Person: Bryson, Bryan
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Bryson
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Bryson, Bryan
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Publication Seq-Well: A Portable, Low-Cost Platform for High-Throughput Single-Cell RNA-Seq of Low-Input Samples(2017) Gierahn, Todd M.; Wadsworth, Marc H.; Hughes, Travis; Bryson, Bryan; Butler, Andrew; Satija, Rahul; Fortune, Sarah; Love, J. Christopher; Shalek, Alex K.Single-cell RNA-Seq can precisely resolve cellular states but application to sparse samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively-parallel single-cell RNA-Seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semi-permeable membrane, enabling efficient cell lysis and transcript capture. We characterize Seq-Well using species-mixing experiments and PBMCs, and profile thousands of primary human macrophages exposed to tuberculosis.Publication Variability in Tuberculosis Granuloma T Cell Responses Exists, but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization(Public Library of Science, 2015) Gideon, Hannah Priyadarshini; Phuah, JiaYao; Myers, Amy J.; Bryson, Bryan; Rodgers, Mark A.; Coleman, M. Teresa; Maiello, Pauline; Rutledge, Tara; Marino, Simeone; Fortune, Sarah; Kirschner, Denise E.; Lin, Philana Ling; Flynn, JoAnne L.Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.Publication RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device(American Chemical Society, 2016) Ma, Sai; Bryson, Bryan; Sun, Chen; Fortune, Sarah M.; Lu, ChangStudies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000–8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10–20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays.Publication Posttranslational modification of a histone-like protein regulates phenotypic resistance to isoniazid in mycobacteria(American Association for the Advancement of Science, 2018) Sakatos, Alexandra; Babunovic, Gregory; Chase, Michael; Dills, Alexander; Leszyk, John; Rosebrock, Tracy; Bryson, Bryan; Fortune, SarahThere is increasing evidence that phenotypically drug-resistant bacteria may be important determinants of antibiotic treatment failure. Using high-throughput imaging, we defined distinct subpopulations of mycobacterial cells that exhibit heritable but semi-stable drug resistance. These subpopulations have distinct transcriptional signatures and growth characteristics at both bulk and single-cell levels, which are also heritable and semi-stable. We find that the mycobacterial histone-like protein HupB is required for the formation of these subpopulations. Using proteomic approaches, we further demonstrate that HupB is posttranslationally modified by lysine acetylation and lysine methylation. Mutation of a single posttranslational modification site specifically abolishes the formation of one of the drug-resistant subpopulations of cells, providing the first evidence in prokaryotes that posttranslational modification of a bacterial nucleoid-associated protein may epigenetically regulate cell state.