Biology subjects

McManus, C. E.

Publications and source records attributed to McManus, C. E..

3 recordsLinked to original sources

bioRxiv2024

Optimizing ex vivo CAR-T cell-mediated cytotoxicity assay through multimodality imaging

CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With the FDAs initiative for advancing alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and inability to automate, among others. To address these limitations, we optimized the assay using multimodality imaging methods, including bioluminescence, impedance tracking, phase contrast, and fluorescence, to track CAR-T cells co-cultured with CD19, CD20, and HER2 luciferase reporter cancer cells in real-time. Additionally, we varied the ratio of CAR-T cells to cancer cells to determine optimal cytotoxicity readouts. Our findings demonstrated that the CAR-T cell group effectively attacked cancer cells, and the optimized assay provided superior temporal and spatial precision measurements of ex vivo CAR-T killing of cancer cells, confirming the reliability, consistency, and high throughput of the optimized assay.

immunology↗

bioRxiv2021

Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling

During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Shep promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two novel enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrated that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.

genomics↗

bioRxiv2018

A simple and effective method to isolate germ nuclei from C. elegans for genomic assays.

The wide variety of specialized permissive and repressive mechanisms by which germ cells regulate developmental gene expression are not well understood genome-wide. Isolation of germ cells with high integrity and purity from living animals is necessary to address these open questions, but no straightforward methods are currently available. Here we present an experimental paradigm that permits the isolation of the nuclei from C. elegans germ cells at quantities sufficient for genomic analyses. We demonstrate that these nuclei represent a very pure population and are suitable for both chromatin immunoprecipitation (ChIP-seq) and transcriptome (RNA-seq) analyses. The method does not require specialized transgenic strains or growth conditions and can be readily adopted by other researchers with minimal troubleshooting. This new capacity removes a major barrier in the field to dissect gene expression mechanisms in the germ line of C. elegans. Consequent discoveries using this technology will be relevant to conserved regulatory mechanisms across species.

genomics↗