A xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases

Aquilegia, Vol. 37 No. 2 - Special Issue, 2013, Newsletter of the Colorado Native Plant Society

https://epublications.regis.edu/aquilegia/1142/thumbnail.jp

Bacterial lipoxygenases: Biochemical characteristics, molecular structure and potential applications

Lipoxygenases (LOXs) are enzymes that catalyze dioxygenation of polyunsaturated fatty acids into fatty acid hydroperoxides. The formed fatty acid hydroperoxides are of interest as they can readily be transformed to a number of value-added compounds. LOXs are widely distributed in both eukaryotic and prokaryotic organisms, including humans, animals, plants, fungi and bacteria. Compared to eukaryotic enzymes, bacterial enzymes are typically easier to produce at industrial scale in a heterologous host. However, many bacterial LOXs were only identified relatively recently and their structure and biochemical characteristics have not been extensively studied. A better understanding of bacterial LOXs' structure and characteristics will lead to the wider application of these enzymes in industrial processes. This review focuses on recent findings on the biochemical characteristics of bacterial LOXs in relation to their molecular structure. The basis of LOX catalysis as well as emerging determinants explaining the regio- and enantioselectivity of different LOXs are also summarized and critically reviewed. Clustering and phylogenetic analyses of bacterial LOX sequences were performed. Finally, the improvement of bacterial LOXs by mutagenesis approaches and their application in chemical synthesis are discussed

Multigram Scale Enzymatic Synthesis of (R)-1-(4′-Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase

The enantioselective oxyfunctionalisation of C−H bonds is a highly interesting reaction, as it provides access to chiral alcohols that are important pharmaceutical building blocks. However, it is hard to achieve using traditional methods. One way in which it can be achieved is through the action of oxidative enzymes. Although many reports of the oxyfunctionalisation capabilities of enzymes at an analytical scale have been