Shane C Burgess

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Research

Veterinary medicine and cancer, virology, proteomics, immunology and bioinformatics research.

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Scholarly Contributions

Journals/Publications

• Blake, J. A., Dolan, M., Drabkin, H., Hill, D. P., Li, N. i., Sitnikov, D., Bridges, S., Burgess, S., Buza, T., McCarthy, F., Peddinti, D., Pillai, L., Carbon, S., Dietze, H., Ireland, A., Lewis, S. E., Mungall, C. J., Gaudet, P., Chisholm, R. L., , Fey, P., et al. (2013). Gene ontology annotations and resources. Nucleic Acids Research, 41(D1), D530-D535.
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  PMID: 23161678;PMCID: PMC3531070;Abstract: The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bio-informatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources. © The Author(s) 2012.
• Nanduri, B., Pendarvis, K., Shack, L. A., Kumar, R., Clymer, J. W., Korvick, D. L., & Burgess, S. C. (2013). Ultrasonic Incisions Produce Less Inflammatory Mediator Response during Early Healing than Electrosurgical Incisions. PLoS ONE, 8(9).
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  PMID: 24058457;PMCID: PMC3776814;Abstract: As the use of laparoscopic surgery has become more widespread in recent years, the need has increased for minimally-invasive surgical devices that effectively cut and coagulate tissue with reduced tissue trauma. Although electrosurgery (ES) has been used for many generations, newly-developed ultrasonic devices (HARMONIC® Blade, HB) have been shown at a macroscopic level to offer better coagulation with less thermally-induced tissue damage. We sought to understand the differences between ES and HB at a microscopic level by comparing mRNA transcript and protein responses at the 3-day timepoint to incisions made by the devices in subcutaneous fat tissue in a porcine model. Samples were also assessed via histological examination. ES-incised tissue had more than twice as many differentially-expressed genes as HB (2,548 vs 1,264 respectively), and more differentially-expressed proteins (508 vs 432) compared to control (untreated) tissue. Evaluation of molecular functions using Gene Ontology showed that gene expression changes for the energized devices reflected the start of wound healing, including immune response and inflammation, while protein expression showed a slightly earlier stage, with some remnants of hemostasis. For both transcripts and proteins, ES exhibited a greater response than HB, especially in inflammatory mediators. These findings were in qualitative agreement with histological results. This study has shown that transcriptomics and proteomics can monitor the wound healing response following surgery and can differentiate between surgical devices. In agreement with clinical observations, electrosurgery was shown to incur a greater inflammatory immune response than an ultrasonic device during initial iatrogenic wound healing. © 2013 Nanduri et al.
• A., J., Braun, E. L., Isberg, S. R., Miles, L. G., Chong, A. Y., Gongora, J., Dalzell, P., Moran, C., Bed'Hom, B., Abzhanov, A., Burgess, S. C., Cooksey, A. M., Castoe, T. A., Crawford, N. G., Densmore, L. D., Drew, J. C., Edwards, S. V., Faircloth, B. C., Fujita, M. K., , Greenwold, M. J., et al. (2012). Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes. Genome Biology, 13(1).
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  PMID: 22293439;PMCID: PMC3334581;Abstract: The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described. © 2012 BioMed Central Ltd.
• Blake, J. A., Dolan, M., Drabkin, H., Hill, D. P., Ni, L., Sitnikov, D., Burgess, S., Buza, T., Gresham, C., McCarthy, F., Pillai, L., Wang, H., Carbon, S., Lewis, S. E., Mungall, C. J., Gaudet, P., Chisholm, R. L., Fey, P., Kibbe, W. A., , Basu, S., et al. (2012). The Gene Ontology: Enhancements for 2011. Nucleic Acids Research, 40(D1), D559-D564.
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  PMID: 22102568;PMCID: PMC3245151;Abstract: The Gene Ontology (GO) (http://www.geneontology .org) is a community bioinformatics resource that represents gene product function through the use of structured, controlled vocabularies. The number of GO annotations of gene products has increased due to curation efforts among GO Consortium (GOC) groups, including focused literature-based annotation and ortholog-based functional inference. The GO ontologies continue to expand and improve as a result of targeted ontology development, including the introduction of computable logical definitions and development of new tools for the streamlined addition of terms to the ontology. The GOC continues to support its user community through the use of e-mail lists, social media and web-based resources. © The Author(s) 2011. Published by Oxford University Press.
• Chouvarine, P., Cooksey, A. M., McCarthy, F. M., Ray, D. A., Baldwin, B. S., Burgess, S. C., & Peterson, D. G. (2012). Transcriptome-based differentiation of closely-related Miscanthus lines. PLoS ONE, 7(1).
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  PMID: 22253803;PMCID: PMC3254643;Abstract: Background: Distinguishing between individuals is critical to those conducting animal/plant breeding, food safety/quality research, diagnostic and clinical testing, and evolutionary biology studies. Classical genetic identification studies are based on marker polymorphisms, but polymorphism-based techniques are time and labor intensive and often cannot distinguish between closely related individuals. Illumina sequencing technologies provide the detailed sequence data required for rapid and efficient differentiation of related species, lines/cultivars, and individuals in a cost-effective manner. Here we describe the use of Illumina high-throughput exome sequencing, coupled with SNP mapping, as a rapid means of distinguishing between related cultivars of the lignocellulosic bioenergy crop giant miscanthus (Miscanthus × giganteus). We provide the first exome sequence database for Miscanthus species complete with Gene Ontology (GO) functional annotations. Results: A SNP comparative analysis of rhizome-derived cDNA sequences was successfully utilized to distinguish three Miscanthus × giganteus cultivars from each other and from other Miscanthus species. Moreover, the resulting phylogenetic tree generated from SNP frequency data parallels the known breeding history of the plants examined. Some of the giant miscanthus plants exhibit considerable sequence divergence. Conclusions: Here we describe an analysis of Miscanthus in which high-throughput exome sequencing was utilized to differentiate between closely related genotypes despite the current lack of a reference genome sequence. We functionally annotated the exome sequences and provide resources to support Miscanthus systems biology. In addition, we demonstrate the use of the commercial high-performance cloud computing to do computational GO annotation. © 2012 Chouvarine et al.
• Kumar, R., Lawrence, M. L., Watt, J., Cooksey, A. M., Burgess, S. C., & Nanduri, B. (2012). RNA-Seq based transcriptional map of bovine respiratory disease pathogen "Histophilus somni 2336". PLoS ONE, 7(1).
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  PMID: 22276113;PMCID: PMC3262788;Abstract: Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify "novel" genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method. The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (~30%) were located in genomic region unique to strain 2336 (~18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations. © 2012 Kumar et al.
• Kumar, S., Kunec, D., Buza, J. J., Chiang, H., Zhou, H., Subramaniam, S., Pendarvis, K., Cheng, H. H., & Burgess, S. C. (2012). Nuclear Factor kappa B is central to Marek's Disease herpesvirus induced neoplastic transformation of CD30 expressing lymphocytes in-vivo. BMC Systems Biology, 6.
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  PMID: 22979947;PMCID: PMC3472249;Abstract: Background: Marek's Disease (MD) is a hyperproliferative, lymphomatous, neoplastic disease of chickens caused by the oncogenic Gallid herpesvirus type 2 (GaHV-2; MDV). Like several human lymphomas the neoplastic MD lymphoma cells overexpress the CD30 antigen (CD30 hi) and are in minority, while the non-neoplastic cells (CD30 lo) form the majority of population. MD is a unique natural in-vivo model of human CD30 hi lymphomas with both natural CD30 hi lymphomagenesis and spontaneous regression. The exact mechanism of neoplastic transformation from CD30 lo expressing phenotype to CD30 hi expressing neoplastic phenotype is unknown. Here, using microarray, proteomics and Systems Biology modeling; we compare the global gene expression of CD30 lo and CD30 hi cells to identify key pathways of neoplastic transformation. We propose and test a specific mechanism of neoplastic transformation, and genetic resistance, involving the MDV oncogene Meq, host gene products of the Nuclear Factor Kappa B (NF-κB) family and CD30; we also identify a novel Meq protein interactome.Results: Our results show that a) CD30 lo lymphocytes are pre-neoplastic precursors and not merely reactive lymphocytes; b) multiple transformation mechanisms exist and are potentially controlled by Meq; c) Meq can drive a feed-forward cycle that induces CD30 transcription, increases CD30 signaling which activates NF-κB, and, in turn, increases Meq transcription; d) Meq transcriptional repression or activation of the CD30 promoter generally correlates with polymorphisms in the CD30 promoter distinguishing MD-lymphoma resistant and susceptible chicken genotypes e) MDV oncoprotein Meq interacts with proteins involved in physiological processes central to lymphomagenesis.Conclusions: In the context of the MD lymphoma microenvironment (and potentially in other CD30 hi lymphomas as well), our results show that the neoplastic transformation is a continuum and the non-neoplastic cells are actually pre-neoplastic precursor cells and not merely immune bystanders. We also show that NF-κB is a central player in MDV induced neoplastic transformation of CD30-expressing lymphocytes in vivo. Our results provide insights into molecular mechanisms of neoplastic transformation in MD specifically and also herpesvirus induced lymphoma in general. © 2012 Kumar et al.; licensee BioMed Central Ltd.
• Reddy, J. S., Kumar, R., Watt, J. M., Lawrence, M. L., Burgess, S. C., & Nanduri, B. (2012). Transcriptome profile of a bovine respiratory disease pathogen: Mannheimia haemolytica PHL213.. BMC bioinformatics, 13 Suppl 15, S4.
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  PMID: 23046475;PMCID: PMC3439734;Abstract: Computational methods for structural gene annotation have propelled gene discovery but face certain drawbacks with regards to prokaryotic genome annotation. Identification of transcriptional start sites, demarcating overlapping gene boundaries, and identifying regulatory elements such as small RNA are not accurate using these approaches. In this study, we re-visit the structural annotation of Mannheimia haemolytica PHL213, a bovine respiratory disease pathogen. M. haemolytica is one of the causative agents of bovine respiratory disease that results in about $3 billion annual losses to the cattle industry. We used RNA-Seq and analyzed the data using freely-available computational methods and resources. The aim was to identify previously unannotated regions of the genome using RNA-Seq based expression profile to complement the existing annotation of this pathogen. Using the Illumina Genome Analyzer, we generated 9,055,826 reads (average length ~76 bp) and aligned them to the reference genome using Bowtie. The transcribed regions were analyzed using SAMTOOLS and custom Perl scripts in conjunction with BLAST searches and available gene annotation information. The single nucleotide resolution map enabled the identification of 14 novel protein coding regions as well as 44 potential novel sRNA. The basal transcription profile revealed that 2,506 of the 2,837 annotated regions were expressed in vitro, at 95.25% coverage, representing all broad functional gene categories in the genome. The expression profile also helped identify 518 potential operon structures involving 1,086 co-expressed pairs. We also identified 11 proteins with mutated/alternate start codons. The application of RNA-Seq based transcriptome profiling to structural gene annotation helped correct existing annotation errors and identify potential novel protein coding regions and sRNA. We used computational tools to predict regulatory elements such as promoters and terminators associated with the novel expressed regions for further characterization of these novel functional elements. Our study complements the existing structural annotation of Mannheimia haemolytica PHL213 based on experimental evidence. Given the role of sRNA in virulence gene regulation and stress response, potential novel sRNA described in this study can form the framework for future studies to determine the role of sRNA, if any, in M. haemolytica pathogenesis.
• Sokale, A., Peebles, E. D., Zhai, W., Pendarvis, K., Burgess, S., & Pechan, T. (2012). Proteome profile of the pipping muscle in broiler embryos. Proteomics, 11(21), 4262-4265.
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  PMID: 21834139;Abstract: This study is the first proteomics analysis of the muscularis complexus (pipping muscle) in chicken (Gallus gallus) broiler embryos. We used differential detergent fractionation and nano-HPLC-MS/MS analysis to identify 676 proteins from all cellular components. The identified proteins were functionally classified in accordance with their involvement in various cellular activities. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Tang, J. D., Perkins, A. D., Sonstegard, T. S., Schroeder, S. G., Burgess, S. C., & Diehl, S. V. (2012). Short-read sequencing for genomic analysis of the brown rot fungus Fibroporia radiculosa. Applied and Environmental Microbiology, 78(7), 2272-2281.
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  PMID: 22247176;PMCID: PMC3302605;Abstract: The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment size) at three different stringency levels and then assembling each data set with Velvet. A simple approach was devised to determine which filter stringency was "best." Venn diagrams identified the regions containing reads that were used in an assembly but were of a low-enough quality to be removed by a filter. By plotting base quality histograms of reads in this region, we judged whether a filter was too stringent or not stringent enough. Our best assembly had a genome size of 33.6 Mb, an N50 of 65.8 kb for a k-mer of 51, and a maximum contig length of 347 kb. Using GeneMark, 9,262 genes were predicted. TargetP and SignalP analyses showed that among the 1,213 genes with secreted products, 986 had motifs for signal peptides and 227 had motifs for signal anchors. Blast2GO analysis provided functional annotation for 5,407 genes. We identified 29 genes with putative roles in copper tolerance and 73 genes for lignocellulose degradation. A search for homologs of these 102 genes showed that F. radiculosa exhibited more similarity to Postia placenta than Serpula lacrymans. Notable differences were found, however, and their involvements in copper tolerance and wood decay are discussed. © 2012, American Society for Microbiology.
• Xianyao, L. i., Swaggerty, C. L., Kogut, M. H., Chiang, H., Wang, Y., Genovese, K. J., Haiqi, H. e., McCarthy, F. M., Burgess, S. C., Pevzner, I. Y., & Zhou, H. (2012). Systemic response to Campylobacter jejuni infection by profiling gene transcription in the spleens of two genetic lines of chickens. Immunogenetics, 64(1), 59-69.
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  PMID: 21748442;Abstract: Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial enteritis worldwide with poultry products being a major source of C. jejuni contamination. The chicken is the natural reservoir of C. jejuni where bacteria colonize the digestive tract of poultry, but rarely cause symptoms of disease. To understand the systemic molecular response mechanisms to C. jejuni infection in chickens, total splenic RNA was isolated and applied to a whole genome chicken microarray for comparison between infected (I) and non-infected (N) chickens within and between genetic lines A and B. There were more total splenic host genes responding to the infection in resistant line A than in susceptible line B. Specifically, genes for lymphocyte activation, differentiation and humoral response, and Ig light and heavy chain were upregulated in the resistant line. In the susceptible line, genes for regulation of erythrocyte differentiation, hemopoiesis, and RNA biosynthetic process were all downregulated. An interaction analysis between genetic lines and treatment demonstrated distinct defense mechanisms between lines: the resistant line promoted apoptosis and cytochrome c release from mitochondria, whereas the susceptible line responded with a downregulation of both functions. This was the first time that such systemic defensive mechanisms against C. jejuni infection have been reported. The results of this study revealed novel molecular mechanisms of the systemic host responses to C. jejuni infection in chickens that warrant further investigation. © Springer-Verlag 2011.
• Zhai, W., Araujo, L. F., Burgess, S. C., Cooksey, A. M., Pendarvis, K., Mercier, Y., & Corzo, A. (2012). Protein expression in pectoral skeletal muscle of chickens as influenced by dietary methionine. Poultry Science, 91(10), 2548-2555.
