Browsing by Author "Dickerman, Allan W."

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    Ancestral Genome Reconstruction in Bacteria
    Yang, Kuan (Virginia Tech, 2012-06-06)
    The rapid accumulation of numerous sequenced genomes has provided a golden opportunity for ancestral state reconstruction studies, especially in the whole genome reconstruction area. However, most ancestral genome reconstruction methods developed so far only focus on gene or replicon sequences instead of whole genomes. They rely largely on either detailed modeling of evolutionary events or edit distance computation, both of which can be computationally prohibitive for large data sets. Hence, most of these methods can only be applied to a small number of features and species. In this dissertation, we describe the design, implementation, and evaluation of an ancestral genome reconstruction system (REGEN) for bacteria. It is the first bacterial genome reconstruction tool that focuses on ancestral state reconstruction at the genome scale instead of the gene scale. It not only reconstructs ancestral gene content and contiguous gene runs using either a maximum parsimony or a maximum likelihood criterion but also replicon structures of each ancestor. Based on the reconstructed genomes, it can infer all major events at both the gene scale, such as insertion, deletion, and translocation, and the replicon scale, such as replicon gain, loss, and merge. REGEN finishes by producing a visual representation of the entire evolutionary history of all genomes in the study. With a model-free reconstruction method at its core, the computational requirement for ancestral genome reconstruction is reduced sufficiently for the tool to be applied to large data sets with dozens of genomes and thousands of features. To achieve as accurate a reconstruction as possible, we also develop a homologous gene family prediction tool for preprocessing. Furthermore, we build our in-house Prokaryote Genome Evolution simulator (PEGsim) for evaluation purposes. The homologous gene family prediction refinement module can refine homologous gene family predictions generated by third party de novo prediction programs by combining phylogeny and local gene synteny. We show that such refinement can be accomplished for up to 80% of homologous gene family predictions with ambiguity (mixed families). The genome evolution simulator, PEGsim, is the first random events based high level bacteria genome evolution simulator with models for all common evolutionary events at the gene, replicon, and genome scales. The concepts of conserved gene runs and horizontal gene transfer (HGT) are also built in. We show the validation of PEGsim itself and the evaluation of the last reconstruction component with simulated data produced by it. REGEN, REconstruction of GENomes, is an ancestral genome reconstruction tool based on the concept of neighboring gene pairs (NGPs). Although it does not cover the reconstruction of actual nucleotide sequences, it is capable of reconstructing gene content, contiguous genes runs, and replicon structure of each ancestor using either a maximum parsimony or a maximum likelihood criterion. Based on the reconstructed genomes, it can infer all major events at both the gene scale, such as insertion, deletion, and translocation, and the replicon scale, such as replicon gain, loss, and merge. REGEN finishes by producing a visual representation of the entire evolutionary history of all genomes in the study.
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    Application of Chromosome Mapping to Understanding Evolutionary History of Anopheles Species
    Kamali, Maryam (Virginia Tech, 2013-06-13)
    Malaria is the main cause of approximately one million deaths every year that mostly affect children in south of Sub-Saharan Africa. The Anopheles gambiae complex consists of seven morphologically indistinguishable sibling species. However, their behavior, ecological adaptations, vectorial capacity, and geographical distribution differ. Studying the phylogenetic relationships among the members of the complex is crucial to understanding the genomic changes that underlie evolving traits. These evolutionary changes can be related to the gain or loss of human blood choice or to other epidemiologically important traits. In order to understand the phylogenetic relationships and evolutionary history of the members of the An. gambiae complex, breakpoints of the 2Ro and 2Rp inversions in An. merus and their homologous sequence in the outgroup species were analyzed using fluorescent in situ hybridization (FISH), library screening, whole-genome mate-paired sequencing and bioinformatics analysis. Molecular phylogenies of breakpoint genes were constructed afterwards. In addition, multigene phylogenetic analyses of African malaria vectors were performed. Our findings revised the chromosomal phylogeny, and demonstrated the ancestry of 2Ro, 2R+p and 2La arrangements.  Our new chromosomal phylogeny strongly suggests that vectorial capacity evolved repeatedly in members of the An. gambiae complex, and the most important vector of malaria in the world, An. gambiae, is more closely related to ancestral species than was previously thought. Our molecular phylogeny data were in agreement with chromosomal phylogeny, indicating that the position of the genetic markers with respect to chromosomal inversion is important for interpretation of the  phylogenetic trees. Multigene phylogenetic analysis revealed that a malaria mosquito from humid savannah and degraded rainforest areas, An. nili, belongs to the basal clade and is more distantly related to other major African malaria vectors than was assumed previously. Finally, for the first time a physical map of 12 microsatellite markers for the Asian malaria vector An. stephensi was developed. Knowledge about the chromosomal position of microsatellites was shown to be important for a proper estimation of population genetic parameters. In conclusion, our study improved understanding of genetics and evolution of some of the major malaria vectors in Africa and Asia.
