Electron tomography reveals the steps in filovirus budding

The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a "submarine-like" budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular "rocketlike" protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses.

Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging

Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRDI as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.

Charged particles produced by corona emission from an overhead high voltage powerline

High voltage powerlines may give rise to corona breakdown, resulting in the release of large concentrations of charged ions into the surrounding environment. These ions quickly attach to aerosols and the resulting charged particles are carried by prevalent winds. This paper describes a study carried out at a site near an overhead double circuit ac transmission voltage powerline to investigate factors that control the rate at which charged particles are produced, and to determine the total particle number concentrations, total particle charge concentrations and vertical dc electric fields in the proximity of the line. Measured mean values of these three parameters at a perpendicular distance of 50m from the line were 1.8 x 103 particle cm-3, 518 ions cm3 and 520 V m-1 respectively. The net electric charge was positive and the electric field was directed downwards. These parameters were correlated with each other and monitored at four different distances from the line. Effects of meteorological parameters such as wind speed and wind direction were also investigated.

Better cars or older cars?: Assessing CO2 emission reduction potential of passenger vehicle replacement programs

The primary motivation for the vehicle replacement schemes that were implemented in many countries was to encourage the purchase of new cars. The basic assumption of these schemes was that these acquisitions would benefit both the economy and the environment as older and less fuel-efficient cars were scrapped and replaced with more fuel-efficient models. In this article, we present a new environmental impact assessment method for assessing the effectiveness of scrappage schemes for reducing CO2 emissions taking into account the rebound effect, driving behavior for older versus new cars and entire lifecycle emissions for during the manufacturing processes of new cars. The assessment of the Japanese scrappage scheme shows that CO2 emissions would only decrease if users of the scheme retained their new gasoline passenger vehicles for at least 4.7 years. When vehicle replacements were restricted to hybrid cars, the reduction in CO2 achieved by the scheme would be 6-8.5 times higher than the emissions resulting from a scheme involving standard, gasoline passenger vehicles. Cost-benefit analysis, based on the emission reduction potential, showed that the scheme was very costly. Sensitivity analysis showed that the Japanese government failed to determine the optimum, or target, car age for scrapping old cars in the scheme. Specifically, scrapping cars aged 13 years and over did not maximize the environmental benefits of the scheme. Consequently, modifying this policy to include a reduction in new car subsidies, focused funding for fuel-efficient cars, and modifying the target car age, would increase environmental benefits. © 2013 Elsevier Ltd.

Testing the effects of the Japanese vehicle emission-control law on the international palladium futures market

This paper tested the effects of the 2005 vehicle emission-control law issued in Japan on the market linkages between the U.S. and Japanese palladium futures markets, To determine these effects, we applied a cointegration test both with and without break points in the time series and found that the market linkages between the two countries changed after the break in October 2005. Our results show that the 2005 long-term regulation of vehicle emissions enacted in Japan influenced the international palladium futures market.

Stature estimation of a contemporary Australian sub-population: An evaluation of the Trotter and Gleser method using computed tomography of the femur

Utilising computed tomography scans to allow a virtual analysis of three-dimensional reconstructions of the femur, this project confirms that the traditional 1952 Trotter and Gleser stature estimation equations are inapplicable for a contemporary Queensland population. Therefore, this study introduces modern stature estimation equations for femoral length and fragmentary femoral remains using Bayesian statistics for application in forensic anthropological casework. In addition, it was found that caution needs to be applied when comparing estimated stature to reported stature on the missing persons database due to inaccuracy in Queensland drivers' licences.

Author response to : Brough et al. in response to the recently published article : Lottering,N., MacGregor,D.M.,Barry,M.D.,Reynolds,M.S., Gregory,L.S., 2014. Introducing standardized protocols for anthropological measurement of virtual sub-adult crania using computed tomography 2(1),34–38

Firstly, we would like to thank Ms. Alison Brough and her colleagues for their positive commentary on our published work [1] and their appraisal of our utility of the “off-set plane” protocol for anthropometric analysis. The standardized protocols described in our manuscript have wide applications, ranging from forensic anthropology and paleodemographic research to clinical settings such as paediatric practice and orthopaedic surgical design. We affirm that the use of geometrically based reference tools commonly found in computer aided design (CAD) programs such as Geomagic Design X® are imperative for more automated and precise measurement protocols for quantitative skeletal analysis. Therefore we stand by our recommendation of the use of software such as Amira and Geomagic Design X® in the contexts described in our manuscript...

Temporal Characterization of Ossification of the Crania in Australian Subadults: New standards for Age Estimation using Computed Tomography

