A Sequence- and Ligation-Independent Cloning (SLIC) Procedure for the Insertion of Genes into a Plasmid Vector.

Molecular cloning is a routine technique for many laboratories with applications from genetic engineering to recombinant protein expression. While restriction-ligation cloning can be slow and inefficient, ligation-independent cloning uses long single-stranded overhangs generated by T4 DNA polymerase's 3' exonuclease activity to anneal the insert and plasmid vector prior to transformation. This chapter describes a fast, high-efficiency protocol for inserting one or more genes into a vector using sequence- and ligation-independent cloning (SLIC).

Predicted wind and solar energy expansion has minimal overlap with multiple conservation priorities across global regions.

Protected areas and renewable energy generation are critical tools to combat biodiversity loss and climate change, respectively. Over the coming decades, expansion of the protected area network to meet conservation objectives will be occurring alongside rapid deployment of renewable energy infrastructure to meet climate targets, driving potential conflict for a finite land resource. Renewable energy infrastructure can have negative effects on wildlife, and co-occurrence may mean emissions targets are met at the expense of conservation objectives. Here, we assess current and projected overlaps of wind and solar photovoltaic installations and important conservation areas across nine global regions using spatially explicit wind and solar data and methods for predicting future renewable expansion. We show similar levels of co-occurrence as previous studies but demonstrate that once area is accounted for, previous concerns about overlaps in the Northern Hemisphere may be largely unfounded, although they are high in some biodiverse countries (e.g., Brazil). Future projections of overlap between the two land uses presented here are generally dependent on priority threshold and region and suggest the risk of future conflict can be low. We use the best available data on protected area degradation to corroborate this level of risk. Together, our findings indicate that while conflicts between renewables and protected areas inevitably do occur, renewables represent an important option for decarbonization of the energy sector that would not significantly affect area-based conservation targets if deployed with appropriate policy and regulatory controls.

Identifying Agricultural Frontiers for Modeling Global Cropland Expansion.

The increasing expansion of cropland is major driver of global carbon emissions and biodiversity loss. However, predicting plausible future global distributions of croplands remains challenging. Here, we show that, in general, existing global data aligned with classical economic theories of expansion explain the current (1992) global extent of cropland reasonably well, but not recent expansion (1992-2015). Deviations from models of cropland extent in 1992 ("frontierness") can be used to improve global models of recent expansion, most likely as these deviations are a proxy for cropland expansion under frontier conditions where classical economic theories of expansion are less applicable. Frontierness is insensitive to the land cover dataset used and is particularly effective in improving models that include mosaic land cover classes and the largely smallholder-driven frontier expansion occurring in such areas. Our findings have important implications as the frontierness approach offers a straightforward way to improve global land use change models.

Impacts of rising temperatures and farm management practices on global yields of 18 crops.

Understanding the impact of changes in temperature and precipitation on crop yields is a vital step in developing policy and management options to feed the world. As most existing studies are limited to a few staple crops, we implemented global statistical models to examine the influence of weather and management practices on the yields of 18 crops, accounting for 70% of crop production by area and 65% by calorific intake. Focusing on the impact of temperature, we found considerable heterogeneity in the responses of yields across crops and countries. Irrigation was found to alleviate negative implications from temperature increases. Countries where increasing temperature causes the most negative impacts are typically the most food insecure, with the lowest calorific food supply and average crop yield. International action must be coordinated to raise yields in these countries through improvement and modernization of agricultural practices to counteract future adverse impacts of climate change.

The influence of the global electric power system on terrestrial biodiversity.

Given its total contribution to greenhouse gas emissions, the global electric power sector will be required to undergo a fundamental transformation over the next decades to limit anthropogenic climate change to below 2 °C. Implications for biodiversity of projected structural changes in the global electric power sector are rarely considered beyond those explicitly linked to climate change. This study uses a spatially explicit consumption-based accounting framework to examine the impact of demand for electric power on terrestrial vertebrate biodiversity globally. We demonstrate that the biodiversity footprint of the electric power sector is primarily within the territory where final demand for electric power resides, although there are substantial regional differences, with Europe displacing its biodiversity threat along international supply chains. The relationship between size of individual components of the electric power sector and threat to biodiversity indicates that a shift to nonfossil sources, such as solar and wind, could reduce pressures on biodiversity both within the territory where demand for power resides and along international supply chains. However, given the current levels of deployment of nonfossil sources of power, there is considerable uncertainty as to how the impacts of structural changes in the global electric power system will scale. Given the strong territorial link between demand and associated biodiversity impacts, development of strong national governance around the electric power sector represents a clear route to mitigate threats to biodiversity associated with efforts to decarbonize society over the coming century.

