Regulation of Precise DNA Repair by Nuclear Actin Polymerization: A Chance for Improving Gene Therapy?

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field.

RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells.

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.

Adaptive and Maladaptive DNA Breaks in Neuronal Physiology and Alzheimer's Disease.

DNA strand breaks excessively accumulate in the brains of patients with Alzheimer's disease (AD). While traditionally considered random, deleterious events, neuron activity itself induces DNA breaks, and these "adaptive" breaks help mediate synaptic plasticity and memory formation. Recent studies mapping the brain DNA break landscape reveal that despite a net increase in DNA breaks in ectopic genomic hotspots, adaptive DNA breaks around synaptic genes are lost in AD brains, and this is associated with transcriptomic dysregulation. Additionally, relationships exist between mitochondrial dysfunction, a hallmark of AD, and DNA damage, such that mitochondrial dysfunction may perturb adaptive DNA break formation, while DNA breaks may conversely impair mitochondrial function. A failure of DNA break physiology could, therefore, potentially contribute to AD pathogenesis.

Toxicological effects of solvent-extracted organic matter associated with PM2.5 on human bronchial epithelial cell line NL-20.

The heterogeneity and complexity of solvent-extracted organic matter associated with PM2.5 (SEOM-PM2.5) is well known; however, there is scarce information on its biological effects in human cells. This work aimed to evaluate the effect of SEOM-PM2.5 collected in northern Mexico City during the cold-dry season (November 2017) on NL-20 cells, a human bronchial epithelial cell line. The SEOM obtained accounted for 15.5% of the PM2.5 mass and contained 21 polycyclic aromatic hydrocarbons (PAHs). The cell viability decreased following exposure to SEOM-PM2.5, and there were noticeable morphological changes such as increased cell size and the presence of cytoplasmic vesicles in cells treated with 5-40 µg/mL SEOM-PM2.5. Exposure to 5 µg/mL SEOM-PM2.5 led to several alterations compared with the control cells, including the induction of double-stranded DNA breaks based (p < 0.001); nuclear fragmentation and an increased mitotic index (p < 0.05); 53BP1 staining, a marker of DNA repair by non-homologous end-joining (p < 0.001); increased BiP protein expression; and reduced ATF6, IRE1α, and PERK gene expression. Conversely, when exposed to 40 µg/mL SEOM-PM2.5, the cells showed an increase in reactive oxygen species formation (p < 0.001), BiP protein expression (p < 0.05), and PERK gene expression (p < 0.05), indicating endoplasmic reticulum stress. Our data suggest concentration-dependent toxicological effects of SEOM-PM2.5 on NL-20 cells, including genotoxicity, genomic instability, and endoplasmic reticulum stress.

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines.

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

Acute exposure and biomarkers assessment in rainbow trout exposed to selected pharmaceuticals.

Pharmaceuticals released from municipal effluents discharges pose a risk to aquatic organisms. The toxicity of 5 pharmaceuticals with distinct therapeutic actions were assessed in rainbow trout: olanzapine (antipsychotic), erythromycin (antibiotic), mycophenoate (immunosuppression), pinaverium (anti-inflammatory) and trazodone (sedative). Juveniles were exposed to these drugs for 96 h at concentrations between 64 µg/L up to 40 mg/L to reach lethality. Survival was determined and a suite of biomarkers was analyzed for drug biotransformation, oxidative stress/damage and metabolic activity at sublethal concentrations. The data revealed the following toxicity: olanzapine >trazodone>mycophenolate>pinaverium∼erythromycin based on mortality. The data also revealed that toxicity was associated to mass, pKa and hydrophobicity and the following sublethal effects: GST, LPO and DNA strand breaks. Pharmaceuticals with lower molecular weight, physiological pKa, moderate hydrophobicity, low biotransformation and DNA strand breaks were generally more toxic to fish. However, this should be considered as a general guide in identifying toxic pharmaceuticals in non-target organisms.

