Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

FoodCAP: acid-labile protein adducts of heterocyclic amines in human blood are not viable biomarkers of HCA dietary exposure

Intake of heterocyclic amines (HCAs, carcinogens produced during cooking of meat/fish, the most abundant being PhIP, DiMeIQx and MeIQx) is influenced by many factors including type/thickness of meat and cooking method/temperature/duration. Thus, assessment of HCA dietary exposure is difficult. Protein adducts of HCAs have been proposed as potential medium-term biomarkers of exposure, e.g. PhIP adducted to serum albumin or haemoglobin. However, evidence is still lacking that HCA adducts are viable biomarkers in humans consuming normal diets. The FoodCAP project, supported by World Cancer Research Fund, developed a highly sensitive mass spectrometric method for hydrolysis, extraction and detection of acid-labile HCAs in blood and assessed their validity as biomarkers of exposure. Multiple acid/alkaline hydrolysis conditions were assessed, followed by liquid-liquid extraction, clean-up by cation-exchange SPE and quantification by UPLC-ESI-MS/ MS. Blood was analysed from volunteers who completed food diaries to estimate HCA intake based on the US National Cancer Institute’s CHARRED database. Standard HCAs were recovered quantitatively from fortified blood. In addition, PhIP/MeIQx adducts bound to albumin and haemoglobin prepared in vitro using a human liver microsome system were also detectable in blood fortified at low ppt concentrations. However, except for one sample (5pg/ml PhIP), acid-labile PhIP, 7,8-DiMeIQx, 4,8-DiMeIQx and MeIQx were not observed above the 2pg/ml limit of detection in plasma (n=35), or in serum, whole blood or purified albumin, even in volunteers with high meat consumption (nominal HCA intake >2µg/day). It is concluded that HCA blood protein adducts are not viable biomarkers of exposure. Untargeted metabolomic analyses may facilitate discovery of suitable markers.

Late Bronze Age casting debris and other base metal finds from Haughey’s Fort

This paper provides a full overview of base metal finds from the excavations conducted at Haughey's Fort between 1987 and 1995. Most of the assemblage consists of waste metal from casting activities relating to the Late Bronze Age occupation of the site. A small minority of objects are of a later date, mostly Iron Age. Both the latter and the vast majority of Late Bronze metal items were recovered from a specific sector of the inner enclosure. Typological parallels, context and chronology of the finds are discussed, and a tentative interpretation of the evidence proposed.

Improving fuel economy application of the organic Rankine Cycle on a hybrid bus

Globally vehicle operators are experiencing rising fuel costs and increased
running expenses as governments around the world attempt to decrease carbon dioxide emissions and fossil fuel consumption, due to global warming and the drive to reduce dependency on fossil fuels. Recent advances in hybrid vehicle design have made great strides towards more efficient operation, with regenerative braking being widely used to capture otherwise lost energy. In this paper a hybrid series bus is developed a step further, by installing another method of energy capture on the vehicle. In this case, it is in the form of the Organic Rankine Cycle (ORC). The waste heat expelled to the exhaust and coolant streams is recovered and converted to electrical energy which is then stored in the hybrid vehicles batteries. The electrical energy can then be used for the auxiliary power circuit or to assist in vehicle propulsion, thus reducing the load on the engine, thereby improving the overall fuel economy of the vehicle and reducing carbon dioxide emissions.

Analytical examination of animal remains from Borneo:the painting of bone and shell

Examination of a selection of shell and bone from archaeological assemblages excavated at Niah Cave and Gua Sireh, both of which are located in Sarawak, Borneo, has revealed the deliberate application of coloured material to one or more surfaces. Small fragments of the surface colourant were analysed using a variety of techniques, including microscopy, energy dispersive microwave analysis and infra-red spectrophotometry. These procedures established that, although red in colour, the applied coating in each instance was not red iron oxide. It is suggested that, based on the chemical components present, this coating was a tree resin or a similar organic substance. The paper further reports the presence of enhanced chloride values in the colourant recovered from the ancient human cranial fragment tested. It is suggested that elevated concentrations of this trace element may indicate that the site, the human remains or ingredients within the colourant were once in close proximity to the sea. (C) 2010 Elsevier Ltd. All rights reserved.

Composite hunting technologies from the Terminal Pleistocene and Early Holocene, Niah Cave, Borneo

Renewed archaeological investigation of the West Mouth of Niah Cave, Borneo has demonstrated that even within lowland equatorial environments depositional conditions do exist where organic remains of late glacial and early post-glacial age can be preserved. Excavations by the Niah Cave Research Project (NCP) (2000-2003) towards the rear of the archaeological reserve produced several bone points and worked stingray spines, which exhibit evidence of hafting mastic and fibrous binding still adhering to their shafts. The position of both gives strong indication of how these cartilaginous points were hafted and gives insight into their potential function. These artefacts were recovered from secure and (14)C dated stratigraphic horizons. The results of this study have implications for our understanding the function of the Terminal Pleistocene and Early Holocene bone tools recovered from other regions of Island Southeast Asia. They demonstrate that by the end the Pleistocene rainforest foragers in Borneo were producing composite technologies that probably included fishing leisters and potentially the bow and arrow. (C) 2009 Elsevier Ltd. All rights reserved.

