Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines. © 2008 Elsevier B.V. All rights reserved.

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Characterisation of sugarcane juice particles that influence the clarification process

Problems associated with processing whole sugarcane crop can be minimised by removing impurities during the clarification stage. As a first step, it is important to understand the colloidal chemistry of juice particles on a molecular level to assist development of strategies for effective clarification performance. This paper presents the composition and surface characteristics of colloidal particles originating from various juice types by using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX), X-ray photoelectron spectroscopy(XPS) and zeta potential measurements. The composition and surface characteristics of colloidal juice particles are reported. The results indicate that there are three types of colloidal particles present, viz. an aluminosilicatecompound, silica and iron oxide, with the latter two being abundant. Proteins, polysaccharides and organic acids were identified on the surface of particles in juice. The overall particle charge varies from -2 mV to -6 mV. In comparison to juice expressed from burnt cane, the zeta potential values were more negative with juice particles originating from whole crop. This in part explains why these juices are difficult to clarify.

A new classification and database for stratospheric dust particles

With the increasing number of stratospheric particles available for study (via the U2 and/or WB57F collections), it is essential that a simple, yet rational, classification scheme be developed for general use. Such a scheme should be applicable to all particles collected from the stratosphere, rather than limited to only extraterrestial or chemical sub-groups. Criteria for the efficacy of such a scheme would include: (a) objectivity , (b) ease of use, (c) acceptance within the broader scientific community and (d) how well the classification provides intrinsic categories which are consistent with our knowledge of particle types present in the stratosphere.

Towards a complete inventory of stratospheric dust particles, with implications for their classification

Several investigators have recently proposed classification schemes for stratospheric dust particles [1-3]. In addition, extraterrestrial materials within stratospheric dust collections may be used as a measure of micrometeorite flux [4]. However, little attention has been given to the problems of the stratospheric collection as a whole. Some of these problems include: (a) determination of accurate particle abundances at a given point in time; (b) the extent of bias in the particle selection process; (c) the variation of particle shape and chemistry with size; (d) the efficacy of proposed classification schemes and (e) an accurate determination of physical parameters associated with the particle collection process (e.g. minimum particle size collected, collection efficiency, variation of particle density with time). We present here preliminary results from SEM, EDS and, where appropriate, XRD analysis of all of the particles from a collection surface which sampled the stratosphere between 18 and 20km in altitude. Determinations of particle densities from this study may then be used to refine models of the behavior of particles in the stratosphere [5].

Auger spectroscopy of stratospheric particles : the influence of aerosols on interplanetary dust

Particle collections from the stratosphere via either the JSC Curatorial Program or the U2 Program (NASA Ames) occur between 16km and 19km altitude and are usually part of ongoing experiments to measure parameters related to the aerosol layer. Fine-grained aerosols (<0.1µm) occur in the stratosphere up to 35km altitude and are concentrated between 15km and 25km altitude[1]. All interplanetary dust particles (IDP's) from these stratospheric collections must pass through this aerosol layer before reaching the collection altitude. The major compounds in this aerosol layer are sulfur rich particulates (<0.1µm) and gases and include H2S04, OCS, S02 and CS2 [2].In order to assess possible surface reactions of interplanetary dust particles (IDP's) with ambient aerosols in the stratosphere, we have initiated a Surface Auger Microprobe (SAM) and electron microscope study of selected particles from the JSC Cosmic Dust Collection.

Porosity of serially sectioned interplanetary dust particles

The application of epoxy embedding and microtomy to individual chondritic interplanetary dust particles (lOP's)(Bradley and Brownlee, 1986a) provides not only higher precision in thin-film elemental analyses (Bradley and Brownlee, 19861:1), but also allows a wealth of other important techniques for the micro-characterization of these primitive extraterrestrial materials. For example, individual sections (e.g. 100 nm thick) or a series of sections, can be examined using image analysis techniques which utilize either transmitted or scanned secondary electron images, or alternatively, secondary X-ray spectra collected concurrently from a given region of sample. Individual particles, or groups of particles with similar image characteristics can then be rapidly identified using conventional grey-scale/particle recognition techniques for each microtomed section of lOP. This type of image analysis provides a suitable method for determination of particle size and shape distribution as well as porosity throughout the aggregate.

