Brain tumour differentiation: rapid stratified serum diagnostics via attenuated total reflection Fourier-transform infrared spectroscopy

The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 μl of human serum, attenuated total reflection - Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100% for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0% sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked. © 2016, The Author(s)

Mechanism of scrapie prion precipitation with phosphotungstate anions.

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions

Structural changes of membrane-anchored native PrPC

Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular β-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases

Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin

<div><p>Mutations in the <i>RHO</i> gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two <i>RHO</i> mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The <i>in vitro</i> biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.</p></div

Correction: Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin.

[This corrects the article DOI: 10.1371/journal.pone.0214639.]