Integration of Acoustic Liquid Handling into Quantitative Analysis of Biological Matrix Samples.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

Novel pyrrolidine heterocycles as CCR1 antagonists.

A novel series of pyrrolidine heterocycles was prepared and found to show potent inhibitory activity of CCR1 binding and CCL3 mediated chemotaxis of a CCR1-expressing cell line. A potent, optimized triazole lead from this series was found to have acceptable pharmacokinetics and microsomal stability in rat and is suitable for further optimization and development.

Discovering biological networks from diverse functional genomic data.

Recent advances in biotechnology have produced a wealth of genomic data, which capture a variety of complementary cellular features. While these data promise to yield key insights into molecular biology, much of the available information remains underutilized because of the lack of scalable approaches for integrating signals across large, diverse data sets. A proper framework for capturing these numerous snapshots of complementary phenomena under a variety of conditions can provide the holistic view necessary for developing precise systems-level hypotheses. Here we describe bioPIXIE, a system for combining information from diverse genomic data sets to predict biological networks. bioPIXIE utilizes a Bayesian framework for probabilistic integration of several high-throughput genomic data types including gene expression, protein-protein interactions, genetic interactions, protein localization, and sequence data to predict biological networks. The main purpose of the system is to support user-driven exploration through the inferred functional network, which is enabled by a public, web-based interface. We describe the features and supporting methods of this integration and discovery framework and present case examples where bioPIXIE has been used to generate specific, testable hypotheses for Saccharomyces cerevisiae, many of which have been confirmed experimentally.

High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye.

We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.

Discovery of biological networks from diverse functional genomic data.

We have developed a general probabilistic system for query-based discovery of pathway-specific networks through integration of diverse genome-wide data. This framework was validated by accurately recovering known networks for 31 biological processes in Saccharomyces cerevisiae and experimentally verifying predictions for the process of chromosomal segregation. Our system, bioPIXIE, a public, comprehensive system for integration, analysis, and visualization of biological network predictions for S. cerevisiae, is freely accessible over the worldwide web.

Expression of NALP1 in cerebellar granule neurons stimulates apoptosis.

NAcht Leucine-rich-repeat Protein 1 (NALP1) contains a putative nucleotide binding site, a region of leucine-rich repeats, and death domain folds at both termini providing protein/protein association functions such as caspase recruitment. We report here that NALP1 gene expression was induced in primary cerebellar granule neurons (CGN) upon injury. Up-regulation of NALP1 was also observed in a model of transient focal ischemia induced by middle cerebral artery occlusion. We investigated the biological consequence of over-expression of NALP1 in both HeLa cells and in CGN. Expression of recombinant NALP1 stimulated cell death in both HeLa cells and CGN by an apoptotic mechanism, demonstrated by the induction of apoptotic nuclear morphology and activation of the apoptotic enzyme caspase-3. Also described here are studies on the mechanism of action studies including deletion analyses and investigations of nucleotide binding, which begin to elucidate a regulatory function for NALP1 in neuronal apoptosis.