Circulating Cell Free DNA and DNA Double-Strand Breakage in Alzheimer's Disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that is characterized by memory loss and multiple cognitive impairments. AD is pathologically characterized by age-dependent accumulation of amyloid-ß protein and the phosphorylation of tau protein in the brains of patients with AD. Clinically, manifestations of AD include cognitive decline, dementia, alterations of high-order brain functions, and movement disorders. Double-stranded DNA breaks are a lethal form of DNA damage and are typically repaired via non-homologous end joining and homologous recombination. However, in AD brain, repair mechanism is disrupted, leading to a cascade of events, cognitive dysfunction, organ failure and reduced lifespan. Increased circulating cell-free DNA in the blood, cerebrospinal fluid, and urine in patients with AD, can be used as early detectable biomarkers for AD. The purpose of our article is to explore the potential uses of cell-free DNA and double-stranded DNA breaks as prognostic markers for AD and examine the recent research on the application of these markers in studies.

Novel Compound Heterozygous Mutations of LIG4 Gene in an Indian LIG4 Syndrome Patient with Severe Microcephaly: Case Report, In-silico Analysis and Systematic Review.

BACKGROUND: LIG4 syndrome, characterized by immunodeficiency, sensitivity to ionizing radiations, intrauterine growth retardation, postnatal growth retardation, and microcephaly, is a rare genetic disorder caused by pathogenic variants of the LIG4 gene. Few patients are presented with no immune dysregulation as well. CASE STUDY: We present here a male child of 2 years and 4 months of age with severe microcephaly and short stature. His birth weight was 1.9 Kg, and his current height, weight, and head circumference are 83.2 cm (z score = -2.37), 9.5 Kg (z score = -2.76), and 36 cm (z score = -9.24), respectively. Possible causative pathogenic compound heterozygous variants of the LIG4 gene, which were inherited from the parents, were identified by whole exome sequencing of the DNA of the patient and his parents. A systematic review of the literature is also performed to summarize the patients of LIG4 syndrome reported worldwide and summarize the associated genetic mutations of the LIG4 gene. Compound heterozygous variants (c.597_600delTCAG/ c.342del) of LIG4 gene were identified. The parents were found to be heterozygous carriers of one variant each. CONCLUSION: The in-silico analysis of identified variants explains their effect on the structure and function of the LIG4 protein hence explaining the genotype-phenotype correlation.

DNA double-stranded breaks, a hallmark of aging, defined at the nucleotide resolution, are increased and associated with transcription in the cardiac myocytes in LMNA-cardiomyopathy.

AIMS: An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. We posit that DSBs would be more prevalent at the genomic sites that are associated with gene expression. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. METHODS AND RESULTS: We identified the genome-wide DSBs in ∼1 million cardiac myocytes per heart in three wild-type and three myocyte-specific LMNA-deficient (Myh6-Cre:LmnaF/F) mice by END-Sequencing. The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively. The END-Seq signals were enriched for 8 and 6764 DSBs in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively (q < 0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the lamin-associated domains (LADs), we defined the LADs in cardiac myocytes by a Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay (N = 5). On average there were 818 LADs per myocyte. Constitutive LADs (cLADs), defined as LADs that were shared by at least three genomes (N = 2572), comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N = 3975) were ∼16-fold lower than those at the non-LAD regions (N = ∼17 778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of-LAD regions, defined as the genomic regions in the Myh6-Cre:LmnaF/F that were juxtaposed to the LAD regions in the wild-type myocytes. CONCLUSION: To our knowledge, this is the first identification of the DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various states and aging.

Combination treatment of zinc and selenium intervention ameliorated BPA-exposed germ cell damage in SD rats: elucidation of molecular mechanisms.

Bisphenol A (BPA) is a commonly used environmental toxicant, is easily exposed to the human body and causes testicular damage, sperm abnormalities, DNA damage and apoptosis, and interferes in the process spermatogenesis and steroidal hormone production along with obstruction in testes and epididymis development. Zinc (Zn), a potent regulator of antioxidant balance, is responsible for cellular homeostasis, enzymes and proteins activities during spermatogenesis for cell defence mechanisms in the testes. Selenium (Se) is required for spermatogenesis, antioxidant action and in the activities of different selenoproteins. Both Zn and Se are essential simultaneously for the proper regulation of spermatogenesis and sperm maturation as well as protection against chemical and disease-associated germ cell toxicity. Thus, the study aimed to understand the importance and beneficial effect of Zn and Se co-treatment against BPA-exposed testicular damage in rats. BPA 100 and 200 mg/kg/day was exposed through an oral gavage. Zn (3 mg/kg/day) i.p. and Se (0.5 mg/kg/day) i.p. were injected for 8 weeks. The testicular toxicity was evaluated by measuring body and organs weight, biochemical investigations, sperm parameters, testicular and epididymal histopathology, quantification DNA damage by halo assay, DNA breaks (TUNEL assay), immunohistochemistry and western blot. Results revealed that Zn and Se co-treatment ameliorated BPA-associated male gonadal toxicity in rat as revealed by decreased SGPT, SGOT and BUN levels in serum, reduced testes and epididymis tissue injury, DNA breaks, apoptosis, expressions of 8-OHdG, γ-H2AX and NFκB with an increased serum testosterone and catalase levels. These findings suggest that Zn and Se co-treatment could be a beneficial and protective option against BPA-exposed testicular and epididymal toxicity.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Loss of Adaptive DNA Breaks in Alzheimer's Disease Brains.

