Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

AbstractCells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5′-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3·CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity

Initial Results from the Variable Intensity Sonic Boom Propagation Database

An extensive sonic boom propagation database with low- to normal-intensity booms (overpressures of 0.08 lbf/sq ft to 2.20 lbf/sq ft) was collected for propagation code validation, and initial results and flight research techniques are presented. Several arrays of microphones were used, including a 10 m tall tower to measure shock wave directionality and the effect of height above ground on acoustic level. A sailplane was employed to measure sonic booms above and within the atmospheric turbulent boundary layer, and the sailplane was positioned to intercept the shock waves between the supersonic airplane and the ground sensors. Sailplane and ground-level sonic boom recordings were used to generate atmospheric turbulence filter functions showing excellent agreement with ground measurements. The sonic boom prediction software PCBoom4 was employed as a preflight planning tool using preflight weather data. The measured data of shock wave directionality, arrival time, and overpressure gave excellent agreement with the PCBoom4-calculated results using the measured aircraft and atmospheric data as inputs. C-weighted acoustic levels generally decreased with increasing height above the ground. A-weighted and perceived levels usually were at a minimum for a height where the elevated microphone pressure rise time history was the straightest, which is a result of incident and ground-reflected shock waves interacting

Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008

BACKGROUND: Methylated gene markers have shown promise in predicting breast cancer outcomes and treatment response. We evaluated whether baseline and changes in tissue and serum methylation levels would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer undergoing preoperative chemotherapy. METHODS: The TBCRC008 trial investigated pCR following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel + vorinostat/placebo (n = 62). We measured methylation of a 10-gene panel by quantitative multiplex methylation-specific polymerase chain reaction and expressed results as cumulative methylation index (CMI). We evaluated association between CMI level [baseline, day 15 (D15), and change] and pCR using univariate and multivariable logistic regression models controlling for treatment and hormone receptor (HR) status, and performed exploratory subgroup analyses. RESULTS: In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with 40% lower chance of obtaining pCR (odds ratio, OR 0.60, 95% CI 0.37-0.97; p = 0.037). Subgroup analyses suggested a significant association between tissue D15 CMI levels and pCR in vorinostat-treated [OR 0.44 (0.20, 0.93), p = 0.03], but not placebo-treated patients. CONCLUSION: In this study investigating the predictive roles of tissue and serum CMI levels in patients with early breast cancer for the first time, we demonstrate that high D15 tissue CMI levels may predict poor response. Larger studies and improved analytical procedures to detect methylated gene markers in early stage breast cancer are needed. TBCRC008 is registered on ClinicalTrials.gov (NCT00616967)

The Utility of Napsin-A in the Identification of Primary and Metastatic Lung Adenocarcinoma Among Cytologically Poorly Differentiated Carcinomas.

BACKGROUND: New developments in the treatment of lung cancer have necessitated the further histologic and cytologic subtyping of nonsmall cell lung carcinomas. Thyroid transcription factor-1 (TTF-1) long has served as the predominant marker for demonstrating lung origin. However, it is also expressed in a variety of other tumors, particularly neuroendocrine neoplasms and, to a much lesser degree, squamous cell carcinoma of the lung. Napsin-A, which is expressed in lung tissue, is a relatively new marker for lung adenocarcinoma. In this study, the authors examined the utility of napsin-A compared with TTF-1 in cytologic specimens of primary and metastatic, poorly differentiated lung adenocarcinomas. METHODS: The archives of the Department of Pathology at The Johns Hopkins Hospital were searched for cytologic cases of poorly differentiated lung adenocarcinoma that were histologically confirmed. In total, 75 patients (cases) along with 95 controls were included, each of whom had adequate cell block material for TTF-1 and napsin-A staining. Tissue microarrays of lung adenocarcinoma also were examined. RESULTS: TTF-1 and napsin-A were detected in 61 of 75 cases (81.3%) and in 49 of 75 cases (65.3%), respectively. The sensitivity and specificity of TTF-1 were 81% each; and napsin-A had a greater specificity of 96%, and sensitivity of 65%. Napsin-A was not detected in small cell carcinomas or in other carcinomas of nonlung origin except for renal cell carcinoma. CONCLUSIONS: Although TTF-1 had a higher sensitivity, napsin-A was useful as a surrogate marker when encountering a poorly differentiated lung adenocarcinoma or an unknown primary tumor, particularly in cytologic specimens and difficult cases. The current results indicate that the dual use of both markers may be necessary to improve diagnostic accuracy

