Potential Environmental Impacts of Oil and Natural Gas Production on a Shortgrass Steppe

Federal lands of the Arapaho and Roosevelt National Forests and Pawnee National Grassland (PNG) lie on the Niobrara play and bring high profits to the State of Colorado. Natural gas development, production, and associated processing activities; however, can be a substantial source of air pollution. Common fugitive emissions on typical PNG Oil and Natural Gas (O&NG) production sites include volatile organic chemicals (VOCs), such as benzene, toluene, ethylbenzene and the xylenes (BTEX). These VOCs can then deposit (wet or dry) onto or transfer (via soil or water) into surrounding vegetation. Minerals, including heavy metals, are also released during the production phase of O&NG development and can deposit near the emission source. There are also impacts beyond pollutants, including habitat loss, fragmentation and the alteration of vegetation communities due to O&NG construction and associated structures. The current study presents novel data related to (1) the ambient levels of common fugitive emissions on typical O&NG production sites (Chapter II) (2) the deposition of these emissions (BTEX) onto proximate flora (Chapter III) (3) the impact on mineral content in proximate flora (Chapter IV) and (4) reclamation success and shifts in plant community structure (Chapter V). In Chapter II, Volatile Organic Compounds (VOCs) were quantified in real-time and used to determine the spatial and temporal windows of exposure for proximate flora and fauna. We found that VOC concentrations generally increased during the 6 hr. day and were predominately the result of O&NG production and not vehicle exhaust. Thirteen of 24 VOCs had statistically significant differences in ambient levels between production groups, frequently above reference standards and thus at biologically relevant levels for shortgrass steppe flora and fauna. The most biologically relevant VOCs found at concentrations exceeding time weighted average permissible exposure limits (TWA PEL), were benzene and acrolein. Generalized Estimating Equations (GEEs) were used to measure the relative quality of statistical models predicting benzene concentrations on sites. For Chapters III-V sites were grouped according to status (PA or PR) and production date (spud date). Groups were as follows: PA = Plugged and abandoned in the 1980s, PR1 = Producing since 1980-1990, PR2 = Producing since 2000-2005, and PR3 = Producing since 2006-2013. We also measured the effects of Distance in all chapters with a maximum distance of 100 m from the wellhead. In Chapter II, Bouteloua gracilis (blue grama) and Bouteloua dactyloides (buffalo grass) leaves were collected and BTEX were quantified in plant tissue. Deposition and accumulation of BTEX onto proximate flora significantly decreased with production age (PA sites). Newer wells and sites with active pumpjacks had significant concentrations of benzene and toluene in vegetation. BTEX were present on every site except one plugged and abandoned site. The average concentration of toluene on all sites combined was 2.32 ppbv. The average concentration for benzene on all sites combined was 13.18 ppbv, but concentrations were as high as 176 ppbv. These concentrations are arguably biologically relevant as organisms within 100 m of O&NG production sites are likely breathing, and if grazing, consuming high levels of BTEX. In Chapter III, concentrations of minerals in Bouteloua leaves were quantified and their effects on foraging quality were determined. Of the macro minerals, K, P, and S were significantly higher in vegetation found at 25 m and 50 m than in those at 100 m. Calcium was highest in vegetation near PA sites, while P and K were highest in vegetation near PR1 and PR2 sites. Shoot concentrations of Cu, Br, Cr, Pb, Sr and Ba were higher further from the wellhead (100 m), indicating impact further than previously expected. There were still impacts near the wellhead, as Hg was significantly higher in vegetation at 25 m. Concentrations of Mn, Fe, and Ba were all highest in vegetation at PR3 sites, whilst Br was highest on PR1 sites and Sr was highest on PA sites. Concentrations of micro minerals in shoots were in the following order Fe \u3e Cl \u3e Pb \u3e Br \u3e Mn \u3e Sr \u3e Ba \u3e Zn \u3e Cu \u3e Se \u3e Ni \u3e Hg \u3e Cr. Concentrations of Se (5.67 ppm), S (0.33%), and K (1.21%) had the potential to exceed max tolerable concentrations for cattle (based on 2 kg daily mass intake). All other nutrient shoot concentrations were potentially appropriate for grazing cattle, depending on specific cattle and grazing characteristics. Toxic elements such as Br (54 ppm) and Sr (46 ppm) were present in shoot samples far below maximum tolerable levels, while concentrations of Hg (1.54 ppm) and Pb (83 ppm) were beyond daily maximum tolerable levels for cattle when considering a 2 kg DM diet. We also compared shoot nutrient levels to data collected by Fresquez et al. (1991) and concentrations of micro minerals were comparable to Bouteloua grown in sludge treated soils, indicating a substantial impact from O&NG production. This impact has had a lasting effect on soils and vegetation as seen with Pb levels on PA sites reclaimed over 30 years ago. In Chapter IV, we characterized proximate vegetation cover, diversity and functionality during well production and following abandonment. In general, PA 100 m sites were distinctly different from all PR sites. As expected, at 20 m and 50 m, sites had substantially more bare ground and introduced plant species than at 100 m and PR3 sites had the highest percentage of bare ground. There were 16.5% introduced plant species on all plots combined and 2% of plant species sampled were invasive. Satisfactory reclamation was achieved at 50 m on PR1 and PR2 sites as vegetation was at 80% total cover when compared to 100 m. Our PA sites were the highest in plant diversity indices and PR3 were the lowest. With the high cover scores and diversity indices on PA sites it seems recovery over time is possible. We did not find high plant functional redundancy on our O&NG sites; instead, we found high plant species diversity and high functional diversity on PA sites. These disturbed plant communities with greater spatial heterogeneity than blue grama-dominated sites are shifting in community structure. We found that novel intensities of O&NG disturbances along with other synergistic disturbances promoted species and functional shifts in vegetation. The PNG has had an exponential increase in O&NG drilling and extraction in the last decade and results indicate production has caused a novel and biologically relevant impact on native flora and fauna