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  PMID: 22991541;Abstract: Effects of dietary methionine (Met) on pectoralis muscle development and the effect that Met as a nutritional substrate has on protein expression of skeletal muscle cells of pectoralis muscle of chickens were evaluated in this study. Broiler chickens received a common pretest diet up to 21 d of age and were subsequently fed either a low (LM) or high Met (HM) diet (0.41 vs. 0.51% of diet) from 21 to 42 d of age. Dietary deficiency was shown in vivo judging by the depression in breast meat weight and yield when broilers were fed the LM diet. Global protein expression was analyzed by quantitative high-performance liquid chromatography nanospray ionization tandem mass spectrometry. Up- and downregulated proteins were analyzed via Ingenuity Pathways Analysis to identify the metabolic pathways affected. Four canonical pathways related to muscle development were identified as being differentially regulated between LM- and HM-fed chickens. These pathways included the citrate cycle and calcium, actin cytoskeleton, and clathrin-mediated endocytosis signaling. The HM diet may have allowed for increased muscle growth by an increased availability of nutrients to muscle cells. Although the Met supplementation was associated with enhanced breast muscle growth, contraction fiber concentrations in muscles decreased and were associated with a lower calcium transportation rate and sensitivity and with a lower energy supply. It is further suggested that increased muscle protein deposition, that was induced by Met supplementation, may have been largely due to sarcoplasmic rather myofibrillar hypertrophy. © 2012 Poultry Science Association Inc.
• Bright, L. A., Mujahid, N., Nanduri, B., McCarthy, F. M., Costa, L. R., Burgess, S. C., & Swiderski, C. E. (2011). Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences. Animal Genetics, 42(4), 395-405.
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  PMID: 21749422;Abstract: The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort. © 2011 The Authors, Animal Genetics.
• Corzo, A., Loar, R. E., Kidd, M. T., & Burgess, S. C. (2011). Dietary protein effects on growth performance, carcass traits and expression of selected jejunal peptide and amino acid transporters in broiler chickens. Revista Brasileira de Ciencia Avicola, 13(2), 139-146.
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  Abstract: The effect of dietary protein on growth, carcass traits and some specific intestinal intestinal peptide and amino acid transporters in broiler chickens was studied. Birds received a common pre-test diet, and were subsequently fed either a standard positive control diet (PC) or a reduced CP diet (RCP) from 21 to 42 d of age. Growth was negatively impacted with feeding of RCP as manifested by an increase in feed consumption and feed conversion ratio. Carcass traits also showed evidence of negative effects of feeding the RCP diet, leading to a reduction in carcass and breast meat yield and an increase in abdominal fat percentage. Blood plasma total protein was reduced when the broilers were fed the RCP diet. Expression of mRNA for one peptide (PepT1) and four AA intestinal transporters (b°,+AT; CAT2; y+LAT2; EAAT3) was measured from the jejunum. Quantified mRNA for the AA transporters y+LAT2 and EAAT3 showed that they were upregulated in chickens fed the RCP-diet. The transport systems PepT1, b°,+AT, and CAT2, were not affected by the dietary treatment imposed. The live and processing data validated the in vivo portion of the study and elucidated the negative impact of feeding the RCP diet, while the responses observed with the expression of the various transporters may help provide some insight on the physiological consequences and adaptations that birds endure when provided diets too low in CP for abnormally extended periods of time.
• Donaldson, J. R., Nanduri, B., Pittman, J. R., Givaruangsawat, S., Burgess, S. C., & Lawrence, M. L. (2011). Proteomic expression profiles of virulent and avirulent strains of Listeria monocytogenes isolated from macrophages. Journal of Proteomics, 74(10), 1906-1917.
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  PMID: 21605710;Abstract: Listeria monocytogenes is able to survive and proliferate within macrophages. In the current study, the ability of three L. monocytogenes strains (serovar 1/2a strain EGDe, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1 was analyzed. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGDe and F2365 proliferated within macrophages for at least 7. h. We further analyzed these strains by comparing their protein expression profiles at 0. h, 3. h, and 5. h post-infection using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. Our results indicated that similar metabolic and cell wall associated proteins were expressed by all three strains at 3. h post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5. h post-infection. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive in macrophages. © 2011 Elsevier B.V.
• Froman, D. P., Feltmann, A. J., Pendarvis, K., Cooksey, A. M., Burgess, S. C., & Rhoads, D. D. (2011). Physiology and endocrinology symposium: A proteome-based model for sperm mobility phenotype. Journal of Animal Science, 89(5), 1330-1337.
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  PMID: 21036929;Abstract: Sperm mobility is defined as sperm movement against resistance at body temperature. Although all mobile sperm are motile, not all motile sperm are mobile. Sperm mobility is a primary determinant of male fertility in the chicken. Previous work explained phenotypic variation at the level of the sperm cell and the mitochondrion. The present work was conducted to determine if phenotypic variation could be explained at the level of the proteome using semen donors from lines of chickens selected for low or high sperm mobility. We began by testing the hypothesis that premature mitochondrial failure, and hence sperm immobility, arose from Ca 2+ overloading. The hypothesis was rejected because staining with a cell permeant Ca 2+-specific dye was not enhanced in the case of low mobility sperm. The likelihood that sperm require little energy before ejaculation and the realization that the mitochondrial permeability transition can be induced by oxidative stress arising from inadequate NADH led to the hypothesis that glycolytic enzymes might differ between lines. This possibility was confirmed by 2-dimensional electrophoresis for aldolase and phosphoglycerate kinase 1. This outcome warranted evaluation of the whole cell proteome by differential detergent fractionation and mass spectrometry. Bioinformatics evaluation of proteins with different expression levels confirmed the likelihood that ATP metabolism and glycolysis differ between lines. This experimental outcome corroborated differences observed between lines in previous work, which include mitochondrial ultrastructure, sperm cell oxygen consumption, and straight line velocity. Although glycolytic proteins were more abundant within highly mobile sperm, quantitative PCR of representative testis RNA, which included mRNA for phosphoglycerate kinase 1, found no difference between lines. In summary, we propose a proteome-based model for sperm mobility phenotype in which a genetic predisposition puts sperm cells at risk of premature mitochondrial failure as they pass through the excurrent ducts of the testis. In other words, we attribute mitochondrial failure to sperm cell and reproductive tract attributes that interact to affect sperm in a stochastic manner before ejaculation. In conclusion, our work provides a starting point for understanding chicken semen quality in terms of gene networks. © 2011 American Society of Animal Science. All rights reserved.
• Kumar, R., Burgess, S. C., Lawrence, M. L., & Nanduri, B. (2011). TAAPP: Tiling array analysis pipeline for prokaryotes. Genomics, Proteomics and Bioinformatics, 9(1-2), 56-62.
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  PMID: 21641563;Abstract: High-density tiling arrays provide closer view of transcription than regular microarrays and can also be used for annotating functional elements in genomes. The identified transcripts usually have a complex overlapping architecture when compared to the existing genome annotation. Therefore, there is a need for customized tiling array data analysis tools. Since most of the initial tiling arrays were conducted in eukaryotes, data analysis methods are well suited for eukaryotic genomes. For using whole-genome tiling arrays to identify previously unknown transcriptional elements like small RNA and antisense RNA in prokaryotes, existing data analysis tools need to be tailored for prokaryotic genome architecture. Furthermore, automation of such custom data analysis workflow is necessary for biologists to apply this powerful platform for knowledge discovery. Here we describe TAAPP, a web-based package that consists of two modules for prokaryotic tiling array data analysis. The transcript generation module works on normalized data to generate transcriptionally active regions (TARs). The feature extraction and annotation module then maps TARs to existing genome annotation. This module further categorizes the transcription profile into potential novel non-coding RNA, antisense RNA, gene expression and operon structures. The implemented workflow is microarray platform independent and is presented as a web-based service. The web interface is freely available for acedemic use at http://lims.lsbi.mafes.msstate.edu/TAAPP-HTML/. © 2011 Beijing Genomics Institute.
• McCarthy, F. M., Gresham, C. R., Buza, T. J., Chouvarine, P., Pillai, L. R., Kumar, R., Ozkan, S., Wang, H., Manda, P., Arick, T., Bridges, S. M., & Burgess, S. C. (2011). AgBase: Supporting functional modeling in agricultural organisms. Nucleic Acids Research, 39(SUPPL. 1), D497-D506.
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  PMID: 21075795;PMCID: PMC3013706;Abstract: AgBase (http://www.agbase.msstate.edu/) provides resources to facilitate modeling of functional genomics data and structural and functional annotation of agriculturally important animal, plant, microbe and parasite genomes. The website is redesigned to improve accessibility and ease of use, including improved search capabilities. Expanded capabilities include new dedicated pages for horse, cat, dog, cotton, rice and soybean. We currently provide 590 240 Gene Ontology (GO) annotations to 105 454 gene products in 64 different species, including GO annotations linked to transcripts represented on agricultural microarrays. For many of these arrays, this provides the only functional annotation available. GO annotations are available for download and we provide comprehensive, species-specific GO annotation files for 18 different organisms. The tools available at AgBase have been expanded and several existing tools improved based upon user feedback. One of seven new tools available at AgBase, GOModeler, supports hypothesis testing from functional genomics data. We host several associated databases and provide genome browsers for three agricultural pathogens. Moreover, we provide comprehensive training resources (including worked examples and tutorials) via links to Educational Resources at the AgBase website. © The Author(s) 2010.
• Paul, D., Kumar, R., Nanduri, B., French, T., Pendarvis, K., Brown, A., Lawrence, M. L., & Burgess, S. C. (2011). Proteome and membrane fatty acid analyses on oligotropha carboxidovorans OM5 grown under chemolithoautotrophic and heterotrophic conditions. PLoS ONE, 6(2).
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  PMID: 21386900;PMCID: PMC3046131;Abstract: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H2 to derive energy for fixation of CO2. Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H2 generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H2 as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes. © 2011 Paul et al.
• Peddinti, D., Memili, E., & Burgess, S. C. (2011). Proteomics in Animal Reproduction and Breeding. Methods in Animal Proteomics, 369-396.
• Sanders, W. S., Johnston, C. I., Bridges, S. M., Burgess, S. C., & Willeford, K. O. (2011). Prediction of Cell Penetrating Peptides by Support Vector Machines. PLoS Computational Biology, 7(7).
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  PMID: 21779156;PMCID: PMC3136433;Abstract: Cell penetrating peptides (CPPs) are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs). We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating. © 2011 Sanders et al.
• Sanders, W. S., Wang, N., Bridges, S. M., Malone, B. M., Dandass, Y. S., McCarthy, F. M., Nanduri, B., Lawrence, M. L., & Burgess, S. C. (2011). The proteogenomic mapping tool. BMC Bioinformatics, 12.
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  PMID: 21513508;PMCID: PMC3107813;Abstract: Background: High-throughput mass spectrometry (MS) proteomics data is increasingly being used to complement traditional structural genome annotation methods. To keep pace with the high speed of experimental data generation and to aid in structural genome annotation, experimentally observed peptides need to be mapped back to their source genome location quickly and exactly. Previously, the tools to do this have been limited to custom scripts designed by individual research groups to analyze their own data, are generally not widely available, and do not scale well with large eukaryotic genomes.Results: The Proteogenomic Mapping Tool includes a Java implementation of the Aho-Corasick string searching algorithm which takes as input standardized file types and rapidly searches experimentally observed peptides against a given genome translated in all 6 reading frames for exact matches. The Java implementation allows the application to scale well with larger eukaryotic genomes while providing cross-platform functionality.Conclusions: The Proteogenomic Mapping Tool provides a standalone application for mapping peptides back to their source genome on a number of operating system platforms with standard desktop computer hardware and executes very rapidly for a variety of datasets. Allowing the selection of different genetic codes for different organisms allows researchers to easily customize the tool to their own research interests and is recommended for anyone working to structurally annotate genomes using MS derived proteomics data. © 2011 Sanders et al; licensee BioMed Central Ltd.
• H., B., McCarthy, F. M., Lamont, S. J., & Burgess, S. C. (2010). Re-annotation is an essential step in systems biology modeling of functional genomics data. PLoS ONE, 5(5).
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  PMID: 20498845;PMCID: PMC2871057;Abstract: One motivation of systems biology research is to understand gene functions and interactions from functional genomics data such as that derived from microarrays. Up-to-date structural and functional annotations of genes are an essential foundation of systems biology modeling. We propose that the first essential step in any systems biology modeling of functional genomics data, especially for species with recently sequenced genomes, is gene structural and functional reannotation. To demonstrate the impact of such re-annotation, we structurally and functionally re-annotated a microarray developed, and previously used, as a tool for disease research. We quantified the impact of this re-annotation on the array based on the total numbers of structural- and functional-annotations, the Gene Annotation Quality (GAQ) score, and canonical pathway coverage. We next quantified the impact of re-annotation on systems biology modeling using a previously published experiment that used this microarray. We show that re-annotation improves the quantity and quality of structural- and functional-annotations, allows a more comprehensive Gene Ontology based modeling, and improves pathway coverage for both the whole array and a differentially expressed mRNA subset. Our results also demonstrate that re-annotation can result in a different knowledge outcome derived from previous published research findings. We propose that, because of this, re-annotation should be considered to be an essential first step for deriving value from functional genomics data. © 2010 van den Berg et al.
• Harhay, G. P., Smith, T. P., Alexander, L. J., Haudenschild, C. D., Keele, J. W., Matukumalli, L. K., Schroeder, S. G., P., C., Gresham, C. R., Bridges, S. M., Burgess, S. C., & Sonstegard, T. S. (2010). An atlas of bovine gene expression reveals novel distinctive tissue characteristics and evidence for improving genome annotation. Genome Biology, 11(10).
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  PMID: 20961407;PMCID: PMC3218658;Abstract: Background: A comprehensive transcriptome survey, or gene atlas, provides information essential for a complete understanding of the genomic biology of an organism. We present an atlas of RNA abundance for 92 adult, juvenile and fetal cattle tissues and three cattle cell lines.Results: The Bovine Gene Atlas was generated from 7.2 million unique digital gene expression tag sequences (300.2 million total raw tag sequences), from which 1.59 million unique tag sequences were identified that mapped to the draft bovine genome accounting for 85% of the total raw tag abundance. Filtering these tags yielded 87,764 unique tag sequences that unambiguously mapped to 16,517 annotated protein-coding loci in the draft genome accounting for 45% of the total raw tag abundance. Clustering of tissues based on tag abundance profiles generally confirmed ontology classification based on anatomy. There were 5,429 constitutively expressed loci and 3,445 constitutively expressed unique tag sequences mapping outside annotated gene boundaries that represent a resource for enhancing current gene models. Physical measures such as inferred transcript length or antisense tag abundance identified tissues with atypical transcriptional tag profiles. We report for the first time the tissue-specific variation in the proportion of mitochondrial transcriptional tag abundance.Conclusions: The Bovine Gene Atlas is the deepest and broadest transcriptome survey of any livestock genome to date. Commonalities and variation in sense and antisense transcript tag profiles identified in different tissues facilitate the examination of the relationship between gene expression, tissue, and gene function. © 2010 Harhay et al.; licensee BioMed Central Ltd.
• Kumar, R., Shah, P., Swiatlo, E., Burgess, S. C., Lawrence, M. L., & Nanduri, B. (2010). Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4 using high-resolution genome tiling arrays. BMC Genomics, 11(1).
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  PMID: 20525227;PMCID: PMC2887815;Abstract: Background: The identification of non-coding transcripts in human, mouse, and Escherichia coli has revealed their widespread occurrence and functional importance in both eukaryotic and prokaryotic life. In prokaryotes, studies have shown that non-coding transcripts participate in a broad range of cellular functions like gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Streptococcus pneumoniae (pneumococcus), an obligate human respiratory pathogen responsible for significant worldwide morbidity and mortality. Tiling microarrays enable genome wide mRNA profiling as well as identification of novel transcripts at a high-resolution.Results: Here, we describe a high-resolution transcription map of the S. pneumoniae clinical isolate TIGR4 using genomic tiling arrays. Our results indicate that approximately 66% of the genome is expressed under our experimental conditions. We identified a total of 50 non-coding small RNAs (sRNAs) from the intergenic regions, of which 36 had no predicted function. Half of the identified sRNA sequences were found to be unique to S. pneumoniae genome. We identified eight overrepresented sequence motifs among sRNA sequences that correspond to sRNAs in different functional categories. Tiling arrays also identified approximately 202 operon structures in the genome.Conclusions: In summary, the pneumococcal operon structures and novel sRNAs identified in this study enhance our understanding of the complexity and extent of the pneumococcal 'expressed' genome. Furthermore, the results of this study open up new avenues of research for understanding the complex RNA regulatory network governing S. pneumoniae physiology and virulence. © 2010 Kumar et al; licensee BioMed Central Ltd.
• Manda, P., Freeman, M. G., Bridges, S. M., Jankun-Kelly, T., Nanduri, B., McCarthy, F. M., & Burgess, S. C. (2010). GOModeler- A tool for hypothesis-testing of functional genomics datasets. BMC Bioinformatics, 11(SUPPL. 6).
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  PMID: 20946613;PMCID: PMC3026376;Abstract: Background: Functional genomics technologies that measure genome expression at a global scale are accelerating biological knowledge discovery. Generating these high throughput datasets is relatively easy compared to the downstream functional modelling necessary for elucidating the molecular mechanisms that govern the biology under investigation. A number of publicly available 'discovery-based' computational tools use the computationally amenable Gene Ontology (GO) for hypothesis generation. However, there are few tools that support hypothesis-based testing using the GO and none that support testing with user defined hypothesis terms.Here, we present GOModeler, a tool that enables researchers to conduct hypothesis-based testing of high throughput datasets using the GO. GOModeler summarizes the overall effect of a user defined gene/protein differential expression dataset on specific GO hypothesis terms selected by the user to describe a biological experiment. The design of the tool allows the user to complement the functional information in the GO with his/her domain specific expertise for comprehensive hypothesis testing.Results: GOModeler tests the relevance of the hypothesis terms chosen by the user for the input gene dataset by providing the individual effects of the genes on the hypothesis terms and the overall effect of the entire dataset on each of the hypothesis terms. It matches the GO identifiers (ids) of the genes with the GO ids of the hypothesis terms and parses the names of those ids that match to assign effects. We demonstrate the capabilities of GOModeler with a dataset of nine differentially expressed cytokine genes and compare the results to those obtained through manual analysis of the dataset by an immunologist. The direction of overall effects on all hypothesis terms except one was consistent with the results obtained by manual analysis. The tool's editing capability enables the user to augment the information extracted. GOModeler is available as a part of the AgBase tool suite (http://www.agbase.msstate.edu).Conclusions: GOModeler allows hypothesis driven analysis of high throughput datasets using the GO. Using this tool, researchers can quickly evaluate the overall effect of quantitative expression changes of gene set on specific biological processes of interest. The results are provided in both tabular and graphical formats. © 2010 Bridges et al; licensee BioMed Central Ltd.
• Nanduri, B., Wang, N., Lawrence, M. L., Bridges, S. M., & Burgess, S. C. (2010). Gene model detection using mass spectrometry.. Methods in molecular biology (Clifton, N.J.), 604, 137-144.
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  PMID: 20013369;Abstract: The utility of a genome sequence in biological research depends entirely on the comprehensive description of all of its functional elements. Analysis of genome sequences is still predominantly gene-centric (i.e., identifying gene models/open reading frames). In this article, we describe a proteomics-based method for identifying open reading frames that are missed by computational algorithms. Mass spectrometry-based identification of peptides and proteins from biological samples provide evidence for the expression of the genome sequence at the protein level. This proteogenomic annotation method combines computationally predicted ORFs and the genome sequence with proteomics to identify novel gene models. We also describe our proteogenomic mapping pipeline - a set of computational tools that automate the proteogenomic annotation work flow. This pipeline is available for download at www.agbase.msstate.edu/tools/ .
• Paul, D., Austin, F. W., Arick, T., Bridges, S. M., Burgess, S. C., Dandass, Y. S., & Lawrence, M. L. (2010). Genome sequence of the solvent-producing bacterium Clostridium carboxidivorans strain P7^T. Journal of Bacteriology, 192(20), 5554-5555.
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  PMID: 20729368;PMCID: PMC2950491;Abstract: Clostridium carboxidivorans strain P7T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO2 fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
• Paul, D., Bridges, S. M., Burgess, S. C., Dandass, Y. S., & Lawrence, M. L. (2010). Complete genome and comparative analysis of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5. BMC Genomics, 11(1).
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  PMID: 20863402;PMCID: PMC3091675;Abstract: Background: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.Results: The genome sequence of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5 consists of a 3.74-Mb chromosome and a 133-kb megaplasmid that contains the genes responsible for utilization of carbon monoxide, carbon dioxide, and hydrogen. To our knowledge, this strain is the first one to be sequenced in the genus Oligotropha, the closest fully sequenced relatives being Bradyrhizobium sp. BTAi and USDA110 and Nitrobacter hamburgiensis X14. Analysis of the O. carboxidovorans genome reveals potential links between plasmid-encoded chemolithoautotrophy and chromosomally-encoded lipid metabolism. Comparative analysis of O. carboxidovorans with closely related species revealed differences in metabolic pathways, particularly in carbohydrate and lipid metabolism, as well as transport pathways.Conclusion: Oligotropha, Bradyrhizobium sp and Nitrobacter hamburgiensis X14 are phylogenetically proximal. Although there is significant conservation of genome organization between the species, there are major differences in many metabolic pathways that reflect the adaptive strategies unique to each species. © 2010 Paul et al; licensee BioMed Central Ltd.
• Peddinti, D., Memili, E., & Burgess, S. C. (2010). Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction. PLoS ONE, 5(6).
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  PMID: 20574525;PMCID: PMC2888582;Abstract: Background: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV) stage are considered essential for proper maturation or 'programming' of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. Methodology/Principal Findings: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. Conclusions/Significance: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level. © 2010 Peddinti et al.
• Thanthrige-Don, N., Parvizi, P., Sarson, A. J., Shack, L. A., Burgess, S. C., & Sharif, S. (2010). Proteomic analysis of host responses to Marek's disease virus infection in spleens of genetically resistant and susceptible chickens. Developmental and Comparative Immunology, 34(7), 699-704.
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  PMID: 20138080;Abstract: Resistance to Marek's disease (MD) in chickens is genetically regulated and there are lines of chickens with differential susceptibility or resistance to this disease. The present study was designed to study comparative changes in the spleen proteomes of MD-susceptible B19 and MD-resistant B21 chickens in response to MDV infection. Spleen proteomes were examined at 4, 7, 14 and 21 days post-infection (d.p.i.) using two-dimensional gel electrophoresis and subsequently the protein spots were identified by one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). On average, there were 520 ± 27 distinct protein spots on each gel and 1.6 ± 0.7% of the spots differed quantitatively in their expression (p≤ 0.05 and fold change ≥2) between infected B19 and B21 chickens. There was one spot at 4. d.p.i. and three spots each at the rest of the time points, which had a qualitative difference in expression. Most of the differentially expressed proteins at 4 and 7. d.p.i. displayed increased expression in B21 chickens; conversely the differentially expressed proteins at 14 and 21. d.p.i. showed an increase in expression in B19 chickens. The differentially expressed proteins identified in the present study included antioxidants, molecular chaperones, proteins involved in the formation of cytoskeleton, protein degradation and antigen presentation, signal transduction, protein translation and elongation, RNA processing and cell proliferation. These findings shed light on some of the underlying processes of genetic resistance or susceptibility to MD. © 2010 Elsevier Ltd.
• Bridges, S. M., Burgess, S. C., & McCarthy, F. M. (2009). Introduction to the Proceedings of the Avian Genomics and Gene Ontology Annotation Workshop. BMC Genomics, 10(SUPPL. 2).
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  PMID: 19607650;PMCID: PMC2966328;Abstract: The Avian Genomics Conference and Gene Ontology Annotation Workshop brought together researchers and students from around the world to present their latest research addressing the delivery of value from the billions of base-pairs of Archosaur sequence that have become available in the last few years. This editorial describes the conference itself and introduces the ten peer-reviewed manuscripts accepted for publications in the proceedings. These manuscripts address issues ranging from the poultry industry view of USDA genomics policy to the genomics of a wide variety of Archeosaur species including chicken, duck, alligator, and condors and their pathogens. © 2009 Bridges et al; licensee BioMed Central Ltd.
• Bright, L. A., Burgess, S. C., Chowdhary, B., Swiderski, C. E., & McCarthy, F. M. (2009). Structural and functional-annotation of an equine whole genome oligoarray. BMC Bioinformatics, 10(SUPPL. 11), S8.
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  PMID: 19811692;PMCID: PMC3226197;Abstract: Background: The horse genome is sequenced, allowing equine researchers to use high-throughput functional genomics platforms such as microarrays; next-generation sequencing for gene expression and proteomics. However, for researchers to derive value from these functional genomics datasets, they must be able to model this data in biologically relevant ways; to do so requires that the equine genome be more fully annotated. There are two interrelated types of genomic annotation: structural and functional. Structural annotation is delineating and demarcating the genomic elements (such as genes, promoters, and regulatory elements). Functional annotation is assigning function to structural elements. The Gene Ontology (GO) is the de facto standard for functional annotation, and is routinely used as a basis for modelling and hypothesis testing, large functional genomics datasets. Results: An Equine Whole Genome Oligonucleotide (EWGO) array with 21,351 elements was developed at Texas A&M University. This 70-mer oligoarray was designed using the approximately 7× assembled and annotated sequence of the equine genome to be one of the most comprehensive arrays available for expressed equine sequences. To assist researchers in determining the biological meaning of data derived from this array, we have structurally annotated it by mapping the elements to multiple database accessions, including UniProtKB, Entrez Gene, NRPD (Non-Redundant Protein Database) and UniGene. We next provided GO functional annotations for the gene transcripts represented on this array. Overall, we GO annotated 14,531 gene products (68.1% of the gene products represented on the EWGO array) with 57,912 annotations. GAQ (GO Annotation Quality) scores were calculated for this array both before and after we added GO annotation. The additional annotations improved the meanGAQ score 16-fold. This data is publicly available at AgBase http://www.agbase.msstate.edu/. Conclusion: Providing additional information about the public databases which link to the gene products represented on the array allows users more flexibility when using gene expression modelling and hypothesis-testing computational tools. Moreover, since different databases provide different types of information, users have access to multiple data sources. In addition, our GO annotation underpins functional modelling for most gene expression analysis tools and enables equine researchers to model large lists of differentially expressed transcripts in biologically relevant ways. © 2009 Bright et al; licensee BioMed Central Ltd.
• Buza, T. J., Kumar, R., Gresham, C. R., Burgess, S. C., & McCarthy, F. M. (2009). Facilitating functional annotation of chicken microarray data. BMC Bioinformatics, 10(SUPPL. 11), S2.
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  PMID: 19811685;PMCID: PMC3226191;Abstract: Background: Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO). However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information. Results: We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (AGOM) tool to help researchers to quickly retrieve corresponding functional information for their dataset. Conclusion: Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using AGOM tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and will be updated on regular basis. © 2009 Buza et al; licensee BioMed Central Ltd.
• Cooksey, A. M., Momen, N., Stocker, R., & Burgess, S. C. (2009). Identifying blood biomarkers and physiological processes that distinguish humans with superior performance under psychological stress. PLoS ONE, 4(12).
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  PMID: 20020041;PMCID: PMC2791215;Abstract: Background: Attrition of students from aviation training is a serious financial and operational concern for the U.S. Navy. Each late stage navy aviator training failure costs the taxpayer over $1,000,000 and ultimately results in decreased operational readiness of the fleet. Currently, potential aviators are selected based on the Aviation Selection Test Battery (ASTB), which is a series of multiple-choice tests that evaluate basic and aviation-related knowledge and ability. However, the ASTB does not evaluate a person's response to stress. This is important because operating sophisticated aircraft demands exceptional performance and causes high psychological stress. Some people are more resistant to this type of stress, and consequently better able to cope with the demands of naval aviation, than others. Methodology/Principal Findings: Although many psychological studies have examined psychological stress resistance none have taken advantage of the human genome sequence. Here we use high-throughput -omic biology methods and a novel statistical data normalization method to identify plasma proteins associated with human performance under psychological stress. We identified proteins involved in four basic physiological processes: innate immunity, cardiac function, coagulation and plasma lipid physiology. Conclusions/Significance: The proteins identified here further elucidate the physiological response to psychological stress and suggest a hypothesis that stress-susceptible pilots may be more prone to shock. This work also provides potential biomarkers for screening humans for capability of superior performance under stress.
• Donaldson, J. R., Nanduri, B., Burgess, S. C., & Lawrence, M. L. (2009). Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD. Applied and Environmental Microbiology, 75(2), 366-373.
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  PMID: 19028911;PMCID: PMC2620715;Abstract: Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins. Copyright © 2009, American Society for Microbiology. All Rights Reserved.
• Hj, B., Konieczka, J. H., McCarthy, F. M., & Burgess, S. C. (2009). ArrayIDer: automated structural re-annotation pipeline for DNA microarrays.. BMC bioinformatics, 10, 30-.
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  PMID: 19166590;PMCID: PMC2636773;Abstract: BACKGROUND: Systems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays. RESULTS: We utilized the Fred Hutchinson Cancer Research Centre 13K chicken cDNA array - a widely-used non-commercial chicken microarray - to demonstrate the principle that ArrayIDer could markedly improve annotation. We structurally re-annotated 55% of the entire array. Moreover, we decreased non-chicken functional annotations by 2 fold. One beneficial consequence of our re-annotation was to identify 290 pseudogenes, of which 66 were previously incorrectly annotated. CONCLUSION: ArrayIDer allows rapid automated structural re-annotation of entire arrays and provides multiple accession types for use in subsequent functional analysis. This information is especially valuable for systems biology modeling in the non-traditional biomedical model organisms.
• Kumar, S., Buza, J. J., & Burgess, S. C. (2009). Genotype-dependent tumor regression in Marek's disease mediated at the level of tumor immunity. Cancer Microenvironment, 2(1), 23-31.
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  PMID: 19308678;PMCID: PMC2787926;Abstract: Marek's disease (MD) of chickens is a unique natural model of Hodgkin's and Non Hodgkin's lymphomas in which the neoplastically-transformed cells over-express CD30 (CD30hi) antigen. All chicken genotypes can be infected with MD virus and develop microscopic lymphomas. From 21 days post infection (dpi) microscopic lymphomas regress in resistant chickens but, in contrast, they progress to gross lymphomas in susceptible chickens. Here we test our hypothesis that in resistant chickens at 21 dpi the tissue microenvironment is pro T-helper (Th)-1 and compatible with cytotoxic T lymphocyte (CTL) immunity but in susceptible lines it is pro Th-2 or pro T-regulatory (T-reg) and antagonistic to CTL immunity. We used the B2, non-MHC-associated, MD resistance/susceptibility system (line [L]61/line [L]72) and quantified the levels of key mRNAs that can be used to define Th-1 (IL-2, IL-12, IL-18, IFNγ), Th-2 (IL-4, IL-10) and T-reg (TGFβ, GPR-83, CTLA-4, SMAD-7) lymphocyte phenotypes. We measured gene expression in both whole tissues (represents tissue microenvironment and tumor microenvironment) and in the lymphoma lesions (tumor microenvironment) themselves. Gene ontology-based modeling of our results shows that the dominant phenotype in whole tissue as well as in microscopic lymphoma lesions, is pro T-reg in both L61 and L72 but a minor pro Th-1 and anti Th-2 tissue microenvironment exists in L61 whereas there is an anti Th-1 and pro Th-2 tissue microenvironment in L72. The tumor microenvironment per se is pro T-reg, anti Th-1 and pro Th-2 in both L61 and L72. Together our data suggests that the neoplastic transformation is essentially the same in both L61 and L72 and that resistance/ susceptibility is mediated at the level of tumor immunity in the tissues. © 2009 Springer Science+Business Media B.V.
