A new lung cancer biomarker--a preliminary report.

OBJECTIVE: To investigate a unique biomarker from the blood plasma and sputum of lung cancer patients based on native fluorescence analysis of body fluids. BACKGROUND: Conventionally, biomarkers indicative of malignancy are identified by biochemical or biophysical processes. Most of the cancer biomarkers, often useful in monitoring disease progression, have sensitivity and specificity in the range of 60%. METHODS: We employed synchronous fluorescence excitation spectroscopy (SFXS) and fluorescence emission spectroscopy for the native fluorescence analysis of blood plasma of 32 normal controls, 32 patients with lung cancer, and 32 patients with other types of cancer. RESULTS: Based on the native fluorescence analysis of blood plasma and sputum, we found that the structural protein elastin, with an excitation peak at 327 nm and an emission peak at 405 nm, is an exclusive biomarker for lung carcinoma with 77% sensitivity and 83% specificity from plasma alone, 92.3% sensitivity and 100% specificity from plasma acetone extract alone, and 66% sensitivity and 100% specificity from sputum alone. CONCLUSION: In this preliminary report with a limited number of lung cancer patients, we have used SFXS of plasma and sputa as the basis for a new technique identifying elastin as an exclusive lung cancer biomarker. This technique has the potential to become a new protocol for rapid and cost-effective screening and diagnosis of lung cancer.

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy.

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.