Mineralogy of chondritic interplanetary dust particles

From a mineralogical survey of approximately 30 chondritic micrometeorites collected from the lower stratosphere and studied in detail using current electron microscopy techniques, it is concluded that these particles represent a unique group of extraterrestrial materials. These micrometeorites differ significantly in form and texture from components of carbonaceous chondrites and contain some mineral assemblages which do not occur in any meteorite class. Electron microscope investigations of chondritic micrometeorites have established that these materials (1) are extraterrestrial in origin, (2) existed in space as small objects, (3) endured minimal alteration by planetary processes since formation, and (4) can suffer minimal pulse heating (<600°C) on entering earth's atmosphere. The probable sources for chondritic interplanetary dust particles (IDPs) are cometary and asteroidal debris and, perhaps to a lesser extent, interstellar regions. These sources have not been conclusively linked to any specific mineralogical subset of IDP, although the chondritic porous (CP) aggregate is considered of likely cometary origin. Chondritic IDPs occur in two predominant mineral assemblages: (1) carbonaceous phases and phyllosilicates and (2) carbonaceous phases and nesosilicates or inosilicates, although particles with both types of silicate assemblages are observed. Olivines, pyroxenes, layer silicates, and carbon-rich phases are the most commonly occurring minerals in many chondritic IDPs. Other phases often observed in variable proportions include sulphides, spinels, metals, metal carbides, carbonates, and minor amounts of sulphates and phosphates. Individual mineral grain sizes range from micrometers (primarily pyroxenes and olivines) to nanometers, with the predominant size for all phases less than 100 nm. Specific mineral characteristics for particular chondritic IDPs provide an indication of processes which may have occurred prior to collection in the earth's stratosphere. For example, pyroxene mineralogy in some chondritic aggregates is consistent with condensation from a vapor phase and, we consider, with condensation in a turbulent solar nebula at relatively low temperatures (<1000°C). Carbonaceous phases present in other CP aggregates have been used to imply low-temperature formation processes such as Fischer-Tropsch synthesis (∼530°C) or carbonization and graphitization (∼315°C). Alteration processes have been implicated in the formation of some layer silicates in CP aggregates and may have involved hydrocryogenic alteration at <0°C. In general, interpretations of transformation processes on submicrometer-size minerals in chondritic IDPs are consistent with formation at a radius equivalent to the asteroid belt or greater during the later stages of solar nebula evolution using currently available models.

Analytical electron microscopy of fine-grained phases in primitive interplanetary dust particles and carbonaceous chondrites

In order to describe the total mineralogical diversity within primitive extraterrestrial materials, individual interplanetary dust particles (IDPs) collected from the stratosphere as part of the JSC Cosmic Dust Curatorial Program were analyzed using a var ...

Grain size distributions of Magneli phases and metallic titanium in chondritic porous interplanetary dust particles

A mineralogical survey of chondritic interplanetary dust particles (IDPs)showed that these micrometeorites differ significantly in form and texture from components of carbonaceous chondrites and contain some mineral assemblages which do not occur in any meteorite class1. Models of chondritic IDP mineral evolution generally ignore the typical (ultra-) fine grain size of consituent minerals which range between 0.002-0.1µm in size2. The chondritic porous (CP) subset of chondritic IDPs is probably debris from short period comets although evidence for a cometary origin is still circumstantial3. If CP IDPs represent dust from regions of the Solar System in which comet accretion occurred, it can be argued that pervasive mineralogical evolution of IDP dust has been arrested due to cryogenic storage in comet nuclei. Thus, preservation in CP IDPs of "unusual meteorite minerals", such as oxides of tin, bismuth and titanium4, should not be dismissed casually. These minerals may contain specific information about processes that occurred in regions of the solar nebula, and early Solar System, which spawned the IDP parent bodies such as comets and C, P and D asteroids6. It is not fully appreciated that the apparent disparity between the mineralogy of CP IDPs and carbonaceous chondrite matrix may also be caused by the choice of electron-beam techniques with different analytical resolution. For example, Mg-Si-Fe distributions of Cl matrix obtained by "defocussed beam" microprobe analyses are displaced towards lower Fe-values when using analytical electron microscope (AEM)data which resolve individual mineral grains of various layer silicates and magnetite in the same matrix6,7. In general, "unusual meteorite minerals" in chondritic IDPs, such as metallic titanium, Tin01-n(Magneli phases) and anatase8 add to the mineral data base of fine-grained Solar System materials and provide constraints on processes that occurred in the early Solar System.

Titanium-oxide magneli phases in four chondritic porous interplanetary dust particles

Detailed analytical electron microscope analyses of four fine-grained chondritic porous interplanetary dust particles (IDPs)reveal the presence of titanium oxide Magneli phases, TinO2n-1 (n=4,5,6), and rare Ti-metal. The titanium minerals are indigenous to these chondritic IDPs. The association of Magneli phases, Ti-metal, and carbonaceous material in chondritic IDPs, along with the grain size distributions support in situ solid carbon gasification in these extraterrestrial particles. The active catalyst in this process is titanium metal that we infer may be of interstellar origin. This favorable catalysis uniquely leads to the formation of Magneli phases. As chondritic IDPs may be solid debris of short-period comets, our data indicate that nuclei of short-period comets may show distinctive chemical reactions that lead to Ti-mineral assemblages that typically include Magneli phases. The proposed model provides a plausible mechnism to explain the higher solid carbon content of chondritic IDPs relative to bulk carbon abundances typical for carbonaceous chondrite matrices that represent another type of more evolved, that is, metamorphosed, undifferentiated solar system bodies.