In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii

Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type

Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional ‘artificial’ mutations, to optimize its in vivo activity

Is Landscape Context Important for Riparian Conservation? Birds in Grassy Woodland

An important challenge for riparian management is to determine the extent to which landscape context influences the faunal assemblages of riparian habitats. We examined this challenge in the variegated landscapes of southeastern Queensland, Australia where riparian vegetation is surrounded by both extensive grazing and intensive cropping. We investigated whether riparian habitats adjacent to different landuses support similar bird assemblages. Three types of riparian habitat condition were sampled (uncleared ungrazed; uncleared grazed; cleared grazed) in four different land-use contexts (ungrazed woodland; grazed woodland; native pasture; crop) although only six of the twelve possible treatment combinations were available. Eighty percent of bird species responded significantly to changes in both riparian habitat condition and landscape context, while fewer than 50% of species were significantly influenced by landscape context alone. The influence of landscape context on the bird assemblage increased as the surrounding land use became more intensive (e.g. woodland to native pasture to crop). Riparian zones have been shown to have consistently high biodiversity values relative to their extent. These findings suggest it is not enough to conserve riparian habitats alone, conservation and restoration plans must also take into consideration landscape context, particularly when that context is intensively used land

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions

Ecosystem resilience despite large-scale altered hydroclimatic conditions

Climate change is predicted to increase both drought frequency and duration, and when coupled with substantial warming, will establish a new hydroclimatological model for many regions. Large-scale, warm droughts have recently occurred in North America, Africa, Europe, Amazonia and Australia, resulting in major effects on terrestrial ecosystems, carbon balance and food security. Here we compare the functional response of above-ground net primary production to contrasting hydroclimatic periods in the late twentieth century (1975-1998), and drier, warmer conditions in the early twenty-first century (2000-2009) in the Northern and Southern Hemispheres. We find a common ecosystem water-use efficiency (WUE e: Above-ground net primary production/ evapotranspiration) across biomes ranging from grassland to forest that indicates an intrinsic system sensitivity to water availability across rainfall regimes, regardless of hydroclimatic conditions. We found higher WUE e in drier years that increased significantly with drought to a maximum WUE e across all biomes; and a minimum native state in wetter years that was common across hydroclimatic periods. This indicates biome-scale resilience to the interannual variability associated with the early twenty-first century drought - that is, the capacity to tolerate low, annual precipitation and to respond to subsequent periods of favourable water balance. These findings provide a conceptual model of ecosystem properties at the decadal scale applicable to the widespread altered hydroclimatic conditions that are predicted for later this century. Understanding the hydroclimatic threshold that will break down ecosystem resilience and alter maximum WUE e may allow us to predict land-surface consequences as large regions become more arid, starting with water-limited, low-productivity grasslands. © 2013 Macmillan Publishers Limited. All rights reserved

Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions

Differential Response to Soil Salinity in Endangered Key Tree Cactus: Implications for Survival in a Changing Climate

Understanding reasons for biodiversity loss is essential for developing conservation and management strategies and is becoming increasingly urgent with climate change. Growing at elevations <1.4 m in the Florida Keys, USA, the endangered Key tree cactus (Pilosocereus robinii) experienced 84 percent loss of total stems from 1994 to 2007. The most severe losses of 99 and 88 percent stems occurred in the largest populations in the Lower Keys, where nine storms with high wind velocities and storm surges, occurred during this period. In contrast, three populations had substantial stem proliferation. To evaluate possible mortality factors related to changes in climate or forest structure, we examined habitat variables: soil salinity, elevation, canopy cover, and habitat structure near 16 dying or dead and 18 living plants growing in the Lower Keys. Soil salinity and elevation were the preliminary factors that discriminated live and dead plants. Soil salinity was 1.5 times greater, but elevation was 12 cm higher near dead plants than near live plants. However, distribution-wide stem loss was not significantly related to salinity or elevation. Controlled salinity trials indicated that salt tolerance to levels above 40 mM NaCl was related to maternal origin. Salt sensitive plants from the Lower Keys had less stem growth, lower root:shoot ratios, lower potassium: sodium ratios and lower recovery rate, but higher δ 13C than a salt tolerant lineage of unknown origin. Unraveling the genetic structure of salt tolerant and salt sensitive lineages in the Florida Keys will require further genetic tests. Worldwide rare species restricted to fragmented, low-elevation island habitats, with little or no connection to higher ground will face challenges from climate change-related factors. These great conservation challenges will require traditional conservation actions and possibly managed relocation that must be informed by studies such as these