After attending this presentation, attendees will gain awareness of the ontogeny of cranial maturation, specifically: (1) the fusion timings of primary ossification centers in the basicranium; and (2) the temporal pattern of closure of the anterior fontanelle, to develop new population-specific age standards for medicolegal death investigation of Australian subadults. This presentation will impact the forensic science community by demonstrating the potential of a contemporary forensic subadult Computed Tomography (CT) database of cranial scans and population data, to recalibrate existing standards for age estimation and quantify growth and development of Australian children. This research welcomes a study design applicable to all countries faced with paucity in skeletal repositories. Accurate assessment of age-at-death of skeletal remains represents a key element in forensic anthropology methodology. In Australian casework, age standards derived from American reference samples are applied in light of scarcity in documented Australian skeletal collections. Currently practitioners rely on antiquated standards, such as the Scheuer and Black1 compilation for age estimation, despite implications of secular trends and population variation. Skeletal maturation standards are population specific and should not be extrapolated from one population to another, while secular changes in skeletal dimensions and accelerated maturation underscore the importance of establishing modern standards to estimate age in modern subadults. Despite CT imaging becoming the gold standard for skeletal analysis in Australia, practitioners caution the application of forensic age standards derived from macroscopic inspection to a CT medium, suggesting a need for revised methodologies. Multi-slice CT scans of subadult crania and cervical vertebrae 1 and 2 were acquired from 350 Australian individuals (males: n=193, females: n=157) aged birth to 12 years. The CT database, projected at 920 individuals upon completion (January 2014), comprises thin-slice DICOM data (resolution: 0.5/0.3mm) of patients scanned since 2010 at major Brisbane Childrens Hospitals. DICOM datasets were subject to manual segmentation, followed by the construction of multi-planar and volume rendering cranial models, for subsequent scoring. The union of primary ossification centers of the occipital bone were scored as open, partially closed or completely closed; while the fontanelles, and vertebrae were scored in accordance with two stages. Transition analysis was applied to elucidate age at transition between union states for each center, and robust age parameters established using Bayesian statistics. In comparison to reported literature, closure of the fontanelles and contiguous sutures in Australian infants occur earlier than reported, with the anterior fontanelle transitioning from open to closed at 16.7±1.1 months. The metopic suture is closed prior to 10 weeks post-partum and completely obliterated by 6 months of age, independent of sex. Utilizing reverse engineering capabilities, an alternate method for infant age estimation based on quantification of fontanelle area and non-linear regression with variance component modeling will be presented. Closure models indicate that the greatest rate of change in anterior fontanelle area occurs prior to 5 months of age. This study complements the work of Scheuer and Black1, providing more specific age intervals for union and temporal maturity of each primary ossification center of the occipital bone. For example, dominant fusion of the sutura intra-occipitalis posterior occurs before 9 months of age, followed by persistence of a hyaline cartilage tongue posterior to the foramen magnum until 2.5 years; with obliteration at 2.9±0.1 years. Recalibrated age parameters for the atlas and axis are presented, with the anterior arch of the atlas appearing at 2.9 months in females and 6.3 months in males; while dentoneural, dentocentral and neurocentral junctions of the axis transitioned from non-union to union at 2.1±0.1 years in females and 3.7±0.1 years in males. These results are an exemplar of significant sexual dimorphism in maturation (p<0.05), with girls exhibiting union earlier than boys, justifying the need for segregated sex standards for age estimation. Studies such as this are imperative for providing updated standards for Australian forensic and pediatric practice and provide an insight into skeletal development of this population. During this presentation, the utility of novel regression models for age estimation of infants will be discussed, with emphasis on three-dimensional modeling capabilities of complex structures such as fontanelles, for the development of new age estimation methods.

Gold nanospikes based microsensor as a highly accurate mercury emission monitoring system

Anthropogenic elemental mercury (Hg0) emission is a serious worldwide environmental problem due to the extreme toxicity of the heavy metal to humans, plants and wildlife. Development of an accurate and cheap microsensor based online monitoring system which can be integrated as part of Hg0 removal and control processes in industry is still a major challenge. Here, we demonstrate that forming Au nanospike structures directly onto the electrodes of a quartz crystal microbalance (QCM) using a novel electrochemical route results in a self-regenerating, highly robust, stable, sensitive and selective Hg0 vapor sensor. The data from a 127 day continuous test performed in the presence of volatile organic compounds and high humidity levels, showed that the sensor with an electrodeposted sensitive layer had 260% higher response magnitude, 3.4 times lower detection limit (,22 mg/m3 or ,2.46 ppbv) and higher accuracy (98% Vs 35%) over a Au control based QCM (unmodified) when exposed to a Hg0 vapor concentration of 10.55 mg/m3 at 1016C. Statistical analysis of the long term data showed that the nano-engineered Hg0 sorption sites on the developed Au nanospikes sensitive layer play a critical role in the enhanced sensitivity and selectivity of the developed sensor towards Hg0 vapor.

Magnetic resonance imaging: An accurate, radiation-free, alternative to computed tomography for the primary imaging and three-dimensional reconstruction of the bony orbit

Purpose: To determine the extent to which the accuracy of magnetic resonance imaging (MRI) based virtual 3-dimensional (3D) models of the intact orbit can approach that of the gold standard, computed tomography (CT) based models. The goal was to determine whether MRI is a viable alternative to CT scans in patients with isolated orbital fractures and penetrating eye injuries, pediatric patients, and patients requiring multiple scans in whom radiation exposure is ideally limited. Materials and Methods: Patients who presented with unilateral orbital fractures to the Royal Brisbane and Women’s Hospital from March 2011 to March 2012 were recruited to participate in this cross-sectional study. The primary predictor variable was the imaging technique (MRI vs CT). The outcome measurements were orbital volume (primary outcome) and geometric intraorbital surface deviations (secondary outcome)between the MRI- and CT-based 3D models. Results: Eleven subjects (9 male) were enrolled. The patients’ mean age was 30 years. On average, the MRI models underestimated the orbital volume of the CT models by 0.50 0.19 cm3 . The average intraorbital surface deviation between the MRI and CT models was 0.34 0.32 mm, with 78 2.7% of the surface within a tolerance of 0.5 mm. Conclusions: The volumetric differences of the MRI models are comparable to reported results from CT models. The intraorbital MRI surface deviations are smaller than the accepted tolerance for orbital surgical reconstructions. Therefore, the authors believe that MRI is an accurate radiation-free alternative to CT for the primary imaging and 3D reconstruction of the bony orbit. �