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5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.

About the British Society for Parasitology. About.Today many researchers and students are very passionate about the fascinating world of infectious diseases, parasites, their complex lifestyles and their associated impact on people and livelihoods.To draw attention to the unique importance of parasitology as a distinct discipline within biology, The British Society for Parasitology was formed in 1962 from the Parasitological Section of the Institute of Biology. Today the Society is the central networking and meeting point for many professional and amateur parasitologists throughout the UK and across the world.Did you know that the UK leads Europe – and Europe leads the world – in parasitology research? No European country publishes more parasitology research than the UK, and UK papers were cited more than those from any other country in the past 5 years (2011-2016, data from Elsevier SciVal, see News item for more details.)As the leading academic society for a country preeminent in parasitology, the remit of the BSP is broad. It promotes and supports the academic study of parasitology in all its many guises. This can be from experimental to theoretical approaches as applied to infection biology and disease research, or from ecological to medical and veterinary studies in global health and international aid. Each year students are given financial support to attend BSP meetings and scholarship schemes are in place to support fieldwork and training events.The membership of the BSP stands at around 1000 in number. Approximately a third of members are from overseas locations. Highlights of the annual BSP calendar include the annual residential meetings in spring and autumn which are focused upon general and specialist aspects of parasitology. The BSP has a close relationship with Cambridge University Press that prints a special issue on our autumn meeting.The BSP Society is a Charitable Incorporated Organisation and managed by the BSP Council. This comprises a President, Honorary Officers and ordinary Council Members, who together act as Company Trustees. Co-opted members of Council also include representation from the student, early career membership and other learned societies where clear synergies are apparent.

There are hundreds of Trypanosoma species that live in the blood and tissue spaces of their vertebrate hosts. The vast majority of these do not have the ornate system of antigenic variation that has evolved in the small number of African trypanosome species, but can still maintain long term infections in the face of the vertebrate adaptive immune system. Trypanosoma theileri is a typical example, it has a restricted host range of cattle and other Bovinae and is only occasionally reported to cause patent disease although no systematic survey of the effect of infection on agricultural productivity has been performed. Here, a detailed genome sequence and a transcriptome analysis of gene expression in bloodstream form T. theileri have been performed. Analysis of the genome sequence and expression showed that T. theileri has a typical kinetoplastid genome structure and allowed a prediction that it is capable of meiotic exchange, gene silencing via RNA interference and, potentially, density-dependent growth control. In particular, the transcriptome analysis has allowed a comparison of two distinct trypanosome cell surfaces, T. brucei and T. theileri, that have each evolved to enable the maintenance of a longterm extracellular infection in cattle. The T. theileri cell surface can be modelled to contain a mixture of proteins encoded by four novel large and divergent gene families and by members of a major surface protease gene family. This surface composition is distinct from the uniform variant surface glycoprotein coat on African trypanosomes providing an insight into a second mechanism used by trypanosome species that proliferate in an extracellular milieu in vertebrate hosts to avoid the adaptive immune response.

Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton., Graphical abstract Image 1, Highlights • Super-resolution imaging of motile cells becomes feasible using hydrogel embedding. • The cytosolic leaflet of the plasma membrane of T. brucei is structured. • Lipid-anchored eYFP preferentially localizes to domains of r = (170 ± 5) nm. • Lipid-anchored eYFP appears diluted in domains of r > (115 ± 15) nm.

Supplementary analyses. (DOCX 17 kb)

Figure S1. Organisation of open reading frames in the E. gracilis genome. Figure S2. Functional analysis of E. gracilis coding capacity by Gene Ontology. Figure S3. Dark adapted cells have altered proteomes and transcriptomes. Figure S4. Orthogroup clusters in E. gracilis and selected eukaryotes. Figure S5. Phylogeny of selected shared large paralog families. Figure S6. Surface families of E. gracilis. Figure S7. The E. gracilis endomembrane system. Figure S8. The E. gracilis nuclear pore and kinetochore complexes. Figure S9. The predicted proteomes of E. gracilis organelles. Figure S10. Metabolism in E. gracilis. Figure S11. Additional assembly features. Figure S12. BUSCO comparisons between the present work and prior transcriptomes. (PDF 10993 kb)

Table S6. Predicted proteomes for the E. gracilis plastid and the mitochondrion. Panels include summaries for each organelle for numbers of genes in functional categories found, annotations for transcripts predicted as mitochondrial or chloroplastic and finally a reconstruction of major mitochondrial complexes and pathways. (DOCX 141 kb)

Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.orgilicensesiby/4.0/)., This work was funded by the DFG (Kr4017/1-1 and Kr4017/1-2 to SK), by the Wellcome trust (grant number 085956/Z/08/Z to MC) and by the Spanish Ministerio de Economia e Innovacion (BFU2014-55193-P to AE). Markus Engstler (University of Wurzburg) is acknowledged for mentoring and providing lab space and research infrastructure. Anti-BiP was kindly provided by Jay Bangs (University of Wisconsin-Madison, Madison, US).

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that approximate to 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency., MN is funded by grants from the Spanish Ministerio de Ciencia e Innovacion, (SAF2012-40029), Junta de Andalucia (CTS-5841) and VI PN de I+D+I 2008-2011, Instituto de Salud Carlos III - Subdireccion General de Redes y Centros de Investigacion Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by 'Fondo de Investigacion Sanitaria' PI10/01128. JR and MC were funded by a Wellcome Trust Project Grant 093008/Z/10/Z. Work in the Dundee laboratory was funded by the Wellcome Trust (program grant 093008/Z/10/Z) and the Medical Research Council.

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms.