Complex patterns of gene fission in the eukaryotic folate biosynthesis pathway

This is the final version. Available on open access from Oxford University Press via the DOI in this recordShared derived genomic characters can be useful for polarizing phylogenetic relationships, for example, gene fusions have been used to identify deep-branching relationships in the eukaryotes. Here, we report the evolutionary analysis of a three-gene fusion of folB, folK, and folP, which encode enzymes that catalyze consecutive steps in de novo folate biosynthesis. The folK-folP fusion was found across the eukaryotes and a sparse collection of prokaryotes. This suggests an ancient derivation with a number of gene losses in the eukaryotes potentially as a consequence of adaptation to heterotrophic lifestyles. In contrast, the folB-folK-folP gene is specific to a mosaic collection of Amorphea taxa (a group encompassing: Amoebozoa, Apusomonadida, Breviatea, and Opisthokonta).Next, we investigated the stability of this character.We identified numerous gene losses and a total of nine gene fission events, either by break up of an open reading frame (four events identified) or loss of a component domain (five events identified). This indicates that this three gene fusion is highly labile. These data are consistent with a growing body of data indicating gene fission events occur at high relative rates. Accounting for these sources of homoplasy, our data suggest that the folB-folK-folP gene fusion was present in the last common an castor of Amoebozoa and Opisthokonta but absent inthe Metazoa including the human genome. Comparative genomic data of these genes provides an important resource for designing therapeutic strategies targeting the de novo folate biosynthesis pathway of a variety of eukaryotic pathogens such as Acanthamoeba castellanii.Society of General MicrobiologyTula Foundation (The Centre for Comparative Genomics and Evolutionary Bioinformatics at Dalhousie University)Gordon and Betty Moore FoundationLeverhulme TrustRoyal SocietyNatural Environment Research Council (NERC)Biotechnology and Biological Sciences Research Council (BBSRC

Diverse molecular signatures for ribosomally 'active' Perkinsea in marine sediments

This is the final published PDF. Available from BMC via the DOI in this record.Background Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity ‘tag’ sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. Results We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. Conclusions These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally ‘active’ and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the ‘seed bank’ microbial community.Marie Curie Intra-European Fellowship grantEMBO Long-Term fellowshipGordon and Betty Moore Foundatio

Intracellular Infection of Diverse Diatoms by an Evolutionary Distinct Relative of the Fungi

This is the final version. Available on open access from Elsevier via the DOI in this recordData and Code Availability: All data and code are available with DOI’s given in the methods section. Specifically, physical and chemical parameters of the water column obtained using CTD ocean water sampling are available at http://biomarks.eu/ctd007 (and replicated here at figshare DOI: 10.6084/m9.figshare.9821936). The phylogenetic tree file, masked and unmasked SSU rDNA alignments are available at Zenodo repository: DOI 10.5281/zenodo.2788876. All sequence data used were derived from the NCBI ‘GenBank’ database and accession numbers are provided in Figure. 1B. The R code used to test statistical association in the FISH data are available at Zenodo repository: DOI 10.5281/zenodo.2788876.The Fungi are a diverse kingdom, dominating terrestrial environments and driving important ecologies. Although fungi, and the related Opisthosporidia, interact with photosynthetic organisms on land and in freshwater as parasites, symbionts, and/or saprotrophic degraders, such interactions in the marine environment are poorly understood. One newly identified uncultured marine lineage has been named novel chytrid-like-clade-1 (NCLC1) or basal-clone-group-I. We use ribosomal RNA (rRNA) encoding gene phylogenies to demonstrate that NCLC1 is a distinct branch within the Opisthosporidia (Holomycota). Opisthosporidia are a diverse and largely uncultured group that form a sister branch to the Fungi or, alternatively, the deepest branch within the Fungi, depending on how the boundary to this kingdom is inferred. Using culture-free lineage-specific rRNA-targeted fluorescent in situ hybridization (FISH) microscopy, we demonstrate that NCLC1 cells form intracellular infection of key diatom species, establishing that intracellular colonization of a eukaryotic host is a consistent lifestyle across the Opisthosporidia. NCLC1 infection-associated loss and/or envelopment of the diatom nuclei infers a necrotrophic-pathogenic interaction. Diatoms are one of the most diverse and ecologically important phytoplankton groups, acting as dominant primary producers and driving carbon fixation and storage in many aquatic environments. Our results provide insight into the diversity of microbial eukaryotes that interact with diatoms. We suggest that such interactions can play a key role in diatom associated ecosystem functions, such as the marine carbon pump through necrotrophic-parasitism, facilitating the export of diatoms to the sediment.Agence Nationale de la Recherche (ANR)Genome CanadaDonald Hill Family FellowshipRoyal SocietyBiodivERsAGordon and Betty Moore Foundatio

