Despite the long held hypothesis that oxidant stress results in accumulated oxidative damage to cellular macromolecules and subsequently to aging and age-related chronic disease, it has been difficult to consistently define and specifically identify markers of oxidant stress that are consistently and directly linked to age and disease status. Inflammation because it is also linked to oxidant stress, aging, and chronic disease also plays an important role in understanding the clinical implications of oxidant stress and relevant markers. Much attention has focused on identifying specific markers of oxidative stress and inflammation that could be measured in easily accessible tissues and fluids (lymphocytes, plasma, serum). The purpose of this review is to discuss markers of oxidant stress used in the field as biomarkers of aging and age-related diseases, highlighting differences observed by race when data is available. We highlight DNA, RNA, protein, and lipid oxidation as measures of oxidative stress, as well as other well-characterized markers of oxidative damage and inflammation and discuss their strengths and limitations. We present the current state of the literature reporting use of these markers in studies of human cohorts in relation to age and age-related disease and also with a special emphasis on differences observed by race when relevant.

A tandem repeat’s (TR) propensity to mutate increases with repeat number, and can become very pronounced beyond a critical boundary, transforming it into a microsatellite (MS). However, a clear understanding of the mutational behavior of different TR classes and motifs and related mechanisms is lacking, as is a consensus on the existence of a boundary separating short TRs (STRs) from MSs. This hinders our understanding of MSs’ mutational properties and their effective use as genetic markers. Using indel calls for 179 individuals from 1000 Genomes Pilot-1 Project, we determined polymorphism incidence for four major TR classes, and formalized its varying relationship with repeat number using segmented regression. We observed a biphasic regime with a transition from a faster to a slower exponential growth at 9, 5, 4, and 4 repeats for mono-, di-, tri-, and tetranucleotide TRs, respectively. We used an in vitro mutagenesis assay to evaluate the contribution of strand slippage errors to mutability. STRs and MSs differ in their absolute polymorphism levels, but more importantly in their rates of mutability growth. Although strand slippage is a major factor driving mononucleotide polymorphism incidence, dinucleotide polymorphism incidence is greater than that expected due to strand slippage alone, indicating that additional cellular factors might be driving dinucleotide mutability in the human genome. Leveraging on hundreds of human genomes, we present the first comprehensive, genome-wide analysis of TR mutational behavior, encompassing several motif sizes and compositions.

Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.

Oxidative DNA damage accumulates with age and is repaired primarily via the base excision repair (BER) pathway. This process is initiated by DNA glycosylases, which remove damaged bases in a substrate-specific manner. The DNA glycosylases human 8-oxoguanine-DNA glycosylase (OGG1) and NEIL1, a mammalian homolog of Escherichia coli endonuclease VIII, have overlapping yet distinct substrate specificity. Recently, we reported that OGG1 binds to the Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein that poly(ADP-ribosyl)ates nuclear proteins in response to DNA damage and other cellular signals. Here, we show that NEIL1 and PARP-1 bind both in vitro and in vivo. PARP-1 binds to the C-terminal-100 amino acids of NEIL1 and NEIL1 binds to the BRCT domain of PARP-1. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. Furthermore, NEIL-deficient fibroblasts have impaired poly(ADP-ribosyl)ation of cellular proteins after DNA damage, which can be rescued by NEIL1 expression. Additionally, PARP-1 inhibits NEIL1 incision activity in a concentration-dependent manner. Consistent with the idea of impaired DNA repair during aging, we observed differential binding of PARP-1 to recombinant NEIL1 in older mice compared to younger mice. These data further support the idea that dynamic interplay between different base excision repair proteins is important for efficient BER.

Brain tissues from Alzheimer’s Disease (AD) patients show increased levels of oxidative DNA damage and 7,8-dihydro-8-oxoguanine (8-oxoG) accumulation. In humans, the base excision repair protein 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme that recognizes and excises the mutagenic DNA base lesion 8-oxoG. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. We characterized the A53T and A288V polymorphic variants and detected a significant reduction in the catalytic activity for both proteins in vitro and in cells. Additionally, the A53T polymorphism has decreased substrate binding, while the A288V polymorphism has reduced AP lyase activity. Both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. We found that OGG1−/− cells expressing A53T and A288V OGG1 were significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein binding activities that could be related to the increase in oxidative damage to DNA found in AD brains.

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC).APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ␤-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADPribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ␤-catenin destruction complex, placing the APC2/ TNKS interaction at the correct intracellular location to regulate ␤-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ␤-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.The Wnt pathway helps direct a myriad of normal developmental and adult homeostasic processes in metazoans, but is also misregulated in several human diseases such as cancer (1, 2). Wnt signaling is regulated through the activity of a multiprotein "destruction complex" that promotes proteolysis of the transcriptional co-activator ␤-catenin (␤cat) 3 by stimulating phosphorylation of the ␤cat phosphodegron (3). Core components of the destruction complex include the scaffold protein Axin, the tumor suppressor adenomatous polyposis coli (APC), and the kinases CK1 and GSK3.While Wnt signaling plays essential roles during development, it is inappropriately activated in a number of cancers, most notably colorectal cancer. Truncating mutations in the tumor suppressor adenomatous polyposis coli (APC) are the initiating mutational event in more than 80% of all colon cancer cases, and these mutations hyperactivate ␤cat signaling (4). Thus small molecule inhibitors of the Wnt pathway should provide an effective therapeutic strategy. Among these strategies, inhibition of oncogenic ␤cat activity would appear to be the most direct approach, as studies have demonstrated that the accumulation of ␤cat is what initiates oncogenesis, and that tumors have a continued reliance on oncogenic ␤cat signaling (5). Indeed, recent promising antagonists have been identified that specifically disrupt ␤cat binding to TCF or th...

It is well accepted that oxidative DNA repair capacity, oxidative damage to DNA and oxidative stress play central roles in aging and disease development. However, the correlation between oxidative damage to DNA, markers of oxidant stress and DNA repair capacity is unclear. In addition, there is no universally accepted panel of markers to assess oxidative stress in humans. Our interest is oxidative damage to DNA and its correlation with DNA repair capacity and other markers of oxidative stress. We present preliminary data from a small comet study that attempts to correlate single strand break (SSB) level with single strand break repair capacity (SSB-RC) and markers of oxidant stress and inflammation. In this limited study of four very small age-matched 24-individual groups of male and female whites and African-Americans aged 30–64 years, we found that females have higher single strand break (SSB) levels than males (p=0.013). There was a significant negative correlation between SSB-RC and SSB level (p=0.041). There was a positive correlation between SSBs in African American males with both heme degradation products (p=0.008) and high-sensitivity C-reactive protein (hs-CRP) (p=0.022). We found a significant interaction between hs-CRP and sex in their effect on residual DNA damage (p=0.002). Red blood cell reduced glutathione concentration was positively correlated with the levels of oxidized bases detected by endonuclease III (p=0.047), heme degradation products (p=0.015) and hs-CRP (p=0.020). However, plasma carbonyl levels showed no significant correlation with other markers. The data from the literature and from our very limited study suggest a complex relationship between measures of oxidative stress and frequently used clinical parameters believed to reflect inflammation or oxidative stress.

Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I interferon. One of several upstream receptor cGAS (cyclic-GMP-AMP synthase) binds to cytosolic DNA and generates di-cyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to Stimulator of Interferon Genes (STING). STING recruits TANK binding kinase 1 (TBK1) which acts as a critical node that allows for efficient activation of interferon regulatory factors (IRFs) to drive the anti-viral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine rich repeat containing protein (NLR) that negatively regulates the type I interferon pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I interferons. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two hybrid screening. Here we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3:STING interaction in the cytosol of human epithelial cells. Furthermore, knock down of IQGAP1 in THP1 and HeLa cells causes significantly more interferon-β production in response to cytosolic nucleic acids. This result phenocopies NLRC3 deficient macrophages and fibroblasts and shRNA knock down of NLRC3 in THP1 cells. Our findings suggest IQGAP1 is a novel regulator of type I interferon production possibly via interacting with NLRC3 in human monocytic and epithelial cells.