DNA-binding dyes are useful in contrasting cell nuclei and chromatin for live cell fluorescence microscopy; however, they may interfere with nuclear and DNA structure and function. We investigated the influence exerted by two DNA dyes on chromatin and nuclear structure, as well as histone-DNA interactions in live HeLa cells. A membranepermeant fluorescent DNA intercalator DRAQ5 (anthracycline derivative), at a concentration of 1 lM, caused microscopically detectable changes of nuclear architecture. Following DRAQ5 intercalation into DNA, chromatin aggregated into distinct areas and foci. The loss of 3D chromatin distribution was exerted via interference with a dynamic exchange of a linker histone (H1), which is a known chromatin stabilizing factor. At higher concentrations (3 and 7.5 lM), DRAQ5 interfered with binding of H2B core histones to DNA. Similar effects resulted from intercalation of chemotherapeutic drugs, adriamycin and daunomycin, but were not observed after binding to DNA of a minor groove binder, Syto17. ' 2008 International Society for Advancement of Cytometry Key terms chromatin structure; histone exchange; DNA intercalator; minor groove binder; DRAQ5; Syto17; fluorescence confocal microscopy CHROMATIN-a complex of DNA, histones, and nonhistone proteins-yields poor contrast in optical microscopy. In standard transmitted light mode, or even in DIC observation, chromatin is barely detectable. Throughout the cell division cycle, chromatin becomes clearly distinguishable only for a relatively short time, when chromosomes condense for mitosis. Thus, chromatin of live cells is commonly visualized using specific fluorescent probes that exhibit affinity to DNA. Most of these probes are intercalators or minor groove binders. A fluorescent probe bound to chromatin reveals the presence of spatial distribution and a local concentration of DNA in nuclei of live cells. However, binding of a probe to DNA may cause changes in chromatin or nuclear structure, which are dependent on the chemical nature and concentration of the probe. In the absence of an independent way of visualizing chromatin, such structural changes remain unknown. Availability of GFP-tagged histones for noninvasive visualization of chromatin in vivo opens a new window of opportunity for investigation of the influence of various DNA-binding dyes on nuclear and chromatin structure.We used HeLa cells expressing eGFP-tagged linker (H1) and core (H2B) histones to investigate the influence of a membrane-permeant DNA intercalator, DRAQ5 (1), and a minor groove binder Syto17, on nuclear and chromatin structure. We describe concentration-dependent aggregation of chromatin and altered interactions between histones and DNA, induced by the bound DRAQ5. We also demonstrate that a minor

The LCA revealed four distinct asthma classes differing in eicosanoid pathways.

During the bronchial challenge, decrease in PGE2 and its metabolite is accompanied by a surge in bronchoconstrictory cysteinyl leukotrienes produced at the expense of LTB4 in AERD subjects. Bronchial PGE2 inhibition in AERD seems specific and sensitive to a low dose of aspirin.

Background: Induced sputum (IS) allows to measure mediators of asthmatic inflammation in bronchial secretions. NSAID-exacerbated respiratory disease (NERD) is recognized as a distinct asthma phenotype, usually with a severe course, eosinophilic airway inflammation, and increased production of pro-inflammatory eicosanoids. A more insightful analysis of NERD patients has shown this phenotype to be nonhomogeneous. Objective:We aimed to identify possible subphenotypes in a cohort of NERD patients with the means of latent class analysis (LCA).Methods: A total of 95 asthma patients with aspirin hypersensitivity underwent sputum induction. High-performance liquid chromatography or gas chromatography coupled with mass spectrometry was used to profile eicosanoids in induced sputum supernatant (ISS). Sixteen variables covering clinical characteristics, IS inflammatory cells, and eicosanoids were considered in the LCA.Results: Three classes (subphenotypes) were distinguished within the NERD cohort.Class 1 subjects had mild-to-moderate asthma, an almost equal distribution of inflammatory cell patterns, the lowest concentrations of eicosanoids, and logLTE 4 /logPGE 2 ratio. Class 2 represented severe asthma with impaired lung function despite high doses of steroids. High sputum eosinophilia was in line with higher pro-inflammatory LTE 4 in ISS and the highest logLTE 4 /logPGE 2 ratio. Class 3 subjects had mild-to-moderate asthma and were also characterized by eosinophilic airway inflammation, yet increased production of pro-(LTE 4 , PGD 2 and 11-dehydro-TBX 2 ) was balanced by

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