Dimethylaniline functionalised pyrene fluorophores; dual colour pH switching in solution and self-assembled monolayers.

A pyrene charge transfer fluorophore with three ionizable N,N-dimethylaniline moieities was explored as an interfacial pH switch. The parent carboxylate compound and the thiolated derivative were shown by spectroscopy combined with DFT calculation to be successively and reversibly protonated. Protonation leads to progressive decrease of intensity of the 550 nm centered N,N-dimethylaniline to pyrene charge transfer emission which on protonation of the third site, leads to extinction of this transition and evolution of an intense blue (450 nm) pyrene-centered emission. Concomitant loss of the charge transfer absorbance was observed and the changes are reversed on neutralization of pH. A self-assembled monolayer of the thiolated derivative was prepared on gold and found from voltammetry of ferricyanide/ferrocyanide probe to form close packed monolayers. The probe voltammetry, label-free electrochemical impedance spectroscopy of the film was monitored as a function of pH and progressive, but reversible protonation steps were reflected in decreasing film resistance. The Stokes shift of the probe prevents self-quenching so a broad, charge transfer fluorescence centered around 540 nm was recorded for the self-assembled monolayer where as per solution, progressive and reversible reduction in intensity was observed. The facile assembly, impedance and optical switching make these materials potentially interesting as on-off or two colour on-off-on fluorescence switches with potential applications in logic gates or in responsive surface applications.

Mega-stokes pyrene ceramide conjugates for STED imaging of lipid droplets in live cells.

Lipid droplets are dynamic subcellular organelles that participate in a range of physiological processes including metabolism, regulation and lipid storage. Their role in disease, such as cancer, where they are involved in metabolism and in chemoresistance, has emerged over recent years. Thus, the value of lipid droplets as diagnostic markers is increasingly apparent where number and size of droplets can be a useful prognostic. Although diverse in size, LDs are typically too small to be easily enumerated by conventional microscopy. The advent of super-resolution microscopy methods offers the prospect of detailed insights but there are currently no commercial STED probes suited to this task and STED, where this method has been used to study LDs it has relied on fixed samples. Here, we report a pyrene-based ceramide conjugate PyLa-C17Cer, that stains lipid droplets with exceptionally high precision in living cells and shows excellent performance in stimulated emission depletion microscopy. The parent compound PyLa comprises a pyrene carboxyl core appended with 3,4-dimethylaminophenyl. The resulting luminophore exhibits high fluorescent quantum yield, mega-Stokes shift and low cytotoxicity. From DFT calculations the Stokes shifted fluorescent state arises from a dimethylaminophenyl to pyrene charge-transfer transition. While the parent compound is cell permeable, it is relatively promiscuous, emitting from both protein and membranous structures within the living mammalian cell. However, on conjugation of C17 ceramide to the free carboxylic acid, the resulting PyLa-C17Cer, remains passively permeable to the cell membrane but targets lipid droplets within the cell through a temperature dependent mechanism, with high selectivity. Targeting was confirmed through colocalisation with the commercial lipid probe Nile Red. PyLa-C17Cer offers outstanding contrast of LDs both in fluorescence intensity and lifetime imaging due to its large Stokes shift and very weak emission from aqueous media. Moreover, because the compound is exceptionally photochemically stable with no detectable triplet emission under low temperature conditions, it can be used as an effective probe for fluorescence correlation spectroscopy (FCS). These versatile fluorophores are powerful multimodal probes for combined STED/FCS/lifetime studies of lipid droplets and domains in live cells.

Linker length in fluorophore-cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells.

Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (Ld) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (Lo) domains. Here, a simple, one-pot approach to probe-cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial Ld marker as a reference. BODIPY-Ar-Chol targets almost exclusively the Ld domains with selectivity of >90% whereas by contrast introducing the C6 linker between the probe and cholesterol drove the probe to Lo with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial Ld marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

Accuracy and Reliability of Internet Resources for Information on Idiopathic Pulmonary Fibrosis.

RATIONALE: Patients commonly use the Internet as a resource for health information; however, no studies have evaluated the online information about idiopathic pulmonary fibrosis (IPF). OBJECTIVES: We sought to determine the readability, content (compared with established guidelines), bias, and quality of online IPF resources. METHODS: We analyzed the first 200 hits for "idiopathic pulmonary fibrosis" in Google, Yahoo, and Bing. Each website was evaluated for content related to IPF features and treatments that are discussed in clinical guidelines. Website quality was assessed using the validated DISCERN instrument. MEASUREMENTS AND MAIN RESULTS: Eligibility criteria were met in 181 websites. The median reading grade level was 12. More content was provided in scientific resources (academic institutions or governmental organizations) and foundation/advocacy organization sites than in personal commentary (blog) sites; however, most sites provided incomplete and/or inaccurate information. Nonindicated and/or harmful pharmacotherapies for IPF were described as potential IPF treatments in 48% of websites and were most often recommended in foundation/advocacy organization websites. Azathioprine and corticosteroids were discussed as potential chronic treatments of IPF in 13.3 and 30.6% of the 98 websites that had been updated after publication of data demonstrating harm from these medications. Website quality (DISCERN score) was poor in all site types but was worse in news/media reports and personal commentary (blog) sites than in sites from scientific and foundation/advocacy organizations. CONCLUSIONS: Patient-directed online information on IPF is frequently incomplete, inaccurate, and outdated. There is no reliable method for patients to identify sites that provide appropriate information on IPF.

Predicting Mortality in Systemic Sclerosis-Associated Interstitial Lung Disease Using Risk Prediction Models Derived From Idiopathic Pulmonary Fibrosis.

BACKGROUND: Mortality risk prediction tools have been developed in idiopathic pulmonary fibrosis, however, it is unknown whether these models accurately estimate mortality in systemic sclerosis-associated interstitial lung disease (SSc-ILD). METHODS: Four baseline risk prediction models--the Composite Physiologic Index, the Interstitial Lung Disease-Gender, Age, Physiology Index, the du Bois index, and the modified du Bois index--were calculated for patients recruited from a specialized SSc-ILD clinic. Each baseline model was assessed using logistic regression analysis with 1-year mortality as the outcome variable. Discrimination was quantified using the area under the receiver operating characteristic curve. Calibration was assessed using the goodness-of-fit test. The incremental prognostic ability of additional predictor variables was determined by adding prespecified variables to each baseline model. RESULTS: The 156 patients with SSc-ILD completed 1,294 pulmonary function tests, 725 6-min walk tests, and 637 echocardiograms. Median survival was 15.0 years from the time of SSc-ILD diagnosis. All baseline models were significant predictors of 1-year mortality in SSc-ILD. The modified du Bois index had an area under the receiver operating characteristic curve of 0.84, compared with 0.77 to 0.81 in the other models. Calibration was acceptable for the modified du Bois index, but was poor for the other models. All baseline models include FVC and 6-min walk distance was identified as an additional independent predictor of 1-year mortality. CONCLUSIONS: The modified du Bois index has good discrimination and calibration for the prediction of 1-year mortality in SSc-ILD. FVC and 6-min walk distance are important independent predictors of 1-year mortality in SSc-ILD.