Cell-free DNA as a biomarker in stroke: Current status, problems and perspectives.

There is currently no proposed stroke biomarker with consistent application in clinical practice. A number of studies have examined cell-free DNA (cfDNA), which circulates in biological fluids during stroke, as a potential biomarker of this disease. The data available suggest that dynamically-determined levels of blood cfDNA may provide new prognostic information for assessment of stroke severity and outcome. However, such an approach has its own difficulties and limitations. This review covers the potential role of cfDNA as a biomarker in stroke, and includes evidence from both animal models and clinical studies, protocols used to analyze cfDNA, and hypotheses on the origin of cfDNA.

Oxidized extracellular DNA as a stress signal that may modify response to anticancer therapy.

An increase in the levels of oxidation is a universal feature of genomic DNA of irradiated or aged or even malignant cells. In case of apoptotic death of stressed cells, oxidized DNA can be released in circulation (cfDNA). According to the results of the studies performed in vitro by our group and other researchers, the oxidized cfDNA serves as a biomarker for a stress and a stress signal that is transmitted from the "stressed" area i.e. irradiated cells or cells with deficient anti-oxidant defenses to distant (bystander) cells. In recipient cells, oxidized DNA stimulates biosynthesis of ROS that is followed up by an increase in the number of single strand and double strand breaks (SSBs and DSBs), and activation of DNA Damage Response (DDR) pathway. Effects of oxidized DNA are considered similar to that of irradiation. It seems that downstream effects of irradiation, in part, depend on the release of oxidized DNA fragments that mediate the effects in distant cells. The responses of normal and tumor cell to oxidized DNA may differ. It seems that tumor cells are more sensitive to oxidized DNA-dependent DNA damage, while developing pronounced adaptive response. This may suggest that in chemotherapy or irradiation-treated human body, the release of oxidized DNA from dying cancer cells may give a boost to remaining malignant cells by augmenting their survival and stress resistance. Further studies of the effects of oxidized DNA in both in vitro and in vivo systems are warranted.

siRNA technology in kidney transplantation: current status and future potential.

Kidney transplantation is one of the most common transplantation operations in the world, accounting for up to 50 % of all transplantation surgeries. To curtail the damage to transplanted organs that is caused by ischemia-reperfusion injury and the recipient's immune system, small interfering RNA (siRNA) technology is being explored. Importantly, the kidney as a whole is a preferential site for non-specific systemic delivery of siRNA. To date, most attempts at siRNA-based therapy for transplantation-related conditions have remained at the in vitro stage, with only a few of them being advanced into animal models. Hydrodynamic intravenous injection of naked or carrier-bound siRNAs is currently the most common route for delivery of therapeutic constructs. To our knowledge, no systematic screens for siRNA targets most relevant for kidney transplantation have been attempted so far. A majority of researchers have arrived at one or another target of interest by analyzing current literature that dissects pathological processes taking place in transplanted organs. A majority of the genes that make up the list of 53 siRNA targets that have been tested in transplantation-related models so far belong to either apoptosis- or immune rejection-centered networks. There is an opportunity for therapeutic siRNA combinations that may be delivered within the same delivery vector or injected at the same time and, by targeting more than one pathway, or by hitting the same pathways within two different key points, will augment the effects of each other.

Oxidized DNA induces an adaptive response in human fibroblasts.

Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA(OX). The levels of cfDNA(OX) are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by Н2О2in vitro (gDNA(OX)) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA(OX) on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA(OX) evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA(OX) leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of РСNА, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA(OX) and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA(OX) inhibits NF-κB signaling. gDNA(OX) is a model for oxidized cfDNA(OX) that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA released from irradiated cells may be responsible for an abscopal effects and a bystander mediated adaptive response seen in some cancer patients. These results indicate the necessity for the further study of the effects of oxidized DNA in both in vitro and in vivo systems.

Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes.

The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA(R) isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA(R) produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA(R) extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.