Genome sequences and great expectations

To assess how automatic function assignment will contribute to genome annotation in the next five years, we have performed an analysis of 31 available genome sequences. An emerging pattern is that function can be predicted for almost two-thirds of the 73,500 genes that were analyzed. Despite progress in computational biology, there will always be a great need for large-scale experimental determination of protein function.     

Natural Transformation-Mediated Transfer of Erythromycin Resistance in Campylobacter coli Strains from Turkeys and Swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10⁻⁵ to 10⁻⁶ for turkey-derived strains but 10⁻⁷ or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 μg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 μg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies

Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, Z_Ca, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the Z_Ca–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.     

Charcoal production in the Mopane woodlands of Mozambique: what are the trade-offs with other ecosystem services?

African woodlands form a major part of the tropical grassy biome and support the livelihoods of millions of rural and urban people. Charcoal production in particular is a major economic activity, but its impact on other ecosystem services is little studied. To address this, our study collected biophysical and social datasets, which were combined in ecological production functions, to assess ecosystem service provision and its change under different charcoal production scenarios in Gaza Province, southern Mozambique. We found that villages with longer histories of charcoal production had experienced declines in wood suitable for charcoal, firewood and construction, and tended to have lower perceived availabilities of these services. Scenarios of future charcoal impacts indicated that firewood and woody construction services were likely to trade-off with charcoal production. However, even under the most extreme charcoal scenario, these services were not completely lost. Other provisioning services, such as wild food, medicinal plants and grass, were largely unaffected by charcoal production. To reduce the future impacts of charcoal production, producers must avoid increased intensification of charcoal extraction by avoiding the expansion of species and sizes of trees used for charcoal production. This is a major challenge to land managers and policymakers in the area.This article is part of the themed issue ‘Tropical grassy biomes: linking ecology, human use and conservation’.     

High affinity interaction between a synthetic, highly negatively charged pentasaccharide and alpha- or beta-antithrombin is predominantly due to nonionic interactions

Idraparinux is a synthetic O-sulfated, O-methylated pentasaccharide that binds tightly to antithrombin (AT) and thereby specifically and efficiently induces the inactivation of the procoagulant protease, factor Xa. In this study, the affinity and kinetics of the interaction of this high-affinity pentasaccharide with alpha- and beta-AT were compared with those of a synthetic pentasaccharide comprising the natural AT-binding sequence of heparin. Dissociation equilibrium constants, Kd, for the interactions of Idraparinux with alpha- and beta-AT were approximately 0.4 and 0.1 nM, respectively, corresponding to an over 100-fold enhancement in affinity compared with that of the normal pentasaccharide. This large enhancement was due to a approximately 400-fold tighter conformationally activated complex formed in the second binding step, whereas the encounter complex established in the first step was approximately 4-fold weaker. The high-affinity and normal pentasaccharides both made a total of four ionic interactions with AT, although the high-affinity saccharide only established one ionic interaction in the first binding step and was compensated by three in the second step, whereas the normal pentasaccharide established two ionic interactions in each step. In contrast, the affinities of the nonionic interactions (Kd approximately 450 and 90 nM for the binding to alpha- and beta-AT, respectively) were considerably higher than those for the normal pentasaccharide and the highest of all AT-saccharide interactions reported so far. The nonionic contribution to the total free energy of the high-affinity pentasaccharide binding to AT thus amounted to approximately 70%. These findings show that nonionic interactions can play a predominant role in the binding of highly charged saccharide ligands to proteins and can be successfully exploited in the design of such biologically active ligands.     

Multiple Comparisons of Treatments with Stable Multivariate Tests in a Two‐Stage Adaptive Design,Including a Test for Non‐Inferiority

The application of stabilized multivariate tests is demonstrated in the analysis of a two‐stage adaptive clinical trial with three treatment arms. Due to the clinical problem, the multiple comparisons include tests of superiority as well as a test for non‐inferiority, where non‐inferiority is (because of missing absolute tolerance limits) expressed as linear contrast of the three treatments. Special emphasis is paid to the combination of the three sources of multiplicity – multiple endpoints, multiple treatments, and two stages of the adaptive design. Particularly, the adaptation after the first stage comprises a change of the a‐priori order of hypotheses.     

Interspecific hybridization causes long‐term phylogenetic discordance between nuclear and mitochondrial genomes in freshwater fishes

Classification, phylogeography and the testing of evolutionary hypotheses rely on correct estimation of species phylogeny. Early molecular phylogenies often relied on mtDNA alone, which acts as a single linkage group with one history. Over the last decade, the use of multiple nuclear sequences has often revealed conflict among gene trees. This observation can be attributed to hybridization, lineage sorting, paralogy or selection. Here, we use 54 groups of fishes from 48 studies to estimate the degree of concordance between mitochondrial and nuclear gene trees in two ecological grades of fishes: marine and freshwater. We test the hypothesis that freshwater fish phylogenies should, on average, show more discordance because of their higher propensity for hybridization in the past. In keeping with this idea, concordance between mitochondrial and nuclear gene trees (as measured by proportion of components shared) is on average 50% higher in marine fishes. We discuss why this difference almost certainly results from introgression caused by greater historical hybridization among lineages in freshwater groups, and further emphasize the need to use multiple nuclear genes, and identify conflict among them, in estimation of species phylogeny.     

Historic Titles and Historic Rights in the Law of the Sea in the Light of the South China Sea Arbitration

Historic titles and historic rights have been a complicated issue in the law of the sea both conceptually and practically. The South China Sea Arbitration between the Philippines and China raised important issues regarding the contemporary relevance and validity of historic claims, and the relationship between the Law of the Sea Convention and historic rights. This articles examines historic rights and historic titles in the law of the sea in the light of the South China Sea Arbitration and evaluates the contribution of the Tribunal's Awards to the clarification of these concepts.     

Hematopoietic differentiation of embryonic stem cells

This review is focused on methods that are used to derive hematopoietic cells from embryonic stem cells (ESCs). One of the strategies that have been recently used to achieve this goal is an approach of mimicking the hematopoietic niche in vitro by using hematopoiesis-supportive feeder cells, cocktails of soluble hematopoietic growth factors and a variety of matrices. While there is clear evidence that it is possible to derive hematopoietic stem cells (HSCs) and subsequently committed hematopoietic progenitors and mature cells from ESCs, there remains the need to address multiple issues including the efficiency of HSCs derivation in vitro and their proper functionality.