In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the b-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Bandwidth considerations for multilevel converters

Multilevel converters can achieve an overall effective switch frequency multiplication and consequent ripple reduction through the cancellation of the lowest order switch frequency terms. This paper investigates the harmonic content and the frequency response of these multimodulator converters. It is shown that the transfer function of uniformly sampled modulators is a bessel function associated with the inherent sampling process. Naturally sampled modulators have a flat transfer function, but multiple switchings per switch cycle will occur unless the input is slew-rate limited. Lower sideband harmonics of the effective carrier frequency and, in uniform converters, harmonics of the input signal also limit the useful bandwidth. Observations about the effect of the number of converters, their type (naturally or uniformly sampled), and the ratio of modulating frequency and switch frequency are made

High efficiency auxiliary power converters

The auxiliary load DC-DC converters of the Sunshark solar car have never been examined. An analysis of the current design reveals it is complicated, and inefficient. Some simple measures to greatly improve the efficiency are present which will achieve an overall worthwhile power saving. Two switch-mode power supply DC-DC converter designs are presented. One is a constant current supply for the LED brake and turn indicators, which allows them to be powered directly from the main DC bus, and switched only as necessary. The second is a low power flyback converter, which employs synchronous rectification among other techniques to achieve good efficiency and regulation over a large range of output powers. Practical results from both converters, and an indication of the overall improvement in system efficiency will be offered.

Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

A cross-discipline and collaborative approach to identifying the predictors of environmentally friendly and health protective behaviour

Background. As a society, our interaction with the environment is having a negative impact on human health. For example, an increase in car use for short trips, over walking or cycling, has contributed to an increase in obesity, diabetes and poor heart health and also contributes to pollution, which is associated with asthma and other respiratory diseases. In order to change the nature of that interaction, to be more positive and healthy, it is recommended that individuals adopt a range of environmentally friendly behaviours (such as walking for transport and reducing the use of plastics). Effective interventions aimed at increasing such behaviours will need to be evidence based and there is a need for the rapid communication of information from the point of research, into policy and practice. Further, a number of health disciplines, including psychology and public health, share a common mission to promote health and well-being. Therefore, the objective of this project is to take a cross-discipline and collaborative approach to reveal psychological mechanisms driving environmentally friendly behaviour. This objective is further divided into three broad aims, the first of which is to take a cross-discipline and collaborative approach to research. The second aim is to explore and identify the salient beliefs which most strongly predict environmentally friendly behaviour. The third aim is to build an augmented model to explain environmentally friendly behaviour. The thesis builds on the understanding that an interdisciplinary collaborative approach will facilitate the rapid transfer of knowledge to inform behaviour change interventions. Methods. The application of this approach involved two surveys which explored the psycho-social predictors of environmentally friendly behaviour. Following a qualitative pilot study, and in collaboration with an expert panel comprising academics, industry professionals and government representatives, a self-administered, Theory of Planned Behaviour (TPB) based, mail survey was distributed to a random sample of 3000 residents of Brisbane and Moreton Bay Region (Queensland, Australia). This survey explored specific beliefs including attitudes, norms, perceived control, intention and behaviour, as well as environmental altruism and green identity, in relation to walking for transport and switching off lights when not in use. Following analysis of the mail survey data and based on feedback from participants and key stakeholders, an internet survey was employed (N=451) to explore two additional behaviours, switching off appliances at the wall when not in use, and shopping with reusable bags. This work is presented as a series of interrelated publications which address each of the research aims. Presentation of Findings. Chapter five of this thesis consists of a published paper which addresses the first aim of the research and outlines the collaborative and multidisciplinary approach employed in the mail survey. The paper argued that forging alliances with those who are in a position to immediately utilise the findings of research has the potential to improve the quality and timely communication of research. Illustrating this timely communication, Chapter six comprises a report presented to Moreton Bay Regional Council (MBRC). This report addresses aim's one and two. The report contains a summary of participation in a range of environmentally friendly behaviours and identifies the beliefs which most strongly predicted walking for transport and switching off lights (from the mail survey). These salient beliefs were then recommended as targets for interventions and included: participants believing that they might save money; that their neighbours also switch off lights; that it would be inconvenient to walk for transport and that their closest friend also walks for transport. Chapter seven also addresses the second aim and presents a published conference paper in which the salient beliefs predicting the four specified behaviours (from both surveys) are identified and potential applications for intervention are discussed. Again, a range of TPB based beliefs, including descriptive normative beliefs, were predictive of environmentally friendly behaviour. This paper was also provided to MBRC, along with recommendations for applying the findings. For example, as descriptive normative beliefs were consistently correlated with environmentally friendly behaviour, local councils could engage in marketing and interventions (workshops, letter box drops, internet promotions) which encourage parents and friends to model, rather than simply encourage, environmentally friendly behaviour. The final two papers, presented in Chapters eight and nine, addresses the third aim of the project. These papers each present two behaviours together to inform a TPB based theoretical model with which to predict environmentally friendly behaviour. A generalised model is presented, which is found to predict the four specific behaviours under investigation. The role of demographics was explored across each of the behaviour specific models. It was found that some behaviour's differ by age, gender, income or education. In particular, adjusted models predicted more of the variance in walking for transport amongst younger participants and females. Adjusted models predicted more variance in switching off lights amongst those with a bachelor degree or higher and predicted more variance in switching off appliances amongst those on a higher income. Adjusted models predicted more variance in shopping with reusable bags for males, people 40 years or older, those on a higher income and those with a bachelor degree or higher. However, model structure and general predictability was relatively consistent overall. The models provide a general theoretical framework from which to better understand the motives and predictors of environmentally friendly behaviour. Conclusion. This research has provided an example of the benefits of a collaborative interdisciplinary approach. It has identified a number of salient beliefs which can be targeted for social marketing campaigns and educational initiatives; and these findings, along with recommendations, have been passed on to a local council to be used as part of their ongoing community engagement programs. Finally, the research has informed a practical model, as well as behaviour specific models, for predicting sustainable living behaviours. Such models can highlight important core constructs from which targeted interventions can be designed. Therefore, this research represents an important step in undertaking collaborative approaches to improving population health through human-environment interactions.

