Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 106 peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 106 PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 106 PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-γ responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-γ response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials. © 2008 SGM.

Automatic particle picking of biological molecules imaged by electron microscopy

One of the next great challenges of cell biology is the determination of the enormous number of protein structures encoded in genomes. In recent years, advances in electron cryo-microscopy and high-resolution single particle analysis have developed to the point where they now provide a methodology for high resolution structure determination. Using this approach, images of randomly oriented single particles are aligned computationally to reconstruct 3-D structures of proteins and even whole viruses. One of the limiting factors in obtaining high-resolution reconstructions is obtaining a large enough representative dataset ($>100,000$ particles). Traditionally particles have been manually picked which is an extremely labour intensive process. The problem is made especially difficult by the low signal-to-noise ratio of the images. This paper describes the development of automatic particle picking software, which has been tested with both negatively stained and cryo-electron micrographs. This algorithm has been shown to be capable of selecting most of the particles, with few false positives. Further work will involve extending the software to detect differently shaped and oriented particles.

A particle-based micromechanics approach to simulate structural changes of plant cells during drying

This paper is concerned with applying a particle-based approach to simulate the micro-level cellular structural changes of plant cells during drying. The objective of the investigation was to relate the micro-level structural properties such as cell area, diameter and perimeter to the change of moisture content of the cell. Model assumes a simplified cell which consists of two basic components, cell wall and cell fluid. The cell fluid is assumed to be a Newtonian fluid with higher viscosity compared to water and cell wall is assumed to be a visco-elastic solid boundary located around the cell fluid. Cell fluid is modelled with Smoothed Particle Hydrodynamics (SPH) technique and for the cell wall; a Discrete Element Method (DEM) is used. The developed model is two-dimensional, but accounts for three-dimensional physical properties of real plant cells. Drying phenomena is simulated as fluid mass reductions and the model is used to predict the above mentioned structural properties as a function of cell fluid mass. Model predictions are found to be in fairly good agreement with experimental data in literature and the particle-based approach is demonstrated to be suitable for numerical studies of drying related structural deformations. Also a sensitivity analysis is included to demonstrate the influence of key model parameters to model predictions.

Accurate stratospheric particle size distributions from two separate flat-plate collection surfaces

Over the past two decades, flat-plate particle collections have revealed the presence of a remarkable variety of both terrestrial and extraterrestrial material in the stratosphere [1-6]. The ratio of terrestrial to extraterrestrial material and the nature of material collected may vary over observable time scales. Variations in particle number density can be important since the earth’s atmospheric radiation balance, and therefore the earth’s climate, can be influenced by articulate absorption and scattering of radiation from the sun and earth [7-9]. In order to assess the number density of solid particles in the stratosphere, we have examined a representative fraction of the so1id particles from two flat-plate collection surfaces, whose collection dates are separated in time by 5 years.

Kaolinite particle sizes in the <2 µM range using laser scattering

The Clay Minerals Society Source Clay kaolinites, Georgia KGa-1 and KGa-2, have been subjected to particle size determinations by 1) conventional sedimentation methods, 2) electron microscopy and image analysis, and 3) laser scattering using improved algorithms for the interaction of light with small particles. Particle shape, size distribution, and crystallinity vary considerably for each kaolinite. Replicate analyses of separated size fractions showed that in the <2 µm range, the sedimentation/centrifugation method of Tanner and Jackson (1947) is reproducible for different kaolinite types and that the calculated size ranges are in reasonable agreement with the size bins estimated from laser scattering. Particle sizes determined by laser scattering must be calculated using Mie theory when the dominant particle size is less than ∼5 µm. Based on this study of two well-known and structurally different kaolinites, laser scattering, with improved data reduction algorithms that include Mie theory, should be considered an internally consistent and rapid technique for clay particle sizing.

Accurate stratospheric particle-size distributions from a flat-plate collection surface

The first representative chemical, structural, and morphological analysis of the solid particles from a single collection surface has been performed. This collection surface sampled the stratosphere between 17 and 19km in altitude in the summer of 1981, and therefore before the 1982 eruptions of El Chichón. A particle collection surface was washed free of all particles with rinses of Freon and hexane, and the resulting wash was directed through a series of vertically stacked Nucleopore filters. The size cutoff for the solid particle collection process in the stratosphere is found to be considerably less than 1 μm. The total stratospheric number density of solid particles larger than 1μm in diameter at the collection time is calculated to be about 2.7×10−1 particles per cubic meter, of which approximately 95% are smaller than 5μm in diameter. Previous classification schemes are expanded to explicitly recognize low atomic number material. With the single exception of the calcium-aluminum-silicate (CAS) spheres all solid particle types show a logarithmic increase in number concentration with decreasing diameter. The aluminum-rich particles are unique in showing bimodal size distributions. In addition, spheres constitute only a minor fraction of the aluminum-rich material. About 2/3 of the particles examined were found to be shards of rhyolitic glass. This abundant volcanic material could not be correlated with any eruption plume known to have vented directly to the stratosphere. The micrometeorite number density calculated from this data set is 5×10−2 micrometeorites per cubic meter of air, an order of magnitude greater than the best previous estimate. At the collection altitude, the maximum collision frequency of solid particles >5μm in average diameter is calculated to be 6.91×10−16 collisions per second, which indicates negligible contamination of extraterrestrial particles in the stratosphere by solid anthropogenic particles.

Layer silicates in a chondritic porous interplanetary dust particle

Analytical electron microscopy on individual grains from a portion of a chondritic porous interplanetary dust particle (aggregate W7029C1 from the NASA Johnson Space Center Cosmic Dust Collection) shows that layer silicates compose 50 percent of the silicate fraction examined. These layer silicates can be classified into two distinct crystallochemical groups: (1) fine-grained, polycrystalline smectite minerals; and (2) well-ordered, single crystals of kaolinite and Mg-poor talc. The layer silicates in this portion of sample W7029(asterisk)A are dissimilar to those described in other chondritic porous aggregates. The predominant layer silicate assemblage in W7029(asterisk)A indicates that heating of the aggregate during atmospheric entry was brief and probably to a temperature less than 300C. Comparison with terrestrial phyllosilicate occurrences suggests that some layer silicates in aggregate W7029(asterisk)A may have been formed by alteratiton from preexisting silicate minerals at low temperatures (less than 25C) after aggregate formation.

Particle formation and interaction

A wide variety of experiments that involve the physics of small particles (μm to cm in size) of planetary significance can be conducted on the Space Station. Processes of interest include nucleation and condensation of particles from a gas, aggregation of small particles into larger ones, and low velocity collisions of particles. Only experiments relevant to planetary processes will be discussed in detail here.

Multidisciplinary design and flight testing of a remote gas/particle airborne sensor system

The main objective of this paper is to describe the development of a remote sensing airborne air sampling system for Unmanned Aerial Systems (UAS) and provide the capability for the detection of particle and gas concentrations in real time over remote locations. The design of the air sampling methodology started by defining system architecture, and then by selecting and integrating each subsystem. A multifunctional air sampling instrument, with capability for simultaneous measurement of particle and gas concentrations was modified and integrated with ARCAA’s Flamingo UAS platform and communications protocols. As result of the integration process, a system capable of both real time geo-location monitoring and indexed-link sampling was obtained. Wind tunnel tests were conducted in order to evaluate the performance of the air sampling instrument in controlled nonstationary conditions at the typical operational velocities of the UAS platform. Once the remote fully operative air sampling system was obtained, the problem of mission design was analyzed through the simulation of different scenarios. Furthermore, flight tests of the complete air sampling system were then conducted to check the dynamic characteristics of the UAS with the air sampling system and to prove its capability to perform an air sampling mission following a specific flight path.

Composition and morphology of particle emissions from in-use aircraft during takeoff and landing

In order to provide realistic data for air pollution inventories and source apportionment at airports, the morphology and composition of ultrafine particles (UFP) in aircraft engine exhaust were measured and characterized. For this purpose, two independent measurement techniques were employed to collect emissions during normal takeoff and landing operations at Brisbane Airport, Australia. PM1 emissions in the airfield were collected on filters and analyzed using the particle-induced X-ray emission (PIXE) technique. Morphological and compositional analyses of individual ultrafine particles in aircraft plumes were performed on silicon nitride membrane grids using transmission electron microscopy (TEM) combined with energy-dispersive X-ray microanalysis (EDX). TEM results showed that the deposited particles were in the range of 5 to 100 nm in diameter, had semisolid spherical shapes and were dominant in the nucleation mode (18 – 20 nm). The EDX analysis showed the main elements in the nucleation particles were C, O, S and Cl. The PIXE analysis of the airfield samples was generally in agreement with the EDX in detecting S, Cl, K, Fe and Si in the particles. The results of this study provide important scientific information on the toxicity of aircraft exhaust and their impact on local air quality.

