Laboratory observations on spawning and embryonic development of a blue-ringed octopus

A female blue-ringed octopus, Hapalochlaena sp. (probably lunulata), was maintained in an aqarium for nearly 3 months, during which time it spawned and cared for the eggs until hatching. The young are planktonic. Embryonic development does not differ markedly from that observed in other octopods, uniting certain features of the development of Octopus vulgaris and Eledone cirrosa. In terms of reproductive biology and development, the species differs from the lesser blue-ringed octopus Hapalochlaena maculosa in its mode of spawning, egg size and mode of life of its young. These differences correspond to conditions recorded for Octopus spp. and Eledone spp

Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co-ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co-ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear-encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N-terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear-encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion

Area minimizing discs in metric spaces

We solve the classical problem of Plateau in the setting of proper metric spaces. Precisely, we prove that among all disc-type surfaces with prescribed Jordan boundary in a proper metric space there exists an area minimizing disc which moreover has a quasi-conformal parametrization. If the space supports a local quadratic isoperimetric inequality for curves we prove that such a solution is locally Hölder continuous in the interior and continuous up to the boundary. Our results generalize corresponding results of Douglas Radò and Morrey from the setting of Euclidean space and Riemannian manifolds to that of proper metric spaces

Multiple FadD Acyl-CoA Synthetases Contribute to Differential Fatty Acid Degradation and Virulence in Pseudomonas aeruginosa

A close interconnection between nutrient metabolism and virulence factor expression contributes to the pathophysiology of Pseudomonas aeruginosa as a successful pathogen. P. aeruginosa fatty acid (FA) degradation is complicated with multiple acyl-CoA synthetase homologs (FadDs) expressed in vivo in lung tissue during cystic fibrosis infections. The promoters of two genetically linked P. aeruginosa fadD genes (fadD1 and fadD2) were mapped and northern blot analysis indicated they could exist on two different transcripts. These FadDs contain ATP/AMP signature and FA-binding motifs highly homologous to those of the Escherichia coli FadD. Upon introduction into an E. coli fadD-/fadR- double mutant, both P. aeruginosa fadDs functionally complemented the E. coli fadD-/fadR- mutant, allowing degradation of different chain-length FAs. Chromosomal mutagenesis, growth analysis, induction studies, and determination of kinetic parameters suggested that FadD1 has a substrate preference for long-chain FAs while FadD2 prefers shorter-chain FAs. When compared to the wild type strain, the fadD2 mutant exhibited decreased production of lipase, protease, rhamnolipid and phospholipase, and retardation of both swimming and swarming motilities. Interestingly, fadD1 mutant showed only increased swarming motility. Growth analysis of the fadD mutants showed noticeable deficiencies in utilizing FAs and phosphatidylcholine (major components of lung surfactant) as the sole carbon source. This defect translated into decreased in vivo fitness of P. aeruginosa in a BALB/c mouse lung infection model, supporting the role of lipids as a significant nutrient source for this bacterium in vivo

High-resolution analysis of multi-copy variant surface glycoprotein gene expression sites in African trypanosomes

BACKGROUND: African trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The expressed VSGs are located at the ends of telomeric, polycistronic transcription units known as VSG expression sites (VSG-ESs). Each cell has many VSG-ESs but only one is transcribed in bloodstream-form parasites and all of them are inactive upon transmission to the insect vector mid-gut; a subset of monocistronic metacyclic VSG-ESs are then activated in the insect salivary gland. Deep-sequence analyses have been informative but assigning sequences to individual VSG-ESs has been challenging because they each contain closely related expression-site associated genes, or ESAGs, thought to contribute to virulence. RESULTS: We utilised ART, an in silico short read simulator to demonstrate the feasibility of accurately aligning reads to VSG-ESs. Then, using high-resolution transcriptomes from isogenic bloodstream and insect-stage Lister 427 Trypanosoma brucei, we uncover increased abundance in the insect mid-gut stage of mRNAs from metacyclic VSG-ESs and of mRNAs from the unusual ESAG, ESAG10. Further, we show that the silencing associated with allelic exclusion involves repression focussed at the ends of the VSG-ESs. We also use the approach to report relative fitness costs following ESAG RNAi from a genome-scale screen. CONCLUSIONS: By assigning sequences to individual VSG-ESs we provide new insights into VSG-ES transcription control, allelic exclusion and impacts on fitness. Thus, deeper insights into the expression and function of regulated multi-gene families are more accessible than previously anticipated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-3154-8) contains supplementary material, which is available to authorized users

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

A Novel Soluble Immune-Type Receptor (SITR) in Teleost Fish: Carp SITR Is Involved in the Nitric Oxide-Mediated Response to a Protozoan Parasite

Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite

Identification and Characterization of an Unusual Class I Myosin Involved in Vesicle Traffic in Trypanosoma brucei

Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of ∼90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites