Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives.

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control) and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

Multiplexing PCR is a simple way to extract genomic regions of interest for various medical and genetic tests. Somatic mutations lead to various diseases including cancer. These mutations are unlikely to be best detected using regular whole genome sequencing. Clinical samples often consist of disease cells, e.g. cancer cells, surrounded by normal cells. Thus, deep sequencing of hundreds to thousands fold coverage is required to detect the mutations. In clinical research many doctors are interested in specific genes or genomic regions and they want to extract the regions from genomic DNA or RNA before sequencing. Many current clinical, forensic, and heretical genetic test workflows start with multiplexing PCR to extract genetic marker carrying regions from whole genomes before running hybridization, sequencing, or electrophoresis tests to identify the markers. Personal medicine and prognosis mostly involve examining sequence variations of a number of targeted genes and metabolic pathway genes so as to predict drug efficacies and drug toxicities. We have developed a new multiplexing PCR approach with a significantly simplified workflow and significantly improved robustness. When applied to sequencing target enrichment application, the workflow for producing amplified targets involves only one hands-on step and one PCR run. The approach is designed to require low sample input and to produce superior amplicon uniformity and sequence specificity. The approach involves a novel primer design and a proprietary reaction composition. A PCR run consists of two functionally separated reaction phases, namely target capture and library amplification, without any hands-on step in between. The performance of the new approach will be demonstrated by a caner panel data. Citation Format: Xiaochuan Zhou, Qi Zhu, Chris Hebel. A simple multiplex PCR approach for target enrichment in next-gen sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3582. doi:10.1158/1538-7445.AM2014-3582

Post-translational modifications (PTMs) of histones play a critical role in diverse biological processes including chromatin compaction, gene expression and cell differentiation. Among a myriad of PMTs, histone methylation catalyzed by histone methyltransferases (HMTs) has been increasingly recognized as an important player responsible for a major signaling mechanism in eukaryotic cells. This suite of epigenetic modifiers represents a new and promising class of therapeutic targets. In cancer, there is a growing body of evidence that suggests changes in the activity of HMTs (a class of chromatin-modifying enzymes) contribute to the uncontrolled cell proliferation that is a hallmark of this devastating disease. The sequence specificity of the substrates of HMTs under a cellular condition are largely unknown but known targets have been mostly identified through a conventional candidate-based approach by using purified HMTs. However, such an experiment frequently does not reflect what could be occurring in cellular contexts or in vivo. In this study, we designed and synthesized a comprehensive histone peptide microarray (PepArray) on a microfluidic chip which contains 3,919 peptides. The peptides contain nine residues with the methylation sites and mutant sites situated in the middle of the sequence so that each peptide has a unique possibility for modification such as methylation or acetylation. We obtained nuclear extract from the breast cancer cell line T47D, and applied the protein lysates to the histone methylation PepArray chip. After incubation of the chip with a methyl-specific antibody, significant signals were detected at the sites containing peptides corresponding to H2AK74, H3K122, and H4K59. We found null signal at mutant sites where the target lysine(K) was replaced with alanine(A). These results reveal the specific activity profiles of HMTs at defined histone sites in a cellular system. Planned further investigation will compare the different histone methylation or acetylation profiles in the various cellular systems, especially in different cancer systems. The current experiment demonstrates an effective solution to comprehensive studies of epigenetic modification. This information may be translated into therapeutic targets of histone methylation inhibition by focusing on identifying inhibitors of specific HMTs as targets for a new generation of therapeutics. Citation Format: Bing Zhu, Ailing Hong, Chris Hebel, Xiaochuan Zhou, Xiaolian Gao. Site specific profiling of histone methyltransferases in cancer cells using histone peptide microarray containing a comprehensive set of histone peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2013-4235