Seasonal Forage Availability and Diet of Reintroduced Elk in the Cumberland Mountains, Tennessee

The Tennessee Wildlife Resources Agency (TWRA) reintroduced elk (Cervus elaphus manitobensis) into the Cumberland Mountains, Tennessee over a 3-year period beginning in December 2000. We radio-collared 160 elk and monitored them by aerial telemetry from February 2001 to June 2003. Locations (n = 1450) were used in a geographic information system (GIS) to develop a core herd home range (789-ha sampling area) to assess elk seasonal forage use and availability. We monitored diet and resource availability from November 2003 to October 2004 by vegetation sampling and microhistological analysis of feces. Tall fescue (Festuca arundinacea; 35.1%) dominated the winter grass diet composition (65.9%). The diet shifted in the spring to a mixture of woody plants (28.1%), forbs (19.4%), and grasses (38.4%). The highest seasonal use of forbs (45%) and legumes (23%) occurred during summer, with jewelweed (Impatiens spp.; 27%) as the dominant plant in the diet. The dominant fall forage class was woody plants (37.4%). Oaks (Quercus spp.; 14.3%) were the most used woody plant and oak acorns comprised 9.7% of the elk diet. Elk herbivory, interspecific competition for key resources (e.g., acorns), and landscape-level changes (e.g., mining) should be monitored. Short-term detrimental effects from mining could be severe for a small, growing elk herd, and demographic effects could affect reintroduction success. We suggest that historic evidence, native grasses, and the elk diet indicate that oak savannas would be an ideal habitat type to manage for on the Royal Blue Wildlife Management Area. Further research will be needed to determine the effects of elk upon the flora and fauna in deciduous forests of eastern Tennessee

Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms

TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

TopBP1 and the Rad9–Rad1–Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR–ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-α to sites of replication stress. Furthermore, we show that pol-α is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-α, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress

Evolution of Th2 responses : Characterization of IL-4/13 in sea bass (Dicentrarchus labrax L.) and studies of expression and biological activity

Acknowledgements This research was funded by the European Commission under the 7th Framework Programme for Research and Technological Development (FP7) of the European Union (Grant Agreement 311993 TARGETFISH). T.W. received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (grant reference number HR09011) and contributing institutions.Peer reviewedPublisher PD

Continued primer synthesis at stalled replication forks contributes to checkpoint activation

An increased number of primer–template junctions generated by PCNA, Pol-δ, and Pol-ε at stalled replication forks activates Chk1

Autoproteolytic Activation of Bacterial Toxins

Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD) in Multifunctional Autoprocessing RTX-like (MARTX) and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6), which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins

Clan CD of cysteine peptidases as an example of evolutionary divergences in related protein families across plant clades

Comparative genomic analyses are powerful tools that can be used to analyze the presence, conservation, and evolution of protein families and to elucidate issues concerning their function. To deal with these questions, we have chosen the clan CD of cysteine peptidases, which is formed by different protein families that play key roles in plants. An evolutionary comparative analysis of clan CD cysteine peptidases in representative species of different taxonomic groups that appeared during the evolution of the Viridiplantae was performed. The results obtained indicates: i) C13 GPI:protein transamidases, C14 metacaspases I, and C50 separases are present in all taxonomic groups; ii) C13 legumains and C14 metacaspases II are absent in some basal algae groups; iii) C11 clostripains have only been found in the two Chlorophyceae species; iv) C25 gingipains and C80 RTX toxins have not been found in plants. Moreover, gene duplication events could have been associated in some families to the increasing complexities acquired in land plants. These findings have demonstrated that comparative genomics is useful to provide valuable insights on the differential evolution of the related peptidase families belonging to clan CD in plant clades. The low number of protein members suggests a restricted physiological role for these peptidase families, mainly in algae species