Increasing access to erectile dysfunction treatment via pharmacies to improve healthcare provider visits and quality of life: Results from a prospective real-world observational study in the United Kingdom

OBJECTIVES: The Medicines and Healthcare Products Regulatory Agency in the United Kingdom (UK) formally reclassified sildenafil citrate 50 mg tablets as a pharmacy medicine (sildenafil-P) in 2017 for adult men with erectile dysfunction (ED). A 1-year prospective real-world observational study was conducted to track men's health behaviour, particularly their healthcare resource utilisation (HCRU) and quality of life (QoL) before and after the availability of sildenafil-P. METHODS: Adult men with ED aged ≥18 years provided data at baseline (prior to launch of sildenafil-P) and every 3 months after the launch. Demographics, health characteristics, treatments at baseline and HCRU, including number of pharmacist and physician/nurse practitioner visits over time are reported. QoL-related outcomes were assessed via the Self-Esteem and Relationship Questionnaire (SEAR), 2-Item Patient Health Questionnaire and ratings of sexual satisfaction. Generalised linear models were used to assess the association of sildenafil-P use with total physician/nurse practitioner and pharmacist visits and QoL-related outcomes at 12 months. RESULTS: Overall, 1162 men completed the survey at all 5 time points. The mean ± SD age was 59.02 ± 12.06 years; 55.42% reported having a moderate-to-severe ED. Hypertension (37.52%) and hypercholesterolaemia (31.50%) were the most common risk factors for ED. At baseline, 62.99% were not using any ED treatment. After adjusting for baseline visits/other covariates, mean physician/nurse practitioner (3.68 vs 2.87; P = .003) and pharmacist visits for any reason (2.10 vs 1.34; P < .001) at 12 months were significantly higher among sildenafil-P users than those who never used sildenafil-P. Sildenafil-P users also had significantly higher SEAR total and domain (sexual relationship and self-esteem) scores at 12 months. CONCLUSION: Following the reclassification to a pharmacy medicine in the UK, sildenafil-P was associated with a higher number of physician/nurse practitioner and pharmacist visits for any reason. Sildenafil-P use was also associated with better QoL, although group differences were small in magnitude

Management of major depression in outpatients attending a cancer centre: a preliminary evaluation of a multicomponent cancer nurse-delivered intervention

A novel nurse-delivered multicomponent intervention for major depressive disorder (MDD) in cancer outpatients was compared with usual care alone in a nonrandomised matched group design (n=30 per group). At the final 6-month outcome, 38.5% (95% CI, 5.4-57%) fewer patients in the intervention group still met the criteria for MDD

Cbx3 inhibits vascular smooth muscle cell proliferation, migration, and neointima formation

This work was supported by British Heart Foundation (FS/09/044/28007, PG/11/40/28891, PG/13/45/30326, PG/15/11/31279, PG/15/86/31723, and PG/16/1/31892 to QX). This work forms part of the research portfolio for the National Institute for Health Research Biomedical Research Centre at Barts

MiRNA-200c-3p promotes endothelial to mesenchymal transition and neointimal hyperplasia in artery bypass grafts

Increasing evidence has suggested a critical role for endothelial‐to‐mesenchymal transition (EndoMT) in a variety of pathological conditions. MicroRNA‐200c‐3p (miR‐200c‐3p) has been implicated in epithelial‐to‐mesenchymal transition. However, the functional role of miR‐200c‐3p in EndoMT and neointimal hyperplasia in artery bypass grafts remains largely unknown. Here we demonstrated a critical role for miR‐200c‐3p in EndoMT. Proteomics and luciferase activity assays revealed that fermitin family member 2 (FERM2) is the functional target of miR‐200c‐3p during EndoMT. FERMT2 gene inactivation recapitulates the effect of miR‐200c‐3p overexpression on EndoMT, and the inhibitory effect of miR‐200c‐3p inhibition on EndoMT was reversed by FERMT2 knockdown. Further mechanistic studies revealed that FERM2 suppresses smooth muscle gene expression by preventing serum response factor nuclear translocation and preventing endothelial mRNA decay by interacting with Y‐box binding protein 1. In a model of aortic grafting using endothelial lineage tracing, we observed that miR‐200c‐3p expression was dramatically up‐regulated, and that EndoMT contributed to neointimal hyperplasia in grafted arteries. MiR‐200c‐3p inhibition in grafted arteries significantly up‐regulated FERM2 gene expression, thereby preventing EndoMT and reducing neointimal formation. Importantly, we found a high level of EndoMT in human femoral arteries with atherosclerotic lesions, and that miR‐200c‐3p expression was significantly increased, while FERMT2 expression levels were dramatically decreased in diseased human arteries. Collectively, we have documented an unexpected role for miR‐200c‐3p in EndoMT and neointimal hyperplasia in grafted arteries. Our findings offer a novel therapeutic opportunity for treating vascular diseases by specifically targeting the miR‐200c‐3p/FERM2 regulatory axis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland

Unexpected mitochondrial genome diversity revealed by targeted single-cell genomics of heterotrophic flagellated protists

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this recordData availability: Complete mtDNA sequences assembled from this study are available at GenBank under the accession numbers MK188935 to MK188947, MN082144 and MN082145. Sequencing data are available under NCBI BioProject PRJNA379597. Reads have been deposited at NCBI Sequence Read Archive with accession number SRP102236. Partial mtDNA contigs and other important contigs mentioned in the text are available from Figshare at https://doi.org/10.6084/m9.figshare.7314728. Nuclear SAG assemblies are available from Figshare at https://doi.org/10.6084/m9.figshare.7352966. A protocol is available from protocols.io at: https://doi.org/10.17504/protocols.io.ywpfxdn.Code availability: The bioinformatic workflow is available at https://doi.org/10.5281/zenodo.192677; additional statistical analysis code is available at https://doi.org/10.6084/m9.figshare.9884309.Most eukaryotic microbial diversity is uncultivated, under-studied and lacks nuclear genome data. Mitochondrial genome sampling is more comprehensive, but many phylogenetically important groups remain unsampled. Here, using a single-cell sorting approach combining tubulin-specific labelling with photopigment exclusion, we sorted flagellated heterotrophic unicellular eukaryotes from Pacific Ocean samples. We recovered 206 single amplified genomes, predominantly from underrepresented branches on the tree of life. Seventy single amplified genomes contained unique mitochondrial contigs, including 21 complete or near-complete mitochondrial genomes from formerly under-sampled phylogenetic branches, including telonemids, katablepharids, cercozoans and marine stramenopiles, effectively doubling the number of available samples of heterotrophic flagellate mitochondrial genomes. Collectively, these data identify a dynamic history of mitochondrial genome evolution including intron gain and loss, extensive patterns of genetic code variation and complex patterns of gene loss. Surprisingly, we found that stramenopile mitochondrial content is highly plastic, resembling patterns of variation previously observed only in plants.Gordon and Betty Moore FoundationLeverhulme TrustDavid and Lucile Packard FoundationRoyal SocietyEuropean Molecular Biology OrganizationCONICYT FONDECYTGenome Canad

Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

This is the final version. Available on open access from the Royal Society via the DOI in this recordData accessibility The raw reads generated during transcriptome and sRNA sequencing are available on the NCBI Sequence Read Archive (accessions: SAMN14932981, SAMN14932982). All other datasets are available on Figshare (https://doi.org/10.6084/m9.figshare.c.5241983.v1), under the relevant headings. Custom scripts for host and endosymbiont transcript binning [80] (https://github.com/fmaguire/dendrogenous, https://doi.org/10.5281/zenodo.4639294) and sRNA read processing [81] (https://github.com/guyleonard/paramecium, https://doi.org/10.5281/zenodo.4638888) are available on GitHub and archived within the Zenodo repository.Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote-eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria-Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down 'paradox' controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria-Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.EMBORoyal SocietyEuropean Research Council (ERC)Wellcome TrustLister institut

Emergent RNA–RNA interactions can promote stability in a facultative phototrophic endosymbiosis

This is the final version. Available on open access from the National Academy of Sciences via the DOI in this recordData Availability: The sequence data, code, and datasets have been deposited in NCBI Sequence Read Archive, GitHub, Figshare, and Zenodo. The raw reads generated during sRNA sequencing are available on the NCBI Sequence Read Archive (accession numbers SAMN14932981 and SAMN14932982). All other datasets are available on Figshare (https://doi.org/10.6084/m9.figshare.c.4978160.v3) under the relevant headings (77). Custom scripts for sRNA read processing (https://github.com/guyleonard/paramecium, https://doi.org/10.5281/zenodo.4638888) and eDicer comparative analysis (https://github.com/fmaguire/eDicer, https://doi.org/10.5281/zenodo.4659378) are available on GitHub and archived within the Zenodo repository.Eukaryote–eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses—between two fundamentally selfish biological organisms—are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont–host RNA–RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term “RNAi collisions,” represents a mechanism that can promote stability in a facultative eukaryote–eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont–host RNA–RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.European Molecular Biology OrganizationRoyal SocietyEuropean Research Council (ERC)Wellcome TrustLister InstituteDonald Hill Family Fellowshi