Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Implications of patterns of carbon pools and fluxes across a semiarid environmental gradient

Landscape scale environmental gradients present variable spatial patterns and ecological processes caused by climate, topography and soil characteristics and, as such, offer candidate sites to study environmental change. Data are presented on the spatial pattern of dominant species, biomass, and carbon pools and the temporal pattern of fluxes across a transitional zone shifting from Great Basin Desert scrub, up through pinyon-juniper woodlands and into ponderosa pine forest and the ecotones between each vegetation type. The mean annual temperature (MAT) difference across the gradient is approximately 3 degrees C from bottom to top (MAT 8.5-5.5) and annual precipitation averages from 320 to 530 mm/yr, respectively. The stems of the dominant woody vegetation approach a random spatial pattern across the entire gradient, while the canopy cover shows a clustered pattern. The size of the clusters increases with elevation according to available soil moisture which in turn affects available nutrient resources. The total density of woody species declines with increasing soil moisture along the gl-adient, but total biomass increases. Belowground carbon and nutrient pools change from a heterogenous to a homogenous distribution on either side of the woodlands. Although temperature controls the: seasonal patterns of carbon efflux from the soils, soil moisture appears to be the primary driving variable, but response differs underneath the different dominant species, Similarly, decomposition of dominant litter occurs faster-at the cooler and more moist sites, but differs within sites due to litter quality of the different species. The spatial pattern of these communities provides information on the direction of future changes, The ecological processes that we documented are not statistically different in the ecotones as compared to the: adjoining communities, but are different at sites above the woodland than those below the woodland. We speculate that an increase in MAT will have a major impact on C pools and C sequestering and release processes in these semiarid landscapes. However, the impact will be primarily related to moisture availability rather than direct effects of an increase in temperature. (C) 1998 Elsevier Science B.V.

Shoot growth dynamics and photosynthetic response to increased nitrogen availability in the alpine willow salix-glauca

Plants subjected to increases in the supply of resource(s) limiting growth may allocate more of those resources to existing leaves, increasing photosynthetic capacity, and/or to production of more leaves, increasing whole-plant photosynthesis. The responses of three populations of the alpine willow, Salix glauca, growing along an alpine topographic sequence representing a gradient in soil moisture and organic matter, and thus potential N supply, to N amendments, were measured over two growing seasons, to elucidate patterns of leaf versus shoot photosynthetic responses. Leaf-(foliar N, photosynthesis rates, photosynthetic N-use efficiency) and shoot-(leaf area per shoot, number of leaves per shoot, stem weight, N resorption efficiency) level measurements were made to examine the spatial and temporal variation in these potential responses to increased N availability. The predominant response of the willows to N fertilization was at the shoot-level, by production of greater leaf area per shoot. Greater leaf area occurred due to production of larger leaves in both years of the experiment and to production of more leaves during the second year of fertilization treatment. Significant leaf-level photosynthetic response occurred only during the first year of treatment, and only in the dry meadow population. Variation in photosynthesis rates was related more to variation in stomatal conductance than to foliar N concentration. Stomatal conductance in turn was significantly related to N fertilization. Differences among the populations in photosynthesis, foliar N, leaf production, and responses to N fertilization indicate N availability may be lowest in the dry meadow population, and highest in the ridge population. This result is contrary to the hypothesis that a gradient of plant available N corresponds with a snowpack/topographic gradient.

The relationship between substrate metabolism, exercise and appetite control : does glycogen availability influence the motivation to eat, energy intake or food choice?

