Broad Bean Stain Virus: Identification, Detectability with ELISA in Faba Bean Leaves and Seeds, Occurrence in West Asia and North Africa and Possible Wild Hosts

Abstract During a survey of faba bean viruses in West Asia and North Africa a virus was identified as broad bean stain virus (BBSV) based on host reactions, electron microscopy, physical properties and serology. An antiserum to a Syrian isolate was prepared. With this antiserum both the direct double antibody sandwich ELISA (DAS-ELISA) and dot-ELISA were very sensitive in detecting BBSV in leaf extracts, ground whole seeds and germi nated embryos. Sens it i vity was not reduced when the two-day procedure was replaced by a one-day procedure. us i ng ELISA the vi rus was detected in 73 out of 589 faba bean samples with virus-like symptoms collected from Egypt (4 out of 70 samples tested), Lebanon (6/44) , Morocco (017), Sudan (19/254), Syria (36/145) and Tunisia (8/69). This is the first report of BBSV infection of faba bean in Lebanon, Sudan, Syria and Tunisia. speci es i ndi genous to Syri a were Fourteen wild legume susceptible to BBSV infection, with only two producing obvious symptoms. The virus was found to be seed transmitted ~n Vicia palaestina.

Can rodents enhance germination rates in rainforest seeds?

The decline of large coevolved frugivorous species within fragmented habitats can have an effect on ecological processes, for example, seed dispersal and germination. It is therefore necessary for more resilient species to ensure essential processes are maintained within the system. This study investigates the influence of two rodent species, Melomys cervinipes (Fawn-footed Melomys) and Rattus fuscipes (Bush Rat), on the germination process of rainforest fruits. Both species are endemic to north Queensland rainforest and commonly found in fragmented habitats in high densities. We found in 85% of fruit species tested, rodent feeding increased seed germination rate by a factor of 3.5. Our results suggest that rodents can play a significant role in enhancing germination rates of fruits in the tropical rainforest of far north Queensland.

Pyrolysis decomposition of mahogony seeds (Swietenia macrophylla) for alternative fuel oil

A fixed bed pyrolysis has been designed and fabricated for obtaining liquid fuel from Mahogany seeds. The major components of the system are fixed bed pyrolysis reactor, liquid condenser and liquid collectors. The Mahogany seed in particle form is pyrolysed in an externally heated 10 cm diameter and 36 cm high fixed bed reactor with nitrogen as the carrier gas. The reactor is heated by means of a biomass source cylindrical heater from 450oC to 600oC. The products are oil, char and gas. The reactor bed temperature, running time and feed particle size are considered as process parameters. A maximum liquid yield of 54wt% of biomass feed is obtained with particle size of 1.18 mm at a reactor bed temperature of 5500C with a running time of 90 minutes. The oil is found to possess favorable flash point and reasonable density and viscosity. The higher calorific value is found to be 39.9 MJ/kg which is higher than other biomass derived pyrolysis oils.

Seeds of cosmopolitan future? Young people and their aspirations for future mobility

Mobility across space is an exemplary characteristic of the global era and an important aspect of the cultural experience of many people in advanced industrialised nations. Mobility evokes powerful images that effectively counter any illusion of stationary life and provide a break from the insularity of the local and parochial. High levels of mobility are simultaneously a fact, a necessity and a cultural aspiration. In recent times, debates about mobility in social theory have considered the relationship between mobility and cosmopolitan culture and identities (Hannerz, 1990, Urry, 1990, 2000, Beck, 2006). Against this backdrop, this paper also draws on some of the more recent discussions about the emergence of globalised and cosmopolitan identities among young people (Beck and Beck-Gernsheim, 2009). Using data from a longitudinal study of young people in Queensland, this paper provides an insight into young people’s aspirations about future mobility. The data affirm Skeggs’ (2004) comment that mobility is an unequal resource, and demonstrate that aspirations of future mobility reflect numerous social, economic and cultural realities of young people’s lives. This inevitably leads us to problematise the established, and often abstract, nexus between cosmopolitanism and mobility in contemporary debates about cosmopolitanism.

