The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations

Pyruvate kinase (PK) is the key control point of glycolysis—the biochemical pathway central to energy metabolism and the production of precursors used in biosynthesis. PK type 1 from Escherichia coli (Ec-PK1) is activated by both fructose-1,6-bisphosphate (FBP) and its substrate, phosphoenol pyruvate (PEP). To date, it has not been possible to determine whether the enzyme is tetrameric at the low concentrations (i.e. low nM range) used to study the steady-state kinetics, or assess whether its allosteric effectors alter the oligomeric state of the enzyme at these concentrations. Employing the new technique of analytical ultracentrifugation with fluorescence detection we have, for the first time, shown that the K_D^4–2 for Ec-PK1 is in the subnanomolar range, well below the concentrations used in kinetic studies. In addition, we show that, unlike some other PK isoenzymes, the modulation of oligomeric state by the allosteric effectors FBP and PEP does not occur at a concentration of 10 nM or above.     

Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Application of the Three Rs to challenge assays used in vaccine testing: Tenth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement

This report aims to facilitate the implementation of the Three Rs (reduction, refinement and replacement) in the testing of vaccines for regulatory and other purposes. The focus is predominantly on identification of reduction and refinement opportunities in batch potency testing but the principles described are widely applicable to other situations that involve experimental infections of animals. The report should also help to interpret the requirements of the European Pharmacopoeia with regard to the use of alternative tests, humane endpoints and other refinements. Two specific worked examples, for batch potency testing of Clostridium chauvoei and canine leptospira, with recommendations for harmonisation of international test requirements for these and other vaccines, are provided as appendices online.     

Habitat Overlap and Facilitation in Tamarisk and Box Elder Stands: Implications for Tamarisk Control Using Native Plants

Invasive plants are typically managed using top‐down control techniques that focus on the removal of the target organism. Bottom‐up control limits the resources available to the undesired species by manipulating disturbance, competition, and successional processes, and thus may prevent reinvasion. Tamarisk species (Tamarix sp.) have invaded riparian areas throughout western North America, resulting in expansive control efforts. A companion study has shown that a native competitor, Box elder (Acer negundo), is capable of outcompeting and killing established Tamarisk through light interception in canyons of Dinosaur National Monument (DNM), Colorado. The goal of this study was to determine the feasibility of using Box elder as a bottom‐up control agent by (1) determining the distributional overlap of the two species in DNM; (2) determining if Tamarisk facilitates Box elder establishment; and (3) analyzing Box elder seedling survival across a range of physical gradients. The distribution of Tamarisk and Box elder overlapped considerably throughout the study area. Box elder seedlings were planted under Tamarisk canopies or areas with the canopy removed. Survival was significantly higher under Tamarisk canopies, indicating that Tamarisk facilitates Box elder seedling survival. Box elder seedling survival was tested across soil texture, litter depth, groundwater depth, and shade intensities indicative of conditions found in the canyons of DNM, and survival was high for all treatments. The manipulation of competitive and successional processes through the promotion of Box elder and other native tree establishment is suggested as a means of bottom‐up Tamarisk control to complement traditional control techniques.     

Virulence for chickens of Clostridium perfringens isolated from poultry and other sources

Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four “major” toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched Cornish × Rock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2^pos), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2^pos, human food poisoning), JGS1936 (genotype A, cpb2^neg, bovine neonatal enteritis), JGS4142 (genotype A, cpb2^pos, bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2^pos, chicken normal flora), JGS1070 (genotype C, cpb2^pos, porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2^pos, porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2^neg, gas gangrene), JGS4151 (strain 13, genotype A, cpb2^pos, canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2^neg, human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease.     

Relative influence of male and female care in determining nestling mass in a migratory songbird

Biparental care is common in birds, with the allocation of effort being highly variable between the sexes. In most songbird species, the female typically provides the most care early in the breeding cycle with both parents providing care when provisioning young. Food provisioning should be directly related to offspring quality; however, the relative influence each parent has on offspring quality has rarely been assessed at the nest level. Consequently, we were interested in assessing the relative influence male and female provisioning has on one measurement of offspring quality, nestling mass, in the black‐throated blue warbler Dendroica caerulescens. Over a six year period, 2003–2008, we collected information on average nestling mass per brood on day 6 of the nestling cycle and parental provisioning rates on day 7 of the nestling cycle from 182 first brood nests on three different study plots. We found that average nestling mass was directly related to male provisioning rate, while it was not related to female provisioning rate. On the other hand, estimated biomass provisioned had little influence on average nestling mass, calling into question its utility in assessing parental quality. Finally, there was some indication that parental influence on average nestling mass was dependent on the other parent's provisioning rate, suggesting that parents work in concert to influence nestling quality.     

