Browsing by Author "Lu, Chang"

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    3-D Bio-inspired Microenvironments for In Vitro Cell Migration
    Hosseini, Seyed Yahya (Virginia Tech, 2015-10-21)
    Cancer metastasis is the leading cause of death related to cancer diseases. Once the cancer cells depart the primary tumor site and enter the blood circulation, they spread through the body and will likely initiate a new tumor site. Therefore, understanding the cell migration and stopping the spread in the initial stage is the utmost of importance. In this dissertation, we have proposed a 3-D microenvironment that (partially) mimics the structures, complexity and circulation of human organs for cell migration studies. We have developed the tools to fabricate 3-D complex geometries in PDMS from our previously developed single-mask, single-etch technology in silicon. In this work, 3-D patterns are transferred from silicon structures to glass following anodic bonding and high temperature glass re-flow processes. Silicon is etched back thoroughly via wet etching and the glass is used as master device to create 3-D PDMS structures for use in dielectrophoresis cell sorting applications. Furthermore, this work has been modified to fabricate 3-D master devices in PDMS to create 3-D structures in collagen hydrogels to mimic native tissue structures. We have studied the interaction of endothelial cells with model geometries of blood vessels in collagen hydrogel at different concentrations to mimic the biomechanical properties of tissues varying from normal to tumor under the growth factor stimulation. Finally, we have designed and fabricated a silicon-based transmigration well with a 30um-thick membrane and 8um pores. This platform includes a deep microfluidic channel on the back-side sealed with a glass wafer. The migratory behavior of highly metastatic breast cancer cells, MDA-MB-231, is tested under different drug treatment conditions. This versatile platform will enable the application of more complex fluidic circulation profile, enhanced integration with other technologies, and running multiple assays simultaneously.
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    BRCA1 mutations attenuate super-enhancer function and chromatin looping in haploinsufficient human breast epithelial cells
    Zhang, Xiaowen; Wang, Yao; Chiang, Huai-Chin; Hsieh, Yuan-Pang; Lu, Chang; Park, Ben H.; Jatoi, Ismail; Jin, Victor X.; Hu, Yanfen; Li, Rong (2019-04-17)
    Background BRCA1-associated breast cancer originates from luminal progenitor cells. BRCA1 functions in multiple biological processes, including double-strand break repair, replication stress suppression, transcriptional regulation, and chromatin reorganization. While non-malignant cells carrying cancer-predisposing BRCA1 mutations exhibit increased genomic instability, it remains unclear whether BRCA1 haploinsufficiency affects transcription and chromatin dynamics in breast epithelial cells. Methods H3K27ac-associated super-enhancers were compared in primary breast epithelial cells from BRCA1 mutation carriers (BRCA1mut/+) and non-carriers (BRCA1+/+). Non-tumorigenic MCF10A breast epithelial cells with engineered BRCA1 haploinsufficiency were used to confirm the H3K27ac changes. The impact of BRCA1 mutations on enhancer function and enhancer-promoter looping was assessed in MCF10A cells. Results Here, we show that primary mammary epithelial cells from women with BRCA1 mutations display significant loss of H3K27ac-associated super-enhancers. These BRCA1-dependent super-enhancers are enriched with binding motifs for the GATA family. Non-tumorigenic BRCA1mut/+ MCF10A cells recapitulate the H3K27ac loss. Attenuated histone mark and enhancer activity in these BRCA1mut/+ MCF10A cells can be partially restored with wild-type BRCA1. Furthermore, chromatin conformation analysis demonstrates impaired enhancer-promoter looping in BRCA1mut/+ MCF10A cells. Conclusions H3K27ac-associated super-enhancer loss is a previously unappreciated functional deficiency in ostensibly normal BRCA1 mutation-carrying breast epithelium. Our findings offer new mechanistic insights into BRCA1 mutation-associated transcriptional and epigenetic abnormality in breast epithelial cells and tissue/cell lineage-specific tumorigenesis.