• Kunec, D., Haren, S. v., Burgess, S. C., & Hanson, L. A. (2009). A Gateway^® recombination herpesvirus cloning system with negative selection that produces vectorless progeny. Journal of Virological Methods, 155(1), 82-86.
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  PMID: 18948138;Abstract: Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines. © 2008 Elsevier B.V. All rights reserved.
• Kunec, D., Nanduri, B., & Burgess, S. C. (2009). Experimental annotation of channel catfish virus by probabilistic proteogenomic mapping. Proteomics, 9(10), 2634-2647.
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  PMID: 19391180;Abstract: Experimental identification of expressed proteins by proteomics constitutes the most reliable approach to identify genomic location and structure of protein-coding genes and substantially complements computational genome annotation. Channel catfish herpesvirus (CCV) is a simple comparative model for understanding herpesvirus biology and the evolution of the Herpesviridae. The canonical CCV genome has 76 predicted ORF and only 12 of these have been confirmed experimentally. We describe a modification of a statistical method, which assigns significance measures, q-values, to peptide identifications based on 2-D LC ESI MS/MS, real-decoy database searches and SEQUEST XCorr and DCn scores. We used this approach to identify CCV proteins expressed during its replication in cell culture, to determine protein composition of mature virions and, consequently, to refine the canonical CCVgenome annotation. To complement trypsin, we used partial proteinase K digestion, which yielded greater proteome coverage. At FDR
• McCarthy, F. M., Cooksey, A. M., & Burgess, S. C. (2009). Sequential detergent extraction prior to mass spectrometry analysis.. Methods in molecular biology (Clifton, N.J.), 528, 110-118.
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  PMID: 19153687;Abstract: Sequential detergent extraction of proteins from eukaryotic cells has been used to increase proteome coverage of 2D-PAGE. We have adapted sequential detergent extraction for use with the high-throughput non-electrophoretic proteomics method of liquid chromatography and electrospray ionisation tandem mass spectrometry. This method of extraction yields comprehensive proteomes that include up to twice as many membrane proteins as other published methods. Two thirds of these membrane proteins have more than one transmembrane domain and many of these have multiple transmembrane domains. Since sequential detergent extraction (SDE) separates proteins based upon their physicochemistry and sub-cellular localisation, this method also provides useful data about cellular localisation.
• McCarthy, F. M., Mahony, T. J., Parcells, M. S., & Burgess, S. C. (2009). Understanding animal viruses using the Gene Ontology. Trends in Microbiology, 17(7), 328-335.
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  PMID: 19577474;Abstract: Understanding the effects of viral infection has typically focused on specific virus-host interactions such as tissue tropism, immune responses and histopathology. However, modeling viral pathogenesis requires information about the functions of gene products from both virus and host, and how these products interact. Recent developments in the functional annotation of genomes using Gene Ontology (GO) and in modeling functional interactions among gene products, together with an increased interest in systems biology, provide an excellent opportunity to generate global interaction models for viral infection. Here, we review how the GO is being used to model viral pathogenesis, with a focus on animal viruses. © 2009 Elsevier Ltd. All rights reserved.
• Nanduri, B., Lawrence, M. L., Vanguri, S., Pechan, T., & Burgess, S. C. (2009). Proteomic analysis using an unfinished bacterial genome: The effects of sub-minimum inhibitory concentrations of antibiotics on Mannheimia haemolytica virulence factor expression (Proteomics 5, 18, (4852-4863) DOI: 10.1002/pmic.200500112). Proteomics, 9(13), 3623-.
• Nanduri, B., Shack, L. A., Burgess, S. C., & Lawrence, M. L. (2009). The transcriptional response of Pasteurella multocida to three classes of antibiotics. BMC Genomics, 10(SUPPL. 2).
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  PMID: 19607655;PMCID: PMC2966327;Abstract: Background: Pasteurella multocida is a gram-negative bacterial pathogen that has a broad host range. One of the diseases it causes is fowl cholera in poultry. The availability of the genome sequence of avian P. multocida isolate Pm70 enables the application of functional genomics for observing global gene expression in response to a given stimulus. We studied the effects of three classes of antibiotics on the P. multocida transcriptome using custom oligonucleotide microarrays from NimbleGen Systems. Hybridizations were conducted with RNA isolated from three independent cultures of Pm70 grown in the presence or absence of sub-minimum inhibitory concentration (sub-MIC) of antibiotics. Differentially expressed (DE) genes were identified by ANOVA and Dunnett's test. Biological modeling of the differentially expressed genes (DE) was conducted based on Clusters of Orthologous (COG) groups and network analysis in Pathway Studio. Results: The three antibiotics used in this study, amoxicillin, chlortetracycline, and enrofloxacin, collectively influenced transcription of 25% of the P. multocida Pm70 genome. Some DE genes identified were common to more than one antibiotic. The overall transcription signatures of the three antibiotics differed at the COG level of the analysis. Network analysis identified differences in the SOS response of P. multocida in response to the antibiotics. Conclusion: This is the first report of the transcriptional response of an avian strain of P. multocida to sub-lethal concentrations of three different classes of antibiotics. We identified common adaptive responses of P. multocida to antibiotic stress. The observed changes in gene expression of known and putative P. multocida virulence factors establish the molecular basis for the therapeutic efficacy of sub-MICs. Our network analysis demonstrates the feasibility and limitations of applying systems modeling to high throughput datasets in 'non-model' bacteria. © 2009 Nanduri et al; licensee BioMed Central Ltd.
• Parvizi, P., Read, L. R., Abdul-Careem, M., Sarson, A. J., Lusty, C., Lambourne, M., Thanthrige-Don, N., Burgess, S. C., & Sharif, S. (2009). Cytokine gene expression in splenic CD4⁺ and CD8⁺ T cell subsets of genetically resistant and susceptible chickens infected with Marek's disease virus. Veterinary Immunology and Immunopathology, 132(2-4), 209-217.
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  PMID: 19615758;Abstract: T cells from the spleens of B19/B19 and B21/B21 chickens infected with MDV JM-16 strain were fractionated by flow cytometry at 4, 10, 21 days post infection (d.p.i.). The expression of cytokine and viral genes (meq and glycoprotein B (gB)) was measured by real-time RT-PCR. It was determined that CD4+ and CD8+ T cells had both become infected with Marek's disease virus (MDV) in both chicken lines. There was significantly higher expression of meq in CD4+ T cells compared to CD8+ T cells at 10 and 21 d.p.i. Furthermore, at 10 and 21 d.p.i., there was a tendency for higher expression of meq in both T cell subsets of B19 chickens compared to those of B21 chickens. There were temporal changes in the expression of cytokines, interferon (IFN)-γ, interleukin (IL)-18, IL-6, and IL-10, in various T cell subsets. Among these changes, there was an increase in IL-10 expression in both T cell subsets at different time points, especially in the susceptible line at 10 and 21 d.p.i. Our results indicate that cytokines could be differentially induced by MDV in CD4+ and CD8+ T cell subsets and that IL-10 may play a role in the modulation of immune response to MDV. However, an association between cytokine gene expression in T cell subsets and resistance or susceptibility to MD was not established. © 2009 Elsevier B.V. All rights reserved.
• Pendarvis, K., Kumar, R., Burgess, S. C., & Nanduri, B. (2009). An automated proteomic data analysis workflow for mass spectrometry. BMC Bioinformatics, 10(SUPPL. 11), S17.
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  PMID: 19811682;PMCID: PMC3226188;Abstract: Background: Mass spectrometry-based protein identification methods are fundamental to proteomics. Biological experiments are usually performed in replicates and proteomic analyses generate huge datasets which need to be integrated and quantitatively analyzed. The Sequest™ search algorithm is a commonly used algorithm for identifying peptides and proteins from two dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2-D LC ESI MS2) data. A number of proteomic pipelines that facilitate high throughput 'post data acquisition analysis' are described in the literature. However, these pipelines need to be updated to accommodate the rapidly evolving data analysis methods. Here, we describe a proteomic data analysis pipeline that specifically addresses two main issues pertinent to protein identification and differential expression analysis: 1) estimation of the probability of peptide and protein identifications and 2) non-parametric statistics for protein differential expression analysis. Our proteomic analysis workflow analyzes replicate datasets from a single experimental paradigm to generate a list of identified proteins with their probabilities and significant changes in protein expression using parametric and non-parametric statistics. Results: The input for our workflow is Bioworks™ 3.2 Sequest (or a later version, including cluster) output in XML format. We use a decoy database approach to assign probability to peptide identifications. The user has the option to select "quality thresholds" on peptide identifications based on the P value. We also estimate probability for protein identification. Proteins identified with peptides at a user-specified threshold value from biological experiments are grouped as either control or treatment for further analysis in ProtQuant. ProtQuant utilizes a parametric (ANOVA) method, for calculating differences in protein expression based on the quantitative measure ΣXcorr. Alternatively ProtQuant output can be further processed using non-parametric Monte-Carlo resampling statistics to calculate P values for differential expression. Correction for multiple testing of ANOVA and resampling P values is done using Benjamini and Hochberg's method. The results of these statistical analyses are then combined into a single output file containing a comprehensive protein list with probabilities and differential expression analysis, associated P values, and resampling statistics. Conclusion: For biologists carrying out proteomics by mass spectrometry, our workflow facilitates automated, easy to use analyses of Bioworks (3.2 or later versions) data. All the methods used in the workflow are peer-reviewed and as such the results of our workflow are compliant with proteomic data submission guidelines to public proteomic data repositories including PRIDE. Our workflow is a necessary intermediate step that is required to link proteomics data to biological knowledge for generating testable hypotheses. © 2009 Pendarvis et al; licensee BioMed Central Ltd.
• Thanthrige-Don, N., Abdul-Careem, M. F., Shack, L. A., Burgess, S. C., & Sharif, S. (2009). Analyses of the spleen proteome of chickens infected with Marek's disease virus. Virology, 390(2), 356-367.
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  PMID: 19540544;Abstract: Marek's disease virus (MDV), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. In the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to MDV infection were studied using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). Comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. In total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (FDR) ≤ 0.05 and fold change ≥ 2) or qualitatively differentially expressed at least once during different sampling points. Overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin-proteasome protein degradation (UPP), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. Notably, early stages of the disease were characterized by changes in the UPP, and antigen presentation. Furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. The present findings provide a basis for further studies aimed at elucidation of the role of these proteins in MDV interactions with its host. © 2009 Elsevier Inc. All rights reserved.
• Wang, N., Burgess, S., Lawrence, M., & Bridges, S. (2009). Proteogenomic mapping for structural annotation of prokaryote genomes. Proceedings - 2009 International Joint Conference on Bioinformatics, Systems Biology and Intelligent Computing, IJCBS 2009, 103-106.
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  Abstract: Structural annotation of genomes is one of major goals of genomics research. Most popular tools for structural annotation of genomes are determined by computational pipelines. It is well-known that these computational methods have a number of shortcomings including false identifications and incorrect identification of gene boundaries. Proteomic data can used to confirm the identification of genes identified by computational methods and correct mistakes. A Proteogenomic mapping method has been developed, which uses peptides identified from mass spectrometry for structural annotation of genomes. Spectra are matched against both a protein database and the genome database translated in all six reading frames. Those peptides that match the genome but not the protein database potentially represent novel protein coding genes, annotation errors. These short experimentally derived peptides are used to discover potential novel protein coding genes called expressed Protein Sequence Tags (ePSTs) by aligning the peptides to the genomic DNA and extending the translation in the 3' and 5' direction. In the paper, an enhanced pipeline, has been designed and developed for discovering and evaluating of potential novel protein coding genes: 1) a distance-based outlier detection method for validating peptides identified from MS/MS, 2) a proteogenomic mapping for discovery of potential novel protein coding genes, 3) collection of evidence from a number of sources and automatically evaluate potential novel protein coding genes by using machine learning techniques, such as Neural Network, Support Vector Machine, Naïve Bayes etc.
• Yueh, F., Zheng, H., Singh, J. P., & Burgess, S. (2009). Preliminary evaluation of laser-induced breakdown spectroscopy for tissue classification. Spectrochimica Acta - Part B Atomic Spectroscopy, 64(10), 1059-1067.
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  Abstract: Laser-induced breakdown spectroscopy (LIBS) is an on-line, real-time technology that can produce immediate information about the elemental contents of tissue samples. We have previously shown that LIBS may be used to distinguish cancerous from non-cancerous tissue. In this work, we study LIBS spectra produced from chicken brain, lung, spleen, liver, kidney and skeletal muscle. Different data processing techniques were used to study if the information contained in these LIBS spectra is able to differentiate between different types of tissue samples and then identify unknown tissues. We have demonstrated a clear distinguishing between each of the known tissue types with only 21 selected analyte lines from each observed LIBS spectrum. We found that in order to produce an analytical model to work well with new sample we need to have representative training data to cover a wide range of spectral variation due to experimental or environmental changes. © 2009 Elsevier B.V. All rights reserved.
• Zhang, S., Crow, J. A., Cooper, R. C., McLaughlin, R. M., Burgess, S., Borazjani, A., & Liao, J. (2009). Detection of myocardial fiber disruption in artificial lesions with 3D DT-MRI tract models. Proceedings of the ASME Summer Bioengineering Conference, SBC2008, 663-664.
• Buza, J. J., & Burgess, S. C. (2008). Different signaling pathways expressed by chicken naïve CD4 ⁺ T cells, CD4 ⁺ lymphocytes activated with staphylococcal enterotoxin B, and those malignantly transformed by marek's disease virus. Journal of Proteome Research, 7(6), 2380-2387.
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  PMID: 18412384;Abstract: Proteomics methods, based on liquid chromatography and tandem mass spectrometry, produce large "shotgun" proteomes that are most appropriately compared not at the level of differentially expressed proteins only but at the more comprehensive level of biological networks and pathways. This is now possible with the emergence of functional annotation databases and tools, databases of canonical pathways and molecular interactions and computational text mining tools. Here, we used shotgun proteomics, and the differential proteomics modeling functionalities available in the Pathwaystudio network modeling program to define the cell physiology of Hodgkin's disease antigen-overexpressing (CD30 hi) CD4 + T cell lymphomas using the unique Marek's disease (MD) natural animal model. CD30 hi lymphoma cells have characteristics of activated T cells but are also fundamentally different from their nontransformed healthy counterparts. We compared the cell physiology of naïve, superantigen-activated and MD-transformed CD4 + T cell proteomes. While the superantigen- activated cells had signaling pathways associated with cell activation, inflammation, proliferation and cell death, the MD-transformed cells had growth factor, cytokine, adhesion, and transcription factor signaling responses associated with oncogenicity, cell proliferation, angiogenesis, motility, and metastasis. © 2008 American Chemical Society.
• Buza, T. J., Mccarthy, F. M., Wang, N., Bridges, S. M., & Burgess, S. C. (2008). Gene Ontology annotation quality analysis in model eukaryotes. Nucleic Acids Research, 36(2).