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    Characterization of activation tagged potato (Solanum tuberosum L.) mutants
    Aulakh, Sukhwinder Singh (Virginia Tech, 2012-09-14)
    Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification.
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    Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis Pathway
    Wattam, Alice R.; Inzana, Thomas J.; Williams, Kelly P.; Mane, Shrinivasrao P.; Shukla, Maulik; Almeida, Nalvo F.; Dickerman, Allan W.; Mason, Steven; Moriyon, Ignacio; O'Callaghan, David; Whatmore, Adrian M.; Sobral, Bruno; Tiller, Rebekah V.; Hoffmaster, Alex R.; Frace, Michael A.; De Castro, Cristina; Molinaro, Antonio; Boyle, Stephen M.; De, Barun K.; Setubal, Joao C. (American Society for Microbiology, 2012-11)
    Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.
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    Comparison of Subterranean Termite (Rhinotermitidae: Reticulitermes) Gut Bacterial Diversity Within and Between Colonies and to Other Termite Species Using Molecular Techniques (ARDRA and 16S rRNA Gene Sequencing)
    Fisher, Marc Lewis (Virginia Tech, 2006-04-25)
    Termites are known to harbor within their gut a diverse assemblage of symbiotic microorganisms. Little work has been done, however, to describe the diversity and function of the bacteria in the economically important eastern subterranean termite, Reticulitermes flavipes. The first object of this study was to characterize the bacterial diversity in the gut of R. flavipes using amplified rDNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. It was determined that ARDRA was an effective technique for characterizing the diversity of the termite gut microbiota. Of the 512 clones analyzed in the ARDRA study, 261 different ARDRA profiles were found. Forty-two 16S rRNA gene sequences were also analyzed, resulting in 33 different ribotypes. Representatives from six major bacterial phyla, Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the newly proposed "Endomicrobia," were discovered. Further analysis indicated that the gut of R. flavipes may harbor as many as 1,318 ribotypes per termite. The second objective was to determine if the gut bacterial diversity could be manipulated by changing the termite's food source. Using ARDRA analysis, I found no evidence that changing the food source affected the termite gut bacterial diversity. In addition, changing the food source did not induce aggression in nestmates fed on different food sources. The third objective was to search for patterns of coevolution between termites and their gut symbiotic bacteria. Using rRNA gene sequences from this study and sequences from public databases (1,450 sequences total), a neighbor-joining tree demonstrated strong evidence for coevolution of termites and their symbiotic bacteria, especially in the phyla Bacteroidetes, Actinobacteria, Spirochaetes, and "Endomicrobia." Many monophyletic clusters were entirely composed of phylotypes specific to Isoptera.
