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SELECTBIO Conferences Innovations in Microfluidics 2023

Richard James's Biography

Richard James, Principal Investigator and Associate Professor, Seattle Children’s Research Institute and University of Washington

Cellular signaling is often thought of as a linear cascade of protein modifications in a single signaling pathway in response to environmental cues. My primary research interest is to understand how disease processes and pharmacological treatments alter cell signaling in immune diseases. To do so, we use a variety of approaches including quantitative proteomics and genome engineering to understand which signaling pathways are up-regulated during disease and in response to therapies. One focus of our lab is to understand how disease-associated exon-coding variants contribute to immune disease and cancer, by way of modeling variant effects in human immune cells. To that end, we have developed a novel approach that for the first time enables high-efficiency gene editing and gene delivery to in primary human B cells. More recently, my lab leveraged our genome engineering expertise and develop a cloning-free method for performing saturation genome editing in a diploid B cell line. This method enabled us to simultaneously query ~2500 variants in a region of CARD11, an adaptor gene associated with atopic immune dys-regulation. We used these data to re-classify several novel variants in this gene in patients from our clinic.

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Integrating Flow Cytometry with Next Generation Sequencing to Find Determinants of Protein Secretion

Friday, 5 May 2023 at 14:30

Add to Calendar ▼2023-05-05 14:30:002023-05-05 15:30:00Europe/LondonIntegrating Flow Cytometry with Next Generation Sequencing to Find Determinants of Protein SecretionInnovations in Microfluidics 2023 in

Protein secretion drives many functions in vivo; however, methods to link secretions with surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human antibody-secreting cells and link this information to surface marker expression and transcriptional profiles from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborated an association between levels of IgG secretion and CD138 expression. Using oligonucleotide-labeled antibodies and droplet-based sequencing, we show that pathways encoding protein localization to the endoplasmic reticulum, NADH complex assembly, and mitochondrial respiration were most associated with high IgG secretion. Altogether, this method links secretion information to cell surface and single-cell sequencing information (SEC-seq) and enables exploration of links between genome and secretory function, laying the foundation for numerous discoveries in immunology, stem cell biology, and beyond.

Add to Calendar ▼2023-05-04 00:00:002023-05-05 00:00:00Europe/LondonInnovations in Microfluidics 2023Innovations in Microfluidics 2023 in