The way in which metabolic fuels are utilised can alter the expression of behaviour in the interests of regulating energy balance and fuel availability. This is consistent with the notion that the regulation of appetite is a psychobiological process, in which physiological mediators act as drivers of behaviour. The glycogenostatic theory suggests that glycogen availability is central in eliciting negative feedback signals to restore energy homeostasis. Due to its limited storage capacity, carbohydrate availability is tightly regulated and its restoration is a high metabolic priority following depletion. It has been proposed that such depletion may act as a biological cue to stimulate compensatory energy intake in an effort to restore availability. Due to the increased energy demand, aerobic exercise may act as a biological cue to trigger compensatory eating as a result of perturbations to muscle and liver glycogen stores. However, studies manipulating glycogen availability over short-term periods (1-3 days) using exercise, diet or both have often produced equivocal findings. There is limited but growing evidence to suggest that carbohydrate balance is involved in the short-term regulation of food intake, with a negative carbohydrate balance having been shown to predict greater ad libitum feeding. Furthermore, a negative carbohydrate balance has been shown to be predictive of weight gain. However, further research is needed to support these findings as the current research in this area is limited. In addition, the specific neural or hormonal signal through which carbohydrate availability could regulate energy intake is at present unknown. Identification of this signal or pathway is imperative if a casual relationship is to be established. Without this, the possibility remains that the associations found between carbohydrate balance and food intake are incidental.

The application of profluorescent nitroxides to detect reactive oxygen species derived from combustion-generated particulate matter: Cigarette smoke – a case study

Reactive oxygen species (ROS) and related free radicals are considered to be key factors underpinning the various adverse health effects associated with exposure to ambient particulate matter. Therefore, measurement of ROS is a crucial factor for assessing the potential toxicity of particles. In this work, a novel profluorescent nitroxide, BPEAnit, was investigated as a probe for detecting particle-derived ROS. BPEAnit has a very low fluorescence emission due to inherent quenching by the nitroxide group, but upon radical trapping or redox activity, a strong fluorescence is observed. BPEAnit was tested for detection of ROS present in mainstream and sidestream cigarette smoke. In the case of mainstream cigarette smoke, there was a linear increase in fluorescence intensity with an increasing number of cigarette puffs, equivalent to an average of 101 nmol ROS per cigarette based on the number of moles of the probe reacted. Sidestream cigarette smoke sampled from an environmental chamber exposed BPEAnit to much lower concentrations of particles, but still resulted in a clearly detectible increase in fluorescence intensity with sampling time. It was calculated that the amount of ROS was equivalent to 50 ± 2 nmol per mg of particulate matter; however, this value decreased with ageing of the particles in the chamber. Overall, BPEAnit was shown to provide a sensitive response related to the oxidative capacity of the particulate matter. These findings present a good basis for employing the new BPEAnit probe for the investigation of particle-related ROS generated from cigarette smoke as well as from other combustion sources.

Vectors and alternative hosts of tobacco yellow dwarf virus in southeastern Australia

Factors that determine the epidemiology of Tobacco yellow dwarf virus (TbYDV), including alternative host plants and insect vector(s), were assessed over three consecutive growing seasons at four field sites in Northeastern Victoria in commercial tobacco growing properties. In addition, these factors were assessed for one growing season at three bean growing properties. Overall, 23 leafhopper species were identified at the 7 sites, with Orosius orientalis as the predominant leafhopper. Of the leafhoppers collected, only O. orientalis and Anzygina zealandica tested positive for TbYDV by polymerase chain reaction (PCR). The population dynamics of O. orientalis was assessed using sweep net sampling over three growing seasons and a trimodal distribution was observed. Despite large numbers of O. orientalis occurring early in the growing season (September–October), TbYDV was only detected in these leafhoppers between late November and end of January. The peaks in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and were associated with warmer temperatures and lower rainfall. Spatial and temporal distribution of vegetation at selected sites was determined using quadrat sampling. Of the 40 plant species identified, TbYDV was detected only in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. The proportion of host and non-host availability for leafhoppers was associated with climatic conditions.

