Maxim Ignatov

Although ionizing radiation quality shapes the spatial structure and complexity of primary DNA lesions, its contribution relative to absorbed dose in the late DNA damage response (DDR) remains poorly quantified. We analyzed γH2AX and pATM foci kinetics in human mesenchymal stromal/stem cells exposed to ⁶⁰Co γ-rays, 150 MeV protons, and 14.1 MeV neutrons at doses of 0.25, 0.5, 1.0, and 2.0 Gy, delivered at dose rates of 0.5 Gy/min for γ-rays, 1 Gy/min for protons, and 46 mGy/min for neutrons, respectively. An effective kinetic model was used to describe the time course of the DDR and incorporated an ultraslow component as a model-based index of long-lasting repair burden. Radiation types differed less in early signal amplitude than in decay rate and late persistence. At every investigated dose, the normalized ultraslow fraction followed the order γ-rays < protons < neutrons. This ordering was preserved in per-dose fits, while parametric bootstrapping supported the γ–neutron separation, although the absolute values of the internal fractions were only partially identifiable. RAD51, analyzed independently from model fitting, showed the same qualitative ordering at late time points, supporting the interpretation that radiation quality is more associated with persistent DDR signaling than absorbed dose within the investigated range. These findings suggest that absorbed dose alone may not fully characterize the late cellular response to different radiation qualities and highlight the importance of radiation quality in comparative radiobiology, radiation protection, and the interpretation of normal-tissue responses to therapeutic irradiation.

The growing use of computed tomography (CT) in medicine requires a better understanding of how low-dose radiation affects human stem cells. This study investigated the long-term consequences of CT-level radiation on the secretory profile of human adipose-derived mesenchymal stem cells (AD-MSCs). AD-MSCs were exposed to radiation regimens simulating a single or multiple head CT scans, or to a single 2 Gy therapeutic dose, and their secretion of 41 cytokines, chemokines, and growth factors was monitored during long-term culture. At the early passage, AD-MSCs receiving a single 2 Gy dose showed a coordinated increase in several lymphocyte-regulating cytokines compared to cells exposed to multiple CT scans. However, these initial differences were not sustained. Long-term culturing led to a progressive and widespread decrease in the secretion of 26 cytokines, chemokines, and growth factors across all groups. By the latest passage, all irradiated cells showed a generalized reduction in secretory function compared to non-irradiated controls. These findings demonstrate that while different radiation regimens trigger distinct immediate responses, long-term culture results in a broad decline of the AD-MSCs secretome, which is accentuated by prior radiation exposure. This underscores the importance of assessing long-term consequences to fully evaluate the functional impact of diagnostic radiation on stem cells.

Цель работы: сравнительный анализ дозовых зависимостей и кинетик пострадиационных изменений количества фокусов белков репарации ДНК (γH2AX и pATM) в мезенхимальных стромальных клетках человека (МСК), подвергшихся воздействию 150 МэВ протонов или гамма-излучения кобальта-60. Материалы и методы. В работе использовали первичную культуру МСК человека, полученную из коллекции ООО "БиолоТ" (Россия). Облучение клеток проводили в МРНЦ им. А.Ф. Цыба – филиале ФГБУ «НМИЦ радиологии» Минздрава России на комплексе протонной терапии «Прометеус» (ФТЦ ФИАН, Россия) пучком 150 МэВ протонов на «пролет» и гамма-терапевтическом аппарате «РОКУС-АМ» (кобальт-60). Для количественной оценки фокусов γН2АХ и pAТМ использовали иммуноцитохимическое окрашивание с использованием антител к γН2АХ и pAТМ, соответственно. Результаты: Показано, что в случае воздействия гамма-излучения количественный выход фокусов на единицу поглощенной дозы составил 18.56±2.64 и 12.58±1.52 фокуса/Гр для белков γH2AX и pATM, соответственно. Количественный выход фокусов на единицу поглощенной дозы после воздействия 150 МэВ протонов составил 19.83±2.07 и 10.79±1.51 фокуса/Гр для белков γH2AX и pATM, соответственно. Проведенные исследования не выявили статистически значимых различий между киниками пострадиационных изменений количества фокусов γН2АХ и pATM в МСК, облученных в дозе 0.5 Гр 150 МэВ протонами или гамма-излучением кобальта-60 Заключение: Сравнительный анализ дозовых зависимостей и кинетик пострадиационных изменений количества фокусов белков репарации ДНК γH2AX и pATM в МСК человека, подвергшихся воздействию 150 МэВ протонов «на пролет» и гамма-излучения кобальта-60 не выявил статически значимых различий между эффектами этих типов излучения. Полученные результаты свидетельствуют о том, что ОБЭ 150 МэВ протонного излучения «на пролет» по отношению к гамма-излучения кобальта-60 для МСК человека равна ~ 1.0. Иммуноцитохимический анализ фокусов белков репарации ДНК представляется весьма перспективным для изучения особенной клеточного отклика на ДНК повреждающее действие ионизирующих излучений с различными физическими характеристиками и биологической микродозиметрии.

