Beschreibung: Background: Cholesterol oxidases are important enzymes for applications such as the analysis of cholesterol in clinical samples, the synthesis of steroid derived drugs and are considered as potential antibacterial drug targets. Results: The gene choA encoding a cholesterol oxidase from Chryseobacterium gleum DSM 16776 was cloned into the pQE-30 expression vector and heterologously expressed in Escherichia coli JM109 co-transformed with pRARE2. The N-terminally His-tagged cholesterol oxidase (CgChoA) was assigned to be a monomer in solution by size exclusion chromatography, showed a temperature optimum of 35[degree sign]C, and a pH optimum at 6.75 using 0.011 M MOPS buffer under the tested conditions. The purified protein showed a maximum activity of 15.5 U/mg. CgChoA showed a Michaelis-Menten kinetic behavior only when the substrate was dissolved in water and taurocholate (apparent Km = 0.5 mM). In addition, the conversion of cholesterol by CgChoA was studied via biocatalytic batches at analytical scale, and cholest-4-en-3-one was confirmed as product by HPLC-MS. Conclusion: CgChoA is a true cholesterol oxidase which activity ranges among the high performing described cholesterol oxidases from other organisms. Thus, the enzyme broadens the available toolbox of cholesterol oxidases for e.g. synthetic and biosensing applications.

Beschreibung: Background: In light of the changes in precipitation and soil water availability expected with climate change, understanding the mechanisms underlying plant responses to water deficit is essential. Toward that end we have conducted an integrative analysis of responses to drought stress in the perennial C4 grass and biofuel crop, Panicum virgatum (switchgrass). Responses to soil drying and re-watering were measured at transcriptional, physiological, and metabolomic levels. To assess the interaction of soil moisture with diel light:dark cycles, we profiled gene expression in drought and control treatments under pre-dawn and mid-day conditions. Results: Soil drying resulted in reduced leaf water potential, gas exchange, and chlorophyll fluorescence along with differential expression of a large fraction of the transcriptome (37%). Many transcripts responded differently depending on time of day (e.g. up-regulation pre-dawn and down-regulation mid-day). Genes associated with C4 photosynthesis were down-regulated during drought, while C4 metabolic intermediates accumulated. Rapid changes in gene expression were observed during recovery from drought, along with increased water use efficiency and chlorophyll fluorescence. Conclusions: Our findings demonstrate that drought responsive gene expression depends strongly on time of day and that gene expression is extensively modified during the first few hours of drought recovery. Analysis of covariation in gene expression, metabolite abundance, and physiology among plants revealed non-linear relationships that suggest critical thresholds in drought stress responses. Future studies may benefit from evaluating these thresholds among diverse accessions of switchgrass and other C4 grasses.

Beschreibung: Background: The importance of consumer trust in the food supply has previously been identified, and dimensions of consumer trust in food---who they trust and the type of trust that they exhibit---has been explored. However, there is a lack of research about the mechanisms through which consumer trust in the food supply is developed, maintained, broken and repaired. This study seeks to address this gap by exploring if, and how, consumer trust in the food supply is considered by the media, food industry and governments when responding to food scares. The aim of the research is to develop models of trust building that can be implemented following food scares. Methods: Semi-structured interviews will be undertaken with media, public relations officials and policy makers in Australia, New Zealand and the United Kingdom. Participants will be recruited through purposive sampling and will be asked to discuss a hypothetical case study outlining a food incident, and any experiences of specific food scares. Models of trust development, maintenance and repair will be developed from interview data. Comment on these models will be sought from experts in food-related organizations through a Delphi study, where participants will be asked to consider the usefulness of the models. Participants' comments will be used to revise the models until consensus is reached on the suitability and usability of the models.DiscussionThis study will contribute to the literature about systems-based trust, and explore trust as a social and regulatory process. The protocol and results will be of interest and use to the food industry, food regulators, consumer advocate groups, media seeking to report food-related issues and policy makers concerned with public health and consumer health and well-being. This research represents an important contribution to the translation of the theoretical conceptualizations of trust into practical use in the context of food.