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    Creating Scientific Software, with Application to Phylogenetics and Oligonucleotide Probe Design
    Nordberg, Eric Kinsley (Virginia Tech, 2015-12-09)
    The demands placed on scientific software are different from those placed on general purpose software, and as a result, creating software for science and for scientists requires a specialized approach. Much of software engineering practices have developed in situations in which a tool is desired to perform some definable task, with measurable and verifiable outcomes. The users and the developers know what the tool "should" do. Scientific software often uses unproven or experimental techniques to address unsolved problems. The software is often run on "experimental" High Performance Computing hardware, adding another layer of complexity. It may not be possible to say what the software should do, or what the results should be, as these may be connected to very scientific questions for which the software is being developed. Software development in this realm requires a deep understanding of the relevent scientific domain area. The present work describes applications resulting from a scientific software development process that builds upon detailed understanding of the scientific domain area. YODA is an application primarily for selecting microarray probe sequences for measuring gene expression. At the time of its development, none of the existing programs for this task satisfied the best-known requirements for microarray probe selection. The question of what makes a good microarray probe was a research area at the time, and YODA was developed to incorporate the latest understanding of these requirements, drawn from the research literature, into a tool that can be used by a research biologist. An appendix examines the response and use in the years since YODA was released. PEPR is a software system for inferring highly resolved whole-genome phylogenies for hundreds of genomes. It encodes a process developed through years of research and collaboration to produce some of the highest quality phylogenies available for large sets of bacterial genomes, with no manual intervention required. This process is described in detail, and results are compared with high quality results from the literature to show that the process is at least as successful as more labor-intensive manual efforts. An appendix presents additional results, including high quality phylogenies for many bacterial Orders.
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    Development and characterization of DNA markers for two avian species
    Kamara, Davida F. (Virginia Tech, 2006-05-22)
    Central to the application of genomics to animal agriculture are DNA markers, especially microsatellites and single nucleotide polymorphisms. These markers are the resources necessary for constructing genetic maps and for determining how improved and unimproved animal breeds are related. Here, DNA markers were developed for two avian species, the turkey, Meleagris gallopavo, and the budgerigar (budgie), Melopsittacus undulatus. Genomic libraries enriched for simple sequence repeats were used to generate about 70 budgie sequences of a total length of 38 kb. From these sequences, 9 primer pairs were designed and used to screen for informativeness in a panel of DNA samples from unrelated budgie samples. All but one of the nine primers evaluated were polymorphic with the number of alleles ranging from two to four. Comparative analysis involving the use of these budgie primers showed moderate sequence similarity to turkey and chicken. The genomic libraries and the comparative sequences provide useful genomic reagents that could be used to construct a budgie genome map. In the turkey, ten previously described microsatellites and a gene-based single nucleotide polymorphism (SNP) were used to evaluate the relatedness of heritage varieties to a commercial strain. Estimates of Nei's genetic distance (D) and genetic differentiation (Rst) between populations using microsatellite markers showed that the commercial strain is genetically more closely related to the Bourbon Red and Narragansett and least related to the Royal palm and Spanish Black. Gene flow (Nm) level was highest between the commercial and Bourbon Red populations. The SNP analysis by PCR-RFLP revealed that the commercial strain was more closely related to the Spanish black and Narragansett and least related to the Bourbon red and Blue slate. Though results of the two marker systems, microsatellite and SNP, were inconsistent, they provide insights into using heritage turkeys to genetically improve commercial populations by introgression. The present thesis investigation showed that DNA markers provide a strong opportunity to develop genomic reagents needed to test hypotheses in little-studied agriculturally important and model avian species.
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    Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa
    Petzold, Herman E. III (Virginia Tech, 2017-11-09)
    The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield.
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    Enabling the use of Heterogeneous Computing for Bioinformatics
    Bijanapalli Chakri, Ramakrishna (Virginia Tech, 2013-10-02)
    The huge amount of information in the encoded sequence of DNA and increasing interest in uncovering new discoveries has spurred interest in accelerating the DNA sequencing and alignment processes. The use of heterogeneous systems, that use different types of computational units, has seen a new light in high performance computing in recent years; However expertise in multiple domains and skills required to program these systems is causing an hindrance to bioinformaticians in rapidly deploying their applications into these heterogeneous systems. This work attempts to make an heterogeneous system, Convey HC-1, with an x86-based host processor and FPGA-based co-processor, accessible to bioinformaticians. First, a highly efficient dynamic programming based Smith-Waterman kernel is implemented in hardware, which is able to achieve a peak throughput of 307.2 Giga Cell Updates per Second (GCUPS) on Convey HC-1. A dynamic programming accelerator interface is provided to any application that uses Smith-Waterman. This implementation is also extended to General Purpose Graphics Processing Units (GP-GPUs), which achieved a peak throughput of 9.89 GCUPS on NVIDIA GTX580 GPU. Second, a well known graphical programming tool, LabVIEW is enabled as a programming tool for the Convey HC-1. A connection is established between the graphical interface and the Convey HC-1 to control and monitor the application running on the FPGA-based co-processor.
