A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g.

Tau reduction affects excitatory and inhibitory neurons differently, reduces excitation/inhibition ratios, and counteracts network hypersynchrony.

The protein tau has been implicated in many brain disorders. In animal models, tau reduction suppresses epileptogenesis of diverse causes and ameliorates synaptic and behavioral abnormalities in various conditions associated with excessive excitation-inhibition (E/I) ratios. However, the underlying mechanisms are unknown.

Biomarkers of nucleic acid oxidation - A summary state-of-the-art.

Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA.

A Contemporary Treatment of an Iatrogenic Root Perforation: A Case Report.

Pulp canal calcification is 1 of the possible outcomes after certain types of dental trauma. This can make endodontic treatment more challenging should it become necessary. Because of the increased degree of difficulty, sometimes procedural incidents do occur during root canal treatment.

Early neuronal accumulation of DNA double strand breaks in Alzheimer's disease.

The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer's disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

Background: Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells.

Myosin II activity is required for structural plasticity at the axon initial segment.

In neurons, axons possess a molecularly defined and highly organised proximal region - the axon initial segment (AIS) - that is a key regulator of both electrical excitability and cellular polarity. Despite existing as a large, dense structure with specialised cytoskeletal architecture, the AIS is surprisingly plastic, with sustained alterations in neuronal activity bringing about significant alterations to its position, length or molecular composition. However, although the upstream activity-dependent signalling pathways that lead to such plasticity have begun to be elucidated, the downstream mechanisms that produce structural changes at the AIS are completely unknown.

Evaluating Tools for Live Imaging of Structural Plasticity at the Axon Initial Segment.

The axon initial segment (AIS) is a specialized neuronal compartment involved in the maintenance of axo-dendritic polarity and in the generation of action potentials. It is also a site of significant structural plasticity-manipulations of neuronal activity and can produce changes in AIS position and/or size that are associated with alterations in intrinsic excitability. However, to date all activity-dependent AIS changes have been observed in experiments carried out on fixed samples, offering only a snapshot, population-wide view of this form of plasticity.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out.

A role for Ca1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions.

Rapid Modulation of Axon Initial Segment Length Influences Repetitive Spike Firing.

Neurons implement a variety of plasticity mechanisms to alter their function over timescales ranging from seconds to days. One powerful means of controlling excitability is to directly modulate the site of spike initiation, the axon initial segment (AIS). However, all plastic structural AIS changes reported thus far have been slow, involving days of neuronal activity perturbation.

Calcineurin signaling mediates activity-dependent relocation of the axon initial segment.

The axon initial segment (AIS) is a specialized neuronal subcompartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarization produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we used dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signaling pathways underlying activity-dependent relocation of the AIS.

Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined.

Immuno-slot blot assay for detection of UVR-mediated DNA damage.

Solar ultraviolet radiation (UVR), through the formation of DNA photolesions, is the primary cause of most skin cancers. A better understanding of the mechanisms of UVR-induced DNA damage may help prevent skin cancer and this may be achieved using methods to quantify DNA damage. The immuno-slot blot (ISB) method is routinely used for detection and quantification of any heat- and alkali-stable DNA adducts for which a sufficiently specific monoclonal antibody is available.

Rapid measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in human biological matrices using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Interaction of reactive oxygen species with DNA results in a variety of modifications, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which has been extensively studied as a biomarker of oxidative stress. Oxidative stress is implicated in a number of pathophysiological processes relevant to obstetrics and gynecology; however, there is a lack of understanding as to the precise role of oxidative stress in these processes. We aimed to develop a rapid, validated assay for the accurate quantification of 8-oxodG in human urine using solid-phase extraction and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and then investigate the levels of 8-oxodG in several fluids of interest to obstetrics and gynecology.

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids.

A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported.

Associations between functional polymorphisms in antioxidant defense genes and urinary oxidative stress biomarkers in healthy, premenopausal women.

Functional polymorphisms in endogenous antioxidant defense genes including manganese superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPX-1) have been linked with risk of cancer at multiple sites. Although it is presumed that these germline variants impact disease risk by altering the host's ability to detoxify mutagenic reactive oxygen species, very few studies have directly examined this hypothesis. Concentrations of 8-isoprostane F2α (8-iso-PGF2α) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoxdG)-sensitive indicators of lipid peroxidation and DNA oxidation, respectively-were measured in 24-h urine samples obtained from 93 healthy, premenopausal women participating in a dietary intervention trial.

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood.

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative.

The alternative splice variant of protein tyrosine kinase 6 negatively regulates growth and enhances PTK6-mediated inhibition of β-catenin.

Protein tyrosine kinase 6 (PTK6), also called breast tumor kinase (BRK), is expressed in epithelial cells of various tissues including the prostate. Previously it was shown that PTK6 is localized to epithelial cell nuclei in normal prostate, but becomes cytoplasmic in human prostate tumors. PTK6 is also primarily cytoplasmic in the PC3 prostate adenocarcinoma cell line.

Non-invasive assessment of oxidatively damaged DNA: liquid chromatography-tandem mass spectrometry analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures.

Salvage of oxidized guanine derivatives in the (2'-deoxy)ribonucleotide pool as source of mutations in DNA.

Recent evidence suggests that salvage of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanine (8-oxoGua) can contribute substantially to levels of 8-oxoGua in DNA and RNA. However, it remains to be determined if this mechanism contributes to mutagenesis and disease. This review covers the predominant methods for detecting 8-oxoGua and its derivatives, summarizes some of the relevant recent DNA repair studies and discusses the mechanisms for metabolism of oxidized guanine derivatives in the (2'-deoxy)ribonucleoside and (2'-deoxy)ribonucleotide pools.

DNA repair and the origins of urinary oxidized 2'-deoxyribonucleosides.

Monitoring oxidative stress in vivo is made easier by the ability to use samples obtained non-invasively, such as urine. The analysis of DNA oxidation, by measurement of oxidized 2'-deoxyribonucleosides in urine, particularly 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), has been reported extensively in the literature in many situations relating to various pathologies, populations and environmental exposures. Understanding the origins of urinary 8-oxodG, other than it simply being a marker of DNA oxidation or its synthetic precursors, is important to being able to effectively interpret differences in baseline urinary 8-oxodG levels between subject groups and changes in excretion.

Recommendations for standardized description of and nomenclature concerning oxidatively damaged nucleobases in DNA.

The field of oxidative stress, and the study of oxidatively damaged DNA, in particular, is a subject of intense, and growing interest. This has, in part, benefited from the availability of kits from commercial suppliers which are advertised as reporting on markers of oxidative stress. Such widespread use has inevitably led to an increase in the number of concerns, amongst experts in the field, editors and referees, over appropriateness of terminology and methodology.

Analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by liquid chromatography-tandem mass spectrometry.

The ability to non-invasively monitor DNA oxidation and its repair has significant utility in large-scale, population-based studies. Such studies could include assessments of the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or inter-individual differences in antioxidant defence and DNA repair. The analysis of urine, or indeed any extracellular matrix, for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic or immunoassay procedures, is by far the most popular method to non-invasively assess oxidative insult to the genome.