Marine biological community baselines in unimpacted tropical ecosystems: spatial and temporal analysis of reefs at Howland and Baker Islands

Howland and Baker Islands are two small, isolated reef and sand islets located near the equator in the central Pacific Ocean that are situated approximately 60 km apart. In 2004 and 2006, species-level monitoring at multiple sites, coupled with towed-diver surveys in 2002, 2004, and 2006 on both of these federally protected islands, revealed diverse fish, coral, macroinvertebrate, and algal assemblages. This study examines inter- and intra-island spatial and temporal differences in community composition among sites and presents baseline biological community parameters for two of the least impacted reef systems in the world. Despite similarities in species composition, permutational multivariate analysis of variance (PERMANOVA) and multidimensional scaling ordinations (nMDS) suggest biological communities at the two islands are distinct with Baker Island containing a greater percent cover of branched Acroporid corals and turf algae and Howland Island containing a greater percent cover of crustose coralline red algae and small, compact genera of coral. Both islands also contained considerable cover of non-invasive macroalgae. PERMANOVA further revealed benthic and fish species composition to differ between forereef and reef shelf sites from different sides of each island. When islands were considered as a whole, temporal changes were not noted between 2004 and 2006; however, temporal changes at select sites did occur, with coral cover decreasing significantly along the west side of Baker Island from 2004 to 2006.     

Coupled mathematical model of tumorigenesis and angiogenesis in vascular tumours

Objectives: Mathematical models are useful for studying vascular and avascular tumours, because these allow for more logical experimental design and provide valuable insights into the underlying mechanisms of their growth and development. The processes of avascular tumour growth and the development of capillary networks through tumour‐induced angiogenesis have already been extensively investigated, albeit separately. Despite the clinical significance of vascular tumours, few studies have combined these approaches to develop a single comprehensive growth and development model. Materials and methods: We develop a continuum‐based mathematical model of vascular tumour growth. In the model, angiogenesis is initiated through the release of angiogenic growth factors (AGFs) by cells in the hypoxic regions of the tumour. The nutrient concentration within the tumour reflects the influence of capillary growth and invasion induced by AGF. Results and conclusions: Parametric and sensitivity studies were performed to evaluate the influence of different model parameters on tumour growth and to identify the parameters with the most influence, which include the rates of proliferation, apoptosis and necrosis, as well as the diffusion of sprout tips and the size of the region affected by angiogenesis. An optimization was performed for values of the model parameters that resulted in the best agreement with published experimental data. The resulting model solution matched the experimental data with a high degree of correlation (r = 0.85).     

The state and conservation of Southeast Asian biodiversity

Southeast Asia is a region of conservation concern due to heavy losses of its native habitats. In this overview, we highlight the conservation importance of Southeast Asia by comparing its degree of species endemism and endangerment, and its rate of deforestation with other tropical regions (i.e., Meso-America, South America, and Sub-Saharan Africa). Southeast Asia contains the highest mean proportion of country-endemic bird (9%) and mammal species (11%). This region also has the highest proportion of threatened vascular plant, reptile, bird, and mammal species. Furthermore, not only is Southeast Asia’s annual deforestation rate the highest in the tropics, but it has also increased between the periods 1990–2000 and 2000–2005. This could result in projected losses of 13–85% of biodiversity in the region by 2100. Secondary habitat restoration, at least in certain countries, would allow for some amelioration of biodiversity loss and thus potentially lower the currently predicted extinction rates. Nonetheless, urgent conservation actions are needed. Conservation initiatives should include public education, sustaining livelihoods, and ways to enhance the sustainability of agriculture and increase the capacity of conservation institutions. Furthermore, these actions should be country-specific and not ignore areas heavily populated by humans, as they can also harbour high numbers of threatened species. We urge that cooperative conservation initiatives be undertaken and support (e.g., capacity-building) be given by more developed countries in the region and beyond.