Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.R ecent work regarding the biological engineering of extremophiles has increased interest in their use as a robust chassis for metabolic engineering. The appeal of using extremophiles in various applications is largely due to their ability to survive conditions toxic to traditional engineering strains. One extremophile considered attractive is Deinococcus radiodurans, a Gram-positive bacteria known for its tolerance to ionizing radiation, heavy metal exposure, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens (1-3). In the context of its applications in bioremediation,

In April 2004, triploid native (Crassostrea virginica) and nonnative (Crassostrea ariakensis) oysters were deployed in cages at four sites along a salinity gradient in Chesapeake Bay. In Maryland, the lowest salinity site was located in the Severn River and two low to mid-salinity sites were located in the Choptank and Patuxent Rivers. The highest salinity site was located in the York River in Virginia. Growth, disease acquisition, and mortality were measured in the deployed oysters through August 2006. Although ANOVA revealed that the nonnative oysters were significantly larger at the end of the experiment than the native oysters at all sites, the differences were much greater at the Virginia site (59 mm) than in Maryland waters (9-23 mm). With the exception of C. ariakensis in the Severn River, Perkinsus marinus infected both species at all sites. Prevalences and weighted prevalences in both species remained relatively low throughout the experiment, but native oysters consistently acquired higher prevalences and weighted prevalences than C. ariakensis by August 2006. With the exception of several mortality-inducing events including winter freezing and hypoxic exposure, mortality was generally low in both species. No disease-related mortality was suspected in either species given the low weighted prevalences observed. In the York River, where a substantial natural spatfall occurred in 2004, more native spat were found on C. ariakensis than on C. virginica. To our knowledge, this is the first comparison of triploid C. ariakensis to triploid C. virginica conducted in the field. Because we did not observe substantial disease-related mortality, it is too soon to draw conclusions regarding the disease tolerance of C. ariakensis in the field or its viability as a replacement for the native species.

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