Mechanism‐based selection of a potent kallikrein‐related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI‐1

Potent and specific enzyme inhibition is a key goal in the development of therapeutic inhibitors targeting proteolytic activity. The backbone-cyclized peptide, Sunflower Trypsin Inhibitor (SFTI-1) affords a scaffold that can be engineered to achieve both these aims. SFTI-1's mechanism of inhibition is unusual in that it shows fast-on/slow-off kinetics driven by cleavage and religation of a scissile bond. This phenomenon was used to select a nanomolar inhibitor of kallikrein-related peptidase 7 (KLK7) from a versatile library of SFTI variants with diversity tailored to exploit distinctive surfaces present in the active site of serine proteases. Inhibitor selection was achieved through the use of size exclusion chromatography to separate protease/inhibitor complexes from unbound inhibitors followed by inhibitor identification according to molecular mass ascertained by mass spectrometry. This approach identified a single dominant inhibitor species with molecular weight of 1562.4 Da, which is consistent with the SFTI variant SFTI-WCTF. Once synthesized individually this inhibitor showed an IC50 of 173.9 ± 7.6 nM against chromogenic substrates and could block protein proteolysis. Molecular modeling analysis suggested that selection of SFTI-WCTF was driven by specific aromatic interactions and stabilized by an enhanced internal hydrogen bonding network. This approach provides a robust and rapid route to inhibitor selection and design.

Anatomical studies of the flower, fruit and seeds of Myrceugenia rufa (Myrtaceae)

Myceugenia rufa is a rare and endemic species from the coast of central Chile. There are no published studies describing flower, fruit or seed anatomy. Forty-two accessions were collected from across the geographic range of the species. Reproductive structures were fixed, dehydrated, embedded in paraffin, sectioned and stained with Safranin O and Fast green. Anatomy of floral buds, mature flowers, fruits and seeds was described. Reproductive anatomy matches that of other Myrtaceae, such as presence of druses, internal phloem and schizogenous secretory cavities in buds, flowers, fruits and seeds. The anatomy and development of reproductive structures of M. rufa might enhance the understanding for future studies regarding natural reproduction and conservation programs.

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Water uptake of aerosols with a focus on seeded aerosols and instrumentation techniques

This thesis focuses on the volatile and hygroscopic properties of mixed aerosol species. In particular, the influence organic species of varying solubility have upon seed aerosols. Aerosol studies were conducted at the Paul Scherrer Institut Laboratory for Atmospheric Chemistry (PSI-LAC, Villigen, Switzerland) and at the Queensland University of Technology International Laboratory for Air Quality and Health (QUT-ILAQH, Brisbane, Australia). The primary measurement tool employed in this program was the Volatilisation and Hygroscopicity Tandem Differential Mobility Analyser (VHTDMA - Johnson et al. 2004). This system was initially developed at QUT within the ILAQH and was completely re-developed as part of this project (see Section 1.4 for a description of this process). The new VHTDMA was deployed to the PSI-LAC where an analysis of the volatile and hygroscopic properties of ammonium sulphate seeds coated with organic species formed from the photo-oxidation of á-pinene was conducted. This investigation was driven by a desire to understand the influence of atmospherically prevalent organics upon water uptake by material with cloud forming capabilities. Of particular note from this campaign were observed influences of partially soluble organic coatings upon inorganic ammonium sulphate seeds above and below their deliquescence relative humidity (DRH). Above the DRH of the seed increasing the volume fraction of the organic component was shown to reduce the water uptake of the mixed particle. Below the DRH the organic was shown to activate the water uptake of the seed. This was the first time this effect had been observed for á-pinene derived SOA. In contrast with the simulated aerosols generated at the PSI-LAC a case study of the volatile and hygroscopic properties of diesel emissions was undertaken. During this stage of the project ternary nucleation was shown, for the first time, to be one of the processes involved in formation of diesel particulate matter. Furthermore, these particles were shown to be coated with a volatile hydrophobic material which prevented the water uptake of the highly hygroscopic material below. This result was a first and indicated that previous studies into the hygroscopicity of diesel emission had erroneously reported the particles to be hydrophobic. Both of these results contradict the previously upheld Zdanovksii-Stokes-Robinson (ZSR) additive rule for water uptake by mixed species. This is an important contribution as it adds to the weight of evidence that limits the validity of this rule.

