Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method

The ann of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous Solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems. (C) 2004 Elsevier B.V. All rights reserved.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Development of forest structure on cleared rainforest land in eastern Australia under different styles of reforestation

Rainforests in eastern Australia have been extensively cleared over the past two centuries. In recent decades, there have been increasing efforts to reforest some of these cleared lands, using a variety of methods, to meet a range of economic and environmental objectives. However, the extent to which the various styles of reforestation restore structure, composition and ecological function to cleared land is not presently understood. In this study, we develop and apply a method for quantifying the structural attributes of reforestation sites in tropical and subtropical Australia. The types of reforestation studied were plantation monocultures, mixed-species cabinet timber plots, diverse restoration plantings and unmanaged regrowth. Two age classes of reforestation were examined: 'young' (5-22 years), incorporating sites from all categories, and 'old' (30-70 years), in which only monoculture plantations and regrowth were represented. A total of 104 sites were surveyed including reference sites in intact rainforest and pasture. Intact rainforest was characterised by a suite of complex structural features including abundant special life forms (vines, epiphytes, hemi-epiphytes and strangler figs), a dense stand of trees in a range of size classes, a closed canopy, a shrubby understorey and a well-developed ground layer of leaf litter and woody debris. These features were lost on conversion to pasture. While all types of reforestation returned some elements of structural complexity to cleared land, young plantation monocultures, cabinet timber plots and young regrowth had a relatively simple structure. These sites typically had a low density of woody stems, a relatively open canopy and grassy ground cover, and lacked large trees, coarse woody debris and most special life forms. Restoration plantings and old regrowth were more complex, with a high density of woody stems, a relatively closed canopy and shrubby understorey. Old monoculture plantations in the tropics had acquired many of the structural attributes of intact forest, however this was not the case in the subtropics, where plantations were subject to more intensive management. The marked differences in structural complexity between sites suggest that the different types of reforestation practiced in eastern Australia are likely to vary considerably in their value as habitat for rainforest biota. (C) 2003 Elsevier Science B.V. All rights reserved.

Transmission electron microscopy sample preparation technique for sintered alloys

Powder metallurgy alloys are typically inhomogeneous with a significant amount of porosity. This complicates conventional transmission electron microscopy sample preparation. However, the use of focused ion beam milling allows site specific transmission electron microscopy samples to be prepared in a short amount of time. This paper presents a method that can be used to produce transmission electron microscopy samples from an Al-Cu-Mg PM alloy. (C) 2003 IoM Communications Ltd. Published by Maney for the Institute of Materials, Minerals and Mining.

A simple, rapid method for mapping bathymetry of small wetland basins

Many tools exist for determining and mapping the bathymetry and topography of aquatic systems, such as freshwater wetlands. However, these tools often require time-consuming survey work to produce accurate maps. In particular, the large quantity of data necessary may be prohibitive for projects where determining bathymetry is not a central focus, but instead a necessary step in achieving some other goal. We present a method to produce bathymetric surface maps with a minimum amount of effort using global positioning system receiver and laser transit survey data. We also demonstrate that this method is surprisingly accurate, given the small amount of data we use to generate the bathymetry maps.

A Two-Step Sol–Gel Method for Synthesis of Nanoporous TiO2

This article reports a study of the effects of synthesis parameters on the preparation and formation of mesoporous titania nanopowders by employing a two-step sol-gel method. These materials displayed crystalline domains characteristic of anatase. The first step of the process involved the hydrolysis of titanium isopropoxide in a basic aqueous solution mediated by neutral surfactant. The solid product obtained from step 1 was then treated in an acidified ethanol solution containing the same titanium precursor to thicken the pore walls. Low pH and higher loading of the Ti precursor in step 2 produced better mesoporosity and crystallinity of titanium dioxide polymorphs. The resultant powder exhibited a high surface area (73.8 m(2)/g) and large pore volume (0.17 cm(3)/g) with uniform mesopores. These materials are envisaged to be used as precursors for mesoporous titania films as a wide band gap semiconductor in dye-sensitized nanocrystalline TiO2 solar cells.

Preparation of immuno-stimulating complexes (ISCOMs) by ether injection

preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles. (C) 2005 Elsevier B.V. All rights reserved.

Development of a DNA-based method for detection and identification of Phytophthora species

Phytophthora diseases cause major losses to agricultural and horticultural production in Australia and worldwide. Most Phytophthora diseases are soilborne and difficult to control, making disease prevention an important component of many disease management strategies. Detection and identification of the causal agent, therefore, is an essential part of effective disease management. This paper describes the development and validation of a DNA-based diagnostic assay that can detect and identify 27 different Phytophthora species. We have designed PCR primers that are specific to the genus Phytophthora. The resulting amplicon after PCR is subjected to digestion by restriction enzymes to yield a specific restriction pattern or fingerprint unique to each species. The restriction patterns are compared with a key comprising restriction patterns of type specimens or representative isolates of 27 different Phytophthora species. A number of fundamental issues, such as genetic diversity within and among species which underpin the development and validation of DNA-based diagnostic assays, are addressed in this paper.

Direct seeding to restore rainforest species: Microsite effects on the early establishment and growth of rainforest tree seedlings on degraded land in the wet tropics of Australia

Reforestation in tropical areas is usually attempted by planting seedlings but, direct seeding (the artificial addition or sowing of seed) may be an alternative way of accelerating forest recovery and successional processes. This study investigated the effects of various sowing treatments (designed to create different microsite conditions for seed germination) and seed sizes on the early establishment and growth of directly sown rainforest tree species in a variety of experimental plots at three sites in the wet tropical region of north-cast Queensland, Australia. The different sowing treatments were found to have significant effects on seedling establishment. Broadcast sowing treatments were ineffective and resulted in very poor seedling establishment and high seed wastage. Higher establishment rates occurred when seeds were buried. Seed size was found to be an important factor affecting establishment in relation to micro-site condition. In general, larger seeded species had higher establishment rates at all three sites than species of small and intermediate seed size, but only in sowing treatments where seeds were buried. Overall these results suggest that direct sowing of seed can be used as a too] to accelerate recolonisation of certain rainforest tree species on degraded tropical lands, but initial success will be dependent on the choice of sowing method and its suitability for the seed types selected. The results also indicate that the recruitment of naturally dispersed tree species at degraded sites is likely to be severely limited by the availability of suitable microsites for seed germination. Consequently the natural recovery of degraded sites via seed rain can be expected to be slow and unpredictable, particularly in areas where soil compaction has occurred. (c) 2006 Elsevier B.V. All rights reserved.

Conservation planning with irreplaceability: does the method matter?

A number of systematic conservation planning tools are available to aid in making land use decisions. Given the increasing worldwide use and application of reserve design tools, including measures of site irreplaceability, it is essential that methodological differences and their potential effect on conservation planning outcomes are understood. We compared the irreplaceability of sites for protecting ecosystems within the Brigalow Belt Bioregion, Queensland, Australia, using two alternative reserve system design tools, Marxan and C-Plan. We set Marxan to generate multiple reserve systems that met targets with minimal area; the first scenario ignored spatial objectives, while the second selected compact groups of areas. Marxan calculates the irreplaceability of each site as the proportion of solutions in which it occurs for each of these set scenarios. In contrast, C-Plan uses a statistical estimate of irreplaceability as the likelihood that each site is needed in all combinations of sites that satisfy the targets. We found that sites containing rare ecosystems are almost always irreplaceable regardless of the method. Importantly, Marxan and C-Plan gave similar outcomes when spatial objectives were ignored. Marxan with a compactness objective defined twice as much area as irreplaceable, including many sites with relatively common ecosystems. However, targets for all ecosystems were met using a similar amount of area in C-Plan and Marxan, even with compactness. The importance of differences in the outcomes of using the two methods will depend on the question being addressed; in general, the use of two or more complementary tools is beneficial.

The water balance on sloping land in rainfed lowland rice ecosystem

Quantifying water losses in paddy fields assists estimation of water availability in rainfed lowland rice ecosystem. There is, however, no definite method for determining the water losses, and little information is available on water balance in different toposequence positions of a sloped rainfed lowland. Therefore, the aim of this work was to quantify percolation and the lateral water flow with special reference to the toposequential variation. Data used for the analysis was collected in Laos and northeast Thailand. Percolation and water tables were measured on a daily basis. The percolator is a steel cylindrical tube with a lid to prevent water loss from evapotranspiration. The water table meter is a short PVC tube for determination of perched water table and a long PVC tube for groundwater table, and the side is perforated with 5-mm diameter holes at 20-mm distance. Percolation rate was determined using linear regression analysis of cumulative percolation. Assuming that the total amount of evaporation and transpiration was equivalent to potential evapotranspiration, the lateral water flow was estimated using the water balance equation. Our results are in agreement with the previously reported findings, and the methodology of estimating water balance components appears reasonably acceptable. With regard to the toposequential variation, the higher position in the topoesquence, the greater potential of the water losses because of higher percolation and lateral flow rates.

Incorporating level set methods in Geographical Information Systems (GIS) for land-surface process modelling

Land-surface processes include a broad class of models that operate at a landscape scale. Current modelling approaches tend to be specialised towards one type of process, yet it is the interaction of processes that is increasing seen as important to obtain a more integrated approach to land management. This paper presents a technique and a tool that may be applied generically to landscape processes. The technique tracks moving interfaces across landscapes for processes such as water flow, biochemical diffusion, and plant dispersal. Its theoretical development applies a Lagrangian approach to motion over a Eulerian grid space by tracking quantities across a landscape as an evolving front. An algorithm for this technique, called level set method, is implemented in a geographical information system (GIS). It fits with a field data model in GIS and is implemented as operators in map algebra. The paper describes an implementation of the level set methods in a map algebra programming language, called MapScript, and gives example program scripts for applications in ecology and hydrology.