An open question in radiobiology concerns whether low doses of radiation are harmful or if cells are able to tolerate such exposure with minimal or no disruption. This issue is relevant for evaluating public health risks associated with the increasing number of medical computed tomography (CT) diagnostic procedures. This study evaluated the impact of CT scan-level exposure on human adipose mesenchymal stem cells (hMSCs) by measuring DNA damage responses (γH2AX, 53BP1, pATM foci), proliferation (Ki-67), senescence (β-galactosidase), and multiple gene expressions. Responses to one or five CT exposures were compared to a 2 Gy X-ray dose at intervals from 1 h to 10 passages post-irradiation. It was shown that CT scan briefly increased DNA damage markers but showed no significant long-term effects. A high dose of 2 Gy X-ray exposure caused sustained DNA damage, decreased proliferation, increased senescence, and significant changes in hundreds of genes even after several cell generations. After a single CT exposure, gene expression changes were minimal, while high-dose exposure led to strong activation of DNA repair and stress response pathways. Five CT scans caused a slight activation of LIF and HSPA1B genes, but these effects were minor compared to the high-dose group. All detected effects from CT scans were not observed by ten cell passages, whereas high-dose effects persisted. In conclusion, typical CT scan exposures have only short-term, mild effects on hMSCs, while high-dose radiation causes lasting cellular and genetic changes.

Prime editing is a promising approach for correcting pathogenic variants, but its efficiency remains variable across genomic contexts. Here, we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region, 4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first, the persistent challenge of AT-rich target sequence correction even with optimized editors, and second, the performance of 4-FEN suggests its potential value for other genomic targets.

The effects of low-dose radiation exposure remain a controversial topic in radiation biology. This study compares early (0.5, 4, 24, 48, and 72 h) and late (5, 10, and 15 cell passages) post-irradiation changes in γH2AX, 53BP1, pATM, and p-p53 (Ser-15) foci, proliferation, autophagy, and senescence in primary fibroblasts exposed to 100 and 2000 mGy X-ray radiation. The results show that exposure to 100 mGy significantly increased γH2AX, 53BP1, and pATM foci only at 0.5 and 4 h post irradiation. There were no changes in p-p53 (Ser-15) foci, proliferation, autophagy, or senescence up to 15 passages post irradiation at the low dose.

DNA repair (DNA damage) foci observed 24 h and later after irradiation are called “residual” in the literature. They are believed to be the repair sites for complex, potentially lethal DNA double strand breaks. However, the features of their post-radiation dose-dependent quantitative changes and their role in the processes of cell death and senescence are still insufficiently studied. For the first time in one work, a simultaneous study of the association of changes in the number of residual foci of key DNA damage response (DDR) proteins (γH2AX, pATM, 53BP1, p-p53), the proportion of caspase-3 positive, LC-3 II autophagic and SA-β-gal senescent cells was carried out 24–72 h after fibroblast irradiation with X-rays at doses of 1–10 Gy. It was shown that with an increase in time after irradiation from 24 h to 72 h, the number of residual foci and the proportion of caspase-3 positive cells decrease, while the proportion of senescent cells, on the contrary, increases. The highest number of autophagic cells was noted 48 h after irradiation. In general, the results obtained provide important information for understanding the dynamics of the development of a dose-dependent cellular response in populations of irradiated fibroblasts.

In experiments on R-irradiated female F1 mice (CBA × C57Bl), the possibility of using rapid tests for the number of double-stranded DNA breaks (γH2AX) in the spleen and micronuclei in polychromatophilic erythrocytes (MN-PCE) of the bone marrow for an approximate evaluation of the effectiveness of antiradiation agents was investigated. Cytokine betaleukin, α-adrenomimetic indraline, and nucleoside riboxin were tested. By the DRF criterion, γH2AX showed a higher sensitivity compared to MN-PCE. Between the two indicators, a significant degree of correlation was revealed.