Non-invasive PD-L1 quantification using [18F]DK222-PET imaging in cancer immunotherapy

Background Combination therapies that aim to improve the clinical efficacy to immune checkpoint inhibitors have led to the need for non-invasive and early pharmacodynamic biomarkers. Positron emission tomography (PET) is a promising non-invasive approach to monitoring target dynamics, and programmed death-ligand 1 (PD-L1) expression is a central component in cancer immunotherapy strategies. [18F]DK222, a peptide-based PD-L1 imaging agent, was investigated in this study using humanized mouse models to explore the relationship between PD-L1 expression and therapy-induced changes in cancer.Methods Cell lines and xenografts derived from three non-small cell lung cancers (NSCLCs) and three urothelial carcinomas (UCs) were used to validate the specificity of [18F]DK222 for PD-L1. PET was used to quantify anti-programmed cell death protein-1 (PD-1) therapy-induced changes in PD-L1 expression in tumors with and without microsatellite instability (MSI) in humanized mice. Furthermore, [18F]DK222-PET was used to validate PD-L1 pharmacodynamics in the context of monotherapy and combination immunotherapy in humanized mice bearing A375 melanoma xenografts. PET measures of PD-L1 expression were used to establish a relationship between pathological and immunological changes. Lastly, spatial distribution analysis of [18F]DK222-PET was developed to assess the effects of different immunotherapy regimens on tumor heterogeneity.Results [18F]DK222-PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high but variable tumor uptake at 60 min that correlated with PD-L1 expression. In MSI tumors treated with anti-PD-1, [18F]DK222 uptake was higher than in control tumors. Moreover, [18F]DK222 uptake was higher in A375 tumors treated with combination therapy compared with monotherapy, and negatively correlated with final tumor volumes. In addition, a higher number of PD-L1+ cells and higher CD8+-to-CD4+ cell ratio was observed with combination therapy compared with monotherapy, and positively correlated with PET. Furthermore, spatial distribution analysis showed higher [18F]DK222 uptake towards the core of the tumors in combination therapy, indicating a more robust and distinct pattern of immune cell infiltration.Conclusion [18F]DK222-PET has potential as a non-invasive tool for monitoring the effects of immunotherapy on tumors. It was able to detect variable PD-L1 expression in tumors of different cancer types and quantify therapy-induced changes in tumors. Moreover, [18F]DK222-PET was able to differentiate the impact of different therapies on tumors

Frequent Inactivation of Cysteine Dioxygenase Type 1 Contributes to Survival of Breast Cancer Cells and Resistance to Anthracyclines

Genome-wide DNA methylation analyses have identified hundreds of candidate DNA-hypermethylated genes in cancer. Comprehensive functional analyses provide an understanding of the biologic significance of this vast amount of DNA methylation data that may allow the determination of key epigenetic events associated with tumorigenesis.info:eu-repo/semantics/publishe

Glycoproteomic Analysis of Bronchoalveolar Lavage (BAL) Fluid Identifies Tumor-Associated Glycoproteins from Lung Adenocarcinoma

Cytological examination of cells from bronchoalveolar lavage (BAL) is commonly used for the diagnosis of lung cancer. Proteins released from lung cancer cells into BAL may serve as biomarkers for cancer detection. In this study, <i>N</i>-glycoproteins in eight cases of BAL fluid, as well as eight lung adenocarcinoma tissues and eight tumor-matched normal lung tissues, were analyzed using the solid-phase extraction of <i>N</i>-glycoprotein (SPEG), iTRAQ labeling, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of 80 glycoproteins found in BAL specimens, 32 were identified in both cancer BAL and cancer tissues, with levels of 25 glycoproteins showing at least a 2-fold difference between cancer and benign BAL. Among them, eight glycoproteins showed greater than 2-fold elevations in cancer BAL, including Neutrophil elastase (NE), Integrin alpha-M, Cullin-4B, Napsin A, lysosome-associated membrane protein 2 (LAMP2), Cathepsin D, BPI fold-containing family B member 2, and Neutrophil gelatinase-associated lipocalin. The levels of Napsin A in cancer BAL were further verified in independently collected 39 BAL specimens using an ELISA assay. Our study demonstrates that potential protein biomarkers in BAL fluid can be detected and quantified