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Search Results: 1 - 10 of 207092 matches for " Scott N Peterson "
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The complexity of simplicity
Scott N Peterson, Claire M Fraser
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-2-comment2002
Abstract: Attempts to define life have touched nearly every scientific discipline and have been a long-standing subject in philosophy. Even when limited to the realm of biology, the definitions of life fall short of universal agreement and remain a widely debated subject. The practice of reductionism, whereby life is considered solely from the perspective of the genetic material, also generates controversy and complications. Many of these complications have been appreciated recently as a result of the large-scale application of whole-genome sequencing to microbes. The specific issue we discuss here is an attempt to define the minimum number of genes or functions necessary to support cellular life. The appeal of this subject lies largely in the fact that it represents a fundamental question in biology.We have approached the concept of a 'minimal genome' in a manner not unlike that taken by physicists interested in understanding the nature of the universe: we have relied on an underlying assumption or guiding force that states that a simple set of rules must exist. The difficulty lies in finding the proper way to strip away the outer layers of complexity in order to uncover what is truly general and therefore satisfactorily describes all things. It is our belief that some set of basic parameters and principles are common to all cellular life on this planet. On the surface, especially in the age of genomics, it would appear that we will indeed be able to draw inferences and define commonalities. The advent of whole-genome shotgun sequencing [1] has changed biology in ways that we will not fully appreciate for many years to come. Advances in functional genomics, structural genomics and computational biology have also contributed greatly to our ability to make 'sense' of the huge quantities of DNA sequence data being generated. As we begin to apply these methodologies to the question of the minimal genome, we are faced with new perspectives which emphasize that, like any good scie
High quality protein microarray using in situ protein purification
Keehwan Kwon, Carissa Grose, Rembert Pieper, Gagan A Pandya, Robert D Fleischmann, Scott N Peterson
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-72
Abstract: Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG) coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST) composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents protein solubility and denaturation problems caused by buffer exchange steps and freeze-thaw cycles, which are associated with resin-based purification, intermittent protein storage and deposition on microarrays.An optimized platform for in situ protein purification on microarray slides using His-tagged recombinant proteins is a desirable tool for the screening of novel protein functions and protein-protein interactions. In the context of immunoproteomics, such protein microarrays are complimentary to approaches using non-recombinant methods to discover and characterize bacterial antigens.Sharp increases in the efficiency of DNA sequencing technology, coupled with dramatic decreases in cost have led to very large quantities of DNA sequences to be deposited into public databases. The number of complete genomes continues to grow. More recently microbial DNA sequences were derived from the environment and those living in the context of the huma
Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis
Keehwan Kwon, Jeremy Hasseman, Saeeda Latham, Carissa Grose, Yu Do, Robert D Fleischmann, Rembert Pieper, Scott N Peterson
BMC Biochemistry , 2011, DOI: 10.1186/1471-2091-12-17
Abstract: In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.Proteases represent one of the largest protein families, and play critical roles in cellular functions and viability in all organisms [1,2]. Proteases have diverse biological roles in signal transduction, post-translational modification, proliferation, apoptosis and pathogenicity through specific processing or non-specific degradation [3-6]. Proteases can be classified into two categories, secreted proteases and intracellular proteases [7]. Secreted proteases such as trypsin, usually cleave at specific short recognition sites of proteins or peptides. In contrast, substrates of intracellular proteases are much more specific, preventing uncon
Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping
Kristine L Bucasas, Gagan A Pandya, Sonal Pradhan, Robert D Fleischmann, Scott N Peterson, John W Belmont
BMC Genetics , 2009, DOI: 10.1186/1471-2156-10-85
Abstract: Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold.We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.Array technologies are designed to rapidly genotype hundreds of thousands of single nucleotide polymorphisms (SNPs) across the genome using a relatively small amount of DNA. Advances in genotyping platforms have allowed cost-effective, whole-genome scans of multiple individuals in large-scale association studies. These studies are aimed at identifying genetic factors affecting many important complex human diseases. DNA samples from carefully characterized populations that are necessary to carry out adequately powered genome-wide association studies (GWAS), however, are often limiting. Collecting a sufficient number of appropriate samples for GWAS can be a complex and expensive collaborative challenge. There are potential alternative sources of genomic DNA for which important medical phenotypes have been recorded but their utility for GWAS has
The Dental Plaque Microbiome in Health and Disease
Scott N. Peterson, Erik Snesrud, Jia Liu, Ana C. Ong, Mogens Kilian, Nicholas J. Schork, Walter Bretz
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058487
Abstract: Dental decay is one of the most prevalent chronic diseases worldwide. A variety of factors, including microbial, genetic, immunological, behavioral and environmental, interact to contribute to dental caries onset and development. Previous studies focused on the microbial basis for dental caries have identified species associated with both dental health and disease. The purpose of the current study was to improve our knowledge of the microbial species involved in dental caries and health by performing a comprehensive 16S rDNA profiling of the dental plaque microbiome of both caries-free and caries-active subjects. Analysis of over 50,000 nearly full-length 16S rDNA clones allowed the identification of 1,372 operational taxonomic units (OTUs) in the dental plaque microbiome. Approximately half of the OTUs were common to both caries-free and caries-active microbiomes and present at similar abundance. The majority of differences in OTU’s reflected very low abundance phylotypes. This survey allowed us to define the population structure of the dental plaque microbiome and to identify the microbial signatures associated with dental health and disease. The deep profiling of dental plaque allowed the identification of 87 phylotypes that are over-represented in either caries-free or caries-active subjects. Among these signatures, those associated with dental health outnumbered those associated with dental caries by nearly two-fold. A comparison of this data to other published studies indicate significant heterogeneity in study outcomes and suggest that novel approaches may be required to further define the signatures of dental caries onset and progression.
Monitoring the Long-Term Molecular Epidemiology of the Pneumococcus and Detection of Potential ‘Vaccine Escape’ Strains
Gagan A. Pandya,M. Catherine McEllistrem,Pratap Venepally,Michael H. Holmes,Behnam Jarrahi,Ravi Sanka,Jia Liu,Svetlana A. Karamycheva,Yun Bai,Robert D. Fleischmann,Scott N. Peterson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015950
Abstract: While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in ‘vaccine escape’ strains.
Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation
Rembert Pieper, Shih-Ting Huang, Prashanth P Parmar, David J Clark, Hamid Alami, Robert D Fleischmann, Robert D Perry, Scott N Peterson
BMC Microbiology , 2010, DOI: 10.1186/1471-2180-10-30
Abstract: Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.Many protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26°C and 37°C), there was little evidence for temperature-specific adaptation processes to iron starvation.Yersinia pestis, a Gram-negative bacterium, is the causative agent of the bubonic and pneumonic plague. The pathogenic lifestyle of this microbe involves two distinct life stages, one in the flea vector, the other in mammalian hosts, primarily rodents [1]. Genome sequencing and analyses have been completed for four major Y. pestis biovars, including the chromosome [2] and three virulence/transmission-associated plasmids [3,4] of the KIM strain, which belongs to the biovar mediaevalis. In addition to plasmid-encoded virulence
In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism
Srilatha Kuntumalla, Quanshun Zhang, John C Braisted, Robert D Fleischmann, Scott N Peterson, Arthur Donohue-Rolfe, Saul Tzipori, Rembert Pieper
BMC Microbiology , 2011, DOI: 10.1186/1471-2180-11-147
Abstract: Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo.Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and Ip
Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome
Moo-Jin Suh, Natalie D. Fedorova, Steven E. Cagas, Susan Hastings, Robert D. Fleischmann, Scott N. Peterson, David S. Perlin, William C. Nierman, Rembert Pieper, Michelle Momany
Proteome Science , 2012, DOI: 10.1186/1477-5956-10-30
Abstract: To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this study (M.W. 7,367). Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for housekeeping functions, particularly translation, respiratory metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle.The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play key roles in host-pathogen interactions, signal transduction during conidial germination, or survival in hostile e
A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection
Brooke L. Deatherage Kaiser, Jie Li, James A. Sanford, Young-Mo Kim, Scott R. Kronewitter, Marcus B. Jones, Christine T. Peterson, Scott N. Peterson, Bryan C. Frank, Samuel O. Purvine, Joseph N. Brown, Thomas O. Metz, Richard D. Smith, Fred Heffron, Joshua N. Adkins
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0067155
Abstract: The potential for commensal microorganisms indigenous to a host (the ‘microbiome’ or ‘microbiota’) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics “systems” approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimurium’s lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.
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