Golden bananas in the field: elevated fruit pro‐vitamin A from the expression of a single banana transgene

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro‐vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA‐biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β‐carotene equivalent (β‐CE) in the fruit. Expression of a Fe'i banana‐derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β‐CE . Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1‐aminocyclopropane‐1‐carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild‐type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate‐limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.     

Evaluating future success of whitebark pine ecosystem restoration under climate change using simulation modeling

Major declines of whitebark pine forests throughout western North America from the combined effects of mountain pine beetle (Dendroctonus ponderosae) outbreaks, fire exclusion policies, and the exotic disease white pine blister rust (WPBR) have spurred many restoration actions. However, projected future warming and drying may further exacerbate the species' decline and possibly compromise long‐term success of today's restoration activities. We evaluated successes of restoration treatments under future climate using a comprehensive landscape simulation experiment. The spatially explicit, ecological process model FireBGCv2 was used to simulate whitebark pine populations on two U.S. Northern Rocky Mountain landscapes over 95 years under two climate, three restoration, and two fire management scenarios. Major findings were that (1) whitebark pine can remain on some high mountain landscapes in a future climate albeit at lower basal areas (50% decrease), (2) restoration efforts, such as thinning and prescribed burning, are vital to ensure future whitebark pine forests, and (3) climate change impacts on whitebark pine vary by local setting. Whitebark pine restoration efforts will mostly be successful in the future but only if future populations are somewhat resistant to WPBR. Results were used to develop general guidelines that address climate change impacts for planning, designing, implementing, and evaluating fine‐scale restoration activities.     

Genetic analysis of emerald ash borer (<Emphasis Type="Italic">Agrilus planipennis</Emphasis> Fairmaire) populations in Asia and North America

Emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an invasive pest of North American ash (Fraxinus spp.) trees first discovered outside of its native range of northeastern Asia in 2002. EAB spread from its initial zone of discovery in the Detroit, Michigan and Windsor, Ontario metropolitan areas, in large part, from inadvertent human-assisted movement of infested ash materials. EAB infestations are now known in 15 US states and two Canadian provinces. The primary goal of this study was to use molecular markers to characterize the population genetic structure of EAB in its native and introduced range. This information may provide valuable insights on the geographic origin, potential host range, invasion potential, and additional biological control agents for ongoing management efforts of this destructive wood-boring beetle. EAB were collected from 17 localities in its native Asian range and from 7 localities in North America, and population structure analyzed using mtDNA gene sequences, AFLP fingerprints, and alleles at 2 microsatellite loci. Analysis of mtDNA cytochrome oxidase subunit I gene (COI; 439 bp) sequences revealed all North American individuals carry a common mtDNA haplotype also found in China and South Korea. Additional mtDNA haplotypes observed in China and South Korea differed from the common haplotype by 1–2 nucleotide substitutions and a single individual from Japan differed by 21 nucleotide changes (4.8%). Analysis using AFLP fingerprints (108 loci) indicated Asian populations were more highly variable, yet had less overall population structure, than the North American populations. North American populations appear most closely related to populations in our sample from the Chinese provinces of Hebei and Tianjin City. Further, population assignment tests assigned 88% of the individual beetles from North America to either Hebei or Tianjin City.     

Summer zooplankton in small rivers in relation to selected conditions

The abundance and the biodiversity of summer zooplankton in the waters of the Drawa drainage (NW Poland) were studied, as was their relation to selected environmental conditions. The conditions upstream, especially in the outlets of lakes, did not affect the zooplankton communities downstream. This was also true of tributaries which had no influence on the shape of the zooplankton communities in the main river. The number of zooplankton in the outlets of eutrophic lakes was greater than in those of mesotrophic lakes. Increased vegetation cover significantly affected mainly the crustacean communities of zooplankton. Larger amounts of zooplankton were observed in rivers where the riparian zone was not covered with vegetation, but this difference was not significant. The hydrological conditions of the rivers and the Secchi depth visibility strongly impacted the composition of the zooplankton. The influence of abiotic factors was most pronounced on the abundance of cladocerans, and least pronounced on the abundance of rotifers.     

At the north-eastern extremity: variation in <Emphasis Type="Italic">Cepaea nemoralis</Emphasis> around Gdańsk,northern Poland

Variation in the shell colour and banding polymorphism in the land snail Cepaea nemoralis was studied in 260 populations in the region of Gdańsk, northern Poland. Unlike in other regions of Poland, many populations contain brown shells. Populations from shaded habitats have higher frequencies of brown than those from open and intermediate habitats, largely at the expense of yellow shells. Nearly all brown shells are also unbanded. Apart from this disequilibrium, banding morphs among yellow and pink shells show no relationship to habitat. There are no broad geographical trends in morph-frequencies, but there are very strong correlations among populations very close together, revealed both by pairwise analysis and Moran’s I. Principal Component Analyses show that these correlations relate to overall genetic similarity at the loci involved. The populations are at the north-eastern limits of the species’ range; habitats are mostly anthropogenic, and comparisons with studies in two urban areas (Wrocław, SW Poland, and Sheffield, central England) suggest that the patterns of variation seen are a product of human transport of propagules followed by local dispersal. The effect of habitat here is much less marked than in regions much further west, but it indicates that natural selection has occurred.     

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.     

Structural basis for executioner caspase recognition of P5 position in substrates

Caspase-3, -6 and -7 cleave many proteins at specific sites to induce apoptosis. Their recognition of the P5 position in substrates has been investigated by kinetics, modeling and crystallography. Caspase-3 and -6 recognize P5 in pentapeptides as shown by enzyme activity data and interactions observed in the crystal structure of caspase-3/LDESD and in a model for caspase-6. In caspase-3 the P5 main-chain was anchored by interactions with Ser209 in loop-3 and the P5 Leu side-chain interacted with Phe250 and Phe252 in loop-4 consistent with 50% increased hydrolysis of LDEVD relative to DEVD. Caspase-6 formed similar interactions and showed a preference for polar P5 in QDEVD likely due to interactions with polar Lys265 and hydrophobic Phe263 in loop-4. Caspase-7 exhibited no preference for P5 residue in agreement with the absence of P5 interactions in the caspase-7/LDESD crystal structure. Initiator caspase-8, with Pro in the P5-anchoring position and no loop-4, had only 20% activity on tested pentapeptides relative to DEVD. Therefore, caspases-3 and -6 bind P5 using critical loop-3 anchoring Ser/Thr and loop-4 side-chain interactions, while caspase-7 and -8 lack P5-binding residues.     

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.     

Biodegradation of Bis(2-Chloroethyl) Ether by Xanthobacter sp. Strain ENV481

Degradation of bis(2-chloroethyl) ether (BCEE) was observed to occur in two bacterial strains. Strain ENV481, a Xanthobacter sp. strain, was isolated by enrichment culturing of samples from a Superfund site located in the northeastern United States. The strain was able to grow on BCEE or 2-chloroethylethyl ether as the sole source of carbon and energy. BCEE degradation in strain ENV481 was facilitated by sequential dehalogenation reactions resulting in the formation of 2-(2-chloroethoxy)ethanol and diethylene glycol (DEG), respectively. 2-Hydroxyethoxyacetic acid was detected as a product of DEG catabolism by the strain. Degradation of BCEE by strain ENV481 was independent of oxygen, and the strain was not able to grow on a mixture of benzene, ethylbenzene, toluene, and xylenes, other prevalent contaminants at the site. Another bacterial isolate, Pseudonocardia sp. strain ENV478 (S. Vainberg et al., Appl. Environ. Microbiol. 72:5218-5224, 2006), degraded BCEE after growth on tetrahydrofuran or propane but was not able to grow on BCEE as a sole carbon source. BCEE degradation by strain ENV478 appeared to be facilitated by a monooxygenase-mediated O-dealkylation mechanism, and it resulted in the accumulation of 2-chloroacetic acid that was not readily degraded by the strain.