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    <title language="eng">Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part - Structuromics</title>
    <parentTitle language="eng">Critical Reviews in Eukaryotic Gene Expression</parentTitle>
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    <author>Martin Falk</author>
    <author>Michael Hausmann</author>
    <author>Emilie Lukasova</author>
    <author>Abin Biswas</author>
    <author>Georg Hildenbrand</author>
    <author>Marie Davidkova</author>
    <author>Evgeny Krasavin</author>
    <author>Zdenek Kleibl</author>
    <author>Iva Falkova</author>
    <author>Lucie Jezkova</author>
    <author>Lenka Stefancikova</author>
    <author>Jan Sevcik</author>
    <author>Michal Hofer</author>
    <author>Alena Bacikova</author>
    <author>Pavel Matula</author>
    <author>Alla Boreyko</author>
    <author>Jana Vachelova</author>
    <author>Anna Michaelidesova</author>
    <author>Stanislav Kozubek</author>
    <subject>
      <language>deu</language>
      <type>swd</type>
      <value>Radiologie</value>
    </subject>
    <collection role="institutes" number="">Medical Engineering and Data Science (Bachelor)</collection>
    <collection role="forschungsschwerpunkte" number="">Material Testing &amp; Sensor Technology</collection>
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    <id>2397</id>
    <completedYear>2022</completedYear>
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    <language>eng</language>
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    <issue>1</issue>
    <volume>14</volume>
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    <title language="eng">Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells</title>
    <abstract language="eng">(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).</abstract>
    <parentTitle language="eng">Pharmaceutics</parentTitle>
    <identifier type="issn">1999-4923</identifier>
    <identifier type="doi">https://doi.org/10.3390/pharmaceutics14010166</identifier>
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    <author>Lucie Dobešová</author>
    <author>Theresa Gier</author>
    <author>Olga Kopecna</author>
    <author>Eva Pagáčová</author>
    <author>Tomáš Vičar</author>
    <author>Felix Bestvater</author>
    <author>Jiří Toufar</author>
    <author>Alena Bačíková</author>
    <author>Pavel Kopel</author>
    <author>Radek Fedr</author>
    <author>Georg Hildenbrand</author>
    <author>Iva Falková</author>
    <author>Martin Falk</author>
    <author>Michael Hausmann</author>
    <subject>
      <language>deu</language>
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      <value>Strahlentherapie</value>
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    <subject>
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      <value>Krebs, Medizin</value>
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    <subject>
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      <value>Tumorzelle</value>
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    <subject>
      <language>deu</language>
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      <value>Gold</value>
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    <subject>
      <language>deu</language>
      <type>swd</type>
      <value>Nanopartikel</value>
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    <edition/>
    <issue>12</issue>
    <volume>19</volume>
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    <title language="eng">Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy</title>
    <abstract language="eng">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>
    <parentTitle language="eng">International Journal of Molecular Sciences</parentTitle>
    <identifier type="issn">1422-0067</identifier>
    <identifier type="doi">https://doi.org/10.3390/ijms19123713</identifier>
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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\u201320 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\u00b0) 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. 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    <author>Martin Falk</author>
    <author>Michael Hausmann</author>
    <author>Emilie Lukasova</author>
    <author>Abin Biswas</author>
    <author>Georg Hildenbrand</author>
    <author>Marie Davidkova</author>
    <author>Evgeny Krasavin</author>
    <author>Zdenek Kleibl</author>
    <author>Iva Falkova</author>
    <author>Lucie Jezkova</author>
    <author>Lenka Stefancikova</author>
    <author>Jan Sevcik</author>
    <author>Michal Hofer</author>
    <author>Alena Bacikova</author>
    <author>Pavel Matula</author>
    <author>Alla Boreyko</author>
    <author>Jana Vachelova</author>
    <author>Anna Michaelidesova</author>
    <author>Stanislav Kozubek</author>
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    <collection role="institutes" number="">Medical Engineering and Data Science (Bachelor)</collection>
    <collection role="forschungsschwerpunkte" number="">Material Testing &amp; Sensor Technology</collection>
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