Multiple Modes of Nrf2 Regulation and Transcriptional Response

Cells have defense systems to deal with chemical insults from the environment. Some examples are chemical scavengers like glutathione and enzymes such as superoxide dismutase that inactivate radicals and other reactive chemicals in the cytoplasm. It is perhaps surprising that these protective systems are not maximally expressed in an unstressed cell. Rather, the ability to inactivate toxic chemicals is tightly regulated and only induced when needed. As a consequence, unstressed cells are usually very sensitive to radicals, but become more resistant as the cellular defense system has been appropriately upregulated after a few hours. The transcription factor Nrf2 is known to be a master regulator of many cytoprotective enzymes and proteins. Chemical inducers of Nrf2 inactivate its repressor, Keap1, when they react with critical cysteine residues in Keap1. The release of Nrf2 from Keap1 results in enhanced expression of genes involved in detoxification. This generates a feedback loop where Nrf2 induces protective enzymes capable of inactivating the chemical that reacted with Keap1. An unproven, but likely, scenario is that Nrf2 transcriptional response can vary depending on the nature of the chemical insult. The aim of this chapter is to examine the mechanisms by which the cell can sense different reactive chemicals and modulate protective responses. It is likely that this knowledge is of vital importance in the development of clinical Nrf2 activators in preventive medicine

Risk of myocardial infarction at specific troponin T levels using the parameter predictive value among lookalikes (PAL)

AbstractBackgroundMyocardial infarction is more likely if the heart damage biomarker cardiac troponin T (cTnT) is elevated in a blood sample, indicating that cardiac damage has occurred. No method allows the clinician to estimate the risk of myocardial infarction at a specific cTnT level in a given patient.MethodsPredictive value among lookalikes (PAL) uses pre-test prevalence, sensitivity and specificity at adjacent cTnT limits based on percentiles. PAL is the pre-test prevalence-adjusted probability of disease between two adjacent cTnT limits. If a chest pain patient's cTnT level is between these limits, the risk of myocardial infarction can be estimated.ResultsThe PAL based on percentiles had an acceptable sampling error when using 100 bootstrapped data of 18 different biomarkers from 38,945 authentic lab measurements. A PAL analysis of an emergency room cohort (n=11,020) revealed that the diagnostic precision of a high-sensitive cTnT assay was similar among chest pain patients at different ages. The higher incidence of false positive results due to non-specific increases in cTnT in the high-age group was counterbalanced by a higher pre-test prevalence of myocardial infarction among older patients, a finding that was missed when using a conventional ROC plot analysis.ConclusionsThe PAL was able to calculate the risk of myocardial infarction at specific cTnT levels and could complement decision limits

Numerical Analysis of Etoposide Induced DNA Breaks

Background: Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs. Methodology/Principal Findings: To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3 % of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10 % of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity. Conclusions/Significance: These results show that only 0.3 % of all strand breaks produced by etoposide activate H2A

Assessment of a multi-marker risk score for predicting cause-specific mortality at three years in older patients with heart failure and reduced ejection fraction

Background: Due to increasing co-morbidity associated with aging, heart failure (HF) has become more prevalent and heterogeneous in older individuals, and non-cardiovascular (CV) mortality has increased. Previously, we defined a multi-marker modality that included cystatin C (CysC), troponin T (TnT), and age. Here, we validated this multi-marker risk score by evalu­ating its predictions of all-cause mortality and CV mortality in an independent population of older individuals with HF and reduced ejection fraction (HFrEF). Methods: This prospective cohort study included 124 patients, median age 73 years, that had HFrEF. We determined all-cause mortality and CV mortality at a 3-year follow-up. We com­pared the risk score to the N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) for predicting all-cause mortality and CV mortality. Results: High risk scores were associated with both all-cause mortality (HR 4.2, 95% CI 2.2–8.1, p < 0.001) and CV mortality (HR 3.6, 95% CI 1.7–8.0, p = 0.0015). Receiver ope­rating characteristics showed similar efficacy for the risk score and NT-proBNP in predicting all-cause mortality (HR 0.74, 95% CI 0.65–0.81 vs. HR 0.74, 95% CI 0.65–0.81, p = 0.99) and CV mortality (HR 0.68, 95% CI 0.59–0.76 vs. HR 0.67, 95% CI 0.58–0.75, p = 0.95). When the risk score was added to the NT-proBNP, the continuous net reclassification impro­vement was 56% for predicting all-cause mortality (95% CI 18–95%, p = 0.004) and 45% for predicting CV mortality (95% CI 2–89%, p = 0.040). Conclusions: In HFrEF, a risk score that included age, TnT, and CysC showed efficacy similar to the NT-proBNP for predicting all-cause mortality and CV mortality in an older population.

Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents

Decreased admissions and hospital costs with a neutral effect on mortality following lowering of the troponin T cutoff point to the 99th percentile

Background: The implementation of high-sensitivity cardiac troponin T (hs-cTnT) assays and a cutoff based on the 99th cTnT percentile in the evaluation of patients with suspected acute coronary syndrome has not been uniform due to uncertain effects on health benefits and utilization of limited resources. Methods: Clinical and laboratory data from patients with chest pain or dyspnea at the emergency de¬partment (ED) were evaluated before (n = 20516) and after (n = 18485) the lowering of the hs-cTnT cutoff point from 40 ng/L to the 99th hs-cTnT percentile of 14 ng/L in February 2012. Myocardial infarction (MI) was diagnosed at the discretion of the attending clinicians responsible for the patient. Results: Following lowering of the hs-cTnT cutoff point fewer ED patients with chest pain or dyspnea as the principal complaint were analyzed with an hs-cTnT sample (81% vs. 72%, p < 0.001). Overall 30-day mortality was unaffected but increased among patients not analyzed with an hs-cTnT sample (5.3% vs. 7.6%, p < 0.001). The MI frequency was unchanged (4.0% vs. 3.9%, p = 0.72) whereas admission rates decreased (51% vs. 45%, p < 0.001) as well as hospital costs. Coronary angiographies were used more frequently (2.8% vs. 3.3%, p = 0.004) but with no corresponding change in coronary interventions. Conclusions: At the participating hospital, lowering of the hs-cTnT cutoff point to the 99th percentile decreased admissions and hospital costs but did not result in any apparent prognostic or treatment benefits for the patients

Analytical considerations in deriving 99th percentile upper reference limits for high-sensitivity cardiac troponin assays: Educational recommendations from the IFCC committee on clinical application of cardiac bio-markers

The International Federation of Clinical Chemistry Committee on Clinical Application of Cardiac Bio-Markers provides evidence-based educational documents to facilitate uniform interpretation and utilization of cardiac biomarkers in clinical laboratories and practice. The committee’s goals are to improve the understanding of certain key analytical and clinical aspects of cardiac biomarkers and how these may interplay in clinical practice. Measurement of high-sensitivity cardiac troponin (hs-cTn) assays is a cornerstone in the clinical evaluation of patients with symptoms and/or signs of acute cardiac ischemia. To define myocardial infarction, the Universal Definition of Myocardial Infarction requires patients who manifest with features suggestive of acute myocardial ischemia to have at least one cTn concentration above the sex-specific 99th percentile upper reference limit (URL) for hs-cTn assays and a dynamic pattern of cTn concentrations to fulfill the diagnostic criteria for MI. This special report provides an overview of how hs-cTn 99th percentile URLs should be established, including recommendations about prescreening and the number of individuals required in the reference cohort, how statistical analysis should be conducted, optimal preanalytical and analytical protocols, and analytical/biological interferences or confounds that can affect accurate determination of the 99th percentile URLs. This document also provides guidance and solutions to many of the issues posed.publishedVersio