The changing performance of multifocal contact lenses compared to monovision.

Purpose:Multifocal contact lenses (MCLs) have been available for decades. A review of the literature suggests that while, historically, these lenses have been partially successful, they have struggled to compete with monovision (MV). More recent publications suggest that there has been an improvement in the performance of these lenses. This study set out to investigate whether the apparent improved lens performance reported in the literature is reflected in clinical practice. Methods:Data collected over the last 5yrs via the International Contact Lens Prescribing Survey Consortium was reviewed for patients over the age of 45yrs. The published reports of clinical trials were reviewed to assess lens performance over the time period. Results:Data review was of 16,680 presbyopic lens fits in 38 countries. The results are that 29% were fit with MCLs, 8% MV and 63% single vision (SV). A previous survey conducted in Australia during 1988-89 reported that 9% of presbyopes were fit with MCLs, 29% MV and 63% SV. The results from our survey for Australia alone were 28% (MV 13%) vs 9% (MV 29%) suggesting an increase in usage of MCLs from 1988-89 to 2010. A review of the literature indicates the reported level of visual acuities with MCLs in comparison to MV has remained equivalent over this time period, yet preference has switch from MV to MCLs. Conclusions:There is evidence that currently more MCLs than MV are being fit to presbyopes, compared to 1988-89. This increased use is likely due to the improved visual performance of these lenses, which is not demonstrated with acuity measures but reported by wearers, suggesting that patient-based subjective ratings are currently the best way to measure visual performance.

Australian baby boomers switched to more environment friendly modes of transport during the global financial crisis

The global financial crisis (GFC) in 2008 rocked local, regional, and state economies throughout the world. Several intermediate outcomes of the GFC have been well documented in the literature including loss of jobs and reduced income. Relatively little research has, however, examined the impacts of the GFC on individual level travel behaviour change. To address this shortcoming, HABITAT panel data were employed to estimate a multinomial logit model to examine mode switching behaviour between 2007 (pre-GFC) and 2009 (post-GFC) of a baby boomers cohort in Brisbane, Australia—a city within a developed country that has been on many metrics the least affected by the GFC. In addition, a Poisson regression model was estimated to model the number of trips made by individuals in 2007, 2008, and 2009. The South East Queensland Travel Survey datasets were used to develop this model. Four linear regression models were estimated to assess the effects of the GFC on time allocated to travel during a day: one for each of the three travel modes including public transport, active transport, less environmentally friendly transport; and an overall travel time model irrespective of mode. The results reveal that individuals were more likely to switch to public transport who lost their job or whose income reduced between 2007 and 2009. Individuals also made significantly fewer trips in 2008 and 2009 compared to 2007. Individuals spent significantly less time using less environmentally friendly transport but more time using public transport in 2009. Baby boomers switched to more environmentally friendly travel modes during the GFC.

Multidirectional effects of Sr, Mg and Si-containing bioceramic coatings with high bonding strength on inflammation, osteoclastogenesis and osteogenesis

Ideal coating materials for implants should be able to induce excellent osseointegration, which requires several important parameters, such as good bonding strength, limited inflammatory reaction, balanced osteoclastogenesis and osteogenesis, to gain well-functioning coated implants with long-term life span after implantation. Bioactive elements, like Sr, Mg and Si, have been found to play important roles in regulating the biological responses. It is of great interest to combine bioactive elements for developing bioactive coatings on Ti-6Al-4V orthopedic implants to elicit multidirectional effects on the osseointegration. In this study, Sr, Mg and Si-containing bioactive Sr2MgSi2O7 (SMS) ceramic coatings on Ti-6Al-4V were successfully prepared by plasma-spray coating method. The prepared SMS coatings have significantly higher bonding strength (~37MPa) than conventional pure hydroxyapatite (HA) coatings (mostly in the range of 15-25 MPa). It was also found that the prepared SMS coatings switch the macrophage phenotype into M2 extreme, inhibiting the inflammatory reaction via the inhibition of Wnt5A/Ca2+ and Toll-like receptor (TLR) pathways of macrophages. In addition, the osteoclastic activities were also inhibited by SMS coatings. The expression of osteoclastogenesis related genes (RANKL and MCSF) in bone marrow derived mesenchymal cells (BMSCs) with the involvement of macrophages was decreased, while OPG expression was enhanced on SMS coatings compared to HA coatings, indicating that SMS coatings also downregulated the osteoclastogenesis. However, the osteogenic differentiation of BMSCs with the involvement of macrophages was comparable between SMS and HA coatings. Therefore, the prepared SMS coatings showed multidirectional effects, such as improving bonding strength, reducing inflammatory reaction and downregulating osteoclastic activities, but maintaining a comparable osteogenesis, as compared with HA coatings. The combination of bioactive elements of Sr, Mg and Si into bioceramic coatings can be a promising method to develop bioactive implants with multifunctional properties for orthopaedic application.

Voltage unbalance reduction in low voltage feeders by dynamic switching of residential customers among three phases

Low voltage distribution feeders with large numbers of single phase residential loads experience severe current unbalance that often causes voltage unbalance problems. The addition of intermittent generation and new loads in the form of roof top photovoltaic generation and electric vehicles makes these problems even more acute. In this paper, an intelligent dynamic residential load transfer scheme is proposed. Residential loads can be transferred from one phase to another phase to minimize the voltage unbalance along the feeder. Each house is supplied through a static transfer switch with three-phase input and single-phase output connection. The main controller, installed at the transformer will observe the power consumption in each load and determine which house(s) should be transferred from one phase to another in order to keep the voltage unbalance in the feeder at a minimum. The efficacy of the proposed load transfer scheme is verified through MATLAB and PSCAD/EMTDC simulations.

Smart Inter-Phase Switching of Residential Loads in Low Voltage Distribution Feeders

In order to dynamically reduce voltage unbalance along a low voltage distribution feeder, a smart residential load transfer system is discussed. In this scheme, residential loads can be transferred from one phase to another to minimize the voltage unbalance along the feeder. Each house is supplied through a static transfer switch and a controller. The master controller, installed at the transformer, observes the power consumption in each house and will determine which house(s) should be transferred from an initially connected phase to another in order to keep the voltage unbalance minimum. The performance of the smart load transfer scheme is demonstrated by simulations.

The resistance of PRESENT-80 against related-key differential attacks

We examine the security of the 64-bit lightweight block cipher PRESENT-80 against related-key differential attacks. With a computer search we are able to prove that for any related-key differential characteristic on full-round PRESENT-80, the probability of the characteristic only in the 64-bit state is not higher than 2−64. To overcome the exponential (in the state and key sizes) computational complexity of the search we use truncated differences, however as the key schedule is not nibble oriented, we switch to actual differences and apply early abort techniques to prune the tree-based search. With a new method called extended split approach we are able to make the whole search feasible and we implement and run it in real time. Our approach targets the PRESENT-80 cipher however,with small modifications can be reused for other lightweight ciphers as well.

The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001–2013

Background The epidemiology of dengue in the South Pacific has been characterized by transmission of a single dominant serotype for 3–5 years, with subsequent replacement by another serotype. From 2001 to 2008 only DENV-1 was reported in the Pacific. In 2008, DENV-4 emerged and quickly displaced DENV-1 in the Pacific, except in New Caledonia (NC) where DENV-1 and DENV-4 co-circulated in 2008–2009. During 2012–2013, another DENV-1 outbreak occurred in NC, the third DENV-1 outbreak in a decade. Given that dengue is a serotype-specific immunizing infection, the recurrent outbreaks of a single serotype within a 10-year period was unexpected. Findings This study aimed to inform this phenomenon by examining the phylogenetic characteristics of the DENV-1 viruses in NC and other Pacific islands between 2001 and 2013. As a result, we have demonstrated that NC experienced introductions of viruses from both the Pacific (genotype IV) and South-east Asia (genotype I). Moreover, whereas genotype IV and I were co-circulating at the beginning of 2012, we observed that from the second half of 2012, i.e. during the major DENV-1 outbreak, all analyzed viruses were genotype I suggesting that a genotype switch occurred. Conclusions Repeated outbreaks of the same dengue serotype, as observed in NC, is uncommon in the Pacific islands. Why the earlier DENV-1 outbreaks did not induce sufficient herd immunity is unclear, and likely multifactorial, but the robust vector control program may have played a role by limiting transmission and thus maintaining a large susceptible pool in the population. Keywords: Dengue; Phylogeny; Genotype; Epidemics; New Caledonia

Photoelectron spectroscopy of HCCN- and HCNC- reveals the quasilinear triplet carbenes, HCCN and HCNC

Negative ion photoelectron spectroscopy has been used to study the HCCN- and HCNC- ions. The electron affinities (EA) of cyanocarbene have been measured to be EA(HCCN (X) over tilde (3)Sigma(-)=2.003+/-0.014 eV and EA(DCCN (X) over tilde (3)Sigma(-))=2.009+/-0.020 eV. Photodetachment of HCCN- to HCCN (X) over tilde (3)Sigma(-) shows a 0.4 eV long vibrational progression in nu(5), the H-CCN bending mode; the HCCN- photoelectron spectra reveal excitations up to 10 quanta in nu(5). The term energies for the excited singlet state are found to be T-0(HCCN (a) over tilde (1)A('))=0.515+/-0.016 eV and T-0(DCCN (a) over tilde (1)A('))=0.518+/-0.027 eV. For the isocyanocarbene, the two lowest states switch and HCNC has a singlet ground state and an excited triplet state. The electron affinities are EA(HCNC (X) over tilde (1)A('))=1.883+/-0.013 eV and EA((X) over tilde (1)A(') DCNC)=1.877+/-0.010 eV. The term energy for the excited triplet state is T-0(HCNC (a) over tilde (3)A("))=0.050+/-0.028 eV and T-0(DCNC (a) over tilde (3)A("))=0.063+/-0.030 eV. Proton transfer kinetics in a flowing afterglow apparatus were used to re-measure the enthalpy of deprotonation of CH3NC to be Delta(acid)H(298)(CH3NC)=383.6+/-0.6 kcal mol(-1). The acidity/EA thermodynamic cycle was used to deduce D-0(H-CHCN)=104+/-2 kcal mol(-1) [Delta(f)H(0)(HCCN)=110+/-4 kcal mol(-1)] and D-0(H-CHNC)=106+/-4 kcal mol(-1) [Delta(f)H(0)(HCNC)=133+/-5 kcal mol(-1)]. (C) 2002 American Institute of Physics.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.