Lyophilisation of lentiviral pseudotypes for the development and distribution of virus neutralisation assay kits for rabies, Marburg and influenza viruses

Purpose: Some conventional serological assays can accurately quantify neutralising antibody responses raised against epitopes on virus glycoproteins, enabling mass vaccine evaluation and serosurveillance studies to take place. However, these assays often necessitate the handling of wild-type virus in expensive high biosafety laboratories, which restricts the scope of their application, particularly in resource-deprived areas. A solution to this issue is the use of lentiviral pseudotype viruses (PVs)—chimeric, replication-deficient virions that imitate the binding and entry mechanisms of their wild-type equivalents. Pseudotype virus neutralisation assays (PVNAs) bypass high biosafety requirements and yield comparable results to established assays. This study explores the potential for using lyophilisation of pseudotypes as a cost-effective, alternative means for production, distribution and storage of a PVNAbased diagnostic kit. Methods & Materials: Rabies, Marburg and H5 subtype Influenza virus pseudotypes were each suspended in cryoprotectant solutions of various molarities and subjected to freeze-drying before incubation at a variety of temperatures, humidities and time periods. Samples were then employed in antibody neutralisation assays using specific sera. Results: High levels of PV titre were retained post-lyophilisation, with acceptable levels of virus activity maintained even after medium-term storage in tropical conditions. Also, the performance of PVs in neutralisation assays was not affected by the lyophilisation process. Conclusion: These results confirm the viability of a freeze-dried PVNA-based diagnostic kit, which could considerably facilitate in-field serology for a number of clinically important viruses

What do gas-rich galaxies actually tell us about modified Newtonian dynamics?

It has recently been claimed that measurements of the baryonic Tully-Fisher relation (BTFR), a power-law relationship between the observed baryonic masses and outer rotation velocities of galaxies, support the predictions of modified Newtonian dynamics for the slope and scatter in the relation, while challenging the cold dark matter (CDM) paradigm. We investigate these claims, and find that: 1) the scatter in the data used to determine the BTFR is in conflict with observational uncertainties on the data; 2) these data do not make strong distinctions regarding the best-fit BTFR parameters; 3) the literature contains a wide variety of measurements of the BTFR, many of which are discrepant with the recent results; and 4) the claimed CDM "prediction" for the BTFR is a gross oversimplification of the complex galaxy-scale physics involved. We conclude that the BTFR is currently untrustworthy as a test of CDM.Comment: 5 pages, 2 figures; minor revisions to match published versio

Thermal decomposition mechanisms of hafnium and zirconium silicates at the atomic scale

The hafnium and zirconium silicates, (MO2)(x)(SiO2)(1-x), with M=Hf/Zr, are being considered as high-k gate dielectrics for field-effect transistors as a compromise between high permittivity and thermal stability during processing. Using atomic-scale models of silicates derived from hafnon/zircon, stability before and after simulated thermal annealing is calculated within a density-functional approach. These silicates are found to be thermodynamically unstable with respect to decomposition into SiO2 and MO2 (M=Hf/Zr). Segregation mechanisms on the atomic scale are studied leading to an insight as to an why SiO2-rich mixtures undergo spinodal decomposition and why, by contrast, MO2-rich phases are metastable, decomposing below typical process temperatures

Development and validation of a chemostat gut model to study both planktonic and biofilm modes of growth of Clostridium difficile and human microbiota

Copyright: 2014 Crowther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. The degree to which composition and function of faecal and mucosal microbiota differ remains unclear. We describe the development and characterisation of an in vitro human gut model, which can be used to facilitate the formation and longitudinal analysis of mature mixed species biofilms. This enables the investigation of the role of biofilms in Clostridium difficile infection (CDI). A well established and validated human gut model of simulated CDI was adapted to incorporate glass rods that create a solid-gaseous-liquid interface for biofilm formation. The continuous chemostat model was inoculated with a pooled human faecal emulsion and controlled to mimic colonic conditions in vivo. Planktonic and sessile bacterial populations were enumerated for up to 46 days. Biofilm consistently formed macroscopic structures on all glass rods over extended periods of time, providing a framework to sample and analyse biofilm structures independently. Whilst variation in biofilm biomass is evident between rods, populations of sessile bacterial groups (log10 cfu/g of biofilm) remain relatively consistent between rods at each sampling point. All bacterial groups enumerated within the planktonic communities were also present within biofilm structures. The planktonic mode of growth of C. difficile and gut microbiota closely reflected observations within the original gut model. However, distinct differences were observed in the behaviour of sessile and planktonic C. difficile populations, with C. difficile spores preferentially persisting within biofilm structures. The redesigned biofilm chemostat model has been validated for reproducible and consistent formation of mixed species intestinal biofilms. This model can be utilised for the analysis of sessile mixed species communities longitudinally, potentially providing information of the role of biofilms in CDI.Peer reviewe

The Optimisation of Pseudotyped Viruses for the Characterisation of Immune Responses to Equine Influenza Virus

Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained. Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from Clostridium perfringens to allow the release of nascent PV particles. Finally, initial characterisation of the reliability of PV neutralisation tests (PVNTs) demonstrated good intra-laboratory repeatability. In conclusion, we have demonstrated that equine influenza PV production can be readily optimised to provide a flexible tool for studying EIV

