Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle.

Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells.

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7-14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Diversion of the Arbuzov reaction: alkylation of C-Cl instead of phosphonic ester formation on the fullerene cage.

We report an "inversed" Arbuzov reaction of the fullerene derivatives C60Ar5Cl with trialkyl phosphites P(OR)3 producing alkylated fullerene derivatives C60Ar5R (R = Me, Et, iPr, nBu) with almost quantitative yields. This reaction provides a convenient synthetic route for the preparation of a large variety of functionalized fullerene derivatives with tailored properties, e.g. water-soluble compounds demonstrating promising antiviral activities against HCMV, HSV1, HIV and several influenza virus strains.

GC-Rich DNA Fragments and Oxidized Cell-Free DNA Have Different Effects on NF-kB and NRF2 Signaling in MSC.

It has been established that cell-free DNA circulating in the bloodstream affects cells. The characteristics of cfDNA depend on the physiological state of the organism. As we showed previously, diseases can cause either GC-enrichment of the cell-free DNA pool or its oxidation. Thus, in cases of cerebral atherosclerosis, heart attack and rheumatic arthritis the cell-free DNA pool is GC-enriched and, in the case of cancer, both GC-enriched and oxidized. Herein we investigated the time-dependent effect of oxidized and GC-rich cell-free DNA on NF-kB and NRF2 signaling pathways in human mesenchymal stem cells and showed that they affect cells in different ways. Oxidized DNA drastically increases expression of NRF2 in a short period of time, but the effect does not last long. GC-rich DNA causes a prolonged increase in mRNA levels of NF-kB and NRF2 which lasts 48 and 24 h, respectively.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls.

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients' blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia.

Oxidized Cell-Free DNA Is a Factor of Stress Signaling in Radiation-Induced Bystander Effects in Different Types of Human Cells.

In pathology or under damaging conditions, the properties of cell-free DNA (cfDNA) change. An example of such change is GC enrichment, which drastically alters the biological properties of cfDNA. GC-rich cfDNA is a factor of stress signaling, whereas genomic cfDNA is biologically inactive. GC-rich cfDNA stimulates TLR9-MyD88-NF-κB signaling cascade, leading to an increase in proinflammatory cytokine levels in the organism. In addition, GC-rich DNA is prone to oxidation and oxidized cfDNA can stimulate secondary oxidative stress. This article is a review of works dedicated to the investigation of a low-dose ionizing radiation effect, a bystander effect, and the role of cfDNA in both of these processes.

Extracellular DNA Containing (dG)n Motifs Penetrates into MCF7 Breast Cancer Cells, Induces the Adaptive Response, and Can Be Expressed.

BACKGROUND: Oxidized human DNA or plasmid DNAs containing human ribosomal genes can easily penetrate into the breast cancer cells MCF7 and stimulate the adaptive response induction. Plasmid DNA containing a CMV promoter, gene EGFP, and the insertion of the human ribosomal genes can be expressed. A hypothesis is proposed: these features of the ribosomal DNA are due to the presence of dGn motifs that are prone to oxidize. METHODS: Cells of MCF7 line were cultured with plasmids which contained a CMV promoter and gene of fluorescent protein EGFP. Genetic construction pEGFP-Gn contains pEGFP vector and a small insertion with dG11 and dG13 motifs that are inclined to oxidation. The accumulation of pEGFP and pEGFP-Gn in MCF7 (qPCR), the levels of ROS in the cells, the content of 8-oxodG in plasmids and cellular DNA (flow cytometry, immunoassay, and fluorescent microscopy), the expression of NOX4 and EGFP, the localization of NOX4 and EGFP in MCF7 (qPCR, flow cytometry, and fluorescent microscopy), and the levels of the cell DNA damage (comet assay) were analyzed. RESULTS: (dG)n insertions in the plasmid pEGFP increase the levels of ROS, the cell DNA oxidation and DNA damage, and the level of transfection of plasmid into the MCF7 cells. NOX4 participates in the oxidation of pEGFP-Gn and pEGFP. The expression of EGFP gene in MCF7 is significantly increased in case of pEGFP-Gn. Stimulation of ROS synthesis (H2O2 40 µM or 10 cGy IR) increases the level of expression of EGFP. CONCLUSIONS: GC-rich DNA fragments containing dGn motifs that are inclined to oxidation penetrate into MCF7 cancer cells, stimulate the adaptive response, and can be expressed. This property of GC-rich cell-free DNA should be considered and/or could potentially be used in therapy of tumors.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome.

Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types.

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.