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  PMID: 18187504;PMCID: PMC2241866;Abstract: Functional analysis using the Gene Ontology (GO) is crucial for array analysis, but it is often difficult for researchers to assess the amount and quality of GO annotations associated with different sets of gene products. In many cases the source of the GO annotations and the date the GO annotations were last updated is not apparent, further complicating a researchers' ability to assess the quality of the GO data provided. Moreover, GO biocurators need to ensure that the GO quality is maintained and optimal for the functional processes that are most relevant for their research community. We report the GO Annotation Quality (GAQ) score, a quantitative measure of GO quality that includes breadth of GO annotation, the level of detail of annotation and the type of evidence used to make the annotation. As a case study, we apply the GAQ scoring method to a set of diverse eukaryotes and demonstrate how the GAQ score can be used to track changes in GO annotations over time and to assess the quality of GO annotations available for specific biological processes. The GAQ score also allows researchers to quantitatively assess the functional data available for their experimental systems (arrays or databases). © 2008 The Author(s).
• Dail, M. B., Shack, L. A., Chambers, J. E., & Burgess, S. C. (2008). Global liver proteomics of rats exposed for 5 days to phenobarbital identifies changes associated with cancer and with CYP metabolism. Toxicological Sciences, 106(2), 556-569.
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  PMID: 18796496;PMCID: PMC2581678;Abstract: A global proteomics approach was applied to model the hepatic response elicited by the toxicologically well-characterized xenobiotic phenobarbital (PB), a prototypical inducer of hepatic xenobiotic metabolizing enzymes and a well-known nongenotoxic liver carcinogen in rats. Differential detergent fractionation two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry and systems biology modeling were used to identify alterations in toxicologically relevant hepatic molecular functions and biological processes in the livers of rats following a 5-day exposure to PB at 80 mg/kg/day or a vehicle control. Of the 3342 proteins identified, expression of 121 (3.6% of the total proteins) was significantly increased and 127 (3.8%) significantly decreased in the PB group compared to controls. The greatest increase was seen for cytochrome P450 (CYP) 2B2 (167-fold). All proteins with statistically significant differences from control were then analyzed using both Gene Ontology (GO) and Ingenuity Pathways Analysis (IPA, 5.0 IPA-Tox) for cellular location, function, network connectivity, and possible disease processes, especially as they relate to CYP-mediated metabolism and nongenotoxic carcinogenesis mechanisms. The GO results suggested that PB's mechanism of nongenotoxic carcinogenesis involves both increased xenobiotic metabolism, especially induction of the 2B subfamily of CYP enzymes, and increased cell cycle activity. Apoptosis, however, also increased, perhaps, as an attempt to counter the rising cancer threat. Of the IPA-mapped proteins, 41 have functions which are procarcinogenic and 14 anticarcinogenic according to the hypothesized nongenotoxic mechanism of imbalance between apoptosis and cellular proliferation. Twenty-two additional IPA nodes can be classified as procarcinogenic by the competing theory of increased metabolism resulting in the formation of reactive oxygen species. Since the systems biology modeling corresponded well to PB effects previously elucidated via more traditional methods, the global proteomic approach is proposed as a new screening methodology that can be incorporated into future toxicological studies. © The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
• Dandass, Y. S., Burgess, S. C., Lawrence, M., & Bridges, S. M. (2008). Accelerating string set matching in FPGA hardware for bioinformatics research. BMC Bioinformatics, 9.
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  PMID: 18412963;PMCID: PMC2374783;Abstract: Background: This paper describes techniques for accelerating the performance of the string set matching problem with particular emphasis on applications in computational proteomics. The process of matching peptide sequences against a genome translated in six reading frames is part of a proteogenomic mapping pipeline that is used as a case-study. The Aho-Corasick algorithm is adapted for execution in field programmable gate array (FPGA) devices in a manner that optimizes space and performance. In this approach, the traditional Aho-Corasick finite state machine (FSM) is split into smaller FSMs, operating in parallel, each of which matches up to 20 peptides in the input translated genome. Each of the smaller FSMs is further divided into five simpler FSMs such that each simple FSM operates on a single bit position in the input (five bits are sufficient for representing all amino acids and special symbols in protein sequences). Results: This bit-split organization of the Aho-Corasick implementation enables efficient utilization of the limited random access memory (RAM) resources available in typical FPGAs. The use of on-chip RAM as opposed to FPGA logic resources for FSM implementation also enables rapid reconfiguration of the FPGA without the place and routing delays associated with complex digital designs. Conclusion: Experimental results show storage efficiencies of over 80% for several data sets. Furthermore, the FPGA implementation executing at 100 MHz is nearly 20 times faster than an implementation of the traditional Aho-Corasick algorithm executing on a 2.67 GHz workstation. © 2008 Dandass et al; licensee BioMed Central Ltd.
• Keshavamurthy, S. S., Leonard, K. M., Burgess, S. C., & Minerick, A. M. (2008). Direct current dielectrophoretic characterization of erythrocytes: Positive ABO blood types. Technical Proceedings of the 2008 NSTI Nanotechnology Conference and Trade Show, NSTI-Nanotech, Nanotechnology 2008, 3, 401-404.
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  Abstract: The adaptation of medical diagnostic applications into micrototal analytical systems (μTAS) has the potential to improve the ease, accessibility and rapidity of medical diagnostics. This work adapts direct current dielectrophoresis (DC-DEP) to a medical diagnostic application of sorting blood cells where an insulating obstacle is used to produce a non-uniform electric field. Initial efforts are focused on achieving separation of positive ABO red blood cells. Two dependencies will simultaneously be explored: blood type and blood cell size. Fluorescent polystyrene particles of three different sizes will be tested and compared against the separation and collection of actual blood cells into different sample bins. Further, continuous separation of red blood cells according to blood types and collection into specific bins will be explored. This developed technique is directly applicable for use in a portable device for easy and rapid blood diagnostics.
• Khijwania, S. K., Kim, C. K., Singh, J. P., & Burgess, S. C. (2008). Optimized fiber optic bioprobe with high spectral contrast exploiting laser-induced fluorescence for malignancy diagnosis. Applied Optics, 47(35), 6615-6624.
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  PMID: 19079471;Abstract: A high spectral contrast is expected to be very important when laser-induced fluorescence (LIF) is employed for cancer diagnosis. We developed a LIF optical fiber sensor to achieve a very high spectral contrast between normal and malignant tissues. A comprehensive experimental investigation was carried out to study the role of two critically important parameters for sensor design, namely, the excitationcollection geometry and the excitation wavelength, and their effect on the autofluorescence spectral contrast. An optimum sensing configuration was determined in order to enhance the small, but consistent, spectral difference between the normal and the malignant tissue for improving the accuracy of LIF-based cancer diagnosis. With the optimum sensor configuration, we realized a spectral contrast of more than 22 times between normal and malignant tissue sample spectra. © 2008 Optical Society of America.
• Kunec, D., Hanson, L. A., Haren, S. V., Nieuwenhuizen, I. F., & Burgess, S. C. (2008). An overlapping bacterial artificial chromosome system that generates vectorless progeny for channel catfish herpesvirus. Journal of Virology, 82(8), 3872-3881.
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  PMID: 18234790;PMCID: PMC2292985;Abstract: Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Metzler, S. A., Pregonero, C. A., Butcher, J. T., Burgess, S. C., & Warnock, J. N. (2008). Cyclic strain regulates pro-inflammatory protein expression in porcine aortic valve endothelial cells. Journal of Heart Valve Disease, 17(5), 571-578.
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  PMID: 18980092;Abstract: Background and aim of the study: The endothelium of diseased heart valves is known to express the adhesion molecules VCAM-1, ICAM-1 and E-selectin, while healthy valves lack these pro-inflammatory proteins. The study aim was to determine if mechanical forces were responsible for the pro-inflammatory reaction in aortic valve endothelial cells. Methods: Isolated porcine aortic valve endothelial cells (PAVEC) were cultured and seeded onto BioFlex™ culture plates. The cells were exposed to equibiaxial cyclic strains of 5,10 and 20% for 24 h in a Flexcell FX-4000T™ Tension Plus system at 1 Hz. Pro-inflammatory protein expression was detected through the use of monoclonal antibodies via fluorescence-assisted cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Results: Pro-inflammatory protein expression was evident at cyclic strains of 5 and 20%, while a 10% strain did not elicit an inflammatory response. Confocal images indicated a disrupted endothelial monolayer, evidence of significant cell death, and the presence of all adhesion molecules at 5% strain. PAVEC exposed to 10% cyclic strain failed to express any of the pro-inflammatory proteins, while the cellular monolayer appeared near-confluent and characteristically similar to cellular images captured prior to cyclic stretching. CLSM images of PAVEC cyclically stretched by 20% demonstrated a similar proinflammatory reaction to those with 5% strain, while the cellular environment also showed decreased monolayer integrity. FACS data showed a significant up-regulation of the membrane-bound VCAM-1-, ICAM-1- and E-selectin-positive cells at 5% and 20% strain, compared to 10% strain and controls. Conclusion: The finding that equibiaxial cyclic strain regulates the pro-inflammatory response in PAVEC suggests that alterations in the mechanical environment of heart valves may contribute to valve pathogenesis. © Copyright by ICR Publishers 2008.
• Nanduri, B., Lawrence, M. L., Peddinti, D. S., & Burgess, S. C. (2008). Effects of subminimum inhibitory concentrations of antibiotics on the pasteurella multocida proteome: A systems approach. Comparative and Functional Genomics, 2008.
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  PMID: 18464924;PMCID: PMC2367384;Abstract: To identify key regulators of subminimum inhibitory concentration (sub-MIC) antibiotic response in the Pasteurella multocida proteome, we applied systems approaches. Using 2D-LC-ESI-MS2, we achieved 53% proteome coverage. To study the differential protein expression in response to sub-MIC antibiotics in the context of protein interaction networks, we inferred P. multocida Pm70 protein interaction network from orthologous proteins. We then overlaid the differential protein expression data onto the P. multocida protein interaction network to study the bacterial response. We identified proteins that could enhance antimicrobial activity. Overall compensatory response to antibiotics was characterized by altered expression of proteins involved in purine metabolism, stress response, and cell envelope permeability.
• Nanduri, B., Shah, P., Ramkumar, M., Allen, E. B., Swiatlo, E., Burgess, S. C., & Lawrence, M. L. (2008). Quantitative analysis of Streptococcus pneumoniae TIGR4 response to in vitro iron restriction by 2-D LC ESI MS/MS. Proteomics, 8(10), 2104-2114.
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  PMID: 18491321;Abstract: Understanding the growth of bacterial pathogens in a micronutrient restricted host environment can identify potential virulence proteins that help overcome this nutritional barrier to productive infection. In this study, we investigated the pneumococcal protein expression response to iron limitation using an in vitro model. We identified S. pneumoniae TIGR4 proteins by 2-D LC ESI MS/MS and determined significant changes in protein expression in response to iron restriction using computer-intensive random resampling methods. Differential protein expression was studied in the context of a S. pneumoniae TIGR4 protein interaction network using Pathway Studio. Our analysis showed that pneumococcal iron restriction response was marked by increased expression of known virulence factors like PsaA. It involved changes in the expression of stress response, and phase variation and biofilm formation proteins. The net effect of changes in all these biological processes could increase the virulence of S. pneumoniae TIGR4 during in vivo infection. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.
• Paul, D., Bridges, S., Burgess, S. C., Dandass, Y., & Lawrence, M. L. (2008). Genome sequence of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5^T. Journal of Bacteriology, 190(15), 5531-5532.
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  PMID: 18539730;PMCID: PMC2493269;Abstract: Oligotropha carboxidovorans OM5T (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium with the capability to utilize carbon monoxide, carbon dioxide, and hydrogen. It is also capable of heterotrophic growth under appropriate environmental conditions. Here we report the annotated genome sequence of the circular chromosome of this organism. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Peddinti, D., Nanduri, B., Kaya, A., Feugang, J. M., Burgess, S. C., & Memili, E. (2008). Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Systems Biology, 2.
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  PMID: 18294385;PMCID: PMC2291030;Abstract: Background: Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. Results: Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. Conclusion: This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype. © 2008 Peddinti et al; licensee BioMed Central Ltd.
• Scott, V. L., Burgess, S. C., Shack, L. A., Lockett, N. N., & Coats, K. S. (2008). Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats. Veterinary Immunology and Immunopathology, 123(1-2), 90-96.
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  PMID: 18295905;PMCID: PMC2464288;Abstract: Feline immunodeficiency virus (FIV) causes a natural infection of domestic cats that resembles HIV-1 in pathogenesis and disease progression. Feline AIDS is characterized by depression of the CD4+ T cell population and fatal opportunistic infections. Maternal-fetal transmission of FIV readily occurs under experimental conditions, resulting in infected viable kittens and resorbed or arrested fetal tissues. Although both FIV and HIV use the chemokine receptor CXCR4 as a co-receptor, FIV does not utilize CD4 as the primary receptor. Rather, CD134 (OX40), a T cell activation antigen and co-stimulatory molecule, is the primary receptor for FIV. We hypothesized that placental expression of CD134 and CXCR4 may render the placenta vulnerable to FIV infection, possibly facilitating efficient vertical transmission of FIV, and impact pregnancy outcome. The purpose of this project was to quantify the relative expression of CD134 and CXCR4 mRNA from the term placentas of three groups of cats: uninfected queens producing viable offspring, experimentally-infected queens producing only viable offspring, and experimentally-infected queens producing viable offspring among mostly non-viable fetuses. Total RNA was extracted from term placental tissues from all groups of cats. Real-time one-step reverse transcriptase-PCR was used to measure gene expression. The FIV receptors CD134 and CXCR4 were expressed in all late term feline placental tissues. Placentas from FIV-infected queens producing litters of only viable offspring expressed more CD134 and CXCR4 mRNA than those from uninfected queens, suggesting that infection may cause upregulation of the receptors. On the other hand, placentas from FIV-infected cats with non-successful pregnancies expressed similar levels of CD134 mRNA and slightly less CXCR4 mRNA than those from uninfected queens. Thus, it appears that cells expressing these receptors may play a role in pregnancy maintenance.
• Shack, L. A., Buza, J. J., & Burgess, S. C. (2008). The neoplastically transformed (CD30^hi) Marek's disease lymphoma cell phenotype most closely resembles T-regulatory cells. Cancer Immunology, Immunotherapy, 57(8), 1253-1262.
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  PMID: 18256827;Abstract: Introduction: Marek's disease (MD), a herpesvirus-induced lymphoma of chickens is a unique natural model of CD30-overexpressing (CD30hi) lymphoma. We have previously proposed that the CD30hi neoplastically transformed CD4+ T cells in MD lymphomas have a phenotype antagonistic to cell mediated immunity. Here were test the hypothesis that the CD30hi neoplastically transformed MD lymphoma cells have a phenotype more closely resembling T-helper (Th)-2 or regulatory T (T-reg) cells. Materials and methods: We separated ex vivo-derived CD30hi, from the CD30lo/- (non-transformed), MD lymphoma cells and then quantified the relative amounts of mRNA and proteins for cytokines and other genes that define CD4+ Th-1, Th-2 or T-reg phenotypes. Results and discussion: Gene Ontology-based modeling of our data shows that the CD30hi MD lymphoma cells having a phenotype more similar to T-reg. Sequences that could be bound by the MD virus putative oncoprotein Meq in each of these genes' promoters suggests that the MD herpesvirus may play a direct role in maintaining this T-reg-like phenotype. © 2008 Springer-Verlag.
• Srivastava, S. K., Daggolu, P. R., Burgess, S. C., & Minerick, A. R. (2008). Dielectrophoretic characterization of erythrocytes: Positive ABO blood types. Electrophoresis, 29(24), 5033-5046.
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  PMID: 19130588;Abstract: Dielectrophoretic manipulation of erythrocytes/red blood cells is investigated as a tool to identify blood type for medical diagnostic applications. Positive blood types of the ABO typing system (A+, B+, AB+ and O+) were tested and cell responses quantified. The dielectrophoretic response of each blood type was observed in a platinum electrode microdevice, delivering a field of 0.025Vpp/μm at 1 MHz. Responses were recorded via video microscopy for 120 s and erythrocyte positions were tabulated at 20-30 s intervals. Both vertical and horizontal motions of erythrocytes were quantified via image object recognition, object tracking in MATLAB, binning into appropriate electric field contoured regions (wedges) and statistical analysis. Cells of O+ type showed relatively attenuated response to the dielectrophoretic field and were distinguished with greater than 95% confidence from all the other three blood types. AB+ cell responses differed from A+ and B+ blood types likely because AB+ erythrocytes express both the A and B glycoforms on their membrane. This research suggests that dielectrophoresis of untreated erythrocytes beyond simple dilution depends on blood type and could be used in portable blood typing devices. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Wang, N., Yuan, C., Burgess, S., Nanduri, B., Lawrence, M., & Bridges, S. (2008). Integrating evidence for evaluation of potential novel protein-coding genes using Bayesian networks. Proceedings of the 2008 International Conference on Bioinformatics and Computational Biology, BIOCOMP 2008, 838-843.
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  Abstract: Evaluating the quality of potential new protein-coding genes that have been predicted by directly searching mass spectrometry against genome sequence is a very challenging task. Many machine learning techniques such as neural networks, decision trees, and support vector machines have been applied to this task. All of these techniques learn a model from a training dataset and predict the quality of potential novel protein-coding genes using various evidential features as inputs. The quality and quantity of the training dataset significantly affect the performance of the learned models. In biological research, data collected is often incomplete and with very few data points. It is desirable to have methods that are robust to noisy data and low sample-size. Furthermore, the models learned by these machine learning techniques typically do not reveal the conditional (in)dependence relations among the evidential features. Gaining insight into the relationships among features is important for biological domains .In this paper, we describe methods for learning Bayesian networks for modeling the conditional (in)dependence relations among features of protein-coding genes and calculating confidence scores for potential novel genes based on their evidential features. Bootstrap methods are applied to assess the confidence measure on the arcs of the learned network structures and to identify a set of robust arcs in order to construct a final model for future predictions. We tested the Bayesian network model learned from our method using a training experimental dataset. The results show that the method significantly improved the accuracy of the learned model in predicting potential novel genes.
• Bridges, S. M., Bryce, G. B., Wang, N., Williams, W. P., Burgess, S. C., & Nanduri, B. (2007). ProtQuant: A tool for the label-free quantification of MudPIT proteomics data. BMC Bioinformatics, 8(SUPPL. 7).
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  PMID: 18047724;PMCID: PMC2099493;Abstract: Background: Effective and economical methods for quantitative analysis of high throughput mass spectrometry data are essential to meet the goals of directly identifying, characterizing, and quantifying proteins from a particular cell state. Multidimensional Protein Identification Technology (MudPIT) is a common approach used in protein identification. Two types of methods are used to detect differential protein expression in MudPIT experiments: those involving stable isotope labelling and the so-called label-free methods. Label-free methods are based on the relationship between protein abundance and sampling statistics such as peptide count, spectral count, probabilistic peptide identification scores, and sum of peptide Sequest XCorr scores (ΣXCorr). Although a number of label-free methods for protein quantification have been described in the literature, there are few publicly available tools that implement these methods. We describe ProtQuant, a Java-based tool for label-free protein quantification that uses the previously published ΣXCorr method for quantification and includes an improved method for handling missing data. Results: ProtQuant was designed for ease of use and portability for the bench scientist. It implements the ΣXCorr method for label free protein quantification from MudPIT datasets. ProtQuant has a graphical user interface, accepts multiple file formats, is not limited by the size of the input files, and can process any number of replicates and any number of treatments. In addition,ProtQuant implements a new method for dealing with missing values for peptide scores used for quantification. The new algorithm, called ΣXCorr, uses "below threshold" peptide scores to provide meaningful non-zero values for missing data points. We demonstrate that ΣXCorr produces an average reduction in false positive identifications of differential expression of 25% compared to ΣXCorr. Conclusion: ProtQuant is a tool for protein quantification built for multi-platform use with an intuitive user interface. ProtQuant efficiently and uniquely performs label-free quantification of protein datasets produced with Sequest and provides the user with facilities for data management and analysis. Importantly, ProtQuant is available as a self-installing executable for the Windows environment used by many bench scientists. © 2007 Bridges et al; licensee BioMed Central Ltd.
• Buza, J. J., & Burgess, S. C. (2007). Modeling the proteome of a Marek's disease transformed cell line: A natural animal model for CD30 overexpressing lymphomas. Proteomics, 7(8), 1316-1326.
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  PMID: 17443643;Abstract: Marek's disease (MD) in the chicken, caused by the highly infectious MD α-herpesvirus (MDV), is both commercially important and a unique, naturally occurring model for human T-cell lymphomas overexpressing the Hodgkin's disease antigen, CD30. Here, we used proteomics as a basis for modeling the molecular functions and biological processes involved in MDV-induced lymphomagenesis. Proteins were extracted from an MDV-transformed cell line and were then identified using 2-D LC-ESI-MS/MS. From the resulting 3870 cellular and 21 MDV proteins we confirm the existence of 3150 "predicted" and 12 "hypothetical" chicken proteins. The UA-01 proteome is proliferative, differentiated, angiogenic, pro-metastatic and pro-immune-escape but anti-programmed cell death, -anergy, -quiescence and -senescence and is consistent with a cancer phenotype. In particular, the pro-metastatic integrin signaling pathway and the ERK/MAPK signaling pathways were the two predominant signaling pathways represented. The cytokines, cytokine receptors, and their related proteins suggest that UA-01 has a regulatory T-cell phenotype. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.
• Buza, T. J., McCarthy, F. M., & Burgess, S. C. (2007). Experimental-confirmation and functional-annotation of predicted proteins in the chicken genome. BMC Genomics, 8.
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  PMID: 18021451;PMCID: PMC2204016;Abstract: Background: The chicken genome was sequenced because of its phylogenetic position as a non-mammalian vertebrate, its use as a biomedical model especially to study embryology and development, its role as a source of human disease organisms and its importance as the major source of animal derived food protein. However, genomic sequence data is, in itself, of limited value; generally it is not equivalent to understanding biological function. The benefit of having a genome sequence is that it provides a basis for functional genomics. However, the sequence data currently available is poorly structurally and functionally annotated and many genes do not have standard nomenclature assigned. Results: We analysed eight chicken tissues and improved the chicken genome structural annotation by providing experimental support for the in vivo expression of 7,809 computationally predicted proteins, including 30 chicken proteins that were only electronically predicted or hypothetical translations in human. To improve functional annotation (based on Gene Ontology), we mapped these identified proteins to their human and mouse orthologs and used this orthology to transfer Gene Ontology (GO) functional annotations to the chicken proteins. The 8,213 orthology-based GO annotations that we produced represent an 8% increase in currently available chicken GO annotations. Orthologous chicken products were also assigned standardized nomenclature based on current chicken nomenclature guidelines. Conclusion: We demonstrate the utility of high-throughput expression proteomics for rapid experimental structural annotation of a newly sequenced eukaryote genome. These experimentally-supported predicted proteins were further annotated by assigning the proteins with standardized nomenclature and functional annotation. This method is widely applicable to a diverse range of species. Moreover, information from one genome can be used to improve the annotation of other genomes and inform gene prediction algorithms. © 2007 Buza et al; licensee BioMed Central Ltd.
• Cogburn, L. A., Porter, T. E., Duclos, M. J., Simon, J., Burgess, S. C., Zhu, J. J., Cheng, H. H., Dodgson, J. B., & Burnside, J. (2007). Functional genomics of the chicken - A model organism. Poultry Science, 86(10), 2059-2094.
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  PMID: 17878436;Abstract: Since the sequencing of the genome and the development of high-throughput tools for the exploration of functional elements of the genome, the chicken has reached model organism status. Functional genomics focuses on understanding the function and regulation of genes and gene products on a global or genome-wide scale. Systems biology attempts to integrate functional information derived from multiple high-content data sets into a holistic view of all biological processes within a cell or organism. Generation of a large collection (∼600K) of chicken expressed sequence tags, representing most tissues and developmental stages, has enabled the construction of high-density microarrays for transcriptional profiling. Comprehensive analysis of this large expressed sequence tag collection and a set of ∼20K full-length cDNA sequences indicate that the transcriptome of the chicken represents approximately 20,000 genes. Furthermore, comparative analyses of these sequences have facilitated functional annotation of the genome and the creation of several bioinformatic resources for the chicken. Recently, about 20 papers have been published on transcriptional profiling with DNA microarrays in chicken tissues under various conditions. Proteomics is another powerful high-throughput tool currently used for examining the dynamics of protein expression in chicken tissues and fluids. Computational analyses of the chicken genome are providing new insight into the evolution of gene families in birds and other organisms. Abundant functional genomic resources now support large-scale analyses in the chicken and will facilitate identification of transcriptional mechanisms, gene networks, and metabolic or regulatory pathways that will ultimately determine the phenotype of the bird. New technologies such as marker-assisted selection, transgenics, and RNA interference offer the opportunity to modify the phenotype of the chicken to fit defined production goals. This review focuses on functional genomics in the chicken and provides a road map for large-scale exploration of the chicken genome. ©2007 Poultry Science Association Inc.
• Dail, M. B., Burgess, S. C., Meek, E. C., Wagner, J., Baravik, J., & Chambers, J. E. (2007). Spatial distribution of CYP2B1/2 messenger RNA within the rat liver acinus following exposure to the inducers phenobarbital and dieldrin. Toxicological Sciences, 99(1), 35-42.
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  PMID: 17517822;Abstract: Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16β-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment. © The Author 2007.
• H., B., Harris, T., McCarthy, F. M., Lamont, S. J., & Burgess, S. C. (2007). Non-electrophoretic differential detergent fractionation proteomics using frozen whole organs. Rapid Communications in Mass Spectrometry, 21(23), 3905-3909.
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  PMID: 17990261;Abstract: Non-electrophoretic methods based on two-dimensional liquid chromatography followed directly by tandem mass spectrometry (2D-LC/MS2) have become the preferred method for high-throughput expression proteomics and are widely applied to fresh tissues. Pre-fractionation techniques are also used in combination with 2D-LC/MS2 to both increase the proteome size and to assign cellular locations. Data from such experiments have become central to systems biology analyses. Here we apply a differential detergent (pre)fractionation (DDF) followed by 2D-LC/MS2 to frozen archival tissues. Our results show that by using frozen archival tissues, we do not lose proteome coverage or the ability to assign proteins to cellular compartments. In addition, we were able to assign 'biological process' Gene Ontology (GO) annotations, which will facilitate systems biological modeling of our proteomics data. Copyright © 2007 John Wiley & Sons, Ltd.
• McCarthy, F. M., Bridges, S. M., & Burgess, S. C. (2007). GOing from functional genomics to biological significance. Cytogenetic and Genome Research, 117(1-4), 278-287.
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  PMID: 17675869;Abstract: The chicken genome is sequenced and this, together with microarray and other functional genomics technologies, makes post-genomic research possible in the chicken. At this time, however, such research is hindered by a lack of genomic structural and functional annotations. Bio-ontologies have been developed for different annotation requirements, as well as to facilitate data sharing and computational analysis, but these are not yet optimally utilized in the chicken. Here we discuss genomic annotation and bio-ontologies. We focus specifically on the Gene Ontology (GO), chicken GO annotations and how these can facilitate functional genomics in the chicken. The GO is the most developed and widely used bio-ontology. It is the de facto standard for functional annotation. Despite its critical importance in analyzing microarray and other functional genomics data, relatively few chicken gene products have any GO annotation. When these are available, the average quality of chicken gene products annotations (defined using evidence code weight and annotation depth) is much less than in mouse. Moreover, tools allowing chicken researchers to easily and rapidly use the GO are either lacking or hard to use. To address all of these problems we developed ChickGO and AgBase. Chicken GO annotations are provided by complementary work at MSU-AgBase and EBI-GOA. The GO tools pipeline at AgBase uses GO to derive functional and biological significance from microarray and other functional genomics data. Not only will improved genomic annotation and tools to use these annotations benefit the chicken research community but they will also facilitate research in other avian species and comparative genomics. Copyright © 2007 S. Karger AG.
• McCarthy, F. M., Bridges, S. M., Wang, N., Magee, G. B., Williams, W. P., Luthe, D. S., & Burgess, S. C. (2007). AgBase: A unified resource for functional analysis in agriculture. Nucleic Acids Research, 35(SUPPL. 1), D599-D603.
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  PMID: 17135208;PMCID: PMC1751552;Abstract: Analysis of functional genomics (transcriptomics and proteomics) datasets is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation. To facilitate systems biology in these species we have established the curated, web-accessible, public resource 'AgBase' (www.agbase.msstate.edu). We have improved the structural annotation of agriculturally important genomes by experimentally confirming the in vivo expression of electronically predicted proteins and by proteogenomic mapping. Proteogenomic data are available from the AgBase proteogenomics link. We contribute Gene Ontology (GO) annotations and we provide a two tier system of GO annotations for users. The 'GO Consortium' gene association file contains the most rigorous GO annotations based solely on experimental data. The 'Community' gene association file contains GO annotations based on expert community knowledge (annotations based directly from author statements and submitted annotations from the community) and annotations for predicted proteins. We have developed two tools for proteomics analysis and these are freely available on request. A suite of tools for analyzing functional genomics datasets using the GO is available online at the AgBase site. We encourage and publicly acknowledge GO annotations from researchers and provide an online mechanism for agricultural researchers to submit requests for GO annotations. © 2007 Oxford University Press.
• Memili, E., Peddinti, D., Shack, L. A., Nanduri, B., McCarthy, F., Sagirkaya, H., & Burgess, S. C. (2007). Bovine germinal vesicle oocyte and cumulus cell proteomics. Reproduction, 133(6), 1107-1120.
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  PMID: 17636165;Abstract: Germinal vesicle (GV) breakdown is fundamental for maturation of fully grown, developmentally competent, mammalian oocytes. Bidirectional communication between oocytes and surrounding cumulus cells (CC) is essential for maturation of a competent oocyte. However, neither the factors involved in this communication nor the mechanisms of their actions are well defined. Here, we define the proteomes of GV oocytes and their surrounding CC, including membrane proteins, using proteomics in a bovine model. We found that 4395 proteins were expressed in the CC and 1092 proteins were expressed in oocytes. Further, 858 proteins were common to both the CC and the oocytes. This first comprehensive proteome analysis of bovine oocytes and CC not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level. Furthermore, some of these proteins may represent molecular biomarkers for developmental potential of oocytes. © 2007 Society for Reproduction and Fertility.
• Sanders, W. S., Bridges, S. M., McCarthy, F. M., Nanduri, B., & Burgess, S. C. (2007). Prediction of peptides observable by mass spectrometry applied at the experimental set level. BMC Bioinformatics, 8(SUPPL. 7).