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    Functional genomics through metabolite profiling and gene expression analysis in Arabidopsis thaliana
    Cortes Bermudez, Diego Fernando (Virginia Tech, 2008-07-25)
    In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. Among the proteins encoded by the Arabidopsis genome are 24 related carboxyl methyltransferases that belong to the SABATH family. Several of the SABATH methyltransferases convert plant hormones, like jasmonic acid, indole-3-acetic acid, salicylic acid, gibberellins, and other plant constituents into methyl esters, thereby regulating the biological activity of these molecules and, consequently, myriad important physiological processes. Our research aims to decipher the function of proteins belonging to the SABATH family by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. Our results, combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights into secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the genome-wide expression and metabolite levels.
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    GeneTrees: a phylogenomics resource for prokaryotes
    Tian, Yuying; Dickerman, Allan W. (2007-01)
    The GeneTrees phylogenomics system pursues comparative genomic analyses from the perspective of gene phylogenies for individual genes. The GeneTrees project has the goal of providing detailed evolutionary models for all protein-coding gene components of the fully sequenced genomes. Currently, a database of alignments and trees for all protein sequences for 325 fully sequenced and annotated prokaryote genomes is available. The prokaryote database contains 890 000 protein sequences organized into over 100 000 alignments, each described by a phylogenetic tree. An original homology group discovery tool assembles sets of related proteins from all versus all pairwise alignments. Multiple alignments for each homology group are stored and subjected to phylogenetic tree inference. A graphical web interface provides visual exploration of the GeneTrees database. Homology groups can be queried by sequence identifiers or annotation terms. Genomes can be browsed visually on a gene map of each chromosome or plasmid. Phylogenetic trees with support values are displayed in conjunction with the associated sequence alignment. A variety of classes of information can be selected to label the tree tips to aid in visual evaluation of annotation and gene function. This web interface is available at http://genetrees.vbi.vt.edu.
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    Methods for Analysis of Prokaryotic Genome Architecture
    Warren, Andrew S. (Virginia Tech, 2017-07-19)
    Research in comparative microbial genomics has largely been organized around the concept of reference genomes. Reference genomes provide a useful comparative touchstone for closely related organisms. However, they do not necessarily represent the biological diversity in a group of genomes. Currently there are more than 96,000 bacterial genomes sequenced and this number is rapidly increasing. Some closely related groups have large numbers of genomes sequenced creating interesting comparative challenges: E. coli more than 5,400 isolates, S. aureus almost 9,000. As this sampling through sequencing becomes both deeper and broader, reference genome based methods become less effective at characterizing groups of organisms. Functional motifs can help explain the organizing principles behind cellular systems in bacteria which have yet to be well understood. Currently there are relatively few bioinformatic tools for analyzing potential patterns at the level of genome organization that do not depend directly on sequence similarity. We present a framework for conducting genomic data mining to look for patterns that currently require human expert designation. We establish new computational methods for identifying patterns in prokaryotic genome construction through a mapping of genomic features, using semantic similarity, independent of a particular corpus to better approximate functional similarity. We also present an algorithm for creating whole genome multiple sequence comparisons and a model for representing the similarities and di erences among sequences as a graph of syntenic gene families. This e ort touches on several di erent research fronts: graph representation of genomes and their alignments, synteny block analysis, whole genome sequence alignment, pan-genome analysis, multiple sequence alignment, and genome rearrangement analysis. Though our approach was originally developed from a pan-genome perspective for prokaryotes, the methods involved have the potential to speed up more expensive computation such as phylogenetic tree construction and SNP analysis. Novel elements include the contextualization of synteny analysis both between and within multi-contig genomes and an analytical framework for detecting genome level evolutionary events such as insertions, inversions, translocations, and fusions.
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    Origin, Evolution, and Genotyping of Emergent Porcine Epidemic Diarrhea Virus Strains in the United States
    Huang, Yao-Wei; Dickerman, Allan W.; Piñyero, Pablo E.; Li, Long; Fang, Li; Kiehne, Ross; Opriessnig, Tanja; Meng, Xiang-Jin (American Society for Microbiology, 2013-09)
    Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U. S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5'-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.
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    The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities
    Davis, James J.; Wattam, Alice R.; Aziz, Ramy K.; Brettin, Thomas; Butler, Ralph; Butler, Rory M.; Chlenski, Philippe; Conrad, Neal; Dickerman, Allan W.; Dietrich, Emily M.; Gabbard, Joseph L.; Gerdes, Svetlana; Guard, Andrew; Kenyon, Ronald W.; Machi, Dustin; Mao, Chunhong; Murphy-Olson, Daniel E.; Nguyen, Marcus; Nordberg, Eric K.; Olsen, Gary J.; Olson, Robert D.; Overbeek, Jamie C.; Overbeek, Ross; Parrello, Bruce; Pusch, Gordon D.; Shukla, Maulik; Thomas, Chris; VanOeffelen, Margo; Vonstein, Veronika; Warren, Andrew S.; Xia, Fangfang; Xie, Dawen; Yoo, Hyunseung; Stevens, Rick L. (2020-01-08)
    The PathoSystems Resource Integration Center (PATRIC) is the bacterial Bioinformatics Resource Center funded by the National Institute of Allergy and Infectious Diseases (https://www.patricbrc.org). PATRIC supports bioinformatic analyses of all bacteria with a special emphasis on pathogens, offering a rich comparative analysis environment that provides users with access to over 250 000 uniformly annotated and publicly available genomes with curated metadata. PATRIC offers web-based visualization and comparative analysis tools, a private workspace in which users can analyze their own data in the context of the public collections, services that streamline complex bioinformatic workflows and command-line tools for bulk data analysis. Over the past several years, as genomic and other omics-related experiments have become more cost-effective and widespread, we have observed considerable growth in the usage of and demand for easy-to-use, publicly available bioinformatic tools and services. Here we report the recent updates to the PATRIC resource, including new web-based comparative analysis tools, eight new services and the release of a command-line interface to access, query and analyze data.
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    PATRIC: The VBI PathoSystems Resource Integration Center
    Snyder, E. E.; Kampanya, N.; Lu, J.; Nordberg, E. K.; Karur, H. R.; Shukla, Maulik; Soneja, J.; Tian, Y.; Xue, T.; Yoo, H.; Zhang, F.; Dharmanolla, C.; Dongre, N. V.; Gillespie, J. J.; Hamelius, J.; Hance, M.; Huntington, K. I.; Jukneliene, D.; Koziski, J.; Mackasmiel, L.; Mane, S. P.; Nguyen, V.; Purkayastha, A.; Shallom, J.; Yu, G.; Guo, Y.; Gabbard, Joseph L.; Hix, D.; Azad, A. F.; Baker, S. C.; Boyle, Stephen M.; Khudyakov, Y.; Meng, Xiang-Jin; Rupprecht, C.; Vinje, J.; Crasta, Oswald R.; Czar, M. J.; Dickerman, Allan W.; Eckart, J. D.; Kenyon, R.; Will, R.; Setubal, Joao C.; Sobral, Bruno (2007-01)
    The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.
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    Polymicrobial Biofilm Interaction Between Histophilus somni and Pasteurella multocida
    Petruzzi, Briana; Dickerman, Allan W.; Lahmers, Kevin K.; Scarratt, William K.; Inzana, Thomas J. (2020-07-10)
    Histophilus somni and Pasteurella multocida are two of multiple agents responsible for bovine respiratory disease (BRD) in cattle. Following respiratory infection of calves withH. somni,P. multocidamay also be isolated from the lower respiratory tract. BecauseH. somnimay form a biofilm during BRD, we sought to determine ifP. multocidacan co-exist withH. somniin a polymicrobial biofilmin vitroandin vivo. Interactions between the two species in the biofilm were characterized and quantified by fluorescencein situhybridization (FISH). The biofilm matrix of each species was examined using fluorescently tagged lectins (FTL) specific for the exopolysaccharide (EPS) using confocal laser scanning microscopy. Bacterial interactions were determined by auto-aggregation and biofilm morphology.Pasteurella multocidaandH. somniwere evenly distributed in thein vitrobiofilm, and both species contributed to the polymicrobial biofilm matrix. The average biomass and biofilm thickness, and the total carbohydrate and protein content of the biofilm, were greatest when both species were present. Polymicrobial bacterial suspensions auto-aggregated faster than single species suspensions, suggesting physical interactions between the two species. Almost 300P. multocidagenes were significantly differentially regulated when the bacteria were in a polymicrobial biofilm compared to a mono-species biofilm, as determined by RNA-sequencing. As expected, host genes associated with inflammation and immune response were significantly upregulated at the infection site followingH. somnichallenge. EncapsulatedP. multocidaisolates not capable of forming a substantial biofilm enhanced anin vitropolymicrobial biofilm withH. somni, indicating they contributed to the polymicrobial biofilm matrix. Indirect evidence indicated that encapsulatedP. multocidaalso contributed to a polymicrobial biofilmin vivo. Only the EPS ofH. somnicould be detected by FTL staining of bovine tissues following challenge withH. somni. However, both species were isolated and an immune response to the biofilm matrix of both species was greater than the response to planktonic cells, suggesting encapsulatedP. multocidamay take advantage of theH. somnibiofilm to persist in the host during chronic BRD. These results may have important implications for the management and prevention of BRD.
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    Probiotics and virulent human rotavirus modulate the transplanted human gut microbiota in gnotobiotic pigs
    Zhang, Husen; Wang, Haifeng; Shepherd, Megan L.; Wen, Ke; Li, Guohua; Yang, Xingdong; Kocher, Jacob; Giri-Rachman, Ernawati; Dickerman, Allan W.; Settlage, Robert E.; Yuan, Lijuan (2014-09-09)
    We generated a neonatal pig model with human infant gut microbiota (HGM) to study the effect of a probiotic on the composition of the transplanted microbiota following rotavirus vaccination and challenge. All the HGM-transplanted pigs received two doses of an oral attenuated rotavirus vaccine. The gut microbiota of vaccinated pigs were investigated for effects of Lactobacillus rhamnosus GG (LGG) supplement and homotypic virulent human rotavirus (HRV) challenge. High-throughput sequencing of V4 region of 16S rRNA genes demonstrated that HGM-transplanted pigs carried microbiota similar to that of the C-section delivered baby. Firmicutes and Proteobacteria represented over 98% of total bacteria in the human donor and the recipient pigs. HRV challenge caused a phylum-level shift from Firmicutes to Proteobacteria. LGG supplement prevented the changes in microbial communities caused by HRV challenge. In particular, members of Enterococcus in LGG-supplemented pigs were kept at the baseline level, while they were enriched in HRV challenged pigs. Taken together, our results suggested that HGM pigs are valuable for testing the microbiota’s response to probiotic interventions for treating infantile HRV infection.
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    Searching Biological Sequence Databases Using Distributed Adaptive Computing
    Pappas, Nicholas Peter (Virginia Tech, 2003-01-24)
    Genetic research projects currently can require enormous computing power to processes the vast quantities of data available. Further, DNA sequencing projects are generating data at an exponential rate greater than that of the development microprocessor technology; thus, new, faster methods and techniques of processing this data are needed. One common type of processing involves searching a sequence database for the most similar sequences. Here we present a distributed database search system that utilizes adaptive computing technologies. The search is performed using the Smith-Waterman algorithm, a common sequence comparison algorithm. To reduce the total search time, an initial search is performed using a version of the algorithm, implemented in adaptive computing hardware, which is designed to efficiently perform the initial search. A final search is performed using a complete version of the algorithm. This two-stage search, employing adaptive and distributed hardware, achieves a performance increase of several orders of magnitude over similar processor based systems.
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    Systems analysis of stress response in plants
    Krishnan, Arjun (Virginia Tech, 2010-09-08)
    The response of plants to environmental stress spans several orders of magnitude in time and space, causing system-wide changes. These changes comprise of both protective responses and adverse reactions in the plant. Stresses like water deficit or drought cause a drastic effect in crop yield, while concomitantly agriculture consumes 1/3rd of the fresh water available to us and there is widespread water scarcity around the world. It is, hence, a fundamental goal of modern biology and applied biotechnology to unravel this complex stress response in laboratory model plants like Arabidopsis and crop models like rice. Such an understanding, especially at the cellular level, will aid in informed engineering of stress tolerance in plants. We have developed and used integrative functional genomics approaches to characterize environmental stress response at various levels of organization including genes, modules and networks in Arabidopsis and rice. We have also applied these methods in problems concerning bioenergy. Since the poor knowledge of the cellular roles of a large portion of plant genes remains a fundamental barrier to using such approaches, we have further explored the problem of 'gene function prediction'. And, finally, as a contribution to the community, we have curated a large mutant resource for the crop model, rice, and established a web resource for exploratory analysis of abiotic stress in this model. All together, this work presents insights into several facets of stress response, offers numerous novel predictions for experimental validation, and provides principled analysis frameworks for systems level analysis of environmental stress response in plants.
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    Theoretical and Statistical Approaches to Understand Human Mitochondrial DNA Heteroplasmy Inheritance
    Wonnapinij, Passorn (Virginia Tech, 2010-04-09)
    Mitochondrial DNA (mtDNA) mutations have been widely observed to cause a variety of human diseases, especially late-onset neurodegenerative disorders. The prevalence of mitochondrial diseases caused by mtDNA mutation is approximately 1 in 5,000 of the population. There is no effective way to treat patients carrying pathogenic mtDNA mutation; therefore preventing transmission of mutant mtDNA became an important strategy. However, transmission of human mtDNA mutation is complicated by a large intergenerational random shift in heteroplasmy level causing uncertainty for genetic counseling. The aim of this dissertation is to gain insight into how human mtDNA heteroplasmy is inherited. By working closely with our experimental collaborators, the computational simulation of mouse embryogenesis has been developed in our lab using their measurements of mouse mtDNA copy number. This experimental-computational interplay shows that the variation of offspring heteroplasmy level has been largely generated by random partition of mtDNA molecules during pre- and early postimplantation development. By adapting a set of probability functions developed to describe the segregation of allele frequencies under a pure random drift process, we now can model mtDNA heteroplasmy distribution using parameters estimated from experimental data. The absence of an estimate of sampling error of mtDNA heteroplasmy variance may largely affect the biological interpretation drawn from this high-order statistic, thereby we have developed three different methods to estimate sampling error values for mtDNA heteroplasmy variance. Applying this error estimation to the comparison of mouse to human mtDNA heteroplasmy variance reveals the difference of the mitochondrial genetic bottleneck between these organisms. In humans, the mothers who carry a high proportion of m.3243A>G mutation tend to have fewer daughters than sons. This offspring gender bias has been revealed by applying basic statistical tests on the human clinical pedigrees carrying this mtDNA mutation. This gender bias may partially determine the mtDNA mutation level among female family members. In conclusion, the application of population genetic theory, statistical analysis, and computational simulation help us gain understanding of human mtDNA heteroplasmy inheritance. The results of these studies would be of benefit to both scientific research and clinical application.