454-Pyrosequencing: A Molecular Battiscope for Freshwater Viral Ecology

Viruses, the most abundant biological entities on the planet, are capable of infecting organisms from all three branches of life, although the majority infect bacteria where the greatest degree of cellular diversity lies. However, the characterization and assessment of viral diversity in natural environments is only beginning to become a possibility. Through the development of a novel technique for the harvest of viral DNA and the application of 454 pyrosequencing, a snapshot of the diversity of the DNA viruses harvested from a standing pond on a cattle farm has been obtained. A high abundance of viral genotypes (785) were present within the virome. The absolute numbers of lambdoid and Shiga toxin (Stx) encoding phages detected suggested that the depth of sequencing had enabled recovery of only ca. 8% of the total virus population, numbers that agreed within less than an order of magnitude with predictions made by rarefaction analysis. The most abundant viral genotypes in the pond were bacteriophages (93.7%). The predominant viral genotypes infecting higher life forms found in association with the farm were pathogens that cause disease in cattle and humans, e.g. members of the Herpesviridae. The techniques and analysis described here provide a fresh approach to the monitoring of viral populations in the aquatic environment, with the potential to become integral to the development of risk analysis tools for monitoring the dissemination of viral agents of animal, plant and human diseases

Tracing the fate of wastewater viruses reveals catchment-scale virome diversity and connectivity

The discharge of wastewater-derived viruses in aquatic environments impacts catchment-scale virome composition and is a potential hazard to human health. Here, we used viromic analysis of RNA and DNA virus-like particle preparations to track virus communities entering and leaving wastewater treatment plants and the connecting river catchment system and estuary. We found substantial viral diversity and geographically distinct virus communities associated with different wastewater treatment plants. River and estuarine water bodies harboured more diverse viral communities in downstream locations, influenced by tidal movement and proximity to wastewater treatment plants. Shellfish and beach sand were enriched in viral communities when compared with the surrounding water, acting as entrapment matrices for virus particles. We reconstructed >40,000 partial viral genomes into 10,149 species-level groups, dominated by dsDNA and (+)ssRNA bacteriophages (Caudovirales and Leviviridae). We identified 73 (partial) genomes comprising six families that could pose a risk to human health; Astroviridae, Caliciviridae (sapovirus), Picornaviridae (cocksackievirus), Reoviridae (rotavirus), Parvoviridae and Circoviridae. Based on the pattern of viral incidence, we observe that wastewater-derived viral genetic material is commonly deposited in the environment, but due to fragemented nature of these viral genomes, the risk to human health is low, and is more likely driven by community transmission, with wastewater-derived viruses subject to cycles of dilution, enrichment and virion degradation influenced by local geography, weather events and tidal effects. Our data illustrate the utility of viromic analyses for wastewater- and environment-based epidemiology, and we present a conceptual model for the circulation of viruses in a freshwater catchment

Viromic Analysis of Wastewater Input to a River Catchment Reveals a Diverse Assemblage of RNA Viruses

Enteric viruses cause gastrointestinal illness and are commonly transmitted through the fecal-oral route. When wastewater is released into river systems, these viruses can contaminate the environment. Our results show that we can use viromics to find the range of potentially pathogenic viruses that are present in the environment and identify prevalent genotypes. The ultimate goal is to trace the fate of these pathogenic viruses from origin to the point where they are a threat to human health, informing reference-based detection methods and water quality management.Detection of viruses in the environment is heavily dependent on PCR-based approaches that require reference sequences for primer design. While this strategy can accurately detect known viruses, it will not find novel genotypes or emerging and invasive viral species. In this study, we investigated the use of viromics, i.e., high-throughput sequencing of the biosphere’s viral fraction, to detect human-/animal-pathogenic RNA viruses in the Conwy river catchment area in Wales, United Kingdom. Using a combination of filtering and nuclease treatment, we extracted the viral fraction from wastewater and estuarine river water and sediment, followed by high-throughput RNA sequencing (RNA-Seq) analysis on the Illumina HiSeq platform, for the discovery of RNA virus genomes. We found a higher richness of RNA viruses in wastewater samples than in river water and sediment, and we assembled a complete norovirus genotype GI.2 genome from wastewater effluent, which was not contemporaneously detected by conventional reverse transcription-quantitative PCR (qRT-PCR). The simultaneous presence of diverse rotavirus signatures in wastewater indicated the potential for zoonotic infections in the area and suggested runoff from pig farms as a possible origin of these viruses. Our results show that viromics can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided appropriate and rigorous controls are included

Transcriptomic Analysis of Shiga-Toxigenic Bacteriophage Carriage Reveals a Profound Regulatory Effect on Acid Resistance in Escherichia coli

Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli

Integrating Tobacco Control and Obesity Prevention Initiatives at Retail Outlets

Tobacco products are sold in approximately 375,000 US retail outlets, including convenience stores and pharmacies, which often sell energy-dense, low-nutrient foods and beverages. The Food and Drug Administration's (FDA's) increased authority over tobacco product sales and marketing, combined with declining smoking rates, provides an opportunity to transition tobacco retailers toward healthier retail environments. Unfortunately, research into improving consumer retail environments is often conducted in isolation by researchers working in tobacco control, nutrition, and physical activity. Interdisciplinary efforts are needed to transform tobacco retailers from stores that are dependent on a declining product category, to the sale and promotion of healthful foods and creating environments conducive to active living. The objective of this article is to describe the potential for interdisciplinary efforts to transition retailers away from selling and promoting tobacco products and toward creating retail environments that promote healthful eating and active living

Retailer Adherence to Family Smoking Prevention and Tobacco Control Act, North Carolina, 2011

IntroductionThe Family Smoking Prevention and Tobacco Control Act regulates the sales and marketing of tobacco products in the United States; poor adherence by tobacco retailers may reduce the effectiveness of the Act’s provisions. The objectives of this study were 1) to assess whether and to which provisions retailers were adherent and 2) to examine differences in adherence by county, retailer neighborhood, and retailer characteristics.MethodsWe conducted multivariate analysis of tobacco retailers’ adherence to 12 point-of-sale provisions of the Tobacco Control Act in 3 North Carolina counties. We conducted observational audits of 324 retailers during 3 months in 2011 to assess adherence. We used logistic regression to assess associations between adherence to provisions and characteristics of each county, retailer neighborhood, and retailer.ResultsWe found 15.7% of retailers did not adhere to at least 1 provision; 84.3% adhered to all provisions. The provisions most frequently violated were the ban on sales of cigarettes with modified-risk labels (eg, “light” cigarettes) (43 [13.3%] retailers nonadherent) and the ban on self-service for cigarettes and smokeless tobacco (6 [1.9%] retailers nonadherent). We found significant differences in rates of nonadherence by county and type of retailer. Pharmacies and drug stores were more than 3 times as likely as grocery stores to be nonadherent.ConclusionMost tobacco retailers have implemented regulatory changes without enforcement by the US Food and Drug Administration. Monitoring rates of adherence by store type and locale (eg, county) may help retailers comply with point-of-sale provisions

Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by 'universal' primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology

Narratives to enhance smoking cessation interventions among African-American smokers, the ACCE project

BACKGROUND: Low-income, African-American smokers are less likely to have resources to aid in quitting smoking. Narrative communication may provide an enhancement to traditional smoking cessation interventions like NRT, medications, or behavioral treatments for this audience. After extensive pilot testing of stories and personal experiences with smoking cessation from African-Americans from a low-income community, we conducted a randomized control trial using stories to augment routine inpatient treatment among African-Americans at an urban Southern hospital (N = 300). RESULTS: Differences in smoking cessation outcomes between the intervention (stories DVD + routine clinical treatment) and control (routine clinical treatment) arms were compared using self-report and carbon monoxide measurement at 6-months. Compared to control, individuals who viewed the intervention stories DVD reported greater intentions to quit. Although continuous quitting marginally favored the intervention, our main result did not reach statistical significance (p = 0.16). CONCLUSION: Narrative communication via storytelling to promote smoking cessation among African-Americans in the South is one method to communicate smoking cessation. Results suggest this may not be sufficient as a stand-alone augmentation of routine clinical treatment for continuous smoking cessation. Smoking cessation efforts need to continually assess different means of communicating to smokers about quitting. CLINICAL TRIALS REGISTRATION: The ClinicalTrials.gov Identifier is NCT00101491. This trial was registered January 10, 2005

Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases

Mouse models of human disease remain the bread and butter of modern biology and therapeutic discovery. Nonetheless, more often than not mouse models do not reproduce the pathophysiology of the human conditions they are designed to mimic. Naturally occurring large animal models have predominantly been found in companion animals or livestock because of their emotional or economic value to modern society and, unlike mice, often recapitulate the human disease state. In particular, numerous models have been discovered in dogs and have a fundamental role in bridging proof of concept studies in mice to human clinical trials. The present article is a review that highlights current canine models of human diseases, including Alzheimer\u27s disease, degenerative myelopathy, neuronal ceroid lipofuscinosis, globoid cell leukodystrophy, Duchenne muscular dystrophy, mucopolysaccharidosis, and fucosidosis. The goal of the review is to discuss canine and human neurodegenerative pathophysiologic similarities, introduce the animal models, and shed light on the ability of canine models to facilitate current and future treatment trials

Decision-making Styles in a Real-Life Decision: Choosing a College Major

Undergraduate students were surveyed at the beginning stages of a potentially life-framing decision: choosing a college major. We investigated the relationships among individual difference variables (decision-making styles, planning proclivities, and epistemological orientations), cognitive measures of performance (e.g., amount of information gathered and considered); and affective reactions to, and descriptive ratings of, the decision-making process. There were few significant relationships between individual differences and performance measures. However, there were significant relationships found between individual differences measures and affective reactions to, or descriptive ratings of, the decision-making process. We suggest that stylistic measures have their effects in the way individuals frame the decision-making process rather than in the way they go about gathering or structuring information