Browsing by Author "Ma, Sai"

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    Cell-type-specific epigenomic variations associated with BRCA1 mutation in pre-cancer human breast tissues
    Hsieh, Yuan-Pang; Naler, Lynette B.; Ma, Sai; Lu, Chang (Oxford University Press, 2022-01-13)
    BRCA1 germline mutation carriers are predisposed to breast cancers. Epigenomic regulations have been known to strongly interact with genetic variations and potentially mediate biochemical cascades involved in tumorigenesis. Due to the cell-type specificity of epigenomic features, profiling of individual cell types is critical for understanding the molecular events in various cellular compartments within complex breast tissue. Here, we produced cell-type-specific profiles of genome-wide histone modifications including H3K27ac and H3K4me3 in basal, luminal progenitor, mature luminal and stromal cells extracted from a small pilot cohort of pre-cancer BRCA1 mutation carriers (BRCA1(mut/+)) and non-carriers (BRCA1(+/+)), using a low-input ChIP-seq technology that we developed. We discovered that basal and stromal cells present the most extensive epigenomic differences between mutation carriers (BRCA1(mut/+)) and non-carriers (BRCA1(+/+)), while luminal progenitor and mature luminal cells are relatively unchanged with the mutation. Furthermore, the epigenomic changes in basal cells due to BRCA1 mutation appear to facilitate their transformation into luminal progenitor cells. Taken together, epigenomic regulation plays an important role in the case of BRCA1 mutation for shaping the molecular landscape that facilitates tumorigenesis.
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    Diffusion-based Microfluidic PCR for "One-pot" Analysis of Cells
    Ma, Sai; Loufakis, Despina N.; Cao, Zhenning; Chang, Yiwen; Achenie, Luke E. K.; Lu, Chang (The Royal Society of Chemistry, 2014-05-28)
    Genetic analysis starting with cell samples often requires multi-step processing including cell lysis, DNA isolation/purification, and polymerase chain reaction (PCR) based assays. When conducted on a microfluidic platform, the compatibility among various steps often demands a complicated procedure and a complex device structure. Here we present a microfluidic device that permits a “one-pot” strategy for multi-step PCR analysis starting from cells. Taking advantage of the diffusivity difference, we replace the smaller molecules in the reaction chamber by diffusion while retaining DNA molecules inside. This simple scheme effectively removes reagents from the previous step to avoid interference and thus permits multi-step processing in the same reaction chamber. Our approach shows high efficiency for PCR and potential for a wide range of genetic analysis including assays based on single cells.
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    Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria
    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina N.; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang (The Royal Society of Chemistry, 2014-08-14)
    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP–PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP–PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm_1 and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.
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    Electroporation-delivered fluorescent protein biosensors for probing molecular activities in cells without genetic encoding
    Sun, Chen; Ouyang, Mingxing; Cao, Zhenning; Ma, Sai; Alqublan, Hamzeh; Sriranganathan, Nammalwar; Wang, Yingxiao; Lu, Chang (The Royal Society of Chemistry, 2014-08-08)
    Fluorescent protein biosensors are typically implemented via genetic encoding which makes the examination of scarce cell samples impractical. By directly delivering the protein form of the biosensor into cells using electroporation, we detected intracellular molecular activity with the sample size down to ~100 cells with high spatiotemporal resolution.
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    Focusing of mammalian cells under an ultrahigh pH gradient created by unidirectional electropulsation in a confined microchamber
    Loufakis, Despina N.; Cao, Zhenning; Ma, Sai; Mittelman, David; Lu, Chang (The Royal Society of Chemistry, 2014-06-09)
    The transport and manipulation of cells in microfluidic structures are often critically required in cellular analysis. Cells typically make consistent movement in a dc electric field in a single direction, due to their electrophoretic mobility or electroosmotic flow or the combination of the two. Here we demonstrate that mammalian cells focus to the middle of a closed microfluidic chamber under the application of unidirectional direct current pulses. With experimental and computational data, we show that under the pulses electrochemical reactions take place in the confined microscale space and create an ultrahigh and nonlinear pH gradient (~2 orders of magnitude higher than the ones in protein isoelectric focusing) at the middle of the chamber. The varying local pH affects the cell surface charge and the electrophoretic mobility, leading to focusing in free solution. Our approach provides a new and simple method for focusing and concentrating mammalian cells at the microscale.
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    Immunomagnetic separation of tumor initiating cells by screening two surface markers
    Sun, Chen; Hsieh, Yuan-Pang; Ma, Sai; Geng, Shuo; Cao, Zhenning; Li, Liwu; Lu, Chang (Springer Nature, 2017-01-11)
    Isolating tumor initiating cells (TICs) often requires screening of multiple surface markers, sometimes with opposite preferences. This creates a challenge for using bead-based immunomagnetic separation (IMS) that typically enriches cells based on one abundant marker. Here, we propose a new strategy that allows isolation of CD44(+)/CD24(-) TICs by IMS involving both magnetic beads coated by anti-CD44 antibody and nonmagnetic beads coated by anti-CD24 antibody (referred to as two-bead IMS). Cells enriched with our approach showed significant enhancement in TIC marker expression (examined by flow cytometry) and improved tumorsphere formation efficiency. Our method will extend the application of IMS to cell subsets characterized by multiple markers.
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    Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex
    Ma, Sai; Hsieh, Yuan-Pang; Ma, Jian; Lu, Chang (AAAS, 2018-04-18)
    Extensive effort is under way to survey the epigenomic landscape of primary ex vivo tissues to establish normal reference data and to discern variation associated with disease. The low abundance of some tissue types and the isolation procedure required to generate a homogenous cell population often yield a small quantity of cells for examination. This difficulty is further compounded by the need to profile a myriad of epigenetic marks. Thus, technologies that permit both ultralow input and high throughput are desired. We demonstrate a simple microfluidic technology, SurfaceChIP-seq, for profiling genome-wide histone modifications using as few as 30 to 100 cells per assay and with up to eight assays running in parallel. We applied the technology to profile epigenomes using nuclei isolated from prefrontal cortex and cerebellum of mouse brain. Our cell type–specific data revealed that neuronal and glial fractions exhibited profound epigenomic differences across the two functionally distinct brain regions.
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    Microfluidics for Genetic and Epigenetic Analysis
    Ma, Sai (Virginia Tech, 2017-06-13)
    Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine.
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    Microfluidics for genome-wide studies involving next generation sequencing
    Ma, Sai; Murphy, Travis W.; Lu, Chang (AIP Publishing, 2017-03-10)
    Next-generation sequencing (NGS) has revolutionized how molecular biology studies are conducted. Its decreasing cost and increasing throughput permit profiling of genomic, transcriptomic, and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this article, we review recent progress on applying microfluidics to facilitate genome-wide studies. We emphasize on several technical aspects of NGS and how they benefit from coupling with microfluidic technology. We also summarize recent efforts on developing microfluidic technology for genomic, transcriptomic, and epigenomic studies, with emphasis on single cell analysis. We envision rapid growth in these directions, driven by the needs for testing scarce primary cell samples from patients in the context of precision medicine.
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    Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells
    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang (Nature, 2016-07-08)
    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples.