Person: Zhang, Huidan
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Zhang
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Huidan
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Zhang, Huidan
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Publication Label-free single-cell protein quantification using a drop-based mix-and-read system(Nature Publishing Group, 2015) Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, DavidQuantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.Publication High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics(Public Library of Science, 2015) Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A.; Schnall-Levin, Michael; Zhang, Huidan; Basu, Anindita; Bernstein, Bradley; Weitz, DavidThe importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.Publication Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition(Nature Publishing Group, 2016) Burger, Jan A.; Landau, Dan A.; Taylor-Weiner, Amaro; Bozic, Ivana; Zhang, Huidan; Sarosiek, Kristopher; Wang, Lili; Stewart, Chip; Fan, Jean; Hoellenriegel, Julia; Sivina, Mariela; Dubuc, Adrian; Fraser, Cameron; Han, Yulong; Li, Shuqiang; Livak, Kenneth J.; Zou, Lihua; Wan, Youzhong; Konoplev, Sergej; Sougnez, Carrie; Brown, Jennifer R.; Abruzzo, Lynne V.; Carter, Scott L.; Keating, Michael J.; Davids, Matthew S.; Wierda, William G.; Cibulskis, Kristian; Zenz, Thorsten; Werner, Lillian; Cin, Paola Dal; Kharchencko, Peter; Neuberg, Donna; Kantarjian, Hagop; Lander, Eric; Gabriel, Stacey; O'Brien, Susan; Letai, Anthony; Weitz, David; Nowak, Martin; Getz, Gad; Wu, CatherineResistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.Publication A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders(Nature Publishing Group, 2016) Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David; Chong, ShaorongDrop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.Publication Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics(American Society for Microbiology, 2015) Zhang, Huidan; Cockrell, Shelley K.; Kolawole, Abimbola O.; Rotem, Assaf; Serohijos, Adrian; Chang, Connie B.; Tao, Ye; Mehoke, Thomas S.; Han, Yulong; Lin, Jeffrey S.; Giacobbi, Nicholas S.; Feldman, Andrew B.; Shakhnovich, Eugene; Weitz, David; Wobus, Christiane E.; Pipas, James M.Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.Publication Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours(Nature Publishing Group, 2012) Glenn, David; Zhang, Huidan; Kasthuri, Narayanan; Schalek, Richard; Lo, P. K.; Trifonov, Alexei; Park, Hongkun; Lichtman, Jeff; Walsworth, RonaldCorrelative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.