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover.

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.

Grassland futures in Great Britain - Productivity assessment and scenarios for land use change opportunities.

To optimise trade-offs provided by future changes in grassland use intensity, spatially and temporally explicit estimates of respective grassland productivities are required at the systems level. Here, we benchmark the potential national availability of grassland biomass, identify optimal strategies for its management, and investigate the relative importance of intensification over reversion (prioritising productivity versus environmental ecosystem services). Process-conservative meta-models for different grasslands were used to calculate the baseline dry matter yields (DMY; 1961-1990) at 1km2 resolution for the whole UK. The effects of climate change, rising atmospheric [CO2] and technological progress on baseline DMYs were used to estimate future grassland productivities (up to 2050) for low and medium CO2 emission scenarios of UKCP09. UK benchmark productivities of 12.5, 8.7 and 2.8t/ha on temporary, permanent and rough-grazing grassland, respectively, accounted for productivity gains by 2010. By 2050, productivities under medium emission scenario are predicted to increase to 15.5 and 9.8t/ha on temporary and permanent grassland, respectively, but not on rough grassland. Based on surveyed grassland distributions for Great Britain in 2010 the annual availability of grassland biomass is likely to rise from 64 to 72milliontonnes by 2050. Assuming optimal N application could close existing productivity gaps of ca. 40% a range of management options could deliver additional 21∗106tonnes of biomass available for bioenergy. Scenarios of changes in grassland use intensity demonstrated considerable scope for maintaining or further increasing grassland production and sparing some grassland for the provision of environmental ecosystem services.

Using Small Angle X-Ray Scattering (SAXS) to Characterize the Solution Conformation and Flexibility of Matrix Metalloproteinases (MMPs).

Small angle X-ray scattering (SAXS) provides information about the conformation and flexibility of proteins in solution, and hence provides complementary structural information to that obtained from X-ray crystallography and nuclear magnetic resonance spectroscopy. In this chapter, we describe the methods for the preparation of matrix metalloproteinase (MMP) samples for SAXS analyses, and for the acquisition, processing and interpretation of the SAXS data.

Potential impacts on ecosystem services of land use transitions to second-generation bioenergy crops in GB.

We present the first assessment of the impact of land use change (LUC) to second-generation (2G) bioenergy crops on ecosystem services (ES) resolved spatially for Great Britain (GB). A systematic approach was used to assess available evidence on the impacts of LUC from arable, semi-improved grassland or woodland/forest, to 2G bioenergy crops, for which a quantitative 'threat matrix' was developed. The threat matrix was used to estimate potential impacts of transitions to either Miscanthus, short-rotation coppice (SRC, willow and poplar) or short-rotation forestry (SRF). The ES effects were found to be largely dependent on previous land uses rather than the choice of 2G crop when assessing the technical potential of available biomass with a transition from arable crops resulting in the most positive effect on ES. Combining these data with constraint masks and available land for SRC and Miscanthus (SRF omitted from this stage due to lack of data), south-west and north-west England were identified as areas where Miscanthus and SRC could be grown, respectively, with favourable combinations of economic viability, carbon sequestration, high yield and positive ES benefits. This study also suggests that not all prospective planting of Miscanthus and SRC can be allocated to agricultural land class (ALC) ALC 3 and ALC 4 and suitable areas of ALC 5 are only minimally available. Beneficial impacts were found on 146 583 and 71 890 ha when planting Miscanthus or SRC, respectively, under baseline planting conditions rising to 293 247 and 91 318 ha, respectively, under 2020 planting scenarios. The results provide an insight into the interplay between land availability, original land uses, bioenergy crop type and yield in determining overall positive or negative impacts of bioenergy cropping on ecosystems services and go some way towards developing a framework for quantifying wider ES impacts of this important LUC.

Impact of alternative metrics on estimates of extent of occurrence for extinction risk assessment.

In International Union for Conservation of Nature (IUCN) Red List assessments, extent of occurrence (EOO) is a key measure of extinction risk. However, the way assessors estimate EOO from maps of species' distributions is inconsistent among assessments of different species and among major taxonomic groups. Assessors often estimate EOO from the area of mapped distribution, but these maps often exclude areas that are not habitat in idiosyncratic ways and are not created at the same spatial resolutions. We assessed the impact on extinction risk categories of applying different methods (minimum convex polygon, alpha hull) for estimating EOO for 21,763 species of mammals, birds, and amphibians. Overall, the percentage of threatened species requiring down listing to a lower category of threat (taking into account other Red List criteria under which they qualified) spanned 11-13% for all species combined (14-15% for mammals, 7-8% for birds, and 12-15% for amphibians). These down listings resulted from larger estimates of EOO and depended on the EOO calculation method. Using birds as an example, we found that 14% of threatened and near threatened species could require down listing based on the minimum convex polygon (MCP) approach, an approach that is now recommended by IUCN. Other metrics (such as alpha hull) had marginally smaller impacts. Our results suggest that uniformly applying the MCP approach may lead to a one-time down listing of hundreds of species but ultimately ensure consistency across assessments and realign the calculation of EOO with the theoretical basis on which the metric was founded.

Identifying the world's most climate change vulnerable species: a systematic trait-based assessment of all birds, amphibians and corals.

Climate change will have far-reaching impacts on biodiversity, including increasing extinction rates. Current approaches to quantifying such impacts focus on measuring exposure to climatic change and largely ignore the biological differences between species that may significantly increase or reduce their vulnerability. To address this, we present a framework for assessing three dimensions of climate change vulnerability, namely sensitivity, exposure and adaptive capacity; this draws on species' biological traits and their modeled exposure to projected climatic changes. In the largest such assessment to date, we applied this approach to each of the world's birds, amphibians and corals (16,857 species). The resulting assessments identify the species with greatest relative vulnerability to climate change and the geographic areas in which they are concentrated, including the Amazon basin for amphibians and birds, and the central Indo-west Pacific (Coral Triangle) for corals. We found that high concentration areas for species with traits conferring highest sensitivity and lowest adaptive capacity differ from those of highly exposed species, and we identify areas where exposure-based assessments alone may over or under-estimate climate change impacts. We found that 608-851 bird (6-9%), 670-933 amphibian (11-15%), and 47-73 coral species (6-9%) are both highly climate change vulnerable and already threatened with extinction on the IUCN Red List. The remaining highly climate change vulnerable species represent new priorities for conservation. Fewer species are highly climate change vulnerable under lower IPCC SRES emissions scenarios, indicating that reducing greenhouse emissions will reduce climate change driven extinctions. Our study answers the growing call for a more biologically and ecologically inclusive approach to assessing climate change vulnerability. By facilitating independent assessment of the three dimensions of climate change vulnerability, our approach can be used to devise species and area-specific conservation interventions and indices. The priorities we identify will strengthen global strategies to mitigate climate change impacts.

Spatial covariation between freshwater and terrestrial ecosystem services.

To inform the design and implementation of land-use policies that consider the variety of goods and services people derive from ecosystems, it is essential to understand spatial patterns of individual services, how multiple services relate to each other, and how these relationships vary across spatial scales and localities. Despite the importance of freshwater as a determinant of regional economic and human demographic patterns, there are surprisingly few studies that map the provision of a range of services associated with the quality of the aquatic environment. Here we examine relationships between indicators of riverine water and associated habitat quality, freshwater biodiversity, three terrestrial ecosystem services, and terrestrial biodiversity across England and Wales. The results indicate strong associations between our indicators of freshwater services. However, a comparison of these indicators of freshwater services with other ecosystem services (carbon storage, agricultural production, recreation) and biodiversity of species of conservation concern in the surrounding terrestrial landscape shows no clear relationships. While there are potential policy "win-wins" for the protection of multiple services shown by associations between indicators of freshwater services and carbon storage in upland areas of Britain, the other ecosystem services showed either negative or no relationships with the indicators of freshwater services. We also consider the influence that spatial scale has on these relationships using River Basin Districts. Our results indicate that relationships between indicators of services can change dramatically depending on the societal pressures and other regional conditions. Thus, the delivery of multiple ecosystem services requires the development of regional strategies, or of national strategies that take account of regional variation.

Component D of chicken hemoglobin and the hemoglobin of the embryonic Tammar wallaby (Macropus eugenii) self-associate upon deoxygenation: Effect on oxygen binding.

Extensive measurements of oxygen binding by some vertebrate hemoglobins (Hbs) have suggested an unusually high degree of cooperativity with reported Hill coefficients, n(H), greater than 4.0. We have reexamined this possibility of "super-cooperativity" with chicken Hb components A (alpha(A) (2)beta(2)) and D (alpha(D) (2)beta(2)). Prior studies have shown that component D but not A self-associates to dimers of tetramers upon deoxygenation. This self-association is reflected in the oxygen equilibrium of Hb D which shows a maximal n(H), greater than 4.0 at approximately 4 mM heme concentration. In contrast, component A has maximal n(H) value below 3. The value of the maximal n(H) for Hb D increases linearly with the fraction of octamer present in the deoxy Hb. We anticipate that deoxygenation-dependent self-association will be shown to be a general property of Hb D from birds and reptiles. Neither oxygen equilibria nor sedimentation measurements show any evidence that components A and D interact to form a complex when deoxygenated. We have also reexamined the oxygen equilibria of Hbs of an embryonic marsupial, the wallaby. The equilibria in red cells have been reported to have Hill coefficients as high as 5-6. Although our oxygen equilibrium measurements of solutions of unfractionated wallaby Hb at a concentration of approximately 1 mM show no n(H) values greater than approximately 3.0, sedimentation velocity measurements provide clear evidence for deoxygenation-dependent self-association.

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation.

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.

The alpha-globin gene family of an Australian marsupial, Macropus eugenii: the long evolutionary history of the theta-globin gene and its functional status in mammals.

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the alpha-globin gene family in mammals by isolating and characterizing alpha-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar alpha-globin family consists of at least four genes including a single adult-expressed gene (alpha), two embryonic/neonatally expressed genes (zeta and zeta'), and theta-globin, each orthologous to the respective alpha-, zeta-, and theta-globin genes of eutherian mammals. The results suggest that the theta-globin lineage arose by duplication of an ancestral adult alpha-globin gene and had already evolved an unusual promoter region, atypical of all known alpha-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that theta-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human theta-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three alpha-like and one beta-like globin genes linked in the order 5'-zeta-alpha-theta-omega-3' existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar zeta-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.

Linkage of the beta-like omega-globin gene to alpha-like globin genes in an Australian marsupial supports the chromosome duplication model for separation of globin gene clusters.

The structure, function, and evolutionary history of globin genes have been the subject of extensive investigation over a period of more than 40 years, yet new globin genes with highly specialized functions are still being discovered and much remains uncertain about their evolutionary history. Here we investigate the molecular evolution of the beta-globin gene family in a marsupial species, the tammar wallaby, Macropus eugenii. We report the complete DNA sequences of two beta-like globin genes and show by phylogenetic analyses that one of these genes is orthologous to embryonically expressed epsilon-globin genes of marsupials and eutherians and the other is orthologous to adult expressed beta-globin genes of marsupials and eutherians. We show that the tammar wallaby contains a third functional beta-like globin gene, omega-globin, which forms part of the alpha-globin gene cluster. The position of omega-globin on the 3' side of the alpha-globin cluster and its ancient phylogenetic history fit the criteria, originally proposed by Jeffreys et al. (1980), of a "fossil" beta-globin gene and suggest that an ancient chromosome or genome duplication preceded the evolution of unlinked clusters of alpha- and beta-globin genes in mammals and avians. In eutherian mammals, such as humans and mice, omega-globin has been silenced or translocated away from the alpha-globin locus, while in marsupials omega-globin is coordinately expressed with the adult alpha-globin gene just prior to birth to produce a functional hemoglobin (alpha2 omega2).