Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response

We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR

ROS-Induced DNA Damage Associates with Abundance of Mitochondrial DNA in White Blood Cells of the Untreated Schizophrenic Patients

Objective. The aim of this study was (1) to examine the leukocyte mtDNA copy number (CN) in unmedicated (SZ (m−)) and medicated (SZ (m+)) male patients with paranoid schizophrenia (SZ) in comparison with the healthy male controls (HC) and (2) to compare the leukocyte mtDNA CN with the content of an oxidation marker 8-oxodG in lymphocytes of the SZ (m−) patients. Methods. We evaluated leukocyte mtDNA CN of 110 subjects with SZ in comparison with 60 male HC by the method qPCR (ratio mtDNA/nDNA (gene B2M) was detected). SZ patients were divided into two subgroups. The patients of the subgroups SZ (m+) (N=55) were treated with standard antipsychotic medications in the hospital. The patients of the subgroup SZ (m−) (N=55) were not treated before venous blood was sampled. To evaluate oxidative DNA damage, we quantified the levels of 8-oxodG in lymphocytes (flow cytometry) of SZ (m−) patients (N=55) and HC (N=30). Results. The leukocyte mtDNA CN showed no significant difference in SZ (m+) patients and HC. The mtDNA CN in the unmedicated subgroup SZ (m−) was significantly higher than that in the SZ (m+) subgroup or in HC group. The level of 8-oxodG in the subgroup SZ (m−) was significantly higher than that in HC group. Conclusion. The leukocytes of the unmedicated SZ male patients with acute psychosis contain more mtDNA than the leukocytes of the male SZ patients treated with antipsychotic medications or the healthy controls. MtDNA content positively correlates with the level of 8-oxodG in the unmedicated SZ patients

Global public perceptions of genomic data sharing: what shapes the willingness to donate DNA and health data?

Analyzing genomic data across populations is central to understanding the role of genetic factors in health and disease. Successful data sharing relies on public support, which requires attention to whether people around the world are willing to donate their data that are then subsequently shared with others for research. However, studies of such public perceptions are geographically limited and do not enable comparison. This paper presents results from a very large public survey on attitudes toward genomic data sharing. Data from 36,268 individuals across 22 countries (gathered in 15 languages) are presented. In general, publics across the world do not appear to be aware of, nor familiar with, the concepts of DNA, genetics, and genomics. Willingness to donate one's DNA and health data for research is relatively low, and trust in the process of data's being shared with multiple users (e.g., doctors, researchers, governments) is also low. Participants were most willing to donate DNA or health information for research when the recipient was specified as a medical doctor and least willing to donate when the recipient was a for-profit researcher. Those who were familiar with genetics and who were trusting of the users asking for data were more likely to be willing to donate. However, less than half of participants trusted more than one potential user of data, although this varied across countries. Genetic information was not uniformly seen as different from other forms of health information, but there was an association between seeing genetic information as special in some way compared to other health data and increased willingness to donate. The global perspective provided by our "Your DNA, Your Say" study is valuable for informing the development of international policy and practice for sharing genomic data. It highlights that the research community not only needs to be worthy of trust by the public, but also urgent steps need to be taken to authentically communicate why genomic research is necessary and how data donation, and subsequent sharing, is integral to this

Таргетная терапия при муковисцидозе

The basic therapy of cystic fibrosis is currently aimed at slowing down the pathological processes associated with a decrease in the CFTR protein activity (cystic fibrosis transmembrane conductance regulator) in the gastrointestinal tract and the respiratory system. The pancreatic insufficiency is well compensated by replacement therapy with microsphere enzyme preparations and a high-calorie diet rich in proteins and fat. Chronic treatment of cystic fibrosis-related lung disease aims to improve the clearance of the bronchial tree, suppressing chronic bacterial infection and local chronic inflammation. However, no therapy was available to correct the defect in the gene or its product until 2012.The aim was to analyze literature on CFTR modulators, including their efficacy and safety, and assess the potential for developing new modulators to treat cystic fibrosis.Materials. The review included literature data (45 publications) on the use of CFTR modulators and international websites’ data.Results. Since the discovery of the CFTR gene in 1989, more than 2000 mutations or variants of the CFTR gene (hereinafter referred to as genetic variants) have been described. They interfere with the synthesis of the CFTR protein, its transport to the apical membrane of the cell, or disrupt its function as a channel for chloride anions. Although it is currently not possible to completely replace the mutant gene with a normal copy, small molecules have been identified that can modify the mutant CFTR protein and amend its function. The potential therapeutic measures are determined by class of the mutation. In clinical practice, pharmacological modeling of ion transport is currently possible only with the use of CFTR modulators: correctors and potentiators. The review defines these groups of drugs and describes 4 licensed CFTR modulators, including molecules of ivacaftor, lumacaftor, tezacaftor, elexacaftor. The data on the promising emerging next generation modulators and the prospects for the personalized selection of drugs using the assays on intestinal organoids are presented.Базисная терапия муковисцидоза (МВ) направлена в настоящее время на замедление патологических процессов, связанных со снижением активности белка CFTR (муковисцидозного трансмембранного регулятора проводимости) в желудочно-кишечном тракте и респираторной системе. Панкреатическая недостаточность хорошо компенсируется заместительной терапией микросферическими ферментами, потреблением высококалорийной, богатой белками и жирами диетой. Постоянное лечение болезни легких, обусловленной МВ, нацелено на улучшение клиренса бронхиального дерева, подавление хронической бактериальной инфекции и местного хронического воспаления. Однако до 2012 г. терапия, исправляющая дефект гена или его продукта, не разработаны.Целью обзора явился анализ данных литературы по применению модуляторов CFTR по их эффективности и безопасности, а также перспективы создания новых модуляторов для терапии МВ.Материалы и методы. Использованы данные литературы (45 статей) по применению CFTR-модуляторов, а также данные международных сайтов.Результаты. Со времени открытия в 1989 г. гена CFTR описано > 2 000 мутаций или вариантов нуклеотидной последовательности гена CFTR (генетические варианты), препятствующие синтезу белка CFTR, его транспорту к апикальной мембране клетки или нарушающие его функцию в качестве канала анионов хлора. Хотя в настоящее время полная замена мутантного гена нормальной копией невозможна, идентифицированы малые молекулы, способные модифицировать мутантный белок CFTR таким образом, что его функция становится близкой к нормальной. Возможность терапевтических мероприятий определяется классом мутации. В клинической практике фармакологическое моделирование ионного транспорта возможно в настоящее время только с использованием модуляторов CFTR – корректоров и потенциаторов.Зключение. В обзоре приводятся определение этих групп препаратов и описание 4 зарегистрированных СFTR-модуляторов, включающих молекулы ивакфтор, лумакофтор, тезакафтор, элексакафтор. Приведены данные о перспективных разработках модуляторов следующего поколения и перспективы персонализированного подбора препаратов с использованием технологии кишечных органоидов

Satellite III (1q12) Copy Number Variation in Cultured Human Skin Fibroblasts from Schizophrenic Patients and Healthy Controls

Background: The chromosome 1q12 region harbors the genome’s largest pericentromeric heterochromatin domain that includes tandemly repeated satellite III DNA [SatIII (1)]. Increased SatIII (1) copy numbers have been found in cultured human skin fibroblasts (HSFs) during replicative senescence. The aim of this study was to analyze the variation in SatIII (1) abundance in cultured HSFs at early passages depending on the levels of endogenous and exogenous stress. Methods: We studied 10 HSF cell lines with either high (HSFs from schizophrenic cases, n = 5) or low (HSFs from healthy controls, n = 5) levels of oxidative stress. The levels of endogenous stress were estimated by the amounts of reactive oxygen species, DNA damage markers (8-hydroxy-2′-deoxyguanosine, gamma-H2A histone family member X), pro- and antioxidant proteins (NADPH oxidase 4, superoxide dismutase 1, nuclear factor erythroid 2-related factor 2), and proteins that regulate apoptosis and autophagy (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein, light chain 3). SatIII (1) copy numbers were measured using the nonradioactive quantitative hybridization technique. For comparison, the contents of telomeric and ribosomal RNA gene repeats were determined. RNASATIII (1 and 9) were quantified using quantitative Polymerase Chain Reaction (PCR). Results: Increased SatIII (1) contents in DNA from confluent HSFs were positively correlated with increased oxidative stress. Confluent cell cultivation without medium replacement and heat shock induced a decrease of SatIII (1) in DNA in parallel with a decrease in RNASATIII (1) and an increase in RNASATIII (9). Conclusions: During HSF cultivation, cells with increased SatIII (1) content accumulated in the cell pool under conditions of exaggerated oxidative stress. This fraction of cells decreased after the additional impact of exogenous stress. The process seems to be oscillatory

Effect of Low-Dose Ionizing Radiation on the Expression of Mitochondria-Related Genes in Human Mesenchymal Stem Cells

The concept of hormesis describes a phenomenon of adaptive response to low-dose ionizing radiation (LDIR). Similarly, the concept of mitohormesis states that the adaptive program in mitochondria is activated in response to minor stress effects. The mechanisms of hormesis effects are not clear, but it is assumed that they can be mediated by reactive oxygen species. Here, we studied effects of LDIR on mitochondria in mesenchymal stem cells. We have found that X-ray radiation at a dose of 10 cGy as well as oxidized fragments of cell-free DNA (cfDNA) at a concentration of 50 ng/mL resulted in an increased expression of a large number of genes regulating the function of the mitochondrial respiratory chain complexes in human mesenchymal stem cells (MSC). Several genes remained upregulated within hours after the exposure. Both X-ray radiation and oxidized cfDNA resulted in upregulation of FIS1 and MFN1 genes, which regulated fusion and fission of mitochondria, within 3–24 h after the exposure. Three hours after the exposure, the number of copies of mitochondrial DNA in cells had increased. These findings support the hypothesis that assumes oxidized cell-free DNA as a mediator of MSC response to low doses of radiation

Genome instability in MCF-7 cells exposed to gDNA^OX at final concentration 50 ng/mL for 24 hours.

<div><p>A – multiple micronuclei [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>], chromatin bridges [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>], M-phase chromatin decondensation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>], non-treated control cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B4" target="_blank">4</a>] (x100). </p> <p>B – proportions of cells with micronuclei in non-treated control cells, cells exposed to gDNA, cells exposed to gDNA<b>^OX</b>. Grey columns: non-confluent, actively proliferating MCF-7 culture. Black columns: MCF-7 cells at high confluency. *p < 0.05 against control group of cells, non-parametric U-test.</p> <p>С - Exposure to gDNA<b>^OX</b> (50 ng/mL, 2 hours) induces formation of 8-oxodG-containing micronuclei (x100). </p></div

The exposure to gDNA^OX leads to an increase in the production of ROS.

<p>А – Microscopy-based evaluation of MCF-7 cells sequentially treated with DNA (50 ng/mL) and H2DCFH-DA (control, gDNA, gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]) and incubated for 30 minutes (x100). Alternatively, MCF-7 cells were incubated with DNA (50 ng/mL) for 1 hour followed by addition of H2DCFH-DA and photography 30 minutes later (gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]). B - MCF-7 cells exposed to gDNA^ox (0.5h; 50ng/mL), were sequentially treated with Mito-tracker TMRM (15 min) and H2DCFH-DA (15 min) (x200). C - Co-detection of labeled probe gDNA^red (50 ng/mL) and DCF after 30 minutes of incubation. D - The results of the quantification of fluorescence using plate reader [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. The time kinetics of fluorescence outputs in cells sequentially treated with H2DCFH-DA and, three minutes later, a DNA sample at final concentration of 5 or 50 ng/mL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. The same for cells pretreated with DNA (final concentration 5 ng/mL) for one hour, with subsequent addition of H2DCFH-DA. *) p < 0.05 against control group of cells, non-parametric U-test.</p