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1.  A Single Whole-Body Low Dose X-Irradiation Does Not Affect L1, B1 and IAP Repeat Element DNA Methylation Longitudinally 
PLoS ONE  2014;9(3):e93016.
The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17–19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen) and a tissue fundamental to the aging process (liver). Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA methylation.
doi:10.1371/journal.pone.0093016
PMCID: PMC3968115  PMID: 24676381
2.  Genome Sequence of the Oleaginous Yeast Rhodotorula glutinis ATCC 204091 
Genome Announcements  2014;2(1):e00046-14.
Rhodotorula glutinis ATCC 204091 is an oleaginous oxidative red yeast that can accumulate lipids to >50% of its biomass when grown with appropriate carbon and nitrogen ratios. It produces a red pigment consisting of useful antioxidants, such as carotenoids, torulene, and torularhodin, when cultivated under carbon-deficient conditions.
doi:10.1128/genomeA.00046-14
PMCID: PMC3924368  PMID: 24526636
3.  Complete Genome Sequence of Channel Catfish Gastrointestinal Septicemia Isolate Edwardsiella tarda C07-087 
Genome Announcements  2013;1(6):e00959-13.
Edwardsiella tarda is a Gram-negative facultative anaerobe causing disease in animals and humans. Here, we announce the complete genome sequence of the channel catfish isolate E. tarda strain C07-87, which was isolated from an outbreak of gastrointestinal septicemia on a commercial catfish farm.
doi:10.1128/genomeA.00959-13
PMCID: PMC3837174  PMID: 24265493
4.  Complete Genome Sequence of a Channel Catfish Epidemic Isolate, Aeromonas hydrophila Strain ML09-119 
Genome Announcements  2013;1(5):e00755-13.
Aeromonas hydrophila is a Gram-negative, rod-shaped, mesophilic bacterium that infects both aquatic poikilothermic animals and mammals, including humans. Here, we present the complete genome sequence of Aeromonas hydrophila strain ML09-119, which represents a clonal group of A. hydrophila isolates causing outbreaks of bacterial septicemia in channel catfish since 2009.
doi:10.1128/genomeA.00755-13
PMCID: PMC3778208  PMID: 24051325
5.  Tricarboxylic Acid Cycle and One-Carbon Metabolism Pathways Are Important in Edwardsiella ictaluri Virulence 
PLoS ONE  2013;8(6):e65973.
Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.
doi:10.1371/journal.pone.0065973
PMCID: PMC3676347  PMID: 23762452
6.  An analysis of potential barriers and enablers to regulating the television marketing of unhealthy foods to children at the state government level in Australia 
BMC Public Health  2012;12:1123.
Background
In Australia there have been many calls for government action to halt the effects of unhealthy food marketing on children's health, yet implementation has not occurred. The attitudes of those involved in the policy-making process towards regulatory intervention governing unhealthy food marketing are not well understood. The objective of this research was to understand the perceptions of senior representatives from Australian state and territory governments, statutory authorities and non-government organisations regarding the feasibility of state-level government regulation of television marketing of unhealthy food to children in Australia.
Method
Data from in-depth semi-structured interviews with senior representatives from state and territory government departments, statutory authorities and non-government organisations (n=22) were analysed to determine participants' views about regulation of television marketing of unhealthy food to children at the state government level. Data were analysed using content and thematic analyses.
Results
Regulation of television marketing of unhealthy food to children was supported as a strategy for obesity prevention. Barriers to implementing regulation at the state level were: the perception that regulation of television advertising is a Commonwealth, not state/territory, responsibility; the power of the food industry and; the need for clear evidence that demonstrates the effectiveness of regulation. Evidence of community support for regulation was also cited as an important factor in determining feasibility.
Conclusions
The regulation of unhealthy food marketing to children is perceived to be a feasible strategy for obesity prevention however barriers to implementation at the state level exist. Those involved in state-level policy making generally indicated a preference for Commonwealth-led regulation. This research suggests that implementation of regulation of the television marketing of unhealthy food to children should ideally occur under the direction of the Commonwealth government. However, given that regulation is technically feasible at the state level, in the absence of Commonwealth action, states/territories could act independently. The relevance of our findings is likely to extend beyond Australia as unhealthy food marketing to children is a global issue.
doi:10.1186/1471-2458-12-1123
PMCID: PMC3572413  PMID: 23272940
Unhealthy food; Regulation; Government; Children; Marketing; Advertising
7.  Genome Sequence of the Fish Pathogen Flavobacterium columnare ATCC 49512 
Journal of Bacteriology  2012;194(10):2763-2764.
Flavobacterium columnare is a Gram-negative, rod-shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512.
doi:10.1128/JB.00281-12
PMCID: PMC3347163  PMID: 22535941
8.  Regulation to Create Environments Conducive to Physical Activity: Understanding the Barriers and Facilitators at the Australian State Government Level 
PLoS ONE  2012;7(9):e42831.
Introduction
Policy and regulatory interventions aimed at creating environments more conducive to physical activity (PA) are an important component of strategies to improve population levels of PA. However, many potentially effective policies are not being broadly implemented. This study sought to identify potential policy/regulatory interventions targeting PA environments, and barriers/facilitators to their implementation at the Australian state/territory government level.
Methods
In-depth interviews were conducted with senior representatives from state/territory governments, statutory authorities and non-government organisations (n = 40) to examine participants': 1) suggestions for regulatory interventions to create environments more conducive to PA; 2) support for preselected regulatory interventions derived from a literature review. Thematic and constant comparative analyses were conducted.
Results
Policy interventions most commonly suggested by participants fell into two areas: 1) urban planning and provision of infrastructure to promote active travel; 2) discouraging the use of private motorised vehicles. Of the eleven preselected interventions presented to participants, interventions relating to walkability/cycling and PA facilities received greatest support. Interventions involving subsidisation (of public transport, PA-equipment) and the provision of more public transport infrastructure received least support. These were perceived as not economically viable or unlikely to increase PA levels. Dominant barriers were: the powerful ‘road lobby’, weaknesses in the planning system and the cost of potential interventions. Facilitators were: the provision of evidence, collaboration across sectors, and synergies with climate change/environment agendas.
Conclusion
This study points to how difficult it will be to achieve policy change when there is a powerful ‘road lobby’ and government investment prioritises road infrastructure over PA-promoting infrastructure. It highlights the pivotal role of the planning and transport sectors in implementing PA-promoting policy, however suggests the need for clearer guidelines and responsibilities for state and local government levels in these areas. Health outcomes need to be given more direct consideration and greater priority within non-health sectors.
doi:10.1371/journal.pone.0042831
PMCID: PMC3459936  PMID: 23028434
9.  Transcriptome profile of a bovine respiratory disease pathogen: Mannheimia haemolytica PHL213 
BMC Bioinformatics  2012;13(Suppl 15):S4.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2105-13-S15-S4
PMCID: PMC3439734  PMID: 23046475
10.  RNA-Seq Based Transcriptional Map of Bovine Respiratory Disease Pathogen “Histophilus somni 2336” 
PLoS ONE  2012;7(1):e29435.
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.
doi:10.1371/journal.pone.0029435
PMCID: PMC3262788  PMID: 22276113
11.  Genome Sequence of Lineage III Listeria monocytogenes Strain HCC23 ▿ 
Journal of Bacteriology  2011;193(14):3679-3680.
More than 98% of reported human listeriosis cases are caused by Listeria monocytogenes serotypes within lineages I and II. Serotypes within lineage III (4a and 4c) are commonly isolated from environmental and food specimens. We report the first complete genome sequence of a lineage III isolate, HCC23, which will be used for comparative analysis.
doi:10.1128/JB.05236-11
PMCID: PMC3133316  PMID: 21602330
13.  Migratory Pathways and Connectivity in Asian Houbara Bustards: Evidence from 15 Years of Satellite Tracking 
PLoS ONE  2011;6(6):e20570.
Information on migratory pathways and connectivity is essential to understanding population dynamics and structure of migrant species. Our manuscript uses a unique dataset, the fruit of 103 individual Asian houbara bustards captured on their breeding grounds in Central Asia over 15 years and equipped with satellite transmitters, to provide a better understanding of migratory pathways and connectivity; such information is critical to the implementation of biologically sound conservation measures in migrant species. At the scale of the distribution range we find substantial migratory connectivity, with a clear separation of migration pathways and wintering areas between western and eastern migrants. Within eastern migrants, we also describe a pattern of segregation on the wintering grounds. But at the local level connectivity is weak: birds breeding within the limits of our study areas were often found several hundreds of kilometres apart during winter. Although houbara wintering in Arabia are known to originate from Central Asia, out of all the birds captured and tracked here not one wintered on the Arabian Peninsula. This is very likely the result of decades of unregulated off-take and severe habitat degradation in this area. At a time when conservation measures are being implemented to safeguard the long-term future of this species, this study provides critical data on the spatial structuring of populations.
doi:10.1371/journal.pone.0020570
PMCID: PMC3110797  PMID: 21687684
14.  The Proteogenomic Mapping Tool 
BMC Bioinformatics  2011;12:115.
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.
doi:10.1186/1471-2105-12-115
PMCID: PMC3107813  PMID: 21513508
15.  Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7T▿  
Journal of Bacteriology  2010;192(20):5554-5555.
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.
doi:10.1128/JB.00877-10
PMCID: PMC2950491  PMID: 20729368
16.  Proteome and Membrane Fatty Acid Analyses on Oligotropha carboxidovorans OM5 Grown under Chemolithoautotrophic and Heterotrophic Conditions 
PLoS ONE  2011;6(2):e17111.
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.
doi:10.1371/journal.pone.0017111
PMCID: PMC3046131  PMID: 21386900
17.  Complete genome and comparative analysis of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5 
BMC Genomics  2010;11:511.
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.
doi:10.1186/1471-2164-11-511
PMCID: PMC3091675  PMID: 20863402
18.  Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon 
BMC Microbiology  2010;10:197.
Background
Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum.
Results
We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies.
Conclusions
This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.
doi:10.1186/1471-2180-10-197
PMCID: PMC2918591  PMID: 20653968
19.  Proteomic analysis of the fish pathogen Flavobacterium columnare 
Proteome Science  2010;8:26.
Background
Flavobacterium columnare causes columnaris disease in cultured and wild fish populations worldwide. Columnaris is the second most prevalent bacterial disease of commercial channel catfish industry in the United States. Despite its economic importance, little is known about the expressed proteins and virulence mechanisms of F. columnare. Here, we report the first high throughput proteomic analysis of F. columnare using 2-D LC ESI MS/MS and 2-DE MALDI TOF/TOF MS.
Results
Proteins identified in this study and predicted from the draft F. columnare genome were clustered into functional groups using clusters of orthologous groups (COGs), and their subcellular locations were predicted. Possible functional relations among the identified proteins were determined using pathway analysis. The total number of unique F. columnare proteins identified using both 2-D LC and 2-DE approaches was 621, of which 10.95% (68) were identified by both methods, while 77.29% (480) and 11.76% (73) were unique in 2-D LC and 2-DE, respectively. COG groupings and subcellular localizations were similar between our data set and proteins predicted from the whole genome. Twenty eight pathways were significantly represented in our dataset (P < 0.05).
Conclusion
Results from this study provide experimental evidence for many proteins that were predicted from the F. columnare genome annotation, and they should accelerate functional and comparative studies aimed at understanding virulence mechanisms of this important pathogen.
doi:10.1186/1477-5956-8-26
PMCID: PMC2890538  PMID: 20525376
20.  Identification of novel non-coding small RNAs from Streptococcus pneumoniae TIGR4 using high-resolution genome tiling arrays 
BMC Genomics  2010;11:350.
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.
doi:10.1186/1471-2164-11-350
PMCID: PMC2887815  PMID: 20525227
21.  High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes▿  
A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogen Edwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to the E. ictaluri genome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.
doi:10.1128/AEM.02449-08
PMCID: PMC2663204  PMID: 19201969
22.  Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells 
The FASEB Journal  2009;23(9):3179-3192.
Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.
doi:10.1096/fj.08-123596
PMCID: PMC2735356  PMID: 19447881
determination; differentiation; melanoma; chondrocyte; Schwann cell
23.  Moving beyond 'rates, roads and rubbish': How do local governments make choices about healthy public policy to prevent obesity? 
While the causes of obesity are well known traditional education and treatment strategies do not appear to be making an impact. One solution as part of a broader complimentary set of strategies may be regulatory intervention at local government level to create environments for healthy nutrition and increased physical activity. Semi structured interviews were conducted with representatives of local government in Australia. Factors most likely to facilitate policy change were those supported by external funding, developed from an evidence base and sensitive to community and market forces. Barriers to change included a perceived or real lack of power to make change and the complexity of the legislative framework. The development of a systematic evidence base to provide clear feedback on the size and scope of the obesity epidemic at a local level, coupled with cost benefit analysis for any potential regulatory intervention, are crucial to developing a regulatory environment which creates the physical and social environment required to prevent obesity.
doi:10.1186/1743-8462-6-20
PMCID: PMC2736971  PMID: 19698170
24.  The transcriptional response of Pasteurella multocida to three classes of antibiotics 
BMC Genomics  2009;10(Suppl 2):S4.
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.
doi:10.1186/1471-2164-10-S2-S4
PMCID: PMC2966327  PMID: 19607655
25.  Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD▿ †  
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.
doi:10.1128/AEM.01847-08
PMCID: PMC2620715  PMID: 19028911

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