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BackgroundThe Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan.ResultsAc encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms.ConclusionsOur analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Generating the raw data for a de novo genome assembly project for a target eukaryotic species is relatively easy. This democratization of access to large-scale data has allowed many research teams to plan to assemble the genomes of non-model organisms. These new genome targets are very different from the traditional, inbred, laboratory-reared model organisms. They are often small, and cannot be isolated free of their environment – whether ingested food, the surrounding host organism of parasites, or commensal and symbiotic organisms attached to or within the individuals sampled. Preparation of pure DNA originating from a single species can be technically impossible, but assembly of mixed-organism DNA can be difficult, as most genome assemblers perform poorly when faced with multiple genomes in different stoichiometries. This class of problem is common in metagenomic datasets that deliberately try to capture all the genomes present in an environment, but replicon assembly is not often the goal of such programs. Here we present an approach to extracting, from mixed DNA sequence data, subsets that correspond to single species’ genomes and thus improving genome assembly. We use both numerical (proportion of GC bases and read coverage) and biological (best-matching sequence in annotated databases) indicators to aid partitioning of draft assembly contigs, and the reads that contribute to those contigs, into distinct bins that can then be subjected to rigorous, optimized assembly, through the use of taxon-annotated GC-coverage plots (TAGC plots). We also present Blobsplorer, a tool that aids exploration and selection of subsets from TAGC-annotated data. Partitioning the data in this way can rescue poorly assembled genomes, and reveal unexpected symbionts and commensals in eukaryotic genome projects. The TAGC plot pipeline script is available from https://github.com/blaxterlab/blobology, and the Blobsplorer tool from https://github.com/mojones/Blobsplorer.

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The synthesis of trans-[RuCl(NO)(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane) can be accomplished by either the addition of cyclam to K2[RuCl5NO] or by the addition of NO to trans-[RuCl(CF3SO3)(cyclam)](CF3-SO3). Crystals of trans-[RuCl(NO)(cyclam)](ClO4)2 form in the monoclinic space group P2(1)/c, with unit cell parameters of a = 7.66500(2) A, b = 24.7244(1) A, c = 16.2871(2) A, beta = 95.2550(10) degrees, and Z = 4. One of the two independent molecules in the unit cell lies disordered on a center of symmetry. For the ion in the general position, the Ru-N and N-O bond distances and the [Ru-N-O]3+ bond angle are 1.747(4) A, 1.128(5) A, 178.0(4) degrees, respectively. In both ions, cyclam adopts the (R,R,S,S) configuration, which is also consistent with 2D COSY 1H NMR studies in aqueous solution. Reduction (E degree = -0.1 V) results in the rapid loss of Cl- by first-order kinetics with k = 1.5 s-1 and the slower loss of NO (k = 6.10 x 10(-4) s-1, delta H++ = 15.3 kcal mol-1, delta S++ = -21.8 cal mol-1 K-1). The slow release of NO following reduction causes trans-[RuCl(NO)(cyclam)]2+ to be a promising controlled-release NO prodrug for vasodilation and other purposes. Unlike the related complex trans-[Ru(NO)(NH3)4(P(OEt)3)](PF6)2, trans-[RuCl(NO)(cyclam)]Cl2 is inactive in modulating evoked potentials recorded from mice hippocampal slices probably because of the slower dissociation of NO following reduction.

Telomeres do not shorten with age in longest-lived bats.

Ruthenium red is a well-known and effective inhibitor of the mitochondrial Ca2+ uniporter; however, Reed and Bygrave [(1974) FEBS Lett. 46, 109-114] tentatively attributed this inhibition to a colorless impurity present in commercial samples of ruthenium red (RR). This component has now been isolated and a derivative, (mu-O) [(HCO2)(NH3)4Ru]2Cl3, structurally characterized. The active species in solution appears to be the symmetrical oxo-bridged ion, [X(NH3)4Ru-O-Ru(NH3)4X]3+, where X = Cl- or OH-. Its absorption spectrum shows a maximum at 360 nm. The dinuclear ruthenium ammine complex inhibits Ca(2+)-stimulated respiration of rat liver mitochondria with an I50 of 3.5 pmol/mg of protein compared to the value of 60 pmol of RR/mg of protein. The inhibition by the dinuclear compound is noncompetitive with Ca2+. Respiration-linked swelling of mitochondria induced by Cd2+ also responds similarly to both the dinuclear complex and RR. A close correlation was observed between binding to mitochondria as monitored with 103Ru-labeled dinuclear complex and inhibition of Ca2+ transport. A Scatchard plot yielded estimates of maximum specific binding and dissociation constant of 7.5 pmol/mg of protein and 1.3 nM, respectively. The inhibitor has the characteristics of a satisfactory affinity ligand for purification of the uniporter.