Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.     

The earliest evidence for anatomically modern humans in northwestern Europe

The earliest anatomically modern humans in Europe are thought to have appeared around 43,000-42,000 calendar years before present (43-42 kyr cal BP), by association with Aurignacian sites and lithic assemblages assumed to have been made by modern humans rather than by Neanderthals. However, the actual physical evidence for modern humans is extremely rare, and direct dates reach no farther back than about 41-39 kyr cal BP, leaving a gap. Here we show, using stratigraphic, chronological and archaeological data, that a fragment of human maxilla from the Kent's Cavern site, UK, dates to the earlier period. The maxilla (KC4), which was excavated in 1927, was initially diagnosed as Upper Palaeolithic modern human. In 1989, it was directly radiocarbon dated by accelerator mass spectrometry to 36.4-34.7 kyr cal BP. Using a Bayesian analysis of new ultrafiltered bone collagen dates in an ordered stratigraphic sequence at the site, we show that this date is a considerable underestimate. Instead, KC4 dates to 44.2-41.5 kyr cal BP. This makes it older than any other equivalently dated modern human specimen and directly contemporary with the latest European Neanderthals, thus making its taxonomic attribution crucial. We also show that in 13 dental traits KC4 possesses modern human rather than Neanderthal characteristics; three other traits show Neanderthal affinities and a further seven are ambiguous. KC4 therefore represents the oldest known anatomically modern human fossil in northwestern Europe, fills a key gap between the earliest dated Aurignacian remains and the earliest human skeletal remains, and demonstrates the wide and rapid dispersal of early modern humans across Europe more than 40 kyr ago.     

An armoured Cambrian lobopodian from China with arthropod-like appendages

Cambrian fossil Lagerst?tten preserving soft-bodied organisms have contributed much towards our understanding of metazoan origins. Lobopodians are a particularly interesting group that diversified and flourished in the Cambrian seas. Resembling 'worms with legs', they have long attracted much attention in that they may have given rise to both Onychophora (velvet worms) and Tardigrada (water bears), as well as to arthropods in general. Here we describe Diania cactiformis gen. et sp. nov. as an 'armoured' lobopodian from the Chengjiang fossil Lagerst?tte (Cambrian Stage 3), Yunnan, southwestern China. Although sharing features with other typical lobopodians, it is remarkable for possessing robust and probably sclerotized appendages, with what appear to be articulated elements. In terms of limb morphology it is therefore closer to the arthropod condition, to our knowledge, than any lobopodian recorded until now. Phylogenetic analysis recovers it in a derived position, close to Arthropoda; thus, it seems to belong to a grade of organization close to the point of becoming a true arthropod. Further, D. cactiformis could imply that arthropodization (sclerotization of the limbs) preceded arthrodization (sclerotization of the body). Comparing our fossils with other lobopodian appendage morphologies--see Kerygmachela, Jianshanopodia and Megadictyon--reinforces the hypothesis that the group as a whole is paraphyletic, with different taxa expressing different grades of arthropodization.     

Attack and brood production by the Douglas-fir beetle (Coleoptera: Scolytidae) in Douglas-fir, Pseudotsuga menziesii var. glauca (Pinaceae), following a wildfire

In 1994 ground fire ignited in forests of Douglas-fir, Pseudotsuga menziesii var. glauca (Mirb.) Franco, on Beaver Mountain, Utah. The Douglas-fir beetle, Dendroctonus pseudotsugae Hopkins, attacked a range of moderately fire-injured host conifers in 1995. Logistic regression models run for 1995 data illustrated that 1 year after the fire event the Douglas-fir beetle selected and attacked large-diameter Douglas-fir with 60%-80% bole char, 60%-80% crown volume scorch, and 50%-70% probability of mortality due to fire. In 1996 beetle preference shifted to smallerdiameter trees with lighter fire injury, because most large, fire-damaged conifers were colonized by beetles in 1995. Although beetle populations did not reach outbreak proportions outside the fire boundary, host selection shifted to green trees in 1997 along the burn perimeter. Log linear analysis indicated that increased brood production was conditioned by increased diameter and moderate fire damage to the trees.     

