Browsing by Author "Lawrence, Christopher B."

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    The Alternaria alternata Mycotoxin Alternariol Suppresses Lipopolysaccharide-Induced Inflammation
    Grover, Shivani; Lawrence, Christopher B. (MDPI, 2017-07-20)
    The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage cell line (RAW264.7) were investigated. During these studies, it was discovered that AOH and to a lesser extent AME potently suppressed lipopolysaccharide (LPS)-induced innate immune responses in a dose-dependent manner. Treatment of BEAS-2B cells with AOH resulted in morphological changes including a detached pattern of growth as well as elongated arms. AOH/AME-related immune suppression and morphological changes were linked to the ability of these mycotoxins to cause cell cycle arrest at the G2/M phase. This model was also used to investigate the AOH/AME mechanism of immune suppression in relation to aryl hydrocarbon receptor (AhR). AhR was not found to be important for the immunosuppressive properties of AOH/AME, but appeared important for the low levels of cell death observed in BEAS-2B cells.
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    The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species
    Dang, Ha X.; Pryor, Barry M.; Peever, Tobin L.; Lawrence, Christopher B. (2015-03-25)
    Background Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. Description We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. Conclusion Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu .
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    Analysis of global gene expression changes in human bronchial epithelial cells exposed to spores of the allergenic fungus, Alternaria alternata
    Babiceanu, Mihaela; Howard, B. A.; Rumore, A. C.; Kita, H.; Lawrence, Christopher B. (Frontiers, 2013-07-19)
    Exposure and sensitivity to ubiquitous airborne fungi such as Alternaria alternata have long been implicated in the development, onset, and exacerbation of chronic allergic airway disorders. This present study is the first to investigate global changes in host gene expression during the interaction of cultured human bronchial epithelial cells and live Alternaria spores. In in vitro experiments human bronchial epithelial cells (BEAS-2B) were exposed to spores or media alone for 24 h. RNA was collected from three biological replicates per treatment and was used to assess changes in gene expression patterns using Affymetrix Human Genome U133 Plus 2.0 Arrays. In cells treated with Alternaria spores compared to controls, 613 probe sets representing 460 individual genes were found differentially expressed (p <= 0.05). In this set of 460 statistically significant, differentially expressed genes, 397 genes were found to be up-regulated and 63 were down-regulated. Of these 397 up-regulated genes, 156 genes were found to be up-regulated >= 2 fold. Interestingly, none of the 63 down-regulated genes were found differentially expressed at <=-2 fold. Differentially expressed genes were identified following statistical analysis and subsequently used for pathway and network evaluation. Interestingly, many cytokine and chemokine immune response genes were up-regulated with a particular emphasis on interferon-inducible genes. Genes involved in cell death, retinoic acid signaling, and TLR3 response pathways were also significantly up-regulated. Many of the differentially up-regulated genes have been shown in other systems to be associated with innate immunity, inflammation and/or allergic airway diseases. This study now provides substantial information for further investigating specific genes and innate immune system pathways activated by Alternaria in the context of allergic airway diseases.