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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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    Computational Analysis of Gene Expression Regulation from Cross Species Comparison to Single Cell Resolution
    Lee, Jiyoung (Virginia Tech, 2020-08-31)
    Gene expression regulation is dynamic and specific to various factors such as developmental stages, environmental conditions, and stimulation of pathogens. Nowadays, a tremendous amount of transcriptome data sets are available from diverse species. This trend enables us to perform comparative transcriptome analysis that identifies conserved or diverged gene expression responses across species using transcriptome data. The goal of this dissertation is to develop and apply approaches of comparative transcriptomics to transfer knowledge from model species to non-model species with the hope that such an approach can contribute to the improvement of crop yield and human health. First, we presented a comprehensive method to identify cross-species modules between two plant species. We adapted the unsupervised network-based module finding method to identify conserved patterns of co-expression and functional conservation between Arabidopsis, a model species, and soybean, a crop species. Second, we compared drought-responsive genes across Arabidopsis, soybean, rice, corn, and Populus in order to explore the genomic characteristics that are conserved under drought stress across species. We identified hundreds of common gene families and conserved regulatory motifs between monocots and dicots. We also presented a BLS-based clustering method which takes into account evolutionary relationships among species to identify conserved co-expression genes. Last, we analyzed single-cell RNA-seq data from monocytes to attempt to understand regulatory mechanism of innate immune system under low-grade inflammation. We identified novel subpopulations of cells treated with lipopolysaccharide (LPS), that show distinct expression patterns from pro-inflammatory genes. The data revealed that a promising therapeutic reagent, sodium 4-phenylbutyrate, masked the effect of LPS. We inferred the existence of specific cellular transitions under different treatments and prioritized important motifs that modulate the transitions using feature selection by a random forest method. There has been a transition in genomics research from bulk RNA-seq to single-cell RNA-seq, and scRNA-seq has become a widely used approach for transcriptome analysis. With the experience we gained by analyzing scRNA-seq data, we plan to conduct comparative single-cell transcriptome analysis across multiple species.
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    The Design of Biodegradable Polyester Nanocarriers for Image-guided Therapeutic Delivery
    Jo, Ami (Virginia Tech, 2018-09-12)
    Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery.
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    Detecting intracellular translocation of native proteins quantitatively at the single cell level
    Cao, Zhenning; Geng, Shuo; Li, Liwu; Lu, Chang (The Royal Society of Chemistry, 2014-04-07)
    The intracellular localization and movement (i.e. translocation) of proteins are critically correlated with the functions and activation states of these proteins. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. In this report, we demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. We sequentially conducted chemical release of cytosolic proteins and fluorescence immunostaining of a targeted protein. The detected fluorescence intensity of cells was shown to be quantitatively correlated to the cytosolic/nuclear localization of the protein. We used our approach to detect the translocation of native NF-_B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. The incorporation of fluorescence immunostaining makes our approach compatible with the analysis of cell samples from lab animals and patients. Our method will dramatically lower the technological hurdle for studying subcellular localization of proteins.
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    Development of Controlled Ring-Opening Polymerization of  O-Carboxyanhydrides
    Zhong, Yongliang (Virginia Tech, 2020-10-27)
    The aim of my Ph.D. thesis is to summarize my research on the development of ring-opening polymerization (ROP) of O-carboxyanhydrides (OCAs) to synthesize functionalized, degradable polyesters. Biodegradable polyesters are promising alternatives to conventional petroleum-based non-degradable polyolefins and they are widely used in everyday applications ranging from clothing and packaging to agriculture and biomedicine. Commercially available polyesters, such as poly(lactic-co-glycolic acid), poly(lactic acid), and polycaprolactone, hydrolyze in physicochemical media. They have been approved by FDA and widely used for medical applications. However, the lack of side-chain functionality in polyesters and in corresponding monomers greatly plagues their utility for applications that demand physicochemical properties such as high stiffness, tensile strength and elasticity. Increasing efforts have been devoted to the introduction of pendant groups along the polymer chain in order to modify and modulate the physicochemical properties of polyesters and thereby to expand their applications. Over the last decade, OCAs have emerged as an alternative class of highly active monomers for polyester polymerization. OCAs are prepared from amino acids and thus have a richer range of side chain functionalities than lactone or lactide. Like lactones, OCAs can undergo ROP to obtain polyesters. Unfortunately, current ROP methods, especially those involving organocatalysts, result in uncontrolled polymerization including epimerization for OCAs bearing electron-withdrawing groups, unpredictable molecular weights (MWs), or slow polymerization kinetics. Based on our recent success of Ni/Ir photoredox catalysis allowing for rapid synthesis of high-MWs polyesters, we further explore new polymerization chemistry to use earth-abundant metal complexes to replace expensive rare-earth metal photocatalysts, and practice the polymerization in moderate and energy-efficient reaction conditions. This thesis introduces novel photoredox and electrochemical earth-abundant metal catalysts that overcome above difficulties in the ROP chemistry of OCAs, and allow for the preparation of stereoregular polyesters bearing abundant side-chain functionalities in a highly controlled manner. Specifically, various highly active metal complexes have been developed for stereoselective ROP of OCAs, either using light or electricity, to synthesize syndiotactic or stereoblock copolymers with different thermal properties. Additionally, simple purification protocols of OCAs have also been initially studied, which potentially paves the way to bulk production of functional monomers. In this thesis, I first describe newly-developed photoredox Co/Zn catalysts to achieve a controlled ROP of enantiopure OCAs under mild reaction conditions (Chapter 2). Such discovery is extended to the combination use of Co catalysts with various Zn/Hf complexes that enable stereoselective controlled ROP of racemic OCAs for the preparation of stereoregular polyesters (Chapter 3). The mechanistic studies of the aforementioned developments lead to the application of such a catalytic system in controlled electrochemical ROP of OCAs (Chapter 4). Such chemistry can also be translated to stereoselectively electrochemical ROP of racemic OCAs to either syndiotactic or stereoblock polyesters, allowing precise control of polyester's tacticity and sequence (Chapter 5). An overview future work has been summarized (Chapter 6).
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    Development of Implantable Optical Fibers for Immunotherapeutics Delivery and Tumor Impedance Measurement
    Chin, Ai Lin (Virginia Tech, 2021-11-30)
    Immune checkpoint blockade antibodies have promising clinical applications but suffer from disadvantages such as severe toxicities and moderate patient-response rates. None of the current delivery strategies, including local administration aiming to avoid systemic toxicities, can sustainably supply drugs over the course of weeks; adjustment of drug dose, either to lower systemic toxicities or to augment therapeutic response, is not possible. Herein, an implantable miniaturized device has been developed using electrode-embedded optical fibers with both local delivery and measurement capabilities over the course of a few weeks. The combination of local immune checkpoint blockade antibodies delivery via this device with photodynamic therapy elicits a sustained anti-tumor immunity in multiple tumor models. Named Implantable Miniature Optical Fiber Device (IMOD), this device uses tumor impedance measurement for timely presentation of treatment outcomes, and allows modifications to the delivered drugs and their concentrations, rendering IMOD as outstandingly valuable for on-demand delivery of potent immunotherapeutics without exacerbating toxicities. Rigorous studies performed using IMOD are presented and discussed in the follow chapters, followed by exploration of proposed work to expand the breadth of functions offered by this implantable biomedical platform.
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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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    Dose-dependent effects of endotoxin on monocyte and the underlying mechanisms
    Pradhan, Kisha (Virginia Tech, 2022-01-24)
    Monocytes are dynamic innate immune cells that respond differently based upon the dose and duration of an infection. While super low dose endotoxin is found in chronic inflammatory diseases such as atherosclerosis, exposure to high dose endotoxin leads to sepsis. However, clear characterization of monocytes and the underlying mechanisms in these disease conditions is lacking. To elucidate the missing information, we conducted two different projects. In the first project, we investigated the role of super low dose endotoxin in polarizing monocytes to a prolonged low-grade inflammatory state with no resolution, disrupting homeostasis. This low grade inflammatory phenotype was confirmed by sustained induction of inflammatory mediators CD40 and CD11a. In addition, low grade inflammatory monocytes influence neighboring T cells by suppressing T cell regulatory functions. Mechanistically, we showed that the non-resolving inflammatory phenotypes in monocytes is dependent on non-traditional TLR4 adaptor called TRAM. In the second project, we focused on the effects of high dose endotoxin on monocyte phenotypes. We reported that high dose endotoxin give rise to a mix of both immunosuppressive and pathogenic inflammatory monocytes, leading to monocyte exhaustion. While thorough research is conducted to study the immunosuppressive monocytes and underlying long term effects, role of pathogenic inflammatory monocytes is not well addressed. Monocyte exhaustion leads to elevated levels of CD38, an inflammatory mediator, elevated ROS levels, depleted NAD+ and mitochondrial respiration. STAT1 and KLF4 are critical transcription factors in sustaining exhausted phenotypes. Indeed, TRAM adaptor molecule also mediates this exhaustion as TRAM deletion restores monocyte health. Taken together, our work defines novel monocyte phenotypes and mechanism in super-low dose or high dose endotoxin environments.