Distribution and abundance of pelagic fish in Lake Powell, Utah, and Lake Mead, Arizona-Nevada

Pelagic fish communities (waters with depths > 20 m) of Lakes Powell and Mead were examined quarterly from 1995 to1998 using vertical gill nets and a scientific echosounder. Nets captured a total of 449 fish consisting of striped bass (57%/45% [Lake Powell/Lake Mead]), threadfin shad (24%/50%), common carp (15%/4%), walleye (3%), channel catfish (2%), and rainbow trout ( -1 ). Reservoirs experienced dramatic seasonal and annual fluctuations in pelagic biomass. Lake Powell's biomass peaked at the Colorado River at 709.7 (± 46.5) kg · ha -1 and Lake Mead's reached 291.9 (± 58.2) kg · ha -1 at Las Vegas Wash. These locations supported estimated fish densities of 124,668 fish · ha -1 and 15,131 fish · ha -1 , respectively. Maximum reservoir biomass peaked in August 1996, with Lake Powell supporting 10,852,738 ± 5,195,556 kg (27.6 × 10 7 fish) and Lake Mead 1,926,697 ± 892.994 kg (10.8 × 10 7 fish). Biomass ebbed in May (1996 and 1997), when Lake Mead supported 65% (296,736 kg vs. 453,097 kg) and 62% (101,016 kg vs. 162,262 kg) of biomass levels found in Lake Powell.     

Influence of water size and type on bat captures in the lower Sonoran Desert

We compared bat use by mist-netting at 4 different types of wildlife water developments in southwestern Arizona during summer 2000 and 2001. Scaling our results by netting effort, we caught bats more frequently and observed higher species diversity at tinajas (modified natural rock pools) with larger open-water area compared with ""guzzler"" type water developments that had less open water and more obstacles to bat flight. We caught the fewest bats at guzzlers with buried concrete vault drinkers, which impede bat access and have the smallest areas of open water. Water development designs that minimize evaporative water loss by reducing the amount of open water apparently reduce use by bats in this area.     

Variation in bitterbrush ( Purshia tridentata Pursh) crude protein in southwestern Montana

The objective of this study was to determine if crude protein varies significantly during late summer and midwinter among stands of bitterbrush ( Purshia tridentata Pursh) in southwestern Montana. A secondary objective was to determine if leaves, when present, contribute significant additional protein in the region. Nine sites with different environmental conditions within a radius of 14.5 km were studied. Bitterbrush leaves and leaders collected in August 1990 and 1991 and February 1991 were used for crude protein and leaf-to-leader ratio determinations. Crude protein differed ( P < 0.001) among sites for both leaves and leaders on individual collection dates. Crude protein in leaves was nearly twice the level found in leaders. Because few leaves were present in February, they increased crude protein in total foliage by only 0.3% over twigs alone. February crude protein levels averaged 6.8% for total foliage, which is below the estimated requirement for wintering deer.     

Populations and habitat relationships of Piute ground squirrels in southwestern Idaho

Piute ground squirrels ( Spermophilus mollis idahoensis ) are normally above ground from late January until late June or early July in the Snake River Birds of Prey National Conservation Area in southwestern Idaho. In 2002 they were rarely seen above ground after early May. Because of the ecological importance of ground squirrels for nesting raptors and other species, we sought to determine the reasons for their early disappearance. We sampled 12 sites from January 2003 through March 2003 to determine if a population crash had occurred in 2002. Tests indicated that Piute ground squirrels had not been exposed to plague within the past year. The presence of yearlings in the population indicated that squirrels reproduced in 2002 and that at least some yearlings survived the winter. Both yearling and adult squirrels appeared to be reproducing at or above normal rates in 2003. The most plausible explanation for the early disappearance of Piute ground squirrels in 2002 is that squirrels entered seasonal torpor early in response to a late spring drought. In addition, the breeding chronology of squirrels may have shifted during the past 2 decades in response to climate change and/or habitat alteration. Shrub habitats provide a more favorable and stable environment for squirrels than grass habitats. Squirrel abundance was higher on live-trapping grids with sagebrush than on grids dominated by grass, and squirrel masses were higher at sites dominated by shrubs and Sandberg bluegrass ( Poa secunda ). Densities in big sagebrush ( Artemisia tridentata ) were within the ranges reported for earlier years, but densities in grass were lower than previously reported. Low densities at grassland sites in 2003 support other findings that drought affects squirrels in altered grass communities more than those in native shrub habitats. Long-term shifts in ground squirrel breeding chronology may have implications for raptors that depend on them for food.