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    Analysis of the Allergenic Potential of the Ubiquitous Airborne Fungus Alternaria Using Bioinformatics
    Babiceanu, Mihaela (Virginia Tech, 2011-06-15)
    Among the environmental airborne fungi one of the most common is Alternaria alternata. From a clinical perspective Alternaria has long been associated with IgE-mediated, histamine-dependent mold allergy, allergic rhinitis, chronic rhinosinusitis (CRS) and asthma. Recently it has been proven that an abnormal immunological response to Alternaria most likely contributes to the pathogenesis of upper respiratory airway disorders. In this body of work, we present for the first time results of several sets of experiments including, 1) the analysis of A. alternata spore germination expressed sequence tags (ESTs), 2) the survey of global allergen homologues in fungal genomes, and 3) the first microarray experiment investigating airway epithelial cell responses to this fungus. In the first project, the analyses of the EST dataset offered a first look into the gene content of A. alternata and represents the beginning of future research of this ubiquitous fungus. Annotation and classification of ESTs revealed a number of genes that could be involved in the immunomodulation process of the human immune response toward fungi. We also discovered that the majority of known allergens are expressed during the spore germination phase of A. alternata. For investigating the allergenic potential of fungi we developed a whole genome approach by querying fungal genome sequences (A. alternata, A. brassicicola, and Aspergillus fumigatus) with a database of all known allergenic proteins from a taxonomically diverse group of organisms. Interestingly, we identified homologues of diverse types of allergens in these fungal genomes and also many homologues of allergens from other organisms including those from pollen, insects, and venoms. Finally, we investigated global gene expression changes of human airway cells in response to A. alternata and an ∆alt a 1 deletion mutant. We found that wild type Alternaria spores induced significant changes in gene expression patterns in human airway epithelial cells, especially known immune response genes. Furthermore, results of these analyses revealed that Alt a 1 is a major factor in inducing epithelial inflammatory responses.
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    The Cell Wall Integrity-Associated Map Kinase Homolog, AbSlt2 in the Necrotrophic fungus Alternaria brassicicola is Required for Pathogenicity of Brassicas
    Scott, Derrick Cornelius (Virginia Tech, 2009-01-22)
    Using the genome database of the phytopathogenic fungus, Alternaria brassicicola, we identified a gene with high homology to the cell wall integrity-associated mitogen-activated protein (MAP) kinase, Slt2 in the yeast, Saccharomyces cerevisiae. This MAP kinase consists of a predicted 1,251-bp open reading frame, and encodes a 416-amino-acid protein weighing 47501 Da. This homolog was designated AbSlt2 (A. brassicicola Slt2) and gene disruption knockout (KO) mutants were generated in an A. brassicicola wild type background. Several altered phenotypes were found in the mutants compared to the wild type. During growth in various liquid and solid media, the abslt2 mutants displayed slightly aberrant hyphal growth and were unable to develop at the same rate as wild type. Furthermore, scanning electron microscopy (SEM) analysis revealed the abslt2 mutants showed decreased penetration ability, underdeveloped appresoria, and altered morphology on the leaf surface of the host plant, Brassica oleracea (cabbage) when compared to wild type. Abslt2 mutant hyphae exhibited slower growth in planta ultimately resulting in highly reduced virulence. Complementation of the disruption mutant with the wild type gene fully restored pathogenicity. Therefore, AbSlt2 is a new pathogenicity and developmental factor in A. brassicicola.
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    Characterizing the Innate Immune Response of Human Airway Cells to the Unique Fungal Allergen Alt a 1
    Hayes, Tristan Alonzo (Virginia Tech, 2017-04-25)
    Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P).
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    Computational approaches for discovery of common immunomodulators in fungal infections: towards broad-spectrum immunotherapeutic interventions
    Kidane, Yared H.; Lawrence, Christopher B.; Murali, T. M. (2013-10-07)
    Background Fungi are the second most abundant type of human pathogens. Invasive fungal pathogens are leading causes of life-threatening infections in clinical settings. Toxicity to the host and drug-resistance are two major deleterious issues associated with existing antifungal agents. Increasing a host’s tolerance and/or immunity to fungal pathogens has potential to alleviate these problems. A host’s tolerance may be improved by modulating the immune system such that it responds more rapidly and robustly in all facets, ranging from the recognition of pathogens to their clearance from the host. An understanding of biological processes and genes that are perturbed during attempted fungal exposure, colonization, and/or invasion will help guide the identification of endogenous immunomodulators and/or small molecules that activate host-immune responses such as specialized adjuvants. Results In this study, we present computational techniques and approaches using publicly available transcriptional data sets, to predict immunomodulators that may act against multiple fungal pathogens. Our study analyzed data sets derived from host cells exposed to five fungal pathogens, namely, Alternaria alternata, Aspergillus fumigatus, Candida albicans, Pneumocystis jirovecii, and Stachybotrys chartarum. We observed statistically significant associations between host responses to A. fumigatus and C. albicans. Our analysis identified biological processes that were consistently perturbed by these two pathogens. These processes contained both immune response-inducing genes such as MALT1, SERPINE1, ICAM1, and IL8, and immune response-repressing genes such as DUSP8, DUSP6, and SPRED2. We hypothesize that these genes belong to a pool of common immunomodulators that can potentially be activated or suppressed (agonized or antagonized) in order to render the host more tolerant to infections caused by A. fumigatus and C. albicans. Conclusions Our computational approaches and methodologies described here can now be applied to newly generated or expanded data sets for further elucidation of additional drug targets. Moreover, identified immunomodulators may be used to generate experimentally testable hypotheses that could help in the discovery of broad-spectrum immunotherapeutic interventions. All of our results are available at the following supplementary website: http://bioinformatics.cs.vt.edu/~murali/supplements/2013-kidane-bmc
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    Discord between morphological and phylogenetic species boundaries: incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen
    Stewart, Jane E.; Timmer, Lavern W.; Lawrence, Christopher B.; Pryor, Barry M.; Peever, Tobin L. (2014-03-03)
    Background Traditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used; however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. Results Coalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia. Conclusions The number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.
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    Dissection of Drought Responses in Arabidopsis
    Harb, Amal Mohammad (Virginia Tech, 2010-07-19)
    Plants as sessile organisms are susceptible to many environmental stresses such as drought, and salinity. They have therefore evolved mechanisms to acclimate and tolerate environmental stresses. Knowledge of the molecular aspects of abiotic stress gleaned from extensive studies in Arabidopsis has provided much information on the complex processes underlying plant response to abiotic stresses. Nevertheless, there is a need for integration of the knowledge gained and a systematic molecular genetic dissection of the complex responses to abiotic stress. In this study in Arabidopsis, comparative expression profiling analysis of progressive (pDr) and moderate (mDr) drought treatments revealed common drought responses, as well as treatment specific signatures responses to drought stress. Under prolonged moderate drought plants develop different mechanisms for acclimation: induction of cell wall loosening at early stage, and a change in hormonal balance (ABA: JA) at late stage of moderate drought. Taking a reverse genetics approach, a MYB transcription factor (MYB109) has been identified as a regulator of growth under drought and salt stress. Global expression profiling showed possible mechanisms of how MYB109 modulates growth under drought conditions: as a regulator of RNA processing and splicing and as a negative regulator of jasmonic acid biosynthesis and signaling. A forward genetics screen for drought and salt tolerance of transposon activation tag (ATag) lines led to the discovery of novel genes, which shed light on unexplored areas of abiotic stress biology. Utilizing this strategy, a potential role for cell wall modification and MATE transporters in response to drought and salt stress has been discovered, which needs further analysis to integrate this information on the role of these biological processes in plant stress biology.
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    Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi
    Ohm, Robin A.; Feau, Nicolas; Henrissat, Bernard; Schoch, Conrad L.; Horwitz, Benjamin A.; Barry, Kerrie W.; Condon, Bradford J.; Copeland, Alex C.; Dhillon, Braham; Glaser, Fabian; Hesse, Cedar N.; Kosti, Idit; LaButti, Kurt; Lindquist, Erika A.; Lucas, Susan; Salamov, Asaf A.; Bradshaw, Rosie E.; Ciuffetti, Lynda; Hamelin, Richard C.; Kema, Gert H. J.; Lawrence, Christopher B.; Scott, James A.; Spatafora, Joseph W.; Turgeon, B. Gillian; de Whit, Pierre J. G. M.; Zhong, Shaobin; Goodwin, Stephen B.; Grigoriev, Igor V. (Public Library of Science, 2012-12-06)
    The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.
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    Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations
    Rouxel, Thierry; Grandaubert, Jonathan; Hane, James K.; Hoede, Claire; van de Wouw, Angela P.; Couloux, Arnaud; Dominguez, Victoria; Anthouard, Veronique; Bally, Pascal; Bourras, Salim; Cozijnsen, Anton J.; Ciuffetti, Lynda M.; Degrave, Alexandre; Dilmaghani, Azita; Duret, Laurent; Fudal, Isabelle; Goodwin, Stephen B.; Gout, Lilian; Glaser, Nicolas; Linglin, Juliette; Kema, Gert H. J.; Lapalu, Nicolas; Lawrence, Christopher B.; May, Kim; Meyer, Michel; Ollivier, Benedicte; Poulain, Julie; Schoch, Conrad L.; Simon, Adeline; Spatafora, Joseph W.; Stachowiak, Anna; Turgeon, B. Gillian; Tyler, Brett M.; Vincent, Delphine; Weissenbach, Jean; Amselem, Joelle; Quesneville, Hadi; Oliver, Richard P.; Wincker, Patrick; Balesdent, Marie-Helene; Howlett, Barbara J. (Springer Nature, 2011-02)
    Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.
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    Engineered microsystems and their application in the culture and characterization of three-dimensional (3D) breast tumor models
    Menon, Nidhi (Virginia Tech, 2021-05-26)
    Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. One of the major challenges in precision and personalized medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation of the limited number of primary cells derived from patients. Therefore, our aims are to 1. Develop a biologically relevant platform for culturing cancer cells and characterize how it influences the cell growth and phenotype compared to conventional 2-dimensional(2D) cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen them on the platform for potential anticancer properties in a preliminary drug discovery pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within these systems. Several biomaterial scaffolds with microscale architectures have been utilized for engineering the tumor extracellular matrix, but very few studies have thoroughly characterized the phenotypic changes in their cell models, which is critical for translational applications of biomaterial systems. The overall objective of these studies is to engineer a biomimetic platform for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with triple-negative breast cancer cells and showed significant changes in characteristics, with cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation. Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer therapy. Furthermore, we were able to show the importance of our biomimetic platform in the process of drug discovery using fungal endophytes and their secondary metabolites as the source for potential anticancer molecules. Additionally, we developed gold nanoparticle and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally on our platform. We were able to show quenching of the green fluorescent fluorophores due to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts are directed towards incorporating the hydrogel system with antigen-dependent sensors in a conceptually-designed microfluidic platform to spatiotemporally quantify the expression of surface proteins in various cells of the tumor stroma. This includes the migration,infiltration, and polarization of specific immune cells. This approach will provide further insight into the heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic platform.
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    The Epigenetic Role of EGR1 during Postnatal Mammalian Brain Development
    Sun, Zhixiong (Virginia Tech, 2018-08-03)
    DNA methylation is an epigenetic mechanism critical for tissue development, cell specification and cellular function. Mammalian brains consist of millions to billions of neurons and glial cells that can be subdivided into many distinct types of cells. We hypothesize that brain methylomes are heterogeneously methylated across different types of cells and the transcription factors play key roles in brain methylome programming. To dissect brain methylome heterogeneity, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) loci which demonstrate bipolar DNA methylation pattern, i.e., hypermethylated in one cell subset but hypomethylated in others. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Importantly, we found that putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the important role of putative CSM loci during brain development, implying that dramatic changes in functions and complexities of the brain may be companied by a rapid change in epigenetic heterogeneity. To explore epigenetic regulatory mechanisms during brain development, as described in Chapter 3, we adopted unbiased data-driven approaches to re-analyze methylomes for human and mouse frontal cortices at different developmental stages. We predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated that thousands of EGR1 binding sites are with cell-type specific methylation patterns established during postnatal frontal cortex development. More specifically, the CpG dinucleotides within these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 programs the brain methylome together with TET1 during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome.
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    Functional Analysis of Secondary Metabolite Biosynthesis-Related Genes in Alternaria brassicicola
    Kim, Kwang Hyung (Virginia Tech, 2009-09-07)
    Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis.
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    Fungus-induced inflammation and eosinophil degranulation
    (United States Patent and Trademark Office, 2010-10-19)
    This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation are provided.
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    Fungus-induced inflammation and eosinophil degranulation
    (United States Patent and Trademark Office, 2010-02-16)
    This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation are provided.
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    Fungus-induced inflammation and eosinophil degranulation
    (United States Patent and Trademark Office, 2011-02-15)
    This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation are provided.
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    Fungus-induced inflammation and eosinophil degranulation
    (United States Patent and Trademark Office, 2011-07-05)
    This document relates to methods and materials involved in fungus-induced inflammation and eosinophil degranulation. For example, isolated nucleic acids encoding fungal polypeptides, fungal polypeptides, methods for assessing fungus-induced inflammation, methods for assessing eosinophil degranulation, and methods for identifying inhibitors of fungus-induced inflammation and/or eosinophil degranulation are provided.
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    Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer
    Hu, Jinnan; Chen, Chenxi; Peever, Tobin L.; Dang, Ha; Lawrence, Christopher B.; Mitchell, Thomas K. (2012-05-06)
    Background Fungal plant pathogens cause serious agricultural losses worldwide. Alternaria arborescens is a major pathogen of tomato, with its virulence determined by the presence of a conditionally dispensable chromosome (CDC) carrying host-specific toxin genes. Genes encoding these toxins are well-studied, however the genomic content and organization of the CDC is not known. Results To gain a richer understanding of the molecular determinants of virulence and the evolution of pathogenicity, we performed whole genome sequencing of A. arborescens. Here we present the de-novo assembly of the CDC and its predicted gene content. Also presented is hybridization data validating the CDC assembly. Predicted genes were functionally annotated through BLAST. Gene ontology terms were assigned, and conserved domains were identified. Differences in nucleotide usage were found between CDC genes and those on the essential chromosome (EC), including GC3-content, codon usage bias, and repeat region load. Genes carrying PKS and NRPS domains were identified in clusters on the CDC and evidence supporting the origin of the CDC through horizontal transfer from an unrelated fungus was found. Conclusions We provide evidence supporting the hypothesis that the CDC in A. arborescens was acquired through horizontal transfer, likely from an unrelated fungus. We also identified several predicted CDC genes under positive selection that may serve as candidate virulence factors.
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    Heparin-based hydrogel scaffolding alters the transcriptomic profile and increases the chemoresistance of MDA-MB-231 triple-negative breast cancer cells
    Menon, Nidhi; Dang, Ha X.; Datla, Udaya Sree; Moarefian, Maryam; Lawrence, Christopher B.; Maher, Christopher A.; Jones, Caroline N. (2020-05-21)
    The tumor microenvironment plays a critical role in the proliferation and chemoresistance of cancer cells. Growth factors (GFs) are known to interact with the extracellular matrix (ECM) via heparin binding sites, and these associations influence cell behavior. In the present study, we demonstrate the ability to define signals presented by the scaffold by pre-mixing growth factors, such as epidermal growth factor, into the heparin-based (HP-B) hydrogel prior to gelation. In the 3D biomimetic microenvironment, breast cancer cells formed spheroids within 24 hours of initial seeding. Despite higher number of proliferating cells in 2D cultures, 3D spheroids exhibited a higher degree of chemoresistance after 72 hours. Further, our RNA sequencing results highlighted the phenotypic changes influenced by solid-phase GF presentation. Wnt/beta-catenin and TGF-beta signaling were upregulated in the cells grown in the hydrogel, while apoptosis, IL2-STAT5 and PI3K-AKT-mTOR signaling were downregulated. With emerging technologies for precision medicine in cancer, this nature of fine-tuning the microenvironment is paramount for cultivation and downstream characterization of primary cancer cells and rare circulating tumor cells (CTCs), and effective screening of chemotherapeutic agents.