Spatial variation of particle number concentration in school microscale environments and its impact on exposure assessment

It has not yet been established whether the spatial variation of particle number concentration (PNC) within a microscale environment can have an effect on exposure estimation results. In general, the degree of spatial variation within microscale environments remains unclear, since previous studies have only focused on spatial variation within macroscale environments. The aims of this study were to determine the spatial variation of PNC within microscale school environments, in order to assess the importance of the number of monitoring sites on exposure estimation. Furthermore, this paper aims to identify which parameters have the largest influence on spatial variation, as well as the relationship between those parameters and spatial variation. Air quality measurements were conducted for two consecutive weeks at each of the 25 schools across Brisbane, Australia. PNC was measured at three sites within the grounds of each school, along with the measurement of meteorological and several other air quality parameters. Traffic density was recorded for the busiest road adjacent to the school. Spatial variation at each school was quantified using coefficient of variation (CV). The portion of CV associated with instrument uncertainty was found to be 0.3 and therefore, CV was corrected so that only non-instrument uncertainty was analysed in the data. The median corrected CV (CVc) ranged from 0 to 0.35 across the schools, with 12 schools found to exhibit spatial variation. The study determined the number of required monitoring sites at schools with spatial variability and tested the deviation in exposure estimation arising from using only a single site. Nine schools required two measurement sites and three schools required three sites. Overall, the deviation in exposure estimation from using only one monitoring site was as much as one order of magnitude. The study also tested the association of spatial variation with wind speed/direction and traffic density, using partial correlation coefficients to identify sources of variation and non-parametric function estimation to quantify the level of variability. Traffic density and road to school wind direction were found to have a positive effect on CVc, and therefore, also on spatial variation. Wind speed was found to have a decreasing effect on spatial variation when it exceeded a threshold of 1.5 (m/s), while it had no effect below this threshold. Traffic density had a positive effect on spatial variation and its effect increased until it reached a density of 70 vehicles per five minutes, at which point its effect plateaued and did not increase further as a result of increasing traffic density.

Study of new particle formation in subtropical urban environment in Brisbane, Australia