What lies beneath? : The pattern and abundance of the subterranean tuber bank of the invasive liana cat’s claw creeper, Macfadyena unguis-cati (Bignoniaceae)

Cat’s claw creeper, Macfadyena unguis-cati (L.) Gentry (Bignoniaceae) is a major environmental weed of riparian areas, rainforest communities and remnant natural vegetation in coastal Queensland and New South Wales, Australia. In densely infested areas, it smothers standing vegetation, including large trees, and causes canopy collapse. Quantitative data on the ecology of this invasive vine are generally lacking. The present study examines the underground tuber traits of M. unguis-cati and explores their links with aboveground parameters at five infested sites spanning both riparian and inland vegetation. Tubers were abundant in terms of density (~1000 per m2), although small in size and low in level of interconnectivity. M. unguis-cati also exhibits multiple stems per plant. Of all traits screened, the link between stand (stem density) and tuber density was the most significant and yielded a promising bivariate relationship for the purposes of estimation, prediction and management of what lies beneath the soil surface of a given M. unguis-cati infestation site. The study also suggests that new recruitment is primarily from seeds, not from vegetative propagation as previously thought. The results highlight the need for future biological-control efforts to focus on introducing specialist seed- and pod-feeding insects to reduce seed-output.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Trailblazers

A recent study in the United Kingdom (Ofsted Report 2008) provides strong evidence that well-organized activities outside the classroom contribute significantly to the quality and depth of children's learning, including their personal, social, and emotional development. Outdoor math trails supply further evidence of such enhanced learning: They are meaningful, stimulating, challenging, and exciting for children. Most important, these trails invite all students, irrespective of their classroom achievement level, to participate successfully in the problem activities and gain a sense of pride in the mathematics they create. Additionally, Math trails empower lifelong learning. Integrating "outside" mathematics with "inside" classroom mathematics can sow the seeds to develop flexible, creative, future-oriented mathematical thinkers and problem solvers. Here, English et al discuss how to design and implement math trails to promote active, meaningful, real-world mathematical learning beyond the classroom walls.

Seed Transmission of Broad Bean Stain Virus in the Wild Legume Vicia Palestina Boiss

Abstract Seed-transmissibility of brood bean stain virus (BBSV) was investigated in a number of wild legume species. Genninating axes of seeds coliected from BBSV -infected plants were tested by the enzyme-linked immunosorbent assay (ELISA). The virus was found to be seedtransmitted in Vida pal«stina.

Can hybridization cause local extinction : the case for demographic swamping of the Australian native, Senecio pinnatifolius, by the invasive, S. madagascariensis?

The outcome of interspecific hybridization between native and invasive species depends on the relative frequencies of parental taxa and viability of hybrid progeny. We investigated individual and population level consequences of hybridization between the Australian native, Senecio pinnatifolius, and the exotic S. madagascariensis, with AFLP markers and used this information to simulate the expected outcome of hybridization.A high frequency (range 8.3-75.6 %) of hybrids was detected in open pollinated seeds of both species, but mature hybrids were absent from sympatric populations indicating that sympatric populations represent tension zones. A hybridization advantage was observed for S. madagascariensis,where significantly more progeny than expected were sired based on proportional representation of the two species in sympatric populations. Simulations indicated S. pinnatifolius could be replaced in sympatric populations if hybridization was density dependent.For this native-exotic pair, prezygotic isolating barriers are weak, but low hybrid viability maintains a strong postzygotic barrier to introgression. Due to asymmetric hybridization, S. pinnatifolius appears under threat from demographic swamping, and local extinction is possible where it occurs in sympatry with S. madagascariensis.

Natural and engineered kallikrein inhibitors: an emerging pharmacopoeia

The kallikreins and kallikrein-related peptidases are serine proteases that control a plethora of developmental and homeostatic phenomena, ranging from semen liquefaction to skin desquamation and blood pressure. The diversity of roles played by kallikreins has stimulated considerable interest in these enzymes from the perspective of diagnostics and drug design. Kallikreins already have well-established credentials as targets for therapeutic intervention and there is increasing appreciation of their potential both as biomarkers and as targets for inhibitor design. Here, we explore the current status of naturally occurring kallikrein protease-inhibitor complexes and illustrate how this knowledge can interface with strategies for rational re-engineering of bioscaffolds and design of small-molecule inhibitors.