The 'human revolution' in lowland tropical Southeast Asia:the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an `intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest. (c) 2006 Elsevier Ltd. All rights reserved.

The ones that come ready made:The identification and use of Sus tusks as tools at prehistoric cave sites in Malaysia

Evidence for osseous technologies has featured in excavation reports from Southeast Asia for almost a century and from archaeological deposits as old as 43,000 years BP. However, in contrast to the significance that is placed on this technology in other parts of the world, until recently, Southeast Asian assemblages have drawn only very limited attention. Concentrating on evidence from Malaysia, the current paper examines one element of this inventory of tools: the deliberate modification of pig canines and the means by which such alteration can be distinguished from patterns of natural tooth wear. Particular attention is paid to the bearded pig (Sus barbatus), as it is one of the two species of wild boar in Malaysia whose tusks are most likely to have been used by prehistoric toolmakers. Reference is also made to wider, regional ethnographic examples of known tusk implements and their accredited uses to further assist in the identification process. Distinguishing criteria for worked tusk are formulated according to the type and extent of modification. These criteria are then applied to archaeological specimens recovered from two prehistoric cave sites in Malaysia, Gua Bintong and Niah Cave.

Field test of multiple 1/10 scale tidal turbines in steady flows

Queen's University Belfast and Wave Barrier Ltd have developed a tidal testing platform to test hydrokinetic turbines at medium scale. Multiple turbines can be pushed through still water conditions, in steady-state pushing tests. Experiments were conducted to evaluate the interactions between two identical, mono-strut, horizontal axis tidal turbines (HATTs) of 1.5 m diameter (D) rotor. Their relative performance when located individually, in-plane and in-line are investigated. The data shows a high consistency in the power curves at different flow speeds, which indicates high repeatability in this Reynolds range. For an individual turbine, there is no performance difference when the rotor is mounted either upstream or downstream of the supporting structure. When placed in-plane, the turbines have no adverse effect on one another. When spaced in-line with 2D separation, there is a 63% reduction in the performance of the downstream turbine. At 6D downstream this performance reduction is still 59%, indicating some wake recovery between 2D and 6D, though the influence from the upstream rotor persists to at least 6D downstream of the first device. In contrast the performance of the downstream turbine when placed at 1.5D offset of the upstream device at 6D downstream is approximately recovered to the individual turbine performance.

CLUSTER LEED - A VERY FAST METHOD FOR SURFACE-STRUCTURE DETERMINATION

A very fast method, cluster low-energy electron diffraction (LEED) is proposed for LEED I-V spectral analysis, in which three appproximations are introduced: the small-atom approximation, omission of the structure factors, and truncation of higher order ( > 2) scattering events. The method has been tested using a total of four sets of I-V spectra calculated by fully dynamic LEED for (i) the simple overlayer system, O on Ni{100}, and (ii) the reconstructed system, Cu on W{100}, and also one set of experimental data from W{100}-c(2 X 2)-Cu. In each case the correct structural parameters are recovered. It is suggested that for complex systems cluster LEED provides an efficient fast route to trial structures, which could be refined by automated tenser LEED.

A Review of Directional Modulation Technology

Directional modulation (DM) is an emerging technology for securing wireless communications at the physical layer. This promising technology, unlike the conventional key-based cryptographic methods and the key-based physical layer security approaches, locks information signals without any requirements of keys. The locked information can only be fully recovered by the legitimate receiver(s) priory known by DM transmitters. This paper reviews the origin of the DM concept and, particularly, its development in recent years, including its mathematical model, assessment metrics, synthesis approaches, physical realizations, and finally its potential aspects for future studies.

Naphthenic acid extraction and speciation from Doba crude oil using carbonate-based ionic liquids

A number of tetraalkylammonium methylcarbonate and hydrogencarbonate based ionic liquids are shown to be capable of reacting with the naphthenic acids contained in Doba crude oil via a neutralisation reaction. Spectral studies show that the ionic liquids neutralisation mechanism involves the formation of an ionic liquid-naphthenate complex, liberating methanol and carbon dioxide. Extraction of the neutralised complex into a separate methanol phase and subsequent regeneration using aqueous carbonic acid results in ∼70% of the ionic liquid being recovered for recycle. Isolation of the naphthenic acids shows that these make up to 0.85 wt% of the crude oil. Speciation of the naphthenic acids shows a mixture of monocyclic, through to tetracyclic structures with carbon numbers in the range C₁₂-C₄₀.

