Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λmax) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages

Aβ40 Oligomers Identified as a Potential Biomarker for the Diagnosis of Alzheimer's Disease

Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD

Gene expression profiling for molecular distinction and characterization of laser captured primary lung cancers

<p>Abstract</p> <p>Methods</p> <p>We examined gene expression profiles of tumor cells from 29 untreated patients with lung cancer (10 adenocarcinomas (AC), 10 squamous cell carcinomas (SCC), and 9 small cell lung cancer (SCLC)) in comparison to 5 samples of normal lung tissue (NT). The European and American methodological quality guidelines for microarray experiments were followed, including the stipulated use of laser capture microdissection for separation and purification of the lung cancer tumor cells from surrounding tissue.</p> <p>Results</p> <p>Based on differentially expressed genes, different lung cancer samples could be distinguished from each other and from normal lung tissue using hierarchical clustering. Comparing AC, SCC and SCLC with NT, we found 205, 335 and 404 genes, respectively, that were at least 2-fold differentially expressed (estimated false discovery rate: < 2.6%). Different lung cancer subtypes had distinct molecular phenotypes, which also reflected their biological characteristics. Differentially expressed genes in human lung tumors which may be of relevance in the respective lung cancer subtypes were corroborated by quantitative real-time PCR.</p> <p>Genetic programming (GP) was performed to construct a classifier for distinguishing between AC, SCC, SCLC, and NT. Forty genes, that could be used to correctly classify the tumor or NT samples, have been identified. In addition, all samples from an independent test set of 13 further tumors (AC or SCC) were also correctly classified.</p> <p>Conclusion</p> <p>The data from this research identified potential candidate genes which could be used as the basis for the development of diagnostic tools and lung tumor type-specific targeted therapies.</p

Infinitely Adaptive Transition-Metal Ordering in Ln2O2MSe2-Type Oxychalcogenides

The origin of transition-metal ordering in Ln2O2MSe2 oxychalcogenides has been revealed by investigating a range of solid solutions. Transition-metal ordering is determined by the relative sizes of [Ln2O2]2+ and [MSe2]2− layers and can be exquisitely tuned by substitution in either layer, leading to an “infinitely adaptive” structural family. The proportion of the inherently smaller corner-shared motif increases as the transition-metal size increases relative to the lanthanide oxide layers

Studies of Early Events of Folding of a Predominately β‑Sheet Protein Using Fluorescence Correlation Spectroscopy and Other Biophysical Methods

The interplay between the early collapse of the unfolded state and the formation of the secondary structure has been the subject of extensive research in protein chemistry. In this study, we used the intestinal fatty acid binding protein (IFABP), a small model protein with predominately β-sheet structure, to study the early events, including the early chain collapse and the formation of the secondary structure. We used a combination offluorescence correlation spectroscopy and far-UV circular dichroism (CD) to understand how these early processes influence the late folding events like the stabilization of the secondary structure and aggregation. Acid-induced unfolded IFABP was found to collapse in the presence of low concentrations of added salt and aggregate at higher concentrations. Both the formation of the collapsed state and aggregation were conveniently probed by fluorescence correlation spectroscopy, a sensitive fluorescence technique with single-molecule resolution. In contrast, the formation of the secondary structure was monitored by far-UV CD. The results suggested that backbone hydrogen bond formation, not only the overall hydrophobicity of IFABP, may play crucial roles in the early collapse. Two mutant proteins positioned at a crucial nucleating site, namely, G80V and L64G, although being opposite in their overall hydrophobicity, collapsed relatively rapidly compared to the wild-type protein. The interconnection among the early collapse, the formation of the secondary structure, and aggregation was similar for these two mutants. Another mutant, G44V, which was identical in its overall hydrophobicity to G80V but situated in a region distant from the hydrophobic core, was found to be very different from G80V and L64G