Atmospheric ultrafine particles play an important role in affecting human health, altering climate and degrading visibility. Numerous studies have been conducted to better understand the formation process of these particles, including field measurements, laboratory chamber studies and mathematical modeling approaches. Field studies on new particle formation found that formation processes were significantly affected by atmospheric conditions, such as the availability of particle precursors and meteorological conditions. However, those studies were mainly carried out in rural areas of the northern hemisphere and information on new particle formation in urban areas, especially those in subtropical regions, is limited. In general, subtropical regions display a higher level of solar radiation, along with stronger photochemical reactivity, than those regions investigated in previous studies. However, based on the results of these studies, the mechanisms involved in the new particle formation process remain unclear, particularly in the Southern Hemisphere. Therefore, in order to fill this gap in knowledge, a new particle formation study was conducted in a subtropical urban area in the Southern Hemisphere during 2009, which measured particle size distribution in different locations in Brisbane, Australia. Characterisation of nucleation events was conducted at the campus building of the Queensland University of Technology (QUT), located in an urban area of Brisbane. Overall, the annual average number concentrations of ultrafine, Aitken and nucleation mode particles were found to be 9.3 x 103, 3.7 x 103 and 5.6 x 103 cm-3, respectively. This was comparable to levels measured in urban areas of northern Europe, but lower than those from polluted urban areas such as the Yangtze River Delta, China and Huelva and Santa Cruz de Tenerife, Spain. Average particle number concentration (PNC) in the Brisbane region did not show significant seasonal variation, however a relatively large variation was observed during the warmer season. Diurnal variation of Aitken and nucleation mode particles displayed different patterns, which suggested that direct vehicle exhaust emissions were a major contributor of Aitken mode particles, while nucleation mode particles originated from vehicle exhaust emissions in the morning and photochemical production at around noon. A total of 65 nucleation events were observed during 2009, in which 40 events were classified as nucleation growth events and the remainder were nucleation burst events. An interesting observation in this study was that all nucleation growth events were associated with vehicle exhaust emission plumes, while the nucleation burst events were associated with industrial emission plumes from an industrial area. The average particle growth rate for nucleation events was found to be 4.6 nm hr-1 (ranging from 1.79-7.78 nm hr-1), which is comparable to other urban studies conducted in the United States, while monthly particle growth rates were found to be positively related to monthly solar radiation (r = 0.76, p <0.05). The particle growth rate values reported in this work are the first of their kind to be reported for the subtropical urban area of Australia. Furthermore, the influence of nucleation events on PNC within the urban airshed was also investigated. PNC was simultaneously measured at urban (QUT), roadside (Woolloongabba) and semi-urban (Rocklea) sites in Brisbane during 2009. Total PNC at these sites was found to be significantly affected by regional nucleation events. The relative fractions of PNC to total daily PNC observed at QUT, Woolloongabba and Rocklea were found to be 12%, 9% and 14%, respectively, during regional nucleation events. These values were higher than those observed as a result of vehicle exhaust emissions during weekday mornings, which ranged from 5.1-5.5% at QUT and Woolloongabba. In addition, PNC in the semi-urban area of Rocklea increased by a factor of 15.4 when it was upwind from urban pollution sources under the influence of nucleation burst events. Finally, we investigated the influence of sulfuric acid on new particle formation in the study region. A H2SO4 proxy was calculated by using [SO2], solar radiation and particle condensation sink data to represent the new particle production strength for the urban, roadside and semi-urban areas of Brisbane during the period June-July of 2009. The temporal variations of the H2SO4 proxies and the nucleation mode particle concentration were found to be in phase during nucleation events in the urban and roadside areas. In contrast, the peak of proxy concentration occurred 1-2 hr prior to the observed peak in nucleation mode particle concentration at the downwind semi-urban area of Brisbane. A moderate to strong linear relationship was found between the proxy and the freshly formed particles, with r2 values of 0.26-0.77 during the nucleation events. In addition, the log[H2SO4 proxy] required to produce new particles was found to be ~1.0 ppb Wm-2 s and below 0.5 ppb Wm-2 s for the urban and semi-urban areas, respectively. The particle growth rates were similar during nucleation events at the three study locations, with an average value of 2.7 ± 0.5 nm hr-1. This result suggested that a similar nucleation mechanism dominated in the study region, which was strongly related to sulphuric acid concentration, however the relationship between the proxy and PNC was poor in the semi-urban area of Rocklea. This can be explained by the fact that the nucleation process was initiated upwind of the site and the resultant particles were transported via the wind to Rocklea. This explanation is also supported by the higher geometric mean diameter value observed for particles during the nucleation event and the time lag relationship between the H2SO4 proxy and PNC observed at Rocklea. In summary, particle size distribution was continuously measured in a subtropical urban area of southern hemisphere during 2009, the findings from which formed the first particle size distribution dataset in the study region. The characteristics of nucleation events in the Brisbane region were quantified and the properties of the nucleation growth and burst events are discussed in detail using a case studies approach. To further investigate the influence of nucleation events on PNC in the study region, PNC was simultaneously measured at three locations to examine the spatial variation of PNC during the regional nucleation events. In addition, the impact of upwind urban pollution on the downwind semi-urban area was quantified during these nucleation events. Sulphuric acid was found to be an important factor influencing new particle formation in the urban and roadside areas of the study region, however, a direct relationship with nucleation events at the semi-urban site was not observed. This study provided an overview of new particle formation in the Brisbane region, and its influence on PNC in the surrounding area. The findings of this work are the first of their kind for an urban area in the southern hemisphere.

Health effects of daily airborne particle dose in children : direct association between personal dose and respiratory health effects

Air pollution is a widespread health problem associated with respiratory symptoms. Continuous exposure monitoring was performed to estimate alveolar and tracheobronchial dose, measured as deposited surface area, for 103 children and to evaluate the long-term effects of exposure to airborne particles through spirometry, skin prick tests and measurement of exhaled nitric oxide (eNO). The mean daily alveolar deposited surface area dose received by children was 1.35×103 mm2. The lowest and highest particle number concentrations were found during sleeping and eating time. A significant negative association was found between changes in pulmonary function tests and individual dose estimates. Significant differences were found for asthmatics, children with allergic rhinitis and sensitive to allergens compared to healthy subjects for eNO. Variation is a child’s activity over time appeared to have a strong impact on respiratory outcomes, which indicates that personal monitoring is vital for assessing the expected health effects of exposure to particles.