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  PMID: 18047723;PMCID: PMC2099492;Abstract: Background: When proteins are subjected to proteolytic digestion and analyzed by mass spectrometry using a method such as 2D LC MS/MS, only a portion of the proteotypic peptides associated with each protein will be observed. The ability to predict which peptides can and cannot potentially be observed for a particular experimental dataset has several important applications in proteomics research including calculation of peptide coverage in terms of potentially detectable peptides, systems biology analysis of data sets, and protein quantification. Results: We have developed a methodology for constructing artificial neural networks that can be used to predict which peptides are potentially observable for a given set of experimental, instrumental, and analytical conditions for 2D LC MS/MS (a.k.a Multidimensional Protein Identification Technology [MudPIT]) datasets. Neural network classifiers constructed using this procedure for two MudPIT datasets exhibit 10-fold cross validation accuracy of about 80%. We show that a classifier constructed for one dataset has poor predictive performance with the other dataset, thus demonstrating the need for dataset specific classifiers. Classification results with each dataset are used to compute informative percent amino acid coverage statistics for each protein in terms of the predicted detectable peptides in addition to the percent coverage of the complete sequence. We also demonstrate the utility of predicted peptide observability for systems analysis to help determine if proteins that were expected but not observed generate sufficient peptides for detection. Conclusion: Classifiers that accurately predict the likelihood of detecting proteotypic peptides by mass spectrometry provide proteomics researchers with powerful new approaches for data analysis. We demonstrate that the procedure we have developed for building a classifier based on an individual experimental data set results in classifiers with accuracy comparable to those reported in the literature based on large training sets collected from multiple experiments. Our approach allows the researcher to construct a classifier that is specific for the experimental, instrument, and analytical conditions of a single experiment and amenable to local, condition-specific, implementation. The resulting classifiers have application in a number of areas such as determination of peptide coverage for protein identification, pathway analysis, and protein quantification. © 2007 Sanders et al; licensee BioMed Central Ltd.
• Betancourt, A. M., Burgess, S. C., & Carr, R. L. (2006). Effect of developmental exposure to chlorpyrifos on the expression of neurotrophin growth factors and cell-specific markers in neonatal rat brain. Toxicological Sciences, 92(2), 500-506.
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  PMID: 16675515;Abstract: Chlorpyrifos (CPS), a known neurotoxicant, is a widely used agricultural organophosphorus insecticide. The effects of postnatal exposure to CPS on the expression of mRNA for two factors critical to brain development, nerve growth factor (NGF) and reelin, were investigated in the forebrain of rats. In addition, the expression of mRNA for the muscarinic acetylcholine receptor (mAChR) M1 subtype and cell-specific markers for developing neurons (β-III tubulin), astrocytes (glial fibrillary acidic protein, GFAP), and oligodendrocytes (myelin-associated glycoprotein, MAG) was also investigated. Oral administration of CPS (1.5 or 3.0 mg/kg) or the corn oil vehicle was performed daily from postnatal days (PNDs) 1 through 6. No signs of overt toxicity or of cholinergic hyperstimulation were observed after CPS administration. Body weight was significantly different from controls on PND7 in both males and females exposed to 3.0 mg/kg CPS. Quantitative PCR was performed on the forebrain. The expression of NGF, reelin, and M1 mAChR mRNA was significantly reduced with both dosages of CPS in both sexes. β-III Tubulin mRNA expression remained unchanged after exposure, whereas MAG mRNA expression was significantly decreased with both dosages of CPS in both sexes, suggesting effects on the developing oligodendrocytes. In contrast, GFAP mRNA levels were significantly increased with both dosages of CPS in both sexes, suggesting increased astrocyte reactivity. Our findings indicate that dosages of CPS which cause significant cholinesterase inhibition but do not exert overt toxicity can adversely affect the expression levels of critical genes involved in brain development during the early postnatal period in the rat. © 2006 Oxford University Press.
• Corzo, A., Kidd, M. T., III, W. D., Shack, L. A., & Burgess, S. C. (2006). Protein expression of pectoralis major muscle in chickens in response to dietary methionine status. British Journal of Nutrition, 95(4), 703-708.
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  PMID: 16571149;Abstract: The present study evaluated the effect of dietary methionine on breast-meat accretion and protein expression in skeletal muscle of broiler chickens in vivo. All broilers received a common pre-test diet up to 21 d of age, and were subsequently fed either a methionine-deficient (MD) or -adequate (MA) diet (3.1 v. 4.5 g/kg diet) from age 21 to 42 d. Dietary cystine levels were 3.7 v. 3.6 g/kg diet for the MD and MA diet, respectively. Detrimental effects on carcass yield (P=0.004), abdominal fat percentage (P=0.001), and breast-meat weight (P=0.001), yield (P=0.001), and uniformity (P=0.002) were observed and validated in birds fed MD diets. Via tandem MS, a total of 190 individual proteins were identified from pectoralis major (PM) muscle tissue. From the former composite, peptides from three proteins were observed to be present exclusively in breast muscle from those chickens fed the MD diet (pyruvate kinase, myosin alkali light chain-1, ribosomal-protein-L-29). No proteins were observed to be uniquely expressed in chickens fed MA diets. Research is warranted to further explore the possibility of the proteins pyruate kinase, myosin alkali light chain-1, or ribosomal protein L-29, as potential biological indicators of differences in protein expression of PM of chickens in response to a dietary methionine deficiency. © The Authors 2006.
• Hamal, K. R., Burgess, S. C., Pevzner, I. Y., & Erf, G. F. (2006). Maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens. Poultry Science, 85(8), 1364-1372.
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  PMID: 16903465;Abstract: Maternal antibodies are transferred from hens to the chicks via the egg. To gain insight into maternal antibody transfer and endogenous production of antibodies in broiler chicks, total IgY, IgA, IgM, as well as anti-Newcastle disease virus (NDV) and anti-infectious bronchitis (IBV) antibody levels were examined in the dams' plasma, egg yolks, egg whites, and chicks' plasma on d 3, 7, 14, and 21. Blood was collected from 39-wk-old breeder hens (line 1, n = 17; line 2, n = 21). Fertile eggs were used for antibody extraction from the egg yolks and egg whites (4 to 5 eggs/dam) and for hatching. Unvaccinated chicks (4 to 5 chicks/dam) were reared in a HEPA-filtered room and were bled on d 3, 7, 14 and 21. Based on ELISA methods, plasma levels of IgY and IgM were higher (P < 0.0001), and those of IgA were similar (P = 0.31), in line 2 compared with line 1. Egg yolk IgY and IgA, as well as egg white IgY, IgA, and IgM levels were higher in line 2 compared with line 1 (P < 0.0001). Independent of line of chicken, the percentage dam-to-chick (3 d) plasma transfer of IgY was estimated to be approximately 30%, with that for IgM and IgA less than 1%. Chicks synthesized IgM first, followed by IgA and IgY. Anti-NDV and anti-IBV antibodies were detected in the dams' plasma, egg yolks, and in the chicks' plasma on d 3 and 7, with line 2 having higher anti-IBV and lower anti-NDV levels than line 1 in all samples (P < 0.0001). In summary, IgY levels, total or antigen-specific, in the dams' plasma or eggs were found to be a direct indicator of maternal antibody transfer to the chicks' circulation, with an expected percentage transfer of approximately 30%. This knowledge, together with the observed time course of endogenous antibody production in broiler chicks, may find direct application in formulating strategies for protecting chicks, especially during the first few weeks of age when their immune system is not yet fully functional. ©2006 Poultry Science Association Inc.
• McCarthy, F. M., Cooksey, A. M., Wang, N., Bridges, S. M., Pharr, G. T., & Burgess, S. C. (2006). Modeling a whole organ using proteomics: The avian bursa of Fabricius. Proteomics, 6(9), 2759-2771.
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  PMID: 16596704;Abstract: While advances in proteomics have improved proteome coverage and enhanced biological modeling, modeling function in multicellular organisms requires understanding how cells interact. Here we used the chicken bursa of Fabricius, a common experimental system for B cell function, to model organ function from proteomics data. The bursa has two major functional cell types: B cells and the supporting stromal cells. We used differential detergent fractionation-multi- dimensional protein identification technology (DDF-MudPIT) to identify 5198 proteins from all cellular compartments. Of these, 1753 were B cell specific, 1972 were stroma specific and 1473 were shared between the two. By modeling programmed cell death (PCD), cell differentiation and proliferation, and transcriptional activation, we have improved functional annotation of chicken proteins and placed chicken-specific death receptors into the PCD process using phylogenetics. We have identified 114 transcription factors (TFs); 42 of the bursal B cell TFs have not been reported before in any B cells. We have also improved the structural annotation of a newly sequenced genome by confirming the in vivo expression of 4006 "predicted", and 6623 ab initio, ORFs. Finally, we have developed a novel method for facilitating structural annotation, "expressed peptide sequence tags" (ePSTs) and demonstrate its utility by identifying 521 potential novel proteins from the chicken "unassigned chromosome". © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.
• McCarthy, F. M., Wang, N., Magee, G. B., Nanduri, B., Lawrence, M. L., Camon, E. B., Barrell, D. G., Hill, D. P., Dolan, M. E., Williams, W. P., Luthe, D. S., Bridges, S. M., & Burgess, S. C. (2006). AgBase: A functional genomics resource for agriculture. BMC Genomics, 7.
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  PMID: 16961921;PMCID: PMC1618847;Abstract: Background: Many agricultural species and their pathogens have sequenced genomes and more are in progress. Agricultural species provide food, fiber, xenotransplant tissues, biopharmaceuticals and biomedical models. Moreover, many agricultural microorganisms are human zoonoses. However, systems biology from functional genomics data is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation and agricultural research communities are smaller with limited funding compared to many model organism communities. Description: To facilitate systems biology in these traditionally agricultural species we have established "AgBase", a curated, web-accessible, public resource http://www.agbase.msstate.edu for structural and functional annotation of agricultural genomes. The AgBase database includes a suite of computational tools to use GO annotations. We use standardized nomenclature following the Human Genome Organization Gene Nomenclature guidelines and are currently functionally annotating chicken, cow and sheep gene products using the Gene Ontology (GO). The computational tools we have developed accept and batch process data derived from different public databases (with different accession codes), return all existing GO annotations, provide a list of products without GO annotation, identify potential orthologs, model functional genomics data using GO and assist proteomics analysis of ESTs and EST assemblies. Our journal database helps prevent redundant manual GO curation. We encourage and publicly acknowledge GO annotations from researchers and provide a service for researchers interested in GO and analysis of functional genomics data. Conclusion: The AgBase database is the first database dedicated to functional genomics and systems biology analysis for agriculturally important species and their pathogens. We use experimental data to improve structural annotation of genomes and to functionally characterize gene products. AgBase is also directly relevant for researchers in fields as diverse as agricultural production, cancer biology, biopharmaceuticals, human health and evolutionary biology. Moreover, the experimental methods and bioinformatics tools we provide are widely applicable to many other species including model organisms. © 2006 McCarthy et al; licensee BioMed Central Ltd.
• Nanduri, B., Lawrence, M. L., Boyle, C. E., Ramkumar, M., & Burgess, S. C. (2006). Effects of subminimum inhibitory concentrations of antibiotics on the Pasteurella multocida proteome. Journal of Proteome Research, 5(3), 572-580.
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  PMID: 16512672;Abstract: Subminimum inhibitory concentrations (sub-MICs) of antibiotics can be therapeutically effective, but the underlying molecular mechanisms are not well-characterized. We analyzed the Pasteurella multocida proteome response to sub-MICs of amoxicillin, chlortetracycline, and enrofloxacin using isotope-coded affinity tags (ICAT). There were parallel effects on inhibition of growth kinetics and suppression of protein expression by clusters of orthologous groups (COG) categories. Potential compensatory mechanisms enabling antibiotic adaptation were identified, including increased RecA expression caused by enrofloxacin. © 2006 American Chemical Society.
• Warnock, J. N., Burgess, S. C., Shack, A., & Yoganathan, A. P. (2006). Differential immediate-early gene responses to elevated pressure in porcine aortic valve interstitial cells. Journal of Heart Valve Disease, 15(1), 34-42.
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  PMID: 16480010;Abstract: Background and aim of the study: Cardiovascular risk factors are believed to play a role in the pathogenesis of aortic valve disease. In the present study the hypothesis was proposed that elevated pressure would cause a change in the expression of prototypical pro-inflammatory genes. Hence, the expression of MCP-1, osteopontin (OPN), VCAM-1, GM-CSF and PAI-1 was examined using semi-quantitative realtime RT-PCR. Methods: Porcine aortic valve interstitial cells at passage 1 were exposed to constant pressures of 100, 140, or 170 mmHg or cyclic pressures of 80-120, 120-160, or 150-190 mmHg for 2 h. Static cultures at atmospheric pressure served as controls. Total RNA from pooled experiments was isolated for analysis of gene expression. Single tube primer-mediated RT-PCR was performed directly on the RNA. Results: Cell s responded differently to constant and cyclic pressure. The most notable response was the expression of OPN, which was significantly up-regulated under steady conditions but down-regulated under cyclic conditions. The opposite was true in VCAM-1 expression, which was significantly downregulated at 170 mmHg static pressure, but up-regulated at 140 and 170 mmHg mean cyclic pressure. There was no clear proportional correlation between pressure magnitude and expression of MCP-1, GM-CSF, or PAI-1. However, elevated cyclic pressure caused a proportional increase in VCAM-1 expression and a proportional decrease in OPN expression. Conclusion: Elevated cyclic pressure is a potent sti mulus for the up-regulation of VCAM-1 expression and the down-regulation of OPN expression. This demonstrates an association between hypertension and aortic valve stenosis and calcification. The regulation of the chemotactic genes MCP-1 and GM-CSF is not correlated to a change in compressive forces. © Copyright by ICR Publishers 2006.
• Corzo, A., Kidd, M. T., Koter, M. D., & Burgess, S. C. (2005). Assessment of dietary amino acid scarcity on growth and blood plasma proteome status of broiler chickens. Poultry Science, 84(3), 419-425.
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  PMID: 15782910;Abstract: Dietary Lys needs for chicks were studied. A titration diet consisting of progressive amounts of dietary Lys from 0.95% up to 1.40% was fed to broiler chicks from 0 to 18 d of age. Optimal dietary Lys level was calculated using regression analysis. Body weight gain and feed conversion were maximized at Lys levels of 1.24% (1.10% digestible) and 1.27% (1.13% digestible) of diet, respectively. Blood samples were then collected from 2 groups: birds fed the lowest Lys level and birds fed dietary Lys nearest the determined requirement level (1.25% Lys). Plasma was analyzed for protein spectra via mass spectrometry and then classified by their functional characteristics. The number of proteins was similar between the 2 samples, but there was a tendency toward increased peptides for specific proteins in plasma from chicks fed adequate Lys levels. Furthermore, after these proteins were classified, more muscle-related proteins were found in plasma samples of birds fed Lys-adequate diets. It would appear that an individual dietary amino acid deficiency does not necessarily translate into decreasing protein synthesis proportionate to body weight, but rather significant changes may be occurring within the types of proteins undergoing anabolism. In conclusion, results herein illustrate the potential for using functional genomics in nutritionally related responses of poultry. ©2005 Poultry Science Association, Inc.
• McCarthy, F. M., Burgess, S. C., H., B., Koter, M. D., & Pharr, G. T. (2005). Differential detergent fractionation for non-electrophoretic eukaryote cell proteomics. Journal of Proteome Research, 4(2), 316-324.
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  PMID: 15822906;Abstract: Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS 2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important. © 2005 American Chemical Society.
• Nanduri, B., Lawrence, M. L., Vanguri, S., & Burgess, S. C. (2005). Proteomic analysis using an unfinished bacterial genome: The effects of subminimum inhibitory concentrations of antibiotics on Mannheimia haemolytica virulence factor expression. Proteomics, 5(18), 4852-4863.