Stress in silicon interlayers at the SiO(x)/Ge interface

Materials such as germanium display an advantage relative to silicon in terms of carrier mobilities but form poor quality interfaces to oxides. By sandwiching silicon layers between a germanium substrate and the oxide, advantages of the silicon oxide/silicon (SiO(x)/Si) interface can be retained combined with the advantage of a high mobility germanium substrate. Using density functional theory calculations, stress within the silicon interlayer is quantified for different interlayer thicknesses revealing that for up to three silicon layers, the stress in the interlayer is compensated for by the energy gained by forming silicon-oxygen bonds at the interface. (c) 2007 American Institute of Physics. (DOI:10.1063/1.2713122

A Complete Spectroscopic Survey of the Milky Way satellite Segue 1: Dark matter content, stellar membership and binary properties from a Bayesian analysis

We introduce a comprehensive analysis of multi-epoch stellar line-of-sight velocities to determine the intrinsic velocity dispersion of the ultrafaint satellites of the Milky Way. Our method includes a simultaneous Bayesian analysis of both membership probabilities and the contribution of binary orbital motion to the observed velocity dispersion within a 14-parameter likelihood. We apply our method to the Segue 1 dwarf galaxy and conclude that Segue 1 is a dark-matter-dominated galaxy at high probability with an intrinsic velocity dispersion of 3.7^{+1.4}_{-1.1} km/sec. The dark matter halo required to produce this dispersion must have an average density of 2.5^{+4.1}_{-1.9} solar mass/pc^3 within a sphere that encloses half the galaxy's stellar luminosity. This is the highest measured density of dark matter in the Local Group. Our results show that a significant fraction of the stars in Segue 1 may be binaries with the most probable mean period close to 10 years, but also consistent with the 180 year mean period seen in the solar vicinity at about 1 sigma. Despite this binary population, the possibility that Segue 1 is a bound star cluster with the observed velocity dispersion arising from the orbital motion of binary stars is disfavored by the multi-epoch stellar velocity data at greater than 99% C.L. Finally, our treatment yields a projected (two-dimensional) half-light radius for the stellar profile of Segue 1 of 28^{+5}_{-4} pc, in excellent agreement with photometric measurements.Comment: 15 pages, 19 figure

Kinematic assessment of hip movement when retrieving an object from the floor

<p>Abstract</p> <p>Background</p> <p>Activities that require extreme hip movement can dislocate hip implants in the early post operative phase. One such activity is retrieving an object from the floor. The aim of this study was to assess hip movement using four different techniques to accomplish this task. This assessment would identify the techniques least likely to cause a hip dislocation.</p> <p>Methods</p> <p>An electromagnetic tracker was used to measure the movement of 50 hips in 25 normal subjects. Sensors were attached over the iliac crest and the mid-shaft of the lateral thigh. Data was then collected for 3 repetitions of each of the following retrieval techniques:-</p> <p indent="1">1. Flexing forward to pick up an object between the feet.</p> <p indent="1">2. Flexing to pick up an object lateral to the foot.</p> <p indent="1">3. Squatting to pick up an object between the feet.</p> <p indent="1">4. Kneeling on one knee to pick up beside the knee.</p> <p>Results</p> <p>Kneeling required a mean movement of 30.4 degree(s) flexion and 7.2 degree(s) external rotation. This was significantly less than all the other techniques (paired t-test, P << 0.001). Squatting required 87.4 degree(s) flexion and 10.1 degree(s) internal rotation.</p> <p>Conclusion</p> <p>The study showed that squatting had the most flexion and internal rotation, whereas kneeling has the least flexion. Thus, to minimise the dislocation risk when retrieving an object from the floor, kneeling should be adopted and squatting should be avoided.</p

A new, high-resolution global mass coral bleaching database

Episodes of mass coral bleaching have been reported in recent decades and have raised concerns about the future of coral reefs on a warming planet. Despite the efforts to enhance and coordinate coral reef monitoring within and across countries, our knowledge of the geographic extent of mass coral bleaching over the past few decades is incomplete. Existing databases, like ReefBase, are limited by the voluntary nature of contributions, geographical biases in data collection, and the variations in the spatial scale of bleaching reports. In this study, we have developed the first-ever gridded, global-scale historical coral bleaching database. First, we conducted a targeted search for bleaching reports not included in ReefBase by personally contacting scientists and divers conducting monitoring in under-reported locations and by extracting data from the literature. This search increased the number of observed bleaching reports by 79%, from 4146 to 7429. Second, we employed spatial interpolation techniques to develop annual 0.04 degrees x 0.04 degrees latitude-longitude global maps of the probability that bleaching occurred for 1985 through 2010. Initial results indicate that the area of coral reefs with a more likely than not (> 50%) or likely (> 66%) probability of bleaching was eight times higher in the second half of the assessed time period, after the 1997/1998 El Nino. The results also indicate that annual maximum Degree Heating Weeks, a measure of thermal stress, for coral reefs with a high probability of bleaching increased over time. The database will help the scientific community more accurately assess the change in the frequency of mass coral bleaching events, validate methods of predicting mass coral bleaching, and test whether coral reefs are adjusting to rising ocean temperatures

CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations

CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (γ2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine β-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine β-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives