QDist--quartet distance between evolutionary trees

SUMMARY: QDist is a program for computing the quartet distance between two unrooted trees, i.e. the number of quartet topology differences between the trees, where a quartet topology is the topological subtree induced by four species. The program is based on an algorithm with running time O(n log2 n), which makes it practical to compare large trees. Available under GNU license. AVAILABILITY: http://www.birc.dk/Software/QDist     

Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans

Heat shock protein (Hsp) 72 is a cytosolic stress protein that is highly inducible by several factors including exercise. Hsp60 is primarily mitochondrial in cellular location, plays a key role in the intracellular protein translocation and cytoprotection, is increased in skeletal muscle by exercise, and is found in the peripheral circulation of healthy humans. Glucose deprivation increases Hsp72 in cultured cells, whereas reduced glycogen availability elevates Hsp72 in contracting human skeletal muscle. To determine whether maintained blood glucose during exercise attenuates the exercise-induced increase in intramuscular and circulating Hsp72 and Hsp60, 6 males performed 120 minutes of semirecumbent cycling at approximately 65% maximal oxygen uptake on 2 occasions while ingesting either a 6.4% glucose (GLU) or sweet placebo (CON) beverage throughout exercise. Muscle biopsies, obtained before and immediately after exercise, were analyzed for Hsp72 and Hsp60 protein expression. Blood samples were simultaneously obtained from a brachial artery, a femoral vein, and the hepatic vein before and during exercise for the analysis of serum Hsp72 and Hsp60. Leg and hepatosplanchnic blood flow were measured to determine Hsp72-Hsp60 flux across these tissue beds. Neither exercise nor glucose ingestion affected the Hsp72 or Hsp60 protein expression in, or their release from, contracting skeletal muscle. Arterial serum Hsp72 increased (P < 0.05) throughout exercise in both trials but was attenuated (P < 0.05) in GLU. This may have been in part because of the increased (P < 0.05) hepatosplanchnic Hsp72 release in CON, being totally abolished (P < 0.05) in GLU. Serum Hsp60 increased (P < 0.05) after 60 minutes of exercise in CON before returning to resting levels at 120 minutes. In contrast, no exercise-induced increase in serum Hsp60 was observed in GLU. We detected neither hepatosplanchnic nor contracting limb Hsp60 release in either trial. In conclusion, maintaining glucose availability during exercise attenuates the circulating Hsp response in healthy humans.     

Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity

Filamentous cyanobacteria of the order Nostocales display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N(2) takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue amiC2 gene in Nostoc punctiforme ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two amiC paralogues from Anabaena sp. strain PCC 7120. The amiC1 (alr0092) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the amiC2 (alr0093) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.     

NetMHCpan,a method for MHC class I binding prediction beyond humans

Binding of peptides to major histocompatibility complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC genomic region (called HLA) is extremely polymorphic comprising several thousand alleles, each encoding a distinct MHC molecule. The potentially unique specificity of the majority of HLA alleles that have been identified to date remains uncharacterized. Likewise, only a limited number of chimpanzee and rhesus macaque MHC class I molecules have been characterized experimentally. Here, we present NetMHCpan-2.0, a method that generates quantitative predictions of the affinity of any peptide–MHC class I interaction. NetMHCpan-2.0 has been trained on the hitherto largest set of quantitative MHC binding data available, covering HLA-A and HLA-B, as well as chimpanzee, rhesus macaque, gorilla, and mouse MHC class I molecules. We show that the NetMHCpan-2.0 method can accurately predict binding to uncharacterized HLA molecules, including HLA-C and HLA-G. Moreover, NetMHCpan-2.0 is demonstrated to accurately predict peptide binding to chimpanzee and macaque MHC class I molecules. The power of NetMHCpan-2.0 to guide immunologists in interpreting cellular immune responses in large out-bred populations is demonstrated. Further, we used NetMHCpan-2.0 to predict potential binding peptides for the pig MHC class I molecule SLA-1*0401. Ninety-three percent of the predicted peptides were demonstrated to bind stronger than 500 nM. The high performance of NetMHCpan-2.0 for non-human primates documents the method’s ability to provide broad allelic coverage also beyond human MHC molecules. The method is available at . Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diffraction-based density restraints for membrane and membrane-peptide molecular dynamics simulations

We have recently shown that current molecular dynamics (MD) atomic force fields are not yet able to produce lipid bilayer structures that agree with experimentally-determined structures within experimental errors. Because of the many advantages offered by experimentally validated simulations, we have developed a novel restraint method for membrane MD simulations that uses experimental diffraction data. The restraints, introduced into the MD force field, act upon specified groups of atoms to restrain their mean positions and widths to values determined experimentally. The method was first tested using a simple liquid argon system, and then applied to a neat dioleoylphosphatidylcholine (DOPC) bilayer at 66% relative humidity and to the same bilayer containing the peptide melittin. Application of experiment-based restraints to the transbilayer double-bond and water distributions of neat DOPC bilayers led to distributions that agreed with the experimental values. Based upon the experimental structure, the restraints improved the simulated structure in some regions while introducing larger differences in others, as might be expected from imperfect force fields. For the DOPC-melittin system, the experimental transbilayer distribution of melittin was used as a restraint. The addition of the peptide caused perturbations of the simulated bilayer structure, but which were larger than observed experimentally. The melittin distribution of the simulation could be fit accurately to a Gaussian with parameters close to the observed ones, indicating that the restraints can be used to produce an ensemble of membrane-bound peptide conformations that are consistent with experiments. Such ensembles pave the way for understanding peptide-bilayer interactions at the atomic level.     

Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases

We have developed a specific and efficient method for complete removal of polyhistidine purification tags (HisTags) from the N-termini of target proteins. The method is based on the use of the aminopeptidase dipeptidyl peptidase I (DPPI), either alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). In both cases, the HisTag is cleaved off by DPPI, which catalyzes a stepwise excision of a wide range of dipeptides from the N-terminus of a peptide chain. Some sequences, however, are resistant to DPPI cleavage and a number of mature proteins have nonsubstrate N-termini which protects them against digestion. For such proteins, HisTags composed of an even number of residues can be cleaved off by treatment with DPPI alone. When the target protein is unprotected against DPPI, a blocking group is generated enzymatically from a glutamine residue inserted between the HisTag and the target protein. A protein with a HisTag-Gln extension is incubated with both DPPI and GCT. As above, the polyhistidine sequence is cleaved off by DPPI, but when the glutamine residue appears in the N-terminus, it is immediately converted into a pyroglutamyl residue by an excess of GCT and further DPPI digestion is prevented. The desired sequence is finally obtained by excision of the pyroglutamyl residue with PGAP. All the enzymes employed can bind to immobilized metal affinity chromatography (IMAC) matrices, and in this paper we demonstrate a simple and highly effective process combining IMAC purification of His-tagged proteins, our aminopeptidase-based method for specific excision of HisTags and use of subtractive IMAC for removing processing enzymes. Typical recoveries were 75-90% for the enzymatic processing and subtractive IMAC. The integrated process holds promises for use in large-scale production of pharmaceutical proteins because of a simple overall design, use of robust and inexpensive matrices, and use of enzymes of either recombinant or plant origin.     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.     

Identification of plant microRNA homologs

MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that regulate gene and protein expression in plants and animals. MiRNAs have so far been identified mostly by specific cloning of small RNA molecules, complemented by computational methods. We present a computational identification approach that is able to identify candidate miRNA homologs in any set of sequences, given a query miRNA. The approach is based on a sequence similarity search step followed by a set of structural filters.     

Environment versus dispersal in the assembly of western Amazonian palm communities

Aim It is a central issue in ecology and biogeography to understand what governs community assembly and the maintenance of biodiversity in tropical rain forest ecosystems. A key question is the relative importance of environmental species sorting (niche assembly) and dispersal limitation (dispersal assembly), which we investigate using a large dataset from diverse palm communities. Location Lowland rain forest, western Amazon River Basin, Peru. Methods We inventoried palm communities, registering all palm individuals and recording environmental conditions in 149 transects of 5 m × 500 m. We used ordination, Mantel tests and indicator species analysis (ISA) to assess compositional patterns, species responses to geographical location and environmental factors. Mantel tests were used to assess the relative importance of geographical distance (as a proxy for dispersal limitation) and environmental differences as possible drivers of dissimilarity in palm species composition. We repeated the Mantel tests for subsets of species that differ in traits of likely importance for habitat specialization and dispersal (height and range size). Results We found a strong relationship between compositional dissimilarity and environmental distance and a weaker but also significant relationship between compositional dissimilarity and geographical distance. Consistent with expectations, relationships with environmental and geographical distance were stronger for understorey species than for canopy species. Geographical distance had a higher correlation with compositional dissimilarity for small‐ranged species compared with large‐ranged species, whereas the opposite was true for environmental distance. The main environmental correlates were inundation and soil nutrient levels. Main conclusions The assembly of palm communities in the western Amazon appears to be driven primarily by species sorting according to hydrology and soil, but with dispersal limitation also playing an important role. The importance of environmental characteristics and geographical distance varies depending on plant height and geographical range size in agreement with functional predictions, increasing our confidence in the inferred assembly mechanisms.     

Nonlinear sorting, curvature generation, and crowding of endophilin N-BAR on tubular membranes

The curvature of biological membranes is controlled by membrane-bound proteins. For example, during endocytosis, the sorting of membrane components, vesicle budding, and fission from the plasma membrane are mediated by adaptor and accessory proteins. Endophilin is a peripherally binding membrane protein that functions as an endocytic accessory protein. Endophilin's membrane tubulation capacity is well known. However, to understand the thermodynamic and mechanical aspects of endophilin function, experimental measurements need to be compared to quantitative theoretical models. We present measurements of curvature sorting and curvature generation of the endophilin A1 N-BAR domain on tubular membranes pulled from giant unilamellar vesicles. At low concentration, endophilin functions primarily as a membrane curvature sensor; at high concentrations, it also generates curvature. We determine the spontaneous curvature induced by endophilin and observe sigmoidal curvature/composition coupling isotherms that saturate at high membrane tensions and protein solution concentrations. The observation of saturation is supported by a strong dependence of lateral diffusion coefficients on protein density on the tether membrane. We develop a nonlinear curvature/composition coupling model that captures our experimental observations. Our model predicts a curvature-induced phase transition among two states with varying protein density and membrane curvature. This transition could act as a switch during endocytosis.