Murine immune responses to virus-like particle-associated pre- and postfusion forms of the respiratory syncytial virus F protein

Virus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, pre-F/F-containing VLPs induced significantly higher neutralizing antibody titers than the post-F/F-containing VLPs or the wild-type F/F-containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by enzyme-linked immunosorbent assay (ELISA) using soluble forms of the prefusion and postfusion forms of the F protein as targets. While both types of VLPs stimulated similar levels of IgG specific for the soluble postfusion F protein, titers of IgG specific for prefusion F induced by the pre-F/F-containing VLPs were higher than those induced by post-F/F-containing VLPs. Thus, VLPs containing a stabilized prefusion form of the RSV F protein represent a promising RSV vaccine candidate. IMPORTANCE: The development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate

Smelting conditions and smelting products: Experimental insights into the development of iron bloomery furnaces

The material record for bloomery furnaces in Iron Age and Roman Britain is fragmentary and, because of this paucity of evidence, the reconstruction of the ceramic structures used in iron production is difficult. Experiments have nevertheless been carried out to explore the working parameters and efficiency of iron smelting in bowl furnaces (small structures with little structure above ground level, interior measuring about 30 cm in height) (Craddock, 1995; Girbal, 2013) and shaft furnaces (height c.1m) (Smith, 2013; Crew, 2013; Doonan and Dungworth, 2013; Tylecote and Merkel, 1985; Tylecote and Wynne, 1958). These experiments aimed to clarify which furnace is more efficient for iron smelting and therefore what method was most likely used in Iron Age and Roman Britain. It is theorised that iron smelting furnaces developed from bowl structures to shaft structures over time, as smelters sought furnaces which could reach higher temperatures and create more reducing atmospheres (Dungworth 2013; Tylecote and Merkel, 1985; Tylecote and Wynne, 1958). These experiments suggest that the shaft furnace was used as it could meet these requirements. This study looks at the working conditions of a shaft furnace at an intermediary height - between that of a bowl furnace and of a shaft furnace - in order to understand its working parameters and to consequently better understand the progression from a bowl to a 1m high shaft structure

Vertical zonation of testate amoebae in the Elatia Mires, northern Greece : palaeoecological evidence for a wetland response to recent climate change or autogenic processes?

The Elatia Mires of northern Greece are unique ecosystems of high conservation value. The mires are climatically marginal and may be sensitive to changing hydroclimate, while northern Greece has experienced a significant increase in aridity since the late twentieth century. To investigate the impact of recent climatic change on the hydrology of the mires, the palaeoecological record was investigated from three near-surface monoliths extracted from two sites. Testate amoebae were analysed as sensitive indicators of hydrology. Results were interpreted using transfer function models to provide quantitative reconstructions of changing water table depth and pH. AMS radiocarbon dates and 210Pb suggest the peats were deposited within the last c. 50 years, but do not allow a secure chronology to be established. Results from all three profiles show a distinct shift towards a more xerophilic community particularly noted by increases in Euglypha species. Transfer function results infer a distinct lowering of water tables in this period. A hydrological response to recent climate change is a tenable hypothesis to explain this change; however other possible explanations include selective test decay, vertical zonation of living amoebae, ombrotrophication and local hydrological change. It is suggested that a peatland response to climatic change is the most probable hypothesis, showing the sensitivity of marginal peatlands to recent climatic change

Protein interactions in Xenopus germ plasm RNP particles

Hermes is an RNA-binding protein that we have previously reported to be found in the ribonucleoprotein (RNP) particles of Xenopus germ plasm, where it is associated with various RNAs, including that encoding the germ line determinant Nanos1. To further define the composition of these RNPs, we performed a screen for Hermes-binding partners using the yeast two-hybrid system. We have identified and validated four proteins that interact with Hermes in germ plasm: two isoforms of Xvelo1 (a homologue of zebrafish Bucky ball) and Rbm24b and Rbm42b, both RNA-binding proteins containing the RRM motif. GFP-Xvelo fusion proteins and their endogenous counterparts, identified with antisera, were found to localize with Hermes in the germ plasm particles of large oocytes and eggs. Only the larger Xvelo isoform was naturally found in the Balbiani body of previtellogenic oocytes. Bimolecular fluorescence complementation (BiFC) experiments confirmed that Hermes and the Xvelo variants interact in germ plasm, as do Rbm24b and 42b. Depletion of the shorter Xvelo variant with antisense oligonucleotides caused a decrease in the size of germ plasm aggregates and loosening of associated mitochondria from these structures. This suggests that the short Xvelo variant, or less likely its RNA, has a role in organizing and maintaining the integrity of germ plasm in Xenopus oocytes. While GFP fusion proteins for Rbm24b and 42b did not localize into germ plasm as specifically as Hermes or Xvelo, BiFC analysis indicated that both interact with Hermes in germ plasm RNPs. They are very stable in the face of RNA depletion, but additive effects of combinations of antisense oligos suggest they may have a role in germ plasm structure and may influence the ability of Hermes protein to effectively enter RNP particles

Sensory cutaneous papillae in the sea lamprey (Petromyzonmarinus L.) : I. Neuroanatomy and physiology

