Honors & Awards
DiMase Fellow, Brown University (2012)
Kou-I Yeh Fellow, Stanford University (2013)
NSF Graduate Research Fellow, Stanford University (2014-2017)
Education & Certifications
Master of Science, Stanford University, BIOE-MS (2015)
B.S., Brown University, Biomedical Engineering (2013)
Stephen Quake, Doctoral Dissertation Advisor (AC)
High-affinity allergen-specific human antibodies cloned from single IgE B cell transcriptomes.
Science (New York, N.Y.)
2018; 362 (6420): 1306–9
Immunoglobulin E (IgE) antibodies protect against helminth infections but can also cause life-threatening allergic reactions. Despite their role in human health, the cells that produce these antibodies are rarely observed and remain enigmatic. We isolated single IgE B cells from individuals with food allergies and used single-cell RNA sequencing to elucidate the gene expression and splicing patterns unique to these cells. We identified a surprising example of convergent evolution in which IgE antibodies underwent identical gene rearrangements in unrelated individuals. Through the acquisition of variable region mutations, these IgE antibodies gained high affinity and unexpected cross-reactivity to the clinically important peanut allergens Ara h 2 and Ara h 3. These findings provide insight into IgE B cell transcriptomics and enable biochemical dissection of this antibody class.
View details for PubMedID 30545888
Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe dengue.
Proceedings of the National Academy of Sciences of the United States of America
Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA-containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.
View details for PubMedID 30530648
High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis
2018; 19: 140
High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards.Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution.Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.
View details for PubMedID 29439658
Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma.
2017; 21 (5): 1399–1410
Glioblastoma (GBM) is the most common primary brain cancer in adults and is notoriously difficult to treat because of its diffuse nature. We performed single-cell RNA sequencing (RNA-seq) on 3,589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumor's genome and transcriptome. We were also able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of information about tumor formation and migration.
View details for PubMedID 29091775
Single fluorophore melting curve analysis for detection of hypervirulent Clostridium difficile
JOURNAL OF MEDICAL MICROBIOLOGY
2016; 65: 62–70
This study demonstrates a novel detection assay able to identify and subtype strains of Clostridium difficile. Primers carefully designed for melting curve analysis amplify DNA from three C. difficile genes, tcdB, tcdC and cdtB, during quantitative (q)PCR. The tcdB gene allows for confirmation of organism presence, whilst the tcdC and cdtB genes allow for differentiation of virulence status, as deletions in the tcdC gene and the concurrent presence of the cdtB gene, which produces binary toxin, are associated with hypervirulence. Following qPCR, subtyping is then achieved by automated, inline melting curve analysis using only a single intercalating dye and verified by microchip electrophoresis. This assay represents a novel means of distinguishing between toxigenic and hypervirulent C. difficile strains NAP1/027/BI and 078 ribotype, which are highly prevalent hypervirulent strains in humans. This methodology can help rapidly detect and identify C. difficile strains that impose a significant health and economic burden in hospitals and other healthcare settings.
View details for DOI 10.1099/jmm.0.000199
View details for Web of Science ID 000372136100009
View details for PubMedID 26516039
Food allergen detection by mass spectrometry: the role of systems biology.
NPJ systems biology and applications
2016; 2: 16022
Food allergy prevalence is rising worldwide, motivating the development of assays that can sensitively and reliably detect trace amounts of allergens in manufactured food. Mass spectrometry (MS) is a promising alternative to commonly employed antibody-based assays owing to its ability to quantify multiple proteins in complex matrices with high sensitivity. In this review, we discuss a targeted MS workflow for the quantitation of allergenic protein in food products that employs selected reaction monitoring (SRM). We highlight the aspects of SRM method development unique to allergen quantitation and identify opportunities for simplifying the process. One promising avenue identified through a comprehensive survey of published MS literature is the use of proteotypic peptides, which are peptides whose presence appears robust to variations in food matrix, sample preparation protocol, and MS instrumentation. We conclude that proteotypic peptides exist for a subset of allergenic milk, egg, and peanut proteins. For less studied allergens such as soy, wheat, fish, shellfish, and tree nuts, we offer guidance and tools for peptide selection and specificity verification as part of an interactive web database, the Allergen Peptide Browser (http://www.AllergenPeptideBrowser.org). With ongoing improvements in MS instrumentation, analysis software, and strategies for targeted quantitation, we expect an increasing role of MS as an analytical tool for ensuring regulatory compliance.
View details for PubMedID 28725476
View details for PubMedCentralID PMC5516885
Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching.
Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state.
View details for DOI 10.7554/eLife.16578
View details for PubMedID 27481325
View details for PubMedCentralID PMC4970870
A platform for retaining native morphology at sub-second time scales in cryogenic transmission electron microscopy
REVIEW OF SCIENTIFIC INSTRUMENTS
2013; 84 (5)
The advantage of cryogenic transmission electron microscopy for morphological analysis of complex fluids is the ability to capture native specimen morphology in solution. This is often limited by available sample preparation devices and procedures, which expose the sample to high shear rates leading to non-native artifacts, are unable to capture evolving samples at a time resolution shorter than a few seconds, and often non-specifically adsorb sample species from suspension resulting in a non-native sample concentration on the grid. In this paper we report the development of a new sample preparation device based on capillary action that overcomes all of these limitations. The use of a removal capillary placed parallel to the grid results in reduced shear and lower absorption of particulate material from the sample. A deposition capillary placed perpendicular to the grid allows for precise and sub-second resolution for time resolved studies. We demonstrate each of the features of this platform using model samples, and where appropriate, compare our results to those prepared using current vitrification platforms. Our results confirm that this new sample vitrification device opens up previously unattainable regimes for sample preparation and imaging and is a powerful new tool for cryogenic transmission electron microscopy.
View details for DOI 10.1063/1.4804648
View details for Web of Science ID 000319999300034
View details for PubMedID 23742558
Hand-Portable Kinematic Viscometer
ASTM Selected Technical Papers
View details for DOI 10.1520/STP156420120119