The Farm Forestry Decision-Support System: Research Outcomes in Relation to Farm Surveys, the AFFFM and the MODSS

This chapter reviews the outcomes of the various components of the project 'A Whole-Farm and Regional Agroforestry Decision-Support System', including the landholder surveys and spatial modelling, and development and application of the decision-support systems (DSSs), and development and testing of the Australian Farm Forestry Financial Model. Achievements of the research and its relevance to decision-making at the farm and regional level are examined. Comments are also made on the viability of the approach of combining a number of separate research groups into a single team

Spherical Tin Oxide, SnO2 Particles Fabricated via Facile Hydrothermal Method for Detection of Mercury (II) Ions.

Smooth-surface spherical tin oxide particles were fabricated via hydrothermal processing route. X-ray diffraction (XRD) revealed that the particles consisted of the rutile phase of SnO2 with tetragonal structure. The spherical morphology was realized with the aid of ammonia. The aggregation of SnO2 particles could be avoided by adjusting the concentration of tin (II) chloride. Bare glassy carbon electrode (GCE) was modified with the hydrothermally prepared SnO2 particles to detect the presence of mercury (II) ions (Hg2+), in the presence of potassium chloride (KCl) as a supporting electrolyte. GCE modified with the spherical SnO2 particles that possessed small crystallite size and smooth surface exhibited significantly enhanced oxidative and reductive current of Hg2+ during cyclic voltammetry compared with its bare counterpart. The reductive current was observed to increase by two fold and the detection limit of 75 nM for Hg 2+ was achieved. This suggests that SnO2 particles are a promising chemical sensor for the detection of Hg2+ in natural waters

The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function

Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis

E2 ubiquitin-conjugating enzymes, UBE2D1 and UBE2D2, regulate VEGFR2 dynamics and endothelial function

Vascular endothelial growth factor receptor 2 (VEGFR2) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination which programs this receptor for trafficking and proteolysis but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen of the human E2 family of ubiquitin-conjugating enzymes to identify gene products which regulate VEGFR2 ubiquitination and proteolysis. We find that depletion of either UBE2D1 or UBE2D2 in endothelial cells cause a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impact on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase C1, and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell surface biotinylation and recycling studies show an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulates endothelial tubulogenesis which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis

Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development

The intracellular target of diphtheria toxin is a modified histidine residue, diphthamide, in the translation elongation factor, eEF2. This enigmatic modification occurs in all eukaryotes, and is produced in yeast by the action of five gene products, DPH1 to DPH5. Sequence homologues of these genes are present in all sequenced eukaryotic genomes and in higher eukaryotes there is functional evidence for DPH1, 2, 3, and 5 acting in diphthamide biosynthesis. We have identified a mouse mutant in the remaining gene, Dph4. Cells derived from homozygous mutant embryos lack the diphthamide modification of EF2 and are resistant to killing by diphtheria toxin. Reporter-tagged DPH4 protein localizes to the cytoskeleton, in contrast to the localization of DPH1, and consistent with evidence that DPH4 is not part of a proposed complex containing DPH1, 2 and 3. Mice homozygous for the mutation are retarded in growth and development and almost always die before birth. Those that survive long enough have preaxial polydactyly, a duplication of digit 1 of the hind foot. This same defect is seen in embryos homozygous for mutation of DPH1, suggesting that lack of diphthamide on eEF2 could result in translational failure of specific proteins, rather than a generalized translation downregulation

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.Peer reviewedFinal Published versio

Development and validation of protein microarray technology for simultaneous inflammatory mediator detection in human sera

Biomarkers, including cytokines, can help in the diagnosis, prognosis, and prediction of treatment response across a wide range of disease settings. Consequently, the recent emergence of protein microarray technology, which is able to quantify a range of inflammatory mediators in a large number of samples simultaneously, has become highly desirable. However, the cost of commercial systems remains somewhat prohibitive. Here we show the development, validation, and implementation of an in-house microarray platform which enables the simultaneous quantitative analysis of multiple protein biomarkers. The accuracy and precision of the in-house microarray system were investigated according to the Food and Drug Administration (FDA) guidelines for pharmacokinetic assay validation. The assay fell within these limits for all but the very low-abundant cytokines, such as interleukin- (IL-) 10. Additionally, there were no significant differences between cytokine detection using our microarray system and the “gold standard” ELISA format. Crucially, future biomarker detection need not be limited to the 16 cytokines shown here but could be expanded as required. In conclusion, we detail a bespoke protein microarray system, utilizing well-validated ELISA reagents, that allows accurate, precise, and reproducible multiplexed biomarker quantification, comparable with commercial ELISA, and allowing customization beyond that of similar commercial microarrays

Mapping the disease-specific LupusQoL to the SF-6D

Purpose To derive a mapping algorithm to predict SF-6D utility scores from the non-preference-based LupusQoL and test the performance of the developed algorithm on a separate independent validation data set. Method LupusQoL and SF-6D data were collected from 320 patients with systemic lupus erythematosus (SLE) attending routine rheumatology outpatient appointments at seven centres in the UK. Ordinary least squares (OLS) regression was used to estimate models of increasing complexity in order to predict individuals’ SF-6D utility scores from their responses to the LupusQoL questionnaire. Model performance was judged on predictive ability through the size and pattern of prediction errors generated. The performance of the selected model was externally validated on an independent data set containing 113 female SLE patients who had again completed both the LupusQoL and SF-36 questionnaires. Results Four of the eight LupusQoL domains (physical health, pain, emotional health, and fatigue) were selected as dependent variables in the final model. Overall model fit was good, with R2 0.7219, MAE 0.0557, and RMSE 0.0706 when applied to the estimation data set, and R2 0.7431, MAE 0.0528, and RMSE 0.0663 when applied to the validation sample. Conclusion This study provides a method by which health state utility values can be estimated from patient responses to the non-preference-based LupusQoL, generalisable beyond the data set upon which it was estimated. Despite concerns over the use of OLS to develop mapping algorithms, we find this method to be suitable in this case due to the normality of the SF-6D data

Improving the representation of adaptation in climate change impact models

Climate change adaptation is a complex human process, framed by uncertainties and constraints, which is difficult to capture in existing assessment models. Attempts to improve model representations are hampered by a shortage of systematic descriptions of adaptation processes and their relevance to models. This paper reviews the scientific literature to investigate conceptualisations and models of climate change adaptation, and the ways in which representation of adaptation in models can be improved. The review shows that real-world adaptive responses can be differentiated along a number of dimensions including intent or purpose, timescale, spatial scale, beneficiaries and providers, type of action, and sector. However, models of climate change consequences for land use and water management currently provide poor coverage of these dimensions, instead modelling adaptation in an artificial and subjective manner. While different modelling approaches do capture distinct aspects of the adaptive process, they have done so in relative isolation, without producing improved unified representations. Furthermore, adaptation is often assumed to be objective, effective and consistent through time, with only a minority of models taking account of the human decisions underpinning the choice of adaptation measures (14%), the triggers that motivate actions (38%) or the time-lags and constraints that may limit their uptake and effectiveness (14%). No models included adaptation to take advantage of beneficial opportunities of climate change. Based on these insights, transferable recommendations are made on directions for future model development that may enhance realism within models, while also advancing our understanding of the processes and effectiveness of adaptation to a changing climate