In Vitro Multiparameter Assay Development Strategy toward Differentiating Macrophage Responses to Inhaled Medicines.

Although foamy macrophages (FMΦ) are commonly observed during nonclinical development of medicines for inhalation, there are no accepted criteria to differentiate adaptive from adverse FMΦ responses in drug safety studies. The purpose of this study was to develop a multiparameter in vitro assay strategy to differentiate and characterize different mechanisms of drug-induced FMΦ. Amiodarone, staurosporine, and poly(vinyl acetate) nanoparticles were used to induce distinct FMΦ phenotypes in J774A.

DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

Background: Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.

Materials And Methods: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO.

Performance testing of a semi-automatic card punch system, using direct STR profiling of DNA from blood samples on FTA™ cards.

The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots.

Profiling of Nutrient Transporter Expression in Human Stem Cell-Derived Cardiomyocytes Exposed to Tyrosine Kinase Inhibitor Anticancer Drugs Using RBD Ligands.

We applied a novel profiling approach using receptor binding domain (RBD) ligands to cell surface domains of a panel of nutrient transporters to characterize the impact of a number of tyrosine kinase inhibitor anticancer drugs on human stem cell-derived cardiomyocytes. High-content screening and flow cytometry analysis showed diagnostic changes in nutrient transporter expression correlating with glycolysis and oxidative phosphorylation-based cell metabolism in glucose and galactose media. Cluster analysis of RBD binding signatures of drug-treated cells cultured in glucose medium showed good correlation with sensitization of mitochondrial toxicity in cells undergoing oxidative phosphorylation in galactose medium.

Adenoviral sensors for high-content cellular analysis.

To maximize the potential of high-content cellular analysis for investigating complex cellular signaling pathways and processes, we have generated a library of adenoviral encoded cellular sensors based on protein translocation and reporter gene activation that enable a diverse set of assays to be applied to lead compound profiling in drug discovery and development. Adenoviral vector transduction is an efficient and technically simple system for expression of cellular sensors in diverse cell types, including primary cells. Adenoviral vector-mediated transient expression of cellular sensors, either as fluorescent protein fusions or live cell gene reporters, allows rapid assay development for profiling the activities of candidate drugs across multiple cellular systems selected for biological and physiological relevance to the target disease state.

Dynamic green fluorescent protein sensors for high-content analysis of the cell cycle.

We have developed two dynamic sensors that report cell cycle position in living mammalian cells. The sensors use well-characterized components from proteins that are spatially and temporally regulated through the cell cycle. Coupling of these components to Enhanced Green Fluorescent Protein (EGFP) has been used to engineer fusion proteins that report G1/S and G2/M transitions during the cell cycle without perturbing cell cycle progression.

Fluorescent proteins and engineered cell lines.

Green fluorescent protein and other fluorescent proteins provide powerful tools for high content analysis of cellular processes. Engineering fluorescent protein sensors for expression in cellular assays requires consideration of a wide range of design factors to produce fusion proteins capable of generating informative and biologically relevant data while meeting the rigorous demands of high content screening. The target protein, fluorescent protein, host cell line, construct components and orientation, expression level, and other factors all contribute to the performance of the sensor.

Acridones and quinacridones: novel fluorophores for fluorescence lifetime studies.

Two new families of fluorescent probe, acridones and quinacridones, whose fluorescence lifetime can be altered to produce a range of lifetimes from 3 ns to 25 ns are described. Both families of fluorophore have fluorescence lifetimes which are unaffected by pH in the range of 5 to 9 and show a marked resistance to photobleaching. The probes have been modified to allow them to be attached to biomolecules and the labelling of a neuropeptide (substance P) is described.

Cy3B: improving the performance of cyanine dyes.

The spectral properties of a rigidified trimethine cyanine dye, Cy3B have been characterised. This probe has excellent fluorescent properties, good water solubility and can be bioconjugated. The emission properties of this fluorophore have also been investigated upon conjugation to an antibody.

Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands.

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands.

Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle.

The cell cycle is the collective mechanism through which all of us develop, exist and in many cases, when it goes wrong, die. Despite enormous progress in unravelling the complexity of the cell cycle through intensive study over the past 100 years, development of new tools to analyse the process and associated cellular events has not kept pace. All standard cell cycle analysis methods preclude real time dynamic analysis of the cell cycle in live cells at single cell resolution.