Background: DNA breaks accumulate in Alzheimer's disease (AD) brains. While their role as true genomic lesions is recognized, DNA breaks also support cognitive function by facilitating the expression of activity-dependent immediate early genes. This process involves TOP2B, a DNA topoisomerase that catalyzes the formation of DNA double-strand breaks. Objective: To characterize how AD impacts adaptive DNA breaks at nervous system genes. Methods: We leveraged the ability of DNA single- and double-strand breaks to activate poly(ADP-ribose) polymerases (PARPs) that conjugate poly(ADP-ribose) (PAR) to adjacent proteins. To characterize the genomic sites harboring DNA breaks in AD brains, nuclei extracted from 3 AD and 3 non-demented autopsy brains (frontal cortex, all male donors, age 78 to 91 years of age) were analyzed through CUT&RUN in which we targeted PAR with subsequent DNA sequencing. Results: Although the AD brains contained 19.9 times more PAR peaks than the non-demented brains, PAR peaks at nervous system genes were profoundly lost in AD brains, and the expression of these genes was downregulated. This result is consistent with our previous CUT&RUN targeting γH2AX, which marks DNA double-strand breaks. In addition, TOP2B expression was significantly decreased in the AD brains. Conclusions: Although AD brains contain a net increase in DNA breaks, adaptive DNA breaks at nervous system genes are lost in AD brains. This could potentially reflect diminished TOP2B expression and contribute to impaired neuron function and cognition in AD patients.

Cellular Alterations Due to Direct and Indirect Interaction of Nanomaterials with Nucleic Acids.

Deoxyribonucleic acid (DNA) represents the main reservoir of genetic information in the cells, which is why it is protected in the nucleus. Entry into the nucleus is, in general, difficult, as the nuclear membrane is a selective barrier to molecules longer than 40 kDa. However, in some cases, the size of certain nanoparticles (NPs) allows their internalization into the nucleus, thus causing a direct effect on the DNA structure. NPs can also induce indirect effects on DNA through reactive oxygen species (ROS) generation. In this context, nanomaterials are emerging as a disruptive tool for the development of novel therapies in a broad range of biomedical fields; although their effect on cell viability is commonly studied, further interactions with DNA or indirect alterations triggered by the internalization of these materials are not always clarified, since the small size of these materials makes them perfectly suitable for interaction with subcellular structures, such as the nucleus. In this context, and using as a reference the predicted interactions presented in a computational model, we describe and discuss the observed direct and indirect effects of the implicated nanomaterials on DNA.

MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks.

BACKGROUND: Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. OBJECTIVES: This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. METHODS: MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. RESULTS: Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. CONCLUSIONS: MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage.

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

DNA strand displacement and TdT-Mediated DNA extension for swift, convenient, and quantitative evaluation of sperm DNA integrity and its clinical implications.

DNA integrity is crucial for the clinical pregnancy outcome and offspring health, while detection methods currently used (comet assay, TUNNEL assay, SCSA, etc.) can only provide the ratio of positive sperms at the cellular level and are unable to quantitatively detect the breakpoints at the DNA molecular level. Herein, we developed a detection system based on terminal deoxynucleotidyl transferase and DNA strand displacement fluorescent probe, which could efficiently and conveniently measure the number of 3'-OH (equivalent to the number of breakpoints). We further investigated the use of this technique in assisted reproduction after completing the principle verification, system optimization, and research on analytical performance. The detection system was shown to have a good linear range from 0.01 nM to 4 nM, using single-stranded DNA with 3'-OH end as the calibrator. The system underwent thorough optimization for stability and accuracy. In comparison to the widely accepted index DFI detected by SCSA, the new system demonstrated reasonable correlation and better prediction efficiency. Its applicability was also proven through its use in assisted reproductive technology procedures.

Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger.

Since the first CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system was developed for creating double-stranded DNA breaks, it has been adapted and improved for different biotechnological applications. In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger. Their technique involved the disruption of selected genes with counter-effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP-dependent DNA helicase II subunit 1 (kusA or ku70)-deletion strain. Multiple copies of the PatE transgene expression cassette were introduced by CRISPR-Cas9-mediated insertion. The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.

Metal Ions Modify In Vitro DNA Damage Yields with High-LET Radiation.

Cu2+ and Co2+ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton-type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu2+ or Co2+ and were irradiated either with X-ray, carbon-ion or iron-ion beams, or they were treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed via gel electrophoresis followed with a band intensity analysis. DNA damage was the greatest when DNA in the solution with either metal was treated with only hydrogen peroxide followed by the DNA damage of DNA in the solution with either metal post irradiation of low-LET (X-Ray) or high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage after treatment with bleomycin. Cu2+ portrayed greater DNA damage than Co2+ following all experimental conditions. The metals' effect caused more DNA damage and was observed to be LET-dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu2+ is more efficient than Co2+ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.

Disturbed Antioxidant Capacity in Patients with Systemic Sclerosis Associates with Lung and Gastrointestinal Symptoms.

The correct balance between reactive oxygen species and antioxidant defense in an organism is disturbed in oxidative stress. To assess oxidative balance in 36 SSc patients and 26 healthy controls (HCs), we measured reactive oxidative metabolites (ROMs), total antioxidant capacity (TAC), lipid peroxidation (measuring 4-HNE), and DNA oxidative damage (measuring 8-OHdG) in serum. Furthermore, DNA breaks in leukocytes of 35 SSc patients and 32 HCs were evaluated using COMET. While we report high ROMs for both SSc patients and age/sex matched HC samples, there was a significant increase in TAC in SSc patients as compared to HCs, and thus also a significantly higher oxidative stress index in SSc patients. TAC was significantly higher in SSc patients with ILD and gastrointestinal involvement, as well as in patients with anti-topoisomerase antibodies. We observe no difference in serum lipid peroxidation status or oxidative DNA damage. However, SSc patients had significantly more leukocyte DNA breaks than HCs; the most damage was observed in patients treated with immunosuppressives. Thus, our study confirms presence of oxidative stress and increased DNA damage in leukocytes of SSc patients; however, it points toward increased antioxidant capacity, which needs to be further studied.

Cytosolic DNA sensing protein pathway is activated in human hearts with dilated cardiomyopathy.

Introduction: The genome is constantly exposed to numerous stressors, which induce DNA lesions, including double-stranded DNA breaks (DSBs). DSBs are the most dangerous, as they induce genomic instability. In response to DNA damage, the cell activates nuclear DNA damage response (DDR) and the cytosolic DNA sensing protein (CDSP) pathways, the latter upon release of the DSBs to the cytosol. The CDSP pathway activates NFκB and IRF3, which induce the expression of the pro-inflammatory genes. There is scant data on the activation of the CDSP pathway in human hearts with dilated cardiomyopathy (DCM). Aim: We aimed to determine expression levels of selected components of the CDSP pathway in human hearts with DCM. Methods: The DNA strand breaks were detected by the single-cell gel electrophoresis or the comet assay and expression of selected proteins by immunoblotting. Transcript levels were quantified in the RNA-Seq data. Results: Single-cell gel electrophoresis showed an approximately 2-fold increase in the number of COMET cells in the DCM hearts. Immunoblotting showed increased levels of cyclic GMP-AMP synthase (CGAS), the canonical CDSP; TANK-binding kinase 1 (TBK1), an intermediary kinase in the pathway; and RELB, P52, and P50 components of the NFκB pathway in human heart samples from patients with DCM. Likewise, transcript levels of over 2 dozen genes involved in inflammatory responses were increased. Conclusions: The findings provide the first set of evidence for the activation of the CDSP pathway in human hearts with DCM. The data in conjunction with the previous evidence of activation of the DDR pathway implicate the DSBs in the pathogenesis of human DCM.