Proteomic characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles produced in HEK-293SF

One of the major concerns associated with the use of influenza virus-like particles (VLPs) produced in cell culture as vaccine candidates is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out with them not only the viral antigenic proteins but also host cell proteins. In addition, the intrinsic nature of the cells to produce membrane derived vesicles which have similar size to the VLPs and can also contain the antigenic proteins, makes the VLP purification process challenging. Certainly, the expression system and the viral recombinant proteins employed will determine the nature of the proteins within the VLPs. To further characterize cell culture produced-influenza VLPs and contribute to enable their approval as vaccine candidates, the composition and biogenesis of VLPs need to be better understood. In this study we have characterized, by nanoscale liquid chromatography tandem mass spectrometry (n-LC-MS/MS), influenza H1N1 Gag-VLPs produced in human embryonic kidney cells adapted to serum-free medium (HEK-293SF). The cells stably express HA and NA, and the VLPs production occurs following transient transfection with a plasmid containing the gag gene of HIV-1 fused to GFP. Extracellular vesicles (EVs) produced by the unmodified HEK-293SF were also characterized by n-LC-MS/MS. A total of 73 host cell proteins were identified in the VLPs, whereas 98 were detected in the extracellular vesicles. From that, 32 host cell proteins were unique to VLPs while 41 proteins were found in both. Importantly, nucleolin was the most abundant host cell differential protein identified in VLPs while lactotransferrin and heat shock protein 90 were the most present in EVs. This study provides a detailed proteomic description of the VLPs and EVs produced in HEK-293SF as well as a critical discussion of the function of each protein incorporated in both nanoparticles species. The outcome of this research also sheds light on unique target proteins differentially identified either in VLPs and EVs that could potentially be exploited for the development of novel purification protocols to separate EVs from VLPs

Time-Varying Potassium in High-Resolution Spectra of the Type Ia Supernova 2014J

We present a time series of the highest resolution spectra yet published for the nearby Type Ia supernova (SN) 2014J in M82. They were obtained at 11 epochs over 33 days around peak brightness with the Levy Spectrograph (resolution R~110,000) on the 2.4m Automated Planet Finder telescope at Lick Observatory. We identify multiple Na I D and K I absorption features, as well as absorption by Ca I H & K and several of the more common diffuse interstellar bands (DIBs). We see no evolution in any component of Na I D, Ca I, or in the DIBs, but do establish the dissipation/weakening of the two most blueshifted components of K I. We present several potential physical explanations, finding the most plausible to be photoionization of circumstellar material, and discuss the implications of our results with respect to the progenitor scenario of SN 2014J.Comment: 11 pages, 8 figures, 3 tables, submitted to Ap

Polar glycosylated and lateral non-glycosylated flagella from Aeromonas hydrophila strain AH-1 (serotype O11)

Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll 'like' receptor 5 TLR5

Aeromonas hydrophila flagella glycosylation: involvement of a lipid carrier.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility

Identification and characterization of glycoproteins on the spore surface of Clostridium difficile

In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-β-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-β-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the β-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages

Polar flagella glycosylation in Aeromonas: genomic characterization and involvement of a specific glycosyltransferase (Fgi-1) in heterogeneous flagella glycosylation

Polar flagella from mesophilic Aeromonas strains have previously been shown to be modified with a range of glycans. Mass spectrometry studies of purified polar flagellins suggested the glycan typically includes a putative pseudaminic acid like derivative; while some strains are modified with this single monosaccharide, others modified with a heterologous glycan. In the current study, we demonstrate that genes involved in polar flagella glycosylation are clustered in highly polymorphic genomic islands flanked by pseudaminic acid biosynthetic genes (pse). Bioinformatic analysis of mesophilic Aeromonas genomes identified three types of polar flagella glycosylation islands (FGIs), denoted Group I, II and III. FGI Groups I and III are small genomic islands present in Aeromonas strains with flagellins modified with a single monosaccharide pseudaminic acid derivative. Group II were large genomic islands, present in strains found to modify polar flagellins with heterogeneous glycan moieties. Group II, in addition to pse genes, contained numerous glycosyltransferases and other biosynthetic enzymes. All Group II strains shared a common glycosyltransferase downstream of luxC that we named flagella glycosylation island 1, fgi-1, in A. piscicola AH-3. We demonstrate that Fgi-1 transfers the first sugar of the heterogeneous glycan to the pseudaminic acid derivative linked to polar flagellins and could be used as marker for polysaccharidic glycosylation of Aeromonas polar flagella

The emergence of social licence necessitates reforms in environmental regulation

The term “social licence to operate” (SLO), popularized in corporate usage over the last 20 years, is frequently used to refer to the level of social approval that exists in relation to the development of natural resources for private or public purposes. However, the theoretical and practical utility of the concept remains contested and it is often used opportunistically to advance individual agendas. Moreover, it remains difficult to assess how an adequate level of SLO can be transparently assessed, or how dialogue can be appropriately achieved. In this paper we argue that the increasing use of the SLO concept is an indication that trust in, and the legitimacy of, formal regulatory processes for natural resource management has eroded and needs to be reimagined. In response, we outline five principles that provide pathways to increase the legitimacy of, and trust in, regulatory approval processes: (i) clear regulatory objectives; (ii) transparent regulatory approval processes; (iii) clear pathways for appeals and reviews of regulatory decisions (iv) early and inclusive collaborative consultation process; and (v) independence of decision-making authorities. By rethinking the basic principles of regulatory and licencing processes in natural resource management, our five principles aim to reduce the need for SLO. This could minimize erratic decision making and inequitable approval processes that are driven by a perceived need for SLO, often only for the corporate sector, which risks the voices of other stakeholders being unevenly represented. We draw upon natural resource management experiences from Tasmania, Australia as illustrative examples to stimulate a discussion on the usefulness of SLO and the need for improved approaches to natural resource management

Assessing the Impact of Stakeholder Engagement in Management Strategy Evaluation

After completing a large, regional, multi-use Management Strategy Evaluation, we attempt to assess the impact of stakeholder engagement on the project. We do so by comparing the original project plan to the actual project development and highlight the changes which can be more directly related to stakeholder engagement aided by the application of a logic model for program evaluation. The impact can be summarised into four broad classes: a) change in the actual project development; b) a measurable change in the network of interactions both stakeholders (which includes researchers); c) changes in how the computer model was developed and run; and d) changes in attitudes among stakeholders (including researchers). We discuss these changes, the way they have been detected and some lessons we learnt which may benefit future Management Strategy Evaluation projects

The polar and lateral flagella from Plesiomonas shigelloides are glycosylated with legionaminic acid

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested