Prion strain discrimination using luminescent conjugated polymers.
ABSTRACT The occurrence of multiple strains of prions may reflect conformational variability of PrP(Sc), a disease-associated, aggregated variant of the cellular prion protein, PrP(C). Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.
SourceAvailable from: Artur Cieslar-Pobuda[Show abstract] [Hide abstract]
ABSTRACT: Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes. © 2015 International Society for Advancement of CytometryCytometry Part A 01/2015; 87(3). DOI:10.1002/cyto.a.22627 · 3.07 Impact Factor
10/2008, Supervisor: Adriano Aguzzi
Nature 08/2014; 512(7512):32-4. DOI:10.1038/512032a · 42.35 Impact Factor