Mitochondrial DNA integrity and function are critical for endothelium-dependent vasodilation in rats with metabolic syndrome.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Improvements in identification and quantitation of alkylated PAHs and forensic ratio sourcing.

Parent and alkylated polycyclic aromatic hydrocarbons (PAHs) are present in a number of different sources in varying proportions depending on the source material and weathering. This range of PAH sources can make it difficult to determine the origin of exposure(s). Ratios of alkylated and parent PAHs have been applied as a forensic tool to distinguish between different sources. However, few studies have examined PAH ratios comprehensively as indicators for sourcing beyond a single study area or matrix type. In this paper, we introduce an expanded analytical method based on ASTM D7363-13a which we adapted for a gas chromatography triple quadrupole mass spectrometry instrument. The modifications increase selectivity and sensitivity compared to the ASTM method. We added five alkylated series to the method. This method has then been applied to 22 independent forensic ratios. We evaluated the method and the forensic ratios with certified reference materials and known environmental samples. This analytical method and thirteen PAH ratios were found to accurately predict sources of PAHs.

Enhanced Mitochondrial DNA Repair Resuscitates Transplantable Lungs Donated After Circulatory Death.

BACKGROUND: Transplantation of lungs procured after donation after circulatory death (DCD) is challenging because postmortem metabolic degradation may engender susceptibility to ischemia-reperfusion (IR) injury. Because oxidative mitochondrial DNA (mtDNA) damage has been linked to endothelial barrier disruption in other models of IR injury, here we used a fusion protein construct targeting the DNA repair 8-oxoguanine DNA glycosylase-1 (OGG1) to mitochondria (mtOGG1) to determine if enhanced repair of mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of lung procurement after DCD. MATERIALS AND METHODS: Lungs excised from donor rats 1 h after cardiac death were cold stored for 2 h after which they were perfused ex vivo in the absence and presence of mt-OGG1 or an inactive mt-OGG1 mutant. Lung endothelial barrier function and mtDNA integrity were determined during and at the end of perfusion, respectively. RESULTS AND CONCLUSIONS: Mitochondria-targeted OGG1 attenuated indices of lung endothelial dysfunction incurred after a 1h post-mortem period. Oxidative lung tissue mtDNA damage as well as accumulation of proinflammatory mtDNA fragments in lung perfusate, but not nuclear DNA fragments, also were reduced by mitochondria-targeted OGG1. A repair-deficient mt-OGG1 mutant failed to protect lungs from the adverse effects of DCD procurement. CONCLUSIONS: These findings suggest that endothelial barrier dysfunction in lungs procured after DCD is driven by mtDNA damage and point to strategies to enhance mtDNA repair in concert with EVLP as a means of alleviating DCD-related lung IR injury.

A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure.

Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10-12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6-12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution.

PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Development of quantitative screen for 1550 chemicals with GC-MS.

With hundreds of thousands of chemicals in the environment, effective monitoring requires high-throughput analytical techniques. This paper presents a quantitative screening method for 1550 chemicals based on statistical modeling of responses with identification and integration performed using deconvolution reporting software. The method was evaluated with representative environmental samples. We tested biological extracts, low-density polyethylene, and silicone passive sampling devices spiked with known concentrations of 196 representative chemicals. A multiple linear regression (R2 = 0.80) was developed with molecular weight, logP, polar surface area, and fractional ion abundance to predict chemical responses within a factor of 2.5. Linearity beyond the calibration had R2 > 0.97 for three orders of magnitude. Median limits of quantitation were estimated to be 201 pg/µL (1.9× standard deviation). The number of detected chemicals and the accuracy of quantitation were similar for environmental samples and standard solutions. To our knowledge, this is the most precise method for the largest number of semi-volatile organic chemicals lacking authentic standards. Accessible instrumentation and software make this method cost effective in quantifying a large, customizable list of chemicals. When paired with silicone wristband passive samplers, this quantitative screen will be very useful for epidemiology where binning of concentrations is common. Graphical abstract A multiple linear regression of chemical responses measured with GC-MS allowed quantitation of 1550 chemicals in samples such as silicone wristbands.

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.

Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage.

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion.

Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers marked cytoprotection in cultured cells and a variety of injury models. In this study, we measured the effect of EndoIII, a fusion protein construct that traffics Endonuclease III, a DNA glycosylase/AP lyase, to the mitochondria, on infarct size in a rat model of myocardial ischemia/reperfusion. Open-chest, anesthetized rats were subjected to 30 min of occlusion of a coronary artery followed by 2 h of reperfusion. An intravenous bolus of EndoIII, 8 mg/kg, just prior to reperfusion reduced infarct size from 43.8 ± 1.4% of the risk zone in control animals to 24.0 ± 1.3% with no detectable hemodynamic effect. Neither EndoIII's vehicle nor an enzymatically inactive EndoIII mutant (K120Q) offered any protection. The magnitude of EndoIII's protection was comparable to that seen with the platelet aggregation inhibitor cangrelor (25.0 ± 1.8% infarction of risk zone). Because loading with a P2Y12 receptor blocker to inhibit platelets is currently the standard of care for treatment of acute myocardial infarction, we tested whether EndoIII could further reduce infarct size in rats treated with a maximally protective dose of cangrelor. The combination reduced infarct size to 15.1 ± 0.9% which was significantly smaller than that seen with either cangrelor or EndoIII alone. Protection from cangrelor but not EndoIII was abrogated by pharmacologic blockade of phosphatidylinositol-3 kinase or adenosine receptors indicating differing cellular mechanisms. We hypothesized that EndoIII protected the heart from spreading necrosis by preventing the release of proinflammatory fragments of mitochondrial DNA (mtDNA) into the heart tissue. In support of this hypothesis, an intravenous bolus at reperfusion of deoxyribonuclease I (DNase I) which should degrade any DNA fragments escaping into the extracellular space was as protective as EndoIII. Furthermore, the combination of EndoIII and DNase I produced additive protection. While EndoIII would maintain mitochondrial integrity in many of the ischemic cardiomyocytes, DNase I would further prevent mtDNA released from those cells that EndoIII could not save from propagating further necrosis. Thus, our mtDNA hypothesis would predict additive protection. Finally to demonstrate the toxicity of mtDNA, isolated hearts were subjected to 15 min of global ischemia. Infarct size doubled when the coronary vasculature was filled with mtDNA fragments during the period of global ischemia. To our knowledge, EndoIII and DNase are the first agents that can both be given at reperfusion and add to the protection of a P2Y12 blocker, and thus should be effective in today's patient with acute myocardial infarction.

Dilution Effect of Intra-articular Injection Administered After Knee Arthroscopy.

The hypothesis was that agents delivered intra-articularly after knee arthroscopy will be diluted by residual arthroscopic fluid. Diagnostic arthroscopy was performed on six cadaver knees. Each procedure was followed by an intra-articular injection of a dye solution. Intra-articular aspirates were gathered from three locations. With significance set at p < .05, the aspirates were compared with the initial dye concentration and with each other. No significant difference was noted among the sites, indicating that no specific knee area was exposed to a higher dye concentration. There was a significant difference in dye concentration of the aspirates when compared with the dye's initial concentration. The concentration of fluid injected intra-articularly after arthroscopy was diluted by 27%. These data indicate that agents injected into the knee postarthroscopy are significantly diluted. In vitro and in vivo experiments evaluating chondrotoxicity of various anesthetic agents may not accurately reflect the actual concentration of the drug within the knee joint unless dilution effects are taken into account.

Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

The role of mitochondrial reactive oxygen species in cartilage matrix destruction.

Upregulation of matrix metalloproteinases (MMPs) is a hallmark of osteoarthritis progression; along with the role reactive oxygen species (ROS) may play in this process. Moreover, mitochondrial DNA damage and dysfunction are also present in osteoarthritic chondrocytes. However, there are no studies published investigating the direct relationship between mitochondrial ROS, mitochondrial DNA damage, and MMP expression. Therefore, the purpose of the present study was to evaluate whether mitochondrial DNA damage and mitochondrial-originated oxidative stress modulates matrix destruction through the upregulation of MMP protein levels. MitoSox red was utilized to observe mitochondrial ROS production while a Quantitative Southern blot technique was conducted to analyze mitochondrial DNA damage. Additionally, Western blot analysis was used to determine MMP protein levels. The results of the present study show that menadione augmented mitochondrial-generated ROS and increased mitochondrial DNA damage. This increase in mitochondrial-generated ROS led to an increase in MMP levels. When a mitochondrial ROS scavenger was added, there was a subsequent reduction in MMP levels. These studies reveal that mitochondrial integrity is essential for maintaining the cartilage matrix by altering MMP levels. This provides new and important insights into the role of mitochondria in chondrocyte function and its potential importance in therapeutic approaches.

Strategies for Recruiting Older Black Men into Aging and Alzheimer's Research.

BACKGROUND: Despite their high risks for Alzheimer's disease, older Black men are minimally represented in Alzheimer's research and clinical trials. The absence of older Black men in Alzheimer's research limits our ability to characterize the changes associated with cognitive impairments in older Black men-a key health disparity concern. METHODS: Drawing on lessons we learned from years of community-based participatory research in Newark, NJ, we highlight recruitment strategies developed alongside community partners to guide our enrollment and retention efforts for Black men. RESULTS: We identified seven recruitment strategies: provide indirect health education through social programming, target older men through the younger men in their lives, go beyond Black churches, use older Black men as trained community ambassadors, enlist the women in Black men's lives, frame research participation as a legacy to leave their sons, and use past and current Black men participants as role models. CONCLUSIONS: These recruitment strategies help us address many barriers to recruiting older Black men. They can be easily implemented by researchers conducting aging and brain health research or interested in working with older Black men and under-represented populations.

Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures.

River regulation and recruitment in a protracted-spawning riverine fish.

We present and test an extension of the "match/mismatch" hypothesis that attempts to explain the persistence, under conditions of flow alteration, of small, short-lived, native, riverine, fish species. The premise is that flow alteration typically changes environmental conditions, such as temperature and prey abundance, which may affect survival during the larval period of fishes. This "window-of-opportunity hypothesis" states that, if optimal conditions for recruitment vary temporally within a year, the probability that a proportion of the larvae of protracted-spawning species will encounter a period of optimal conditions is greater than for larvae with only a brief spawning period, and so the former will have a recruitment advantage. We determined whether all hatching events contributed equally to juvenile recruitment of the protracted-spawning Australian smelt (Retropinna semoni) during one breeding season in three pairs of heavily regulated and largely free-flowing unregulated rivers in the Murray-Darling Basin, Australia, and related patterns in the hatch dates of recruits to temperature or prey biomass for one pair. For all rivers, heavily regulated or not, recruits present at the end of the breeding season most commonly hatched in the latter part of the breeding season. Mortality of those fish hatched in the first part of the season likely explains this trend. Furthermore, while hatching times were similar for all rivers, each river showed a distinct pattern of hatching and recruitment, which may relate to the temperature range within which epigenetic processes are aligned. Patterns of zooplankton biomass differed between the largely free-flowing ovens and regulated Goulburn rivers and likely had different sources: within the channel and within the storage lake, respectively. For the Ovens River, recruits hatched subsequent to the period when the first significant increase in zooplankton biomass occurred. We hypothesize that temperature may largely influence the "window" during which recruitment can take place but that prey density, responding to river-specific interactions between temperature and discharge, plays a role in the timing and magnitude of recruitment of Australian smelt. We conclude that the match/mismatch hypothesis may be applicable to rivers, that the window-of-opportunity hypothesis has some currency and deserves further investigation, and that river regulation may have significant impacts on fish recruitment.

An analytical investigation of 24 oxygenated-PAHs (OPAHs) using liquid and gas chromatography-mass spectrometry.

We developed two independent approaches for separation and quantitation of 24 oxygenated polycyclic aromatic hydrocarbons (OPAHs) using both liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry (LC-APCI/MS) and gas chromatography-electron impact/mass spectrometry (GC-EI/MS). Building on previous OPAH research, we examined laboratory stability of OPAHs, improved existing method parameters, and compared quantification strategies using standard addition and an internal standard on an environmental sample. Of 24 OPAHs targeted in this research, 19 compounds are shared between methods, with 3 uniquely quantitated by GC-EI/MS and 2 by LC-APCI/MS. Using calibration standards, all GC-EI/MS OPAHs were within 15 % of the true value and had less than 15 % relative standard deviations (RSDs) for interday variability. Similarly, all LC-APCI/MS OPAHs were within 20 % of the true value and had less than 15 % RSDs for interday variability. Instrument limits of detection ranged from 0.18 to 36 ng mL(-1) on the GC-EI/MS and 2.6 to 26 ng mL(-1) on the LC-APCI/MS. Four standard reference materials were analyzed with each method, and we report some compounds not previously published in these materials, such as perinaphthenone and xanthone. Finally, an environmental passive sampling extract from Portland Harbor Superfund, OR was analyzed by each method using both internal standard and standard addition to compensate for potential matrix effects. Internal standard quantitation resulted in increased precision with similar accuracy to standard addition for most OPAHs using 2-fluoro-fluorenone-(13)C as an internal standard. Overall, this work improves upon OPAH analytical methods and provides some considerations and strategies for OPAHs as focus continues to expand on this emerging chemical class.

Microsatellite instability in non-endometrioid ovarian epithelial tumors: a study of 400 cases comparing immunohistochemistry, PCR, and NGS based testing with mutation status of MMR genes.

Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D)/microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, 6 cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in 2 cases, the mutation was germline (Lynch syndrome). An additional 5 cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other nonendometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.

The effects of day-to-day variability of physiological data on operator functional state classification.

The application of pattern classification techniques to physiological data has undergone rapid expansion. Tasks as varied as the diagnosis of disease from magnetic resonance images, brain-computer interfaces for the disabled, and the decoding of brain functioning based on electrical activity have been accomplished quite successfully with pattern classification. These classifiers have been further applied in complex cognitive tasks to improve performance, in one example as an input to adaptive automation. In order to produce generalizable results and facilitate the development of practical systems, these techniques should be stable across repeated sessions. This paper describes the application of three popular pattern classification techniques to EEG data obtained from asymptotically trained subjects performing a complex multitask across five days in one month. All three classifiers performed well above chance levels. The performance of all three was significantly negatively impacted by classifying across days; however two modifications are presented that substantially reduce misclassifications. The results demonstrate that with proper methods, pattern classification is stable enough across days and weeks to be a valid, useful approach.

Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs.

In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.