Molecules present in an animal's environment can indicate the presence of predators,food, or sexual partners and consequently, induce migratory, reproductive, foraging,or escape behaviors. Three sensory systems, the olfactory, gustatory, and solitarychemosensory cell (SCC) systems detect chemical stimuli in vertebrates. While agreat deal of research has focused on the olfactory and gustatory system over theyears, it is only recently that significant attention has been devoted to the SCC sys-tem. The SCCs are microvillous cells that were first discovered on the skin of fish,and later in amphibians, reptiles, and mammals. Lampreys also possess SCCs that areparticularly numerous on cutaneous papillae. However, little is known regarding theirprecise distribution, innervation, and function. Here, we show that sea lampreys(Petromyzon marinus L.) have cutaneous papillae located around the oral disk, nostril,gill pores, and on the dorsal fins and that SCCs are particularly numerous on thesepapillae. Tract-tracing experiments demonstrated that the oral and nasal papillae areinnervated by the trigeminal nerve, the gill pore papillae are innervated by branchialnerves, and the dorsal fin papillae are innervated by spinal nerves. We also character-ized the response profile of gill pore papillae to some chemicals and showed thattrout-derived chemicals, amino acids, and a bile acid produced potent responses.Together with a companion study (Suntres et al., Journal of Comparative Neurology,this issue), our results provide new insights on the function and evolution of the SCCsystem in vertebrates

Development of a 2,4-diaminothiazole series for the treatment of human African trypanosomiasis highlights the importance of static-cidal screening of analogues

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues

Testing peatland testate amoeba transfer functions: Appropriate methods for clustered training-sets

Transfer functions are widely used in palaeoecology to infer past environmental conditions from fossil remains of many groups of organisms. In contrast to traditional training-set design with one observation per site, some training-sets, including those for peatland testate amoeba-hydrology transfer functions, have a clustered structure with many observations from each site. Here we show that this clustered design causes standard performance statistics to be overly optimistic. Model performance when applied to independent data sets is considerably weaker than suggested by statistical cross-validation. We discuss the reasons for these problems and describe leave-one-site-out cross-validation and the cluster bootstrap as appropriate methods for clustered training-sets. Using these methods we show that the performance of most testate amoeba-hydrology transfer functions is worse than previously assumed and reconstructions are more uncertain

Testing peatland water-table depth transfer functions using high-resolution hydrological monitoring data

Transfer functions are now commonly used to reconstruct past environmental variability from palaeoecological data. However, such approaches need to be critically appraised. Testate amoeba-based transfer functions are an established method for the quantitative reconstruction of past water-table variations in peatlands, and have been applied to research questions in palaeoclimatology, peatland ecohydrology and archaeology. We analysed automatically-logged peatland water-table data from dipwells located in England, Wales and Finland and a suite of three year, one year and summer water-table statistics were calculated from each location. Surface moss samples were extracted from beside each dipwell and the testate amoebae community composition was determined. Two published transfer functions were applied to the testate-amoeba data for prediction of water-table depth (England and Europe). Our results show that estimated water-table depths based on the testate amoeba community reflect directional changes, but that they are poor representations of the real mean or median water-table magnitudes for the study sites. We suggest that although testate amoeba-based reconstructions can be used to identify past shifts in peat hydrology, they cannot currently be used to establish precise hydrological baselines such as those needed to inform management and restoration of peatlands. One approach to avoid confusion with contemporary water-table determinations is to use residuals or standardised values for peatland water-table reconstructions. We contend that our test of transfer functions against independent instrumental data sets may be more powerful than relying on statistical testing alone

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

BACKGROUND:The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research. METHODOLOGY/PRINCIPAL FINDINGS:We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor gamma-chain knockout (NOD-scid IL2rgamma(null)) mice engrafted with human hematopoietic stem cells. Human CD45(+) cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rgamma(null) mice with HLA-A2(+) human cord blood hematopoietic stem cells, were able to secrete IFN-gamma, IL-2 and TNF-alpha in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353((111-119)), NS4b 2423((181-189)), and NS4a 2148((56-64)). CONCLUSIONS/SIGNIFICANCE:This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections

Charting the course for a Blue Economy in Peru: A Research Agenda

Ocean- and coastal-based economic activities are increasingly recognised as key drivers for supporting global economies. This move towards the “blue economy” is becoming globally widespread, with the recognition that if ocean-based activities are to be sustainable, they will need to move beyond solely extractive and exploitative endeavours, aligning more closely with marine conservation and effective marine spatial planning. In this paper we define the “blue economy” as a “platform for strategic, integrated and participatory coastal and ocean development and protection that incorporates a low carbon economy, the ecosystem approach and human well-being through advancing regional industries, services and activities”. In Peru, while the seas contribute greatly to the national economy, the full potential of the blue economy has yet to be realised. This paper presents the findings of an early career scientist workshop in Lima, Peru, in March 2016. The workshop “Advancing Green Growth in Peru” brought together researchers to identify challenges and opportunities for green growth across three Peruvian economic sectors—tourism, transport and the blue economy with this paper exploring in detail the priorities generated from the “blue economy” stream. These priorities include themes such as marine spatial planning, detailed evaluations of existing maritime industries (e.g. guano collection and fisheries), development of an effective MPA network, support for sustainable coastal tourism, and better inclusion of social science disciplines in understanding societal and political support for a Peruvian blue economy. In addition, the paper discusses the research requirements associated with these priorities. While not a comprehensive list, these priorities provide a starting point for future dialogue on a co-ordinated scientific platform supporting the blue growth agenda in Peru, and in other regions working towards a successful “blue economy”