The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds

This is the publisher's version, also available electronically from "http://www.molbiolcell.org".he cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of gamma-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope

Predatory Organisms with Untapped Biosynthetic Potential:Descriptions of Novel Corallococcus Species C. aberystwythensis sp. nov., C. carmarthensis sp. nov., C. exercitus sp. nov., C. interemptor sp. nov., C. llansteffanensis sp. nov., C. praedator sp. nov., C. sicarius sp. nov., and C. terminator sp. nov

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously (Corallococcus coralloides and Corallococcus exiguus). A polyphasic approach was taken to characterise antimicrobial, biochemical and phenotypic properties of eight Corallococcus spp. strains and the two type strains. We also report here the genome sequence of the C. exiguus type strain (DSM 14696T). The genomes of the eight candidate strains, C. exiguus DSM 14696T and C. coralloides DSM 2259T, had an average nucleotide identity below 95% and digital DNA-DNA hybridisation scores less than the 70% lower bound for species identity, indicating they belong to distinct species. All ten strains, including the two type strains, were thoroughly characterised, including biochemical analysis of their fatty acid methyl esters, substrate utilisation and sugar assimilation. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species: Corallococcus exercitus sp. nov. (AB043AT = DSM 108849T = NBRC 113887T), Corallococcus interemptor sp. nov. (AB047AT = DSM 108843T = NBRC 113888T), Corallococcus aberystwythensis sp. nov. (AB050AT = DSM 108846T = NBRC 114019T), Corallococcus praedator sp. nov. (CA031BT = DSM 108841T = NBRC 113889T), Corallococcus sicarius sp. nov. (CA040BT = DSM 108850T = NBRC 113890T), Corallococcus carmarthenensis sp. nov. (CA043DT = DSM 108842T = NBRC 113891T), Corallococcus llansteffanensis sp. nov. (CA051BT = DSM 108844T = NBRC 114100T) and Corallococcus terminator sp. nov. (CA054AT = DSM 108848T = NBRC 113892T)

Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

Fasciola hepatica NP-40 released antigens (FhTeg) exhibit potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhTeg, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) is discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study

Towards delineating functions within the fasciola secreted cathepsin L protease family by integrating in vivo based sub-proteomics and phylogenetics

BACKGROUND: Fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family. METHODOLOGY/PRINCIPAL FINDINGS: The sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. CONCLUSIONS/SIGNIFICANCE: We have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase

Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host

The Sigma Class glutathione transferase from the liver fluke Fasciola hepatica

BACKGROUND: Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. CONCLUSIONS/SIGNIFICANCE: We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity

FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a nonperturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking

Special considerations for studies of extracellular vesicles from parasitic helminths: a community-led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member

What Counts as ‘World Class’? : Global University Rankings and Shifts in Institutional Strategies

Global university rankings have emerged as a benchmark of institutional success, setting standards for higher education policymaking and institutional practices. Nevertheless, only a marginal share of higher education institutions (HEI) are in a realistic position to be ranked as a ‘world-class’ institutions. In the European context, the global rankings have been used to highlight a performance gap between European and North American institutions. Here the focus has been on the HEIs in the top-100 positions, causing concerns over European higher education. This has also become a marker of world-class university. We analyze the strategies of 27 Northern European universities in different tiers to learn how they have adjusted to the reality of ranking. We conclude that the references to global rankings have increased between 2014 and 2018. At the same time, the references to rankings have become more implicit in nature. Nevertheless, we find that the discourse of global comparison and excellence has become more common in the strategies. There are also emerging references to the regional role of universities, which are apparent in the strategies of universities that are clearly outside the top-100 ranked institutions. However, this is also a reflection of the discourse of world-class university.Peer reviewe