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.

• Klíčová slova
• 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,
• Publikační typ
• časopisecké články MeSH

In our work, we developed the synthesis of new polyfunctional pegylated trehalose derivatives and evaluated their cryoprotective effect using flow cytometry. We showed that new compounds (modified trehaloses) bound to appropriate extracellular polymeric cryoprotectants could be helpful as a chemical tool for the evaluation of their potential toxic cell membrane influences. Our aim was to form a chemical tool for the evaluation of cryoprotectant cell membrane influences, which are still not easily predicted during the freezing/thawing process. We combined two basic cryoprotectants: polyethyleneglycols (PEGs) and trehalose in the new chemical compounds-pegylated trehalose hybrids. If PEG and trehalose are chemically bound and trehalose is adsorbed on the cell surface PEGs molecules which are, due to the chemical bonding with trehalose, close to the cell surface, can remove the cell surface hydration layer which destabilizes the cell membrane. This was confirmed by the comparison of new material, PEG, trehalose, and their mixture cryoprotective capabilities.

• Klíčová slova
• click-chemistry, cryoprotection, pegylation,
• MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• click chemie MeSH
• dimethylsulfoxid farmakologie   MeSH
• kryoprezervace MeSH
• kryoprotektivní látky farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polyethylenglykoly chemie   farmakologie   MeSH
• průtoková cytometrie MeSH
• trehalosa analogy a deriváty   chemická syntéza   chemie   farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• zmrazování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dimethylsulfoxid MeSH
• kryoprotektivní látky MeSH
• polyethylenglykoly MeSH
• trehalosa MeSH

From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.

• Klíčová slova
• DNA damage, DNA double strand breaks (DSBs), DNA repair, DNA repair foci, cancer radiotherapy, damage to lysosomes, metal nanoparticles, single-molecule localization microscopy (SMLM), super-resolution microscopy, tumor cell radiosensitization,
• MeSH
• dvouřetězcové zlomy DNA účinky záření   MeSH
• gadolinium chemie   MeSH
• HeLa buňky MeSH
• konfokální mikroskopie MeSH
• kovové nanočástice chemie   terapeutické užití   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• poškození DNA účinky záření   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• gadolinium MeSH
• zlato 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 účinky léků   genetika   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dvouřetězcové zlomy DNA účinky léků   MeSH
• fibroblasty MeSH
• kryoprezervace metody   MeSH
• kryoprotektivní látky farmakologie   MeSH
• kůže cytologie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• S fáze účinky léků   MeSH
• viabilita buněk účinky léků   genetika   MeSH
• zmrazování škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• chromatin MeSH
• dimethylsulfoxid MeSH
• kryoprotektivní látky MeSH

Cryopreservation of cells (mouse embryonic fibroblasts) is a fundamental task for wide range of applications. In practice, cells are protected against damage during freezing by applications of specific cryoprotectants and freezing/melting protocols. In this study by using AFM and fluorescence microscopy we showed how selected cryoprotectants (dimethyl sulfoxide and polyethylene glycol) affected the cryopreserved cells mechanical properties (stiffness) and how these parameters are correlated with cytoskeleton damage and reconstruction. We showed how cryopreserved (frozen and thawed) cells' stiffness change according to type of applied cryoprotectant and its functionality in extracellular or intracellular space. We showed that AFM can be used as technique for investigation of cryopreserved cells surfaces state and development ex vivo. Our results offer a new perspective on the monitoring and characterization of frozen cells recovery by measuring changes in elastic properties by nanoindentation technique. This may lead to a new and detailed way of investigating the post-thaw development of cryopreserved cells which allows to distinguish between different cell parts.

• Klíčová slova
• AFM, DMSO, PEG, cell stiffness, cryopreservation, fluorescence microscopy,
• Publikační typ
• časopisecké články MeSH