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    Droplet-Based Microfluidics for High-Throughput Single-Cell Omics Profiling
    Zhang, Qiang (Virginia Tech, 2022-09-06)
    Droplet-based microfluidics is a powerful tool permitting massive-scale single-cell analysis in pico-/nano-liter water-in-oil droplets. It has been integrated into various library preparation techniques to accomplish high-throughput scRNA-seq, scDNA-seq, scATAC-seq, scChIP-seq, as well as scMulti-omics-seq. These advanced technologies have been providing unique and novel insights into both normal differentiation and disease development at single-cell level. In this thesis, we develop four new droplet-based tools for single-cell omics profiling. First, the developed Drop-BS is the first droplet-based platform to construct single-cell bisulfite sequencing libraries for DNA methylome profiling and allows production of BS library of 2,000-10,000 single cells within 2 d. We applied the technology to separately profile mixed cell lines, mouse brain tissues, and human brain tissues to reveal cell type heterogeneity. Second, the new Drop-ChIP platform only requires two steps of droplet generation to achieve multiple steps of reactions in droplets such as single-cell lysis, chromatin fragmentation, ChIP, and barcoding. Third, we aim to establish a droplet-based platform to accomplish high-throughput full-length RNA-seq (Drop-full-seq), which both current tube-based and droplet-based methods cannot realize. Last, we constructed an in-house droplet-based tool to assist single-cell ATAC-seq library preparation (Drop-ATAC), which provided a low-cost and facile protocol to conduct scATAC-seq in laboratories without the expensive instrument.
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    Dynamics of Micro-Particles in Complex Environment
    Yang, Fengchang (Virginia Tech, 2017-07-21)
    Micro-particles are ubiquitous in microsystems. The effective manipulation of micro-particles is often crucial for achieving the desired functionality of microsystems and requires a fundamental understanding of the particle dynamics. In this dissertation, the dynamics of two types of micro-particles, Janus catalytic micromotors (JCMs) and magnetic clusters, in complex environment are studied using numerical simulations. The self-diffusiophoresis of JCMs in a confined environment is studied first. Overall, the translocation of a JCM through a short pore is slowed down by pore walls, although the slowdown is far weaker than the transport of particles through similar pores driven by other mechanisms. A JCM entering a pore with its axis not aligned with the pore axis can execute a self-alignment process and similar phenomenon is found for JCMs already inside the pore. Both hydrodynamic effect and 'chemical effect', i.e., the modification of the concentration of chemical species around JCMs by walls and other JCMs, play a key role in the observed dynamics of JCMs in confined and crowded environment. The dynamics of bubbles and JCMs in liquid films covering solid substrates are studied next. A simple criterion for the formation of bubbles on isolated JCMs is developed and validated. The anomalous bubble growth law (r~t^0.7) is rationalized by considering the relative motion of growing bubbles and their surrounding JCMs. The experimentally observed ultra-fast collapse of bubbles is attributed to the coalescence of the bubble with the liquid film-air interface. It is shown that the collective motion of JCMs toward a bubble growing on a solid substrate is caused by the evaporation-induced Marangoni flow near the bubble. The actuation of magnetic clusters using non-uniform alternating magnetic fields is studied next. It is discovered that the clusters' clockwise, out-of-plane rotation is a synergistic effect of the magnetophoresis force, the externally imposed magnetic torque and the hydrodynamic interactions between the cluster and the substrate. Such a rotation enables the cluster to move as a surface walker and leads to unique dynamics, e.g., the cluster moves away from the magnetic source and its trajectory exhibits a periodic fluctuation with a frequency twice of the field frequency.
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    Effects of Electric Fields on Forces between Dielectric Particles in Air
    Chiu, Ching-Wen (Virginia Tech, 2013-05-01)
    We developed a quantitative measurement technique using atomic force microscopy (AFM) to study the effects of both DC and AC external electric fields on the forces between two dielectric microspheres. In this work we measured the DC and AC electric field-induced forces and adhesion force between two barium titanate (BaTiO?) glass microspheres in a low humidity environment by this technique. The objective here is to find out the correlation between these measured forces and applied field strength, frequency, and the separation distance between the two spheres was studied. Since the spheres would oscillate under an AC field, the AC field-induced force was divided into dynamic component (i.e., time-varying term) and static component (i.e., time-averaged term) to investigate. The oscillatory response occurs at a frequency that is twice the drive frequency since the field-induced force is theoretically proportional to the square of the applied field. This behavior can be observed in the fast Fourier transformation (FFT) spectra of the time series of the deflection signal. The magnitude of the vibration response increases when the frequency of the drive force is near resonant frequency of the particle-cantilever probe. The amplitude of this vibration increases with proximity of the two particles, and ultimately causes the particles to repeatedly hit each other as in tapping mode AFM. The effect of the Maxwell-Wagner interfacial relaxation on the DC electric field-induced force was discovered by monitoring the variation of the field-induced force with time. The static component of the AC electric field-induced force does not vary with the applied frequency in the range from 1 to 100 kHz, suggesting that the crossover frequency may equal to or less than 1 kHz and the permittivities of the BaTiO? glass microspheres and medium dominate the field-3 induced force. The AC field-induced force is proportional to the square of the applied electric field strength. This relationship persists even when the separation between the spheres is much smaller than the diameter of the microspheres. The large magnitude of the force at small separations suggests that the local field is distorted by the presence of a second particle, and the continued dependence on the square of the field but the measured force is much larger than the theoretical results, suggesting that the local electric field around the closely spaced spheres is distorted and enhanced but the effects of the local field distortion may have not much to with the applied electric field. Compared with the calculated results from different models, our results demonstrate that the field-induced force is much more long-range than expected in theory. In addition, the DC field-induced adhesion force is larger than the AC field-induced one due to the interfacial charge accumulation, agreeing with the discovery of the Maxwell-Wagner interfacial relaxation effect on the DC field-induced force. No obvious correlation between the field-induced adhesion and the applied frequency is found. However, both the DC and AC field-induced adhesion forces display the linearity with the square of the applied electric field strength as well.
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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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    Enzymatic fuel cells via synthetic pathway biotransformation
    Zhu, Zhiguang (Virginia Tech, 2013-06-11)
    Enzyme-catalyzed biofuel cells would be a great alternative to current battery technology, as they are clean, safe, and capable of using diverse and abundant renewable biomass with high energy densities, at mild reaction conditions. However, currently, three largest technical challenges for emerging enzymatic fuel cell technologies are incomplete oxidation of most fuels, limited power output, and short lifetime of the cell. Synthetic pathway biotransformation is a technology of assembling a number of enzymes coenzymes for producing low-value biocommodities. In this work, it was applied to generate bioelectricity for the first time. Non-natural enzymatic pathways were developed to utilize maltodextrin and glucose in enzymatic fuel cells. Three immobilization approaches were compared for preparing enzyme electrodes. Thermostable enzymes from thermophiles were cloned and expressed for improving the lifetime and stability of the cell. To further increase the power output, non-immobilized enzyme system was demonstrated to have higher power densities compared to those using immobilized enzyme system, due to better mass transfer and retained native enzyme activities. With the progress on pathway development and power density/stability improvement in enzymatic fuel cells, a high energy density sugar-powered enzymatic fuel cell was demonstrated. The enzymatic pathway consisting of 13 thermostable enzymes enabled the complete oxidation of glucose units in maltodextrin to generate 24 electrons, suggesting a high energy density of such enzymatic fuel cell (300 Wh/kg), which was several folds higher than that of a lithium-ion battery. Maximum power density was 0.74 mW/cm2 at 50 deg C and 20 mM fuel concentration, which was sufficient to power a digital clock or a LED light. These results suggest that enzymatic fuel cells via synthetic pathway biotransformation could achieve high energy density, high power density and increased lifetime. Future efforts should be focused on further increasing power density and enzyme stability in order to make enzymatic fuel cells commercially applicable.
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    Epigenomic and transcriptomic analyses reveal differences between low-grade inflammation and severe exhaustion in LPS-challenged murine monocytes
    Naler, Lynette B.; Hsieh, Yuan-Pang; Geng, Shuo; Zhou, Zirui; Li, Liwu; Lu, Chang (Nature Portfolio, 2022-01-28)
    Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies. Lynette Naler and Yuan-Pang Hsieh et al. evaluate epigenomic and transcriptomic differences in mouse bone marrow-derived macrophages following exposure to high or low doses of LPS. Their results suggest that both low- and high-grade inflammation involve TRAM-dependent pathways.
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    Epigenomic and Transcriptomic Changes in the Onset of Disease
    Naler, Lynette Brigitte (Virginia Tech, 2021-05-19)
    Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies.
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    Epigenomic tomography for probing spatially defined chromatin state in the brain
    Liu, Zhengzhi; Deng, Chengyu; Zhou, Zirui; Ya, Xiao; Jiang, Shan; Zhu, Bohan; Naler, Lynette B.; Jia, Xiaoting; Yao, Danfeng (Daphne); Lu, Chang (Cell Press, 2024-03-25)
    Spatially resolved epigenomic profiling is critical for understanding biology in the mammalian brain. Singlecell spatial epigenomic assays were developed recently for this purpose, but they remain costly and labor intensive for examining brain tissues across substantial dimensions and surveying a collection of brain samples. Here, we demonstrate an approach, epigenomic tomography, that maps spatial epigenomes of mouse brain at the scale of centimeters. We individually profiled neuronal and glial fractions of mouse neocortex slices with 0.5 mm thickness. Tri-methylation of histone 3 at lysine 27 (H3K27me3) or acetylation of histone 3 at lysine 27 (H3K27ac) features across these slices were grouped into clusters based on their spatial variation patterns to form epigenomic brain maps. As a proof of principle, our approach reveals striking dynamics in the frontal cortex due to kainic-acid-induced seizure, linked with transmembrane ion transporters, exocytosis of synaptic vesicles, and secretion of neurotransmitters. Epigenomic tomography provides a powerful and cost-effective tool for characterizing brain disorders based on the spatial epigenome.
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    Finding Typhoid Mary: Identifying Latent Carriers of Salmonella enterica serovar Typhimurium
    Schroeder, Betsy (Virginia Tech, 2020-09-16)
    Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important human pathogen. The Centers for Disease Control and Prevention (CDC) estimates that 1,027,561 people become ill with nontyphoidal Salmonellosis annually, and S. Typhimurium is one of the most common disease causing serovars. Quantification of the true number of cases of salmonellosis is hampered by the presence of a carrier state. These carriers are animals and humans that carry the pathogens for a variable period of time without showing any clinical signs. One of the biggest barriers to controlling and preventing salmonellosis in a population is identification of these carriers. Identifying these latent carriers of chronic infections is vital to preventing such disease transmission and creating avenues for novel control and treatments. In my dissertation research, we developed a cell culture model to study latent Salmonella infections. By activating human monocytes with retinoic acid and vitamin D3, we were able to isolate Salmonella from such cells 45 days after inoculation. We subsequently used this model to identify genes that were upregulated in this chronic infection model. We found that aceA, a gene that codes for isocitrate lyase, is significantly upregulated on days 10 and 30 post infection. Isocitrate lyase is part of the glyloxylate cycle. Some bacterial species have developed a mechanism to utilize acetone as a carbon source to synthesize tricarboxylic acid (TCA) cycle intermediates. This anaplerotic reaction allows organisms to conserve carbon and use alternative carbon sources. This cycle is one way in which bacteria can adapt and survive in an intracellular environment. This intracellular survival is key to latent infections persisting within a host. It is biologically plausible that, in order to survive in a latent state, S. Typhimurium would up-regulate genes that would facilitate intracellular survival. After establishing the cell culture model, we tested the hypothesis that aceA is upregulated in latent infections of S. Typhimurium in a mouse model. We orally challenged mice that were resistant to Salmonella infection, collected their feces, and collected tissue specimens at several time points up to 135 days post-challenge. These samples were cultured and tested using quantitative polymerase chain reaction (qPCR). The qPCR results showed that tissue samples from inoculated mice had increased aceA expression 95 days after challenge. Finally, we examined whether aceA expression could be detected in cattle lymph node samples. Supra-mammary lymph nodes from 40 dairy cattle and mesenteric lymph nodes from 100 culled cattle were sampled and submitted for culture and qPCR. None of the supra-mammary lymph nodes were positive for Salmonella via culture or aceA qPCR; however, 11 mesenteric lymph nodes showed increased aceA expression in qPCR compared to 5 culture positive lymph nodes. Further research is necessary, but these results demonstrate some of the advantages of using genetic primers to identify latent Salmonella infections in clinically normal cattle. In addition, the assay may be able to differentiate between latent and active salmonellosis, and could be used to provide targeted drug delivery.