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  PMID: 16247735;Abstract: Here we identify, using nonelectrophoretic proteomics, effects of subminimum inhibitory concentrations (subMIC) of two antibiotic preparations, chlortetracycline (CTC), and chlortetracycline-sulfamethazine (CTC + SMZ), on protein expression in the bovine respiratory pathogen Mannheimia haemolytica. The M. haemolytica genome is currently in draft form, and annotation is incomplete. Relying on the principle of gene sequence conservation across species, we used annotated genomes from closely related species to identify, confirm, and functionally annotate 495 M. haemolytica proteins. To conduct quantitative comparative proteomics, we developed a protein quantitation method based on the cross correlation function of the SEQUEST algorithm. When M. haemolytica was cultivated in the presence of 1/4 MIC of CTC and CTC + SMZ, expression of proteins involved in energy production, nucleotide metabolism, translation, and the bacterial stress response (chaperones) were affected. The most notable subMIC effect was a significant decrease in the expression of leukotoxin A, which is an important M. haemolytica virulence factor. Reduction in leukotoxin expression could be one of the molecular mechanisms responsible for the efficacy of these antibiotics against bovine respiratory disease. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.
• Scott, T. R., Messersmith, A. R., McCrary, W. J., Herlong, J. L., & Burgess, S. C. (2005). Hematopoietic prostaglandin D₂ synthase in the chicken Harderian gland. Veterinary Immunology and Immunopathology, 108(3-4), 295-306.
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  PMID: 16046238;Abstract: The Harderian gland (HG), a sero-mucous secreting organ in the eye orbit, has long been recognized as immunologically important in chickens. During experimentation to characterize immune components of the gland, proteomics analysis revealed the presence of hematopoietic prostaglandin D synthase (H-PGDS). Extraction of total RNA followed by RT-PCR produced cDNA of 597 base pairs. DNA sequencing revealed nucleic acid and predicted amino acid sequences that were 99% aligned with the one published sequence for chicken H-PGDS of the spleen. Alignment with murine, rat, and human H-PGDS were 69, 69, and 66%, respectively. Ocular vaccination of chickens with a Newcastle Disease/Infectious Bronchitis vaccine (Mass.-Ark. Strain) induced an increase in H-PGDS expression determined by real-time PCR. Furthermore, immunohistochemistry of frozen HG sections showed positive stained cells for both H-PGDS and mast cell tryptase in the sub-epithelial cell layers of the HG ducts. Based on the potent vasoactive role of PGD2, it appears that the chicken HG is a site of active mucosal immunity partially mediated by PGD2 synthesized by H-PGDS in the gland. © 2005 Elsevier B.V. All rights reserved.
• Weaver, C. C., Burgess, S. C., Nelson, P. D., Wilkinson, M., Ryan, P. L., Nail, C. A., Kelly-Quagliana, K., May, M. L., Reeves, R. K., Boyle, C. R., & Coats, K. S. (2005). Placental immunopathology and pregnancy failure in the FIV-infected cat. Placenta, 26(2-3), 138-147.
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  PMID: 15708115;Abstract: Placental HIV infections frequently result in infected babies or miscarriage. Aberrant placental cytokine expression during HIV infections may facilitate transplacental viral transmission or pregnancy perturbation. The feline immunodeficiency virus (FIV)-infected cat is a model for HIV infections due to similarities in biology and clinical disease. The purpose of this study was to evaluate placental immunomodulator expression and reproductive outcome using the FIV-infected cat model. Kittens were cesarean delivered from FIV-B-2542-infected and control queens near term; placental and fetal tissues were collected. Real-time RT-PCR was used to measure expression of representative placental Th1 cytokines, interleukin-1β (IL-1β) and interferon-γ (IFN-γ), a Th2 cytokine, IL-10, and chemokine receptor CXCR4. On average, control queens delivered 3.8 kittens/litter; 1 of 31 kittens (3.2%) was non-viable. FIV-infected queens produced 2.7 kittens/litter; 15 of 25 concepti (60%) were non-viable. FIV was detected in 14 of 15 placentas (93%) and 21 of 22 fetuses (95%) using PCR. Placental immunomodulator expression did not differ significantly when placentas from infected cats were compared to those of control cats. However, elevated expression of Th1 cytokines and increased Th1/Th2 ratios (IL-1β/IL-10) occurred in placentas from resorptions. Therefore, increased placental Th1 cytokine expression was associated with pregnancy failure in the FIV-infected cat. © 2004 Elsevier Ltd. All rights reserved.
• Burgess, S. C. (2004). Proteomics in the chicken: Tools for understanding immune responses to avian diseases. Poultry Science, 83(4), 552-573.
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  PMID: 15109053;Abstract: The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the ~"sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.
• Burgess, S. C., Young, J. R., Baaten, B. J., Hunt, L., Ross, L. N., Parcells, M. S., Kumar, P. M., Tregaskes, C. A., Lee, L. F., & Davison, T. F. (2004). Marek's disease is a natural model for lymphomas overexpressing Hodgkin's disease antigen (CD30). Proceedings of the National Academy of Sciences of the United States of America, 101(38), 13879-13884.
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  PMID: 15356338;PMCID: PMC518847;Abstract: Animal models are essential for elucidating the molecular mechanisms of carcinogenesis. Hodgkin's and many diverse non-Hodgkin's lymphomas overexpress the Hodgkin's disease antigen CD30 (CD30hi), a tumor necrosis factor receptor II family member. Here we show that chicken Marek's disease (MD) lymphoma cells are also CD30hi and are a unique natural model for CD30hi lymphoma. Chicken CD30 resembles an ancestral form, and we identify a previously undescribed potential cytoplasmic signaling domain conserved in chicken, human, and mouse CD30. Our phylogeneic analysis defines a relationship between the structures of human and mouse CD30 and confirms that mouse CD30 represents the ancestral mammalian gene structure. CD30 expression by MD virus (MDV)-transformed lymphocytes correlates with expression of the MDV Meq putative oncogene (a c-Jun homologue) in vivo. The chicken CD30 promoter has 15 predicted high-stringency Meq-binding transcription factor recognition motifs, and Meg enhances transcription from the CD30 promoter in vitro. Plasma proteomics identified a soluble form of CD30. CD30 overexpression is evolutionarily conserved and defines one class of neoplastic transformation events, regardless of etiology. We propose that CD30 is a component of a critical intracellular signaling pathway perturbed in neoplastic transformation. Specific anti-CD30 Igs occurred after infection of genetically MD-resistant chickens with oncogenic MDV, suggesting immunity to CD30 could play a role in MD lymphoma regression.
• Corzo, A., Kidd, M. T., Pharr, G. T., & Burgess, S. C. (2004). Initial mapping of the chicken blood plasma proteome. International Journal of Poultry Science, 3(3), 157-162.
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  Abstract: Proteomics is the study of the entire protein compliment of an organism. The blood plasma is the only tissue in which an organism's entire proteome may be potentially represented. First results toward mapping the broiler plasma proteome are presented here. Blood was taken from eight 18 day-old representative commercial broiler chickens. Plasma was isolated from each sample and pooled. For initial sample fractioning a 0.4 μl aliquot of the pooled plasma was run on one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on relative amounts of protein, the gel was divided into three fractions. The proteins were in-gel digested with trypsin. Two-dimensional liquid chromatography in-line with electrospray ionization tandem mass spectrometry was then used for "shot-gun" qualitative plasma proteomics. The resulting tandem mass spectra were then searched against the non-redundant chicken protein database. Generally accepted high stringency statistical criteria for protein identification were used. Eighty-four chicken proteins were identified. Our work demonstrates the future potential for plasma proteomics for identifying biomarkers of disease and production in chickens. © Asian Network for Scientific Information, 2004.
• Hale, L. L., Pharr, G. T., Burgess, S. C., Corzo, A., & Kidd, M. T. (2004). Isoleucine needs of thirty- to forty-day-old female chickens: Immunity. Poultry Science, 83(12), 1979-1985.
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  PMID: 15615010;Abstract: Broilers fed diets with reduced amino acid levels may be limiting in isoleucine. Because research addressing daily Ile needs for broiler immunity is sparse, Ile responses for immunity in female broilers were evaluated in 2 experiments in broilers from 30 to 42 d of age. Cellular and humoral immunity were evaluated in diets limiting in Ile and diets varying in Ile from deficient to adequate. Pen was the experimental unit in both experiments. Treatments in experiment 1 consisted of 2 levels of Ile (0.42 vs. 0.72% total of diet) and 3 strains of broilers, Arbor Acres+, Ross 508, Ross 708 (6 treatments; 5 pens each). In experiment 1, measurements consisted of: a cutaneous basophil hypersensitivity test to phytohemagglutinin-P (PHA-P) on d 37 and 38; cell quantification of CD4+, CD8+, and BU-1+ lymphocytes at d 41 and 42; and relative immune organ weights at 42 d. No Ile x strain interaction occurred. Feeding an Ile-deficient diet to broilers suppressed the cell mediated response to PHA-P, and reduced thymus weight and the percentage of CD8+ T cells. There were no significant differences between strains. In experiment 2, gradations of Ile (0.42, 0.50, 0.58, 0.66, 0.74, and 0.82% total of diet) were fed to one strain (Ross 508) of female broilers (7 pens per diet). A control diet containing 0.70% Ile (6 pens) was compared with an Ile surfeit concentration. Measurements in experiment 2 consisted of a hypersensitivity test to PHA-P on d 35 and 36; a primary antibody response to SRBC from 35 to 42 d; cell quantification of CD8+ α, β, and T cell receptor (TCR)-1 (δ/γ) lymphocytes on d 41 and 42; and immune organ weights at 42 d. Immunity measurements in birds fed surfeit Ile in the titration diets were equal to birds fed the control diet. A linear response to increasing Ile was obtained for relative bursa, but no Ile quadratic responses were noted for other measurements in experiment 2. Although feeding broilers a diet deficient in Ile suppressed some immune criteria, it does not appear that a marginal Ile deficiency will compromise immunity in growing female broilers.
• Kumar, A., Yueh, F., Singh, J. P., & Burgess, S. (2004). Characterization of malignant tissue cells by laser-induced breakdown spectroscopy. Applied Optics, 43(28), 5399-5403.
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  PMID: 15495432;Abstract: Cancer diagnosis and classification is extremely complicated and, for the most part, relies on subjective interpretation of biopsy material. Such methods are laborious and in some cases might result in different results depending on the histopathologist doing the examination. Automated, real-time diagnostic procedures would greatly facilitate cancer diagnosis and classification. Laser-induced breakdown spectroscopy (LIBS) is used for the first time to our knowledge to distinguish normal and malignant tumor cells from histological sections. We found that the concentration of trace elements in normal and tumor cells was significantly different. For comparison, the tissue samples were also analyzed by an inductively coupled plasma emission spectroscopy (ICPES) system. The results from the LIBS measurement and ICPES analysis were in good agreement. © 2004 Optical Society of America.
• Barrow, A. D., Burgess, S. C., Baigent, S. J., Howes, K., & Nair, V. K. (2003). Infection of macrophages by a lymphotropic herpesvirus: A new tropism for Marek's disease virus. Journal of General Virology, 84(10), 2635-2645.
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  PMID: 13679597;Abstract: Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B, αβT and γδT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophages in vivo and, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or 'hypervirulent' MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38+. It is demonstrated that macrophages are pp38+ because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4+, pp38+ and gB+, and ICP4 had nuclear localization denoting infection. Finally, MDV pp38+ macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.
• Barrow, A. D., Burgess, S. C., Howes, K., & Nair, V. K. (2003). Monocytosis is associated with the onset of leukocyte and viral infiltration of the brain in chickens infected with the very virulent Marek's disease virus strain C12/130. Avian Pathology, 32(2), 183-191.
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  PMID: 12745364;Abstract: Marek's disease (MDV) virus is mainly known for the induction of visceral lymphomas and lymphoid infiltration of peripheral nerves. Recently, additional tropism for the central nervous system has been recognised as a distinct feature of disease induced by very virulent MDV isolates. During the analysis of changes in the peripheral blood leukocyte subpopulations in chickens infected with either a virulent (HPRS-16)or a very virulent (C12/130) strain of MDV, we observed a marked monocytosis in chickens infected with C12/130. Perivascular cuffing in brain and mononuclear cell infiltration into the meninges-of chickens infected with C12/130 were associated with the appearance of the monocytosis from 6-10 days post-infection. Our results show that a peripheral blood monocytosis may be a contributory factor in establishing or accelerating the severity of mononuclear infiltration into the meninges and perivascular spaces in the brain during infection by very virulent C12/130 strain of MDV.
• Levy, A. M., Burgess, S. C., Davidson, I., Underwood, G., Leitner, G., & Heller, E. D. (2003). Interferon-Containing Supernatants Increase Marek's Disease Herpesvirus Genomes and Gene Transcription Levels, but Not Virion Replication in Vitro. Viral Immunology, 16(4), 501-509.
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  PMID: 14733737;Abstract: Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Marek's disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.
• Levy, A. M., Davidson, I., Burgess, S. C., & Heller, E. D. (2003). Major histocompatibility complex class I is downregulated in Marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon. Comparative Immunology, Microbiology and Infectious Diseases, 26(3), 189-198.
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  PMID: 12581748;Abstract: The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor-ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Marek's disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect. © 2002 Elsevier Science Ltd. All rights reserved.
• Burgess, S. C., & Davison, T. F. (2002). Identification of the neoplastically transformed cells in Marek's disease herpesvirus-induced lymphomas: recognition by the monoclonal antibody AV37.. Journal of Virology, 76(14), 7276-7292.
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  PMID: 12072527;PMCID: PMC136297;Abstract: Understanding the interactions between herpesviruses and their host cells and also the interactions between neoplastically transformed cells and the host immune system is fundamental to understanding the mechanisms of herpesvirus oncology. However, this has been difficult as no animal models of herpesvirus-induced oncogenesis in the natural host exist in which neoplastically transformed cells are also definitively identified and may be studied in vivo. Marek's disease (MD) herpesvirus (MDV) of poultry, although a recognized natural oncogenic virus causing T-cell lymphomas, is no exception. In this work, we identify for the first time the neoplastically transformed cells in MD as the CD4(+) major histocompatibility complex (MHC) class I(hi), MHC class II(hi), interleukin-2 receptor alpha-chain-positive, CD28(lo/-), phosphoprotein 38-negative (pp38(-)), glycoprotein B-negative (gB(-)), alphabeta T-cell-receptor-positive (TCR(+)) cells which uniquely overexpress a novel host-encoded extracellular antigen that is also expressed by MDV-transformed cell lines and recognized by the monoclonal antibody (MAb) AV37. Normal uninfected leukocytes and MD lymphoma cells were isolated directly ex vivo and examined by flow cytometry with MAb recognizing AV37, known leukocyte antigens, and MDV antigens pp38 and gB. CD28 mRNA was examined by PCR. Cell cycle distribution and in vitro survival were compared for each lymphoma cell population. We demonstrate for the first time that the antigen recognized by AV37 is expressed at very low levels by small minorities of uninfected leukocytes, whereas particular MD lymphoma cells uniquely express extremely high levels of the AV37 antigen; the AV37(hi) MD lymphoma cells fulfill the accepted criteria for neoplastic transformation in vivo (protection from cell death despite hyperproliferation, presence in all MD lymphomas, and not supportive of MDV production); the lymphoma environment is essential for AV37(+) MD lymphoma cell survival; pp38 is an antigen expressed during MDV-productive infection and is not expressed by neoplastically transformed cells in vivo; AV37(+) MD lymphoma cells have the putative immune evasion mechanism of CD28 down-regulation; AV37(hi) peripheral blood leukocytes appear early after MDV infection in both MD-resistant and -susceptible chickens; and analysis of TCR variable beta chain gene family expression suggests that MD lymphomas have polyclonal origins. Identification of the neoplastically transformed cells in MD facilitates a detailed understanding of MD pathogenesis and also improves the utility of MD as a general model for herpesvirus oncology.