vectors under missing most orbital parameter exactly, a simulation method is developed that is only based on a few basic parameters, easily measurable even in extrasolar planetary systems. Such a method as the one presented here allows finding a relation between the tidal acceleration vectors and potential heating inside celestial objects. Using the values and results of our model approach to our solar system as a "gold standard" for feasibility allowed us to classify this heating in relation to different forms of volcanism. This "gold standard" approach gave us a classification measure for the relevance of tidal heating in other extrasolar systems with a reduced availability of exact physical parameters. We help to estimate conditions for the identification of potential candidates for further sophisticated investigations by more complex established methods such as viscoelastic multi-body theories. As a first example, we applied the procedures developed here to the extrasolar planetary system TRAPPIST-1 as an example to check our working hypothesis. [ABSTRACT FROM AUTHOR]

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair. [ABSTRACT FROM AUTHOR]

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. Bymeans of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors inmembranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. [ABSTRACT FROM AUTHOR]

Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements—dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA–DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets. [ABSTRACT FROM AUTHOR]

In genome analysis, k-mer-based comparison methods have become standard tools. However, even though they are able to deliver reliable results, other algorithms seem to work better in some cases. To improve k-mer-based DNA sequence analysis and comparison, we successfully checked whether adding positional resolution is beneficial for finding and/or comparing interesting organizational structures. A simple but efficient algorithm for extracting and saving local k-mer spectra (frequency distribution of k-mers) was developed and used. The results were analyzed by including positional information based on visualizations as genomic maps and by applying basic vector correlation methods. This analysis was concentrated on small word lengths (1 ⩽ k ⩽ 4) on relatively small viral genomes of Papillomaviridae and Herpesviridae, while also checking its usability for larger sequences, namely human chromosome 2 and the homologous chromosomes (2A, 2B) of a chimpanzee. Using this alignment-free analysis, several regions with specific characteristics in Papillomaviridae and Herpesviridae formerly identified by independent, mostly alignment-based methods, were confirmed. Correlations between the k-mer content and several genes in these genomes have been found, showing similarities between classified and unclassified viruses, which may be potentially useful for further taxonomic research. Furthermore, unknown k-mer correlations in the genomes of Human Herpesviruses (HHVs), which are probably of major biological function, are found and described. Using the chromosomes of a chimpanzee and human that are currently known, identities between the species on every analyzed chromosome were reproduced. This demonstrates the feasibility of our approach for large data sets of complex genomes. Based on these results, we suggest k-mer analysis with positional resolution as a method for closing a gap between the effectiveness of alignment-based methods (like NCBI BLAST) and the high pace of standard k-mer analysis. [ABSTRACT FROM AUTHOR]

Several strongly conserved DNA sequence patterns in and between introns and intergenic regions (IIRs) consisting of short tandem repeats (STRs) with repeat lengths <3 bp have already been described in the kingdom of Animalia. In this work, we expanded the search and analysis of conserved DNA sequence patterns to a wider range of eukaryotic genomes. Our aims were to confirm the conservation of these patterns, to support the hypothesis on their functional constraints and/or the identification of unknown patterns. We pairwise compared genomic DNA sequences of genes, exons, CDS, introns and intergenic regions of 34 Embryophyta (land plants), 30 Protista and 29 Fungi using established k-mer-based (alignment-free) comparison methods. Additionally, the results were compared with values derived for Animalia in former studies. We confirmed strong correlations between the sequence structures of IIRs spanning over the entire domain of Eukaryotes. We found that the high correlations within introns, intergenic regions and between the two are a result of conserved abundancies of STRs with repeat units ≤2 bp (e.g., (AT)n). For some sequence patterns and their inverse complementary sequences, we found a violation of equal distribution on complementary DNA strands in a subset of genomes. Looking at mismatches within the identified STR patterns, we found specific preferences for certain nucleotides stable over all four phylogenetic kingdoms. We conclude that all of these conserved patterns between IIRs indicate a shared function of these sequence structures related to STRs. [ABSTRACT FROM AUTHOR]

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10–20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site. [ABSTRACT FROM AUTHOR]

In this study, we pairwise-compared multiple genome regions, including genes, exons, coding DNA sequences (CDS), introns, and intergenic regions of 39 Animalia genomes, including Deuterostomia (27 species) and Protostomia (12 species), by applying established k-mer-based (alignment-free) comparison methods. We found strong correlations between the sequence structure of introns and intergenic regions, individual organisms, and within wider phylogenetical ranges, indicating the conservation of certain structures over the full range of analyzed organisms. We analyzed these sequence structures by quantifying the contribution of different sets of DNA words to the average correlation value by decomposing the correlation coefficients with respect to these word sets. We found that the conserved structures within introns, intergenic regions, and between the two were mainly a result of conserved tandem repeats with repeat units ≤ 2 bp (e.g., (AT)n), while other conserved sequence structures, such as those found between exons and CDS, were dominated by tandem repeats with repeat unit sizes of 3 bp in length and more complex DNA word patterns. We conclude that the conservation between intron and intergenic regions indicates a shared function of these sequence structures. Also, the similar differences in conserved structures with known origin, especially to the conservation between exons and CDS resulting from DNA codons, indicate that k-mer composition-based functional properties of introns and intergenic regions may differ from those of exons and CDS. [ABSTRACT FROM AUTHOR]