Forensic analysis of wooden safety matches - A case study

In this case, an individual was suspected of attempting to burn materials potentially relating to a murder case. A number of spent and unspent matches were seized at the scene by police for forensic examination. Coincidentally, a police raid at the suspect's house revealed a number of matchboxes, all of the same brand, containing matches that had a visual similarity to those recovered at the scene. Stable Isotope Profiling (SIP) was used to assess whether matches could either be distinguished or shown to be indistinguishable by 13C and 2H isotopic composition. These results were then compared to those from the X-ray diffraction (XRD) analysis of match heads and microscopy of the wood. SIP showed the scene matches and seized matches to be different, which was confirmed by XRD and microscopy analyses.

Deglacial mesophotic reef demise on the Great Barrier Reef

Submerged reefs are important recorders of palaeo-environments and sea-level change, and provide a substrate for modern mesophotic (deep-water, light-dependent) coral communities. Mesophotic reefs are rarely, if ever, described from the fossil record and nothing is known of their long-term record on Great Barrier Reef (GBR). Sedimentological and palaeo-ecological analyses coupled with 67 14C AMS and U–Th radiometric dates from dredged coral, algae and bryozoan specimens, recovered from depths of 45 to 130 m, reveal two distinct generations of fossil mesophotic coral community development on the submerged shelf edge reefs of the GBR. They occurred from 13 to 10 ka and 8 ka to present. We identified eleven sedimentary facies representing both autochthonous (in situ) and allochthonous (detrital) genesis, and their palaeo-environmental settings have been interpreted based on their sedimentological characteristics, biological assemblages, and the distribution of similar modern biota within the dredges. Facies on the shelf edge represent deep sedimentary environments, primarily forereef slope and open platform settings in palaeo-water depths of 45–95 m. Two coral–algal assemblages and one non-coral encruster assemblage were identified: 1) Massive and tabular corals including Porites, Montipora and faviids associated with Lithophylloids and minor Mastophoroids, 2) platy and encrusting corals including Porites, Montipora and Pachyseris associated with melobesioids and Sporolithon, and 3) Melobesiods and Sporolithon with acervulinids (foraminifera) and bryozoans. Based on their modern occurrence on the GBR and Coral Sea and modern specimens collected in dredges, these are interpreted as representing palaeo-water depths of < 60 m, < 80–100 m and > 100 m respectively. The first mesophotic generation developed at modern depths of 85–130 m from 13 to 10.2 ka and exhibit a deepening succession of < 60 to > 100 m palaeo-water depth through time. The second generation developed at depths of 45–70 m on the shelf edge from 7.8 ka to present and exhibit stable environmental conditions through time. The apparent hiatus that interrupted the mesophotic coral communities coincided with the timing of modern reef initiation on the GBR as well as a wide-spread flux of siliciclastic sediments from the shelf to the basin. For the first time we have observed the response of mesophotic reef communities to millennial scale environmental perturbations, within the context of global sea-level rise and environmental changes.

Systematic Uncertainties Associated with the Cosmological Analysis of the First PAN-STARRS1 Type Ia Supernova Sample

We probe the systematic uncertainties from the 113 Type Ia supernovae (SN Ia) in the Pan-STARRS1 (PS1) sample along with 197 SN Ia from a combination of low-redshift surveys. The companion paper by Rest et al. describes the photometric measurements and cosmological inferences from the PS1 sample. The largest systematic uncertainty stems from the photometric calibration of the PS1 and low-z samples. We increase the sample of observed Calspec standards from 7 to 10 used to define the PS1 calibration system. The PS1 and SDSS-II calibration systems are compared and discrepancies up to ∼0.02 mag are recovered. We find uncertainties in the proper way to treat intrinsic colors and reddening produce differences in the recovered value of w up to 3%. We estimate masses of host galaxies of PS1 supernovae and detect an insignificant difference in distance residuals of the full sample of 0.037 ± 0.031 mag for host galaxies with high and low masses. Assuming flatness and including systematic uncertainties in our analysis of only SNe measurements, we find w = -1.120^+0.360_-0.206(Stat)^+0.269_-0.291(Sys). With additional constraints from Baryon acoustic oscillation, cosmic microwave background (CMB) (Planck) and H₀ measurements, we find w = -1.166^+0.072_-0.069 and Ω_m = 0.280^+0.013_-0.012 (statistical and systematic errors added in quadrature). The significance of the inconsistency with w = -1 depends on whether we use Planck or Wilkinson Microwave Anisotropy Probe measurements of the CMB: w_{BAO+H0+SN+WMAP} = -1.124^+0.083_-0.065.