BIOFRAG: A new database for analysing BIOdiversity responses to forest FRAGmentation

Habitat fragmentation studies are producing inconsistent and complex results across which it is nearly impossible to synthesise. Consistent analytical techniques can be applied to primary datasets, if stored in a flexible database that allows simple data retrieval for subsequent analyses. Method: We developed a relational database linking data collected in the field to taxonomic nomenclature, spatial and temporal plot attributes and further environmental variables (e.g. information on biogeographic region. Typical field assessments include measures of biological variables (e.g. presence, abundance, ground cover) of one species or a set of species linked to a set of plots in fragments of a forested landscape. Conclusion: The database currently holds records of 5792 unique species sampled in 52 landscapes in six of eight biogeographic regions: mammals 173, birds 1101, herpetofauna 284, insects 2317, other arthropods: 48, plants 1804, snails 65. Most species are found in one or two landscapes, but some are found in four. Using the huge amount of primary data on biodiversity response to fragmentation becomes increasingly important as anthropogenic pressures from high population growth and land demands are increasing. This database can be queried to extract data for subsequent analyses of the biological response to forest fragmentation with new metrics that can integrate across the components of fragmented landscapes. Meta-analyses of findings based on consistent methods and metrics will be able to generalise over studies allowing inter-comparisons for unified answers. The database can thus help researchers in providing findings for analyses of trade-offs between land use benefits and impacts on biodiversity and to track performance of management for biodiversity conservation in human-modified landscapes.Fil: Pfeifer, Marion. Imperial College London; Reino UnidoFil: Lefebvre, Veronique. Imperial College London; Reino UnidoFil: Gardner, Toby A.. Stockholm Environment Institute; SueciaFil: Arroyo Rodríguez, Víctor. Universidad Nacional Autónoma de México; MéxicoFil: Baeten, Lander. University of Ghent; BélgicaFil: Banks Leite, Cristina. Imperial College London; Reino UnidoFil: Barlow, Jos. Lancaster University; Reino UnidoFil: Betts, Matthew G.. State University of Oregon; Estados UnidosFil: Brunet, Joerg. Swedish University of Agricultural Sciences; SueciaFil: Cerezo Blandón, Alexis Mauricio. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Métodos Cuantitativos y Sistemas de Información; ArgentinaFil: Cisneros, Laura M.. University of Connecticut; Estados UnidosFil: Collard, Stuart. Nature Conservation Society of South Australia; AustraliaFil: D´Cruze, Neil. The World Society for the Protection of Animals; Reino UnidoFil: Da Silva Motta, Catarina. Ministério da Ciência, Tecnologia, Inovações. Instituto Nacional de Pesquisas da Amazônia; BrasilFil: Duguay, Stephanie. Carleton University; CanadáFil: Eggermont, Hilde. University of Ghent; BélgicaFil: Eigenbrod, Félix. University of Southampton; Reino UnidoFil: Hadley, Adam S.. State University of Oregon; Estados UnidosFil: Hanson, Thor R.. No especifíca;Fil: Hawes, Joseph E.. University of East Anglia; Reino UnidoFil: Heartsill Scalley, Tamara. United State Department of Agriculture. Forestry Service; Puerto RicoFil: Klingbeil, Brian T.. University of Connecticut; Estados UnidosFil: Kolb, Annette. Universitat Bremen; AlemaniaFil: Kormann, Urs. Universität Göttingen; AlemaniaFil: Kumar, Sunil. State University of Colorado - Fort Collins; Estados UnidosFil: Lachat, Thibault. Swiss Federal Institute for Forest; SuizaFil: Lakeman Fraser, Poppy. Imperial College London; Reino UnidoFil: Lantschner, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Laurance, William F.. James Cook University; AustraliaFil: Leal, Inara R.. Universidade Federal de Pernambuco; BrasilFil: Lens, Luc. University of Ghent; BélgicaFil: Marsh, Charles J.. University of Leeds; Reino UnidoFil: Medina Rangel, Guido F.. Universidad Nacional de Colombia; ColombiaFil: Melles, Stephanie. University of Toronto; CanadáFil: Mezger, Dirk. Field Museum of Natural History; Estados UnidosFil: Oldekop, Johan A.. University of Sheffield; Reino UnidoFil: Overal , Williams L.. Museu Paraense Emílio Goeldi. Departamento de Entomologia; BrasilFil: Owen, Charlotte. Imperial College London; Reino UnidoFil: Peres, Carlos A.. University of East Anglia; Reino UnidoFil: Phalan, Ben. University of Southampton; Reino UnidoFil: Pidgeon, Anna Michle. University of Wisconsin; Estados UnidosFil: Pilia, Oriana. Imperial College London; Reino UnidoFil: Possingham, Hugh P.. Imperial College London; Reino Unido. The University Of Queensland; AustraliaFil: Possingham, Max L.. No especifíca;Fil: Raheem, Dinarzarde C.. Royal Belgian Institute of Natural Sciences; Bélgica. Natural History Museum; Reino UnidoFil: Ribeiro, Danilo B.. Universidade Federal do Mato Grosso do Sul; BrasilFil: Ribeiro Neto, Jose D.. Universidade Federal de Pernambuco; BrasilFil: Robinson, Douglas W.. State University of Oregon; Estados UnidosFil: Robinson, Richard. Manjimup Research Centre; AustraliaFil: Rytwinski, Trina. Carleton University; CanadáFil: Scherber, Christoph. Universität Göttingen; AlemaniaFil: Slade, Eleanor M.. University of Oxford; Reino UnidoFil: Somarriba, Eduardo. Centro Agronómico Tropical de Investigación y Enseñanza; Costa RicaFil: Stouffer, Philip C.. State University of Louisiana; Estados UnidosFil: Struebig, Matthew J.. University of Kent; Reino UnidoFil: Tylianakis, Jason M.. University College London; Estados Unidos. Imperial College London; Reino UnidoFil: Teja, Tscharntke. Universität Göttingen; AlemaniaFil: Tyre, Andrew J.. Universidad de Nebraska - Lincoln; Estados UnidosFil: Urbina Cardona, Jose N.. Pontificia Universidad Javeriana; ColombiaFil: Vasconcelos, Heraldo L.. Universidade Federal de Uberlandia; BrasilFil: Wearn, Oliver. Imperial College London; Reino Unido. The Zoological Society of London; Reino UnidoFil: Wells, Konstans. University of Adelaide; AustraliaFil: Willig, Michael R.. University of Connecticut; Estados UnidosFil: Wood, Eric. University of Wisconsin; Estados UnidosFil: Young, Richard P.. Durrell Wildlife Conservation Trust; Reino UnidoFil: Bradley, Andrew V.. Imperial College London; Reino UnidoFil: Ewers, Robert M.. Imperial College London; Reino Unid

Trading between healthy food, alcohol and physical activity behaviours

BACKGROUND: While recent lifestyle studies have explored the role that food, alcohol or physical activity have on health and wellbeing, few have explored the interplay between these behaviours and the impact this has on a healthy lifestyle. Given the long term health advantages associated with leading healthier lifestyles, this study seeks to: 1) explore the interplay between the food, alcohol and physical activity behaviours of young adults (aged 19–26 years) in the North East of England; 2) explore the trade-offs young adults make between their food, alcohol and physical activity behaviours; and 3) recognise the positive and negative associations between the three behaviours. METHODS: Qualitative self-reported lifestyle diaries and in-depth interviews were conducted with 50 young adults from the North East of England between February and June 2008. Qualitative thematic analysis was undertaken using Nvivo QSR software, and diary coding using Windiets software. RESULTS: Young adults who attempt to achieve a ‘healthy lifestyle’ make trade-offs between the food and alcohol they consume, and the amounts of physical activity they undertake. There are negative reasons and positive consequences associated with these trade-offs. Young adults recognise the consequences of their behaviours and as a result are prepared to undertake healthy behaviours to compensate for unhealthy behaviours. They prefer certain strategies to promote healthier behaviours over others, in particular those that relate to personalised advice and support, more affordable ways to be healthier and easily-accessed advice from a range of media sources. CONCLUSIONS: Young adults seek to compensate unhealthy behaviours (e.g. binge drinking) with healthy behaviours (e.g. physical activity). Creative solutions may be required to tackle these trade-offs and promote a balance across the food, alcohol and physical activity behaviours of this age group. Solutions that may be effective with this age group include environmental changes (e.g. green spaces and increasing the price of alcohol) designed to encourage and facilitate young people making healthier choices and improving their access to, and lowering the price of, healthy food products. Solutions must recognise these trade-offs and in particular, the strong reluctance of young adults to alter their higher-than-recommended levels of alcohol consumption