Molecular characterisation of six badnavirus species associated with leaf streak disease of banana in East Africa

Banana leaf streak disease, caused by several species of Banana streak virus (BSV), is widespread in East Africa. We surveyed for this disease in Uganda and Kenya, and used rolling-circle amplification (RCA) to detect the presence of BSV in banana. Six distinct badnavirus sequences, three from Uganda and three from Kenya, were amplified for which only partial sequences were previously available. The complete genomes were sequenced and characterised. The size and organisation of all six sequences was characteristic of other badnaviruses, including conserved functional domains present in the putative polyprotein encoded by open reading frame (ORF) 3. Based on nucleotide sequence analysis within the reverse transcriptase/ribonuclease H-coding region of open reading frame 3, we propose that these sequences be recognised as six new species and be designated as Banana streak UA virus, Banana streak UI virus, Banana streak UL virus, Banana streak UM virus, Banana streak CA virus and Banana streak IM virus. Using PCR and species-specific primers to test for the presence of integrated sequences, we demonstrated that sequences with high similarity to BSIMV only were present in several banana cultivars which had tested negative for episomal BSV sequences.

Evaluation of spectral and texture features for object-based vegetation species classification using Support Vector Machines

The use of appropriate features to characterize an output class or object is critical for all classification problems. This paper evaluates the capability of several spectral and texture features for object-based vegetation classification at the species level using airborne high resolution multispectral imagery. Image-objects as the basic classification unit were generated through image segmentation. Statistical moments extracted from original spectral bands and vegetation index image are used as feature descriptors for image objects (i.e. tree crowns). Several state-of-art texture descriptors such as Gray-Level Co-Occurrence Matrix (GLCM), Local Binary Patterns (LBP) and its extensions are also extracted for comparison purpose. Support Vector Machine (SVM) is employed for classification in the object-feature space. The experimental results showed that incorporating spectral vegetation indices can improve the classification accuracy and obtained better results than in original spectral bands, and using moments of Ratio Vegetation Index obtained the highest average classification accuracy in our experiment. The experiments also indicate that the spectral moment features also outperform or can at least compare with the state-of-art texture descriptors in terms of classification accuracy.

Color and texture feature fusion using kernel PCA with application to object-based vegetation species classification

A good object representation or object descriptor is one of the key issues in object based image analysis. To effectively fuse color and texture as a unified descriptor at object level, this paper presents a novel method for feature fusion. Color histogram and the uniform local binary patterns are extracted from arbitrary-shaped image-objects, and kernel principal component analysis (kernel PCA) is employed to find nonlinear relationships of the extracted color and texture features. The maximum likelihood approach is used to estimate the intrinsic dimensionality, which is then used as a criterion for automatic selection of optimal feature set from the fused feature. The proposed method is evaluated using SVM as the benchmark classifier and is applied to object-based vegetation species classification using high spatial resolution aerial imagery. Experimental results demonstrate that great improvement can be achieved by using proposed feature fusion method.

Alkaline hydrothermal treatment of titanate nanostructures

Since its initial proposal in 1998, alkaline hydrothermal processing has rapidly become an established technology for the production of titanate nanostructures. This simple, highly reproducible process has gained a strong research following since its conception. However, complete understanding and elucidation of nanostructure phase and formation have not yet been achieved. Without fully understanding phase, formation, and other important competing effects of the synthesis parameters on the final structure, the maximum potential of these nanostructures cannot be obtained. Therefore this study examined the influence of synthesis parameters on the formation of titanate nanostructures produced by alkaline hydrothermal treatment. The parameters included alkaline concentration, hydrothermal temperature, the precursor material‘s crystallite size and also the phase of the titanium dioxide precursor (TiO2, or titania). The nanostructure‘s phase and morphology was analysed using X-ray diffraction (XRD), Raman spectroscopy and transmission electron microscopy. X-ray photoelectron spectroscopy (XPS), dynamic light scattering (non-invasive backscattering), nitrogen sorption, and Rietveld analysis were used to determine phase, for particle sizing, surface area determinations, and establishing phase concentrations, respectively. This project rigorously examined the effect of alkaline concentration and hydrothermal temperature on three commercially sourced and two self-prepared TiO2 powders. These precursors consisted of both pure- or mixed-phase anatase and rutile polymorphs, and were selected to cover a range of phase concentrations and crystallite sizes. Typically, these precursors were treated with 5–10 M sodium hydroxide (NaOH) solutions at temperatures between 100–220 °C. Both nanotube and nanoribbon morphologies could be produced depending on the combination of these hydrothermal conditions. Both titania and titanate phases are comprised of TiO6 units which are assembled in different combinations. The arrangement of these atoms affects the binding energy between the Ti–O bonds. Raman spectroscopy and XPS were therefore employed in a preliminary study of phase determination for these materials. The change in binding energy from a titania to a titanate binding energy was investigated in this study, and the transformation of titania precursor into nanotubes and titanate nanoribbons was directly observed by these methods. Evaluation of the Raman and XPS results indicated a strengthening in the binding energies of both the Ti (2p3/2) and O (1s) bands which correlated to an increase in strength and decrease in resolution of the characteristic nanotube doublet observed between 320 and 220 cm.1 in the Raman spectra of these products. The effect of phase and crystallite size on nanotube formation was examined over a series of temperatures (100.200 �‹C in 20 �‹C increments) at a set alkaline concentration (7.5 M NaOH). These parameters were investigated by employing both pure- and mixed- phase precursors of anatase and rutile. This study indicated that both the crystallite size and phase affect nanotube formation, with rutile requiring a greater driving force (essentially �\harsher. hydrothermal conditions) than anatase to form nanotubes, where larger crystallites forms of the precursor also appeared to impede nanotube formation slightly. These parameters were further examined in later studies. The influence of alkaline concentration and hydrothermal temperature were systematically examined for the transformation of Degussa P25 into nanotubes and nanoribbons, and exact conditions for nanostructure synthesis were determined. Correlation of these data sets resulted in the construction of a morphological phase diagram, which is an effective reference for nanostructure formation. This morphological phase diagram effectively provides a .recipe book�e for the formation of titanate nanostructures. Morphological phase diagrams were also constructed for larger, near phase-pure anatase and rutile precursors, to further investigate the influence of hydrothermal reaction parameters on the formation of titanate nanotubes and nanoribbons. The effects of alkaline concentration, hydrothermal temperature, crystallite phase and size are observed when the three morphological phase diagrams are compared. Through the analysis of these results it was determined that alkaline concentration and hydrothermal temperature affect nanotube and nanoribbon formation independently through a complex relationship, where nanotubes are primarily affected by temperature, whilst nanoribbons are strongly influenced by alkaline concentration. Crystallite size and phase also affected the nanostructure formation. Smaller precursor crystallites formed nanostructures at reduced hydrothermal temperature, and rutile displayed a slower rate of precursor consumption compared to anatase, with incomplete conversion observed for most hydrothermal conditions. The incomplete conversion of rutile into nanotubes was examined in detail in the final study. This study selectively examined the kinetics of precursor dissolution in order to understand why rutile incompletely converted. This was achieved by selecting a single hydrothermal condition (9 M NaOH, 160 °C) where nanotubes are known to form from both anatase and rutile, where the synthesis was quenched after 2, 4, 8, 16 and 32 hours. The influence of precursor phase on nanostructure formation was explicitly determined to be due to different dissolution kinetics; where anatase exhibited zero-order dissolution and rutile second-order. This difference in kinetic order cannot be simply explained by the variation in crystallite size, as the inherent surface areas of the two precursors were determined to have first-order relationships with time. Therefore, the crystallite size (and inherent surface area) does not affect the overall kinetic order of dissolution; rather, it determines the rate of reaction. Finally, nanostructure formation was found to be controlled by the availability of dissolved titanium (Ti4+) species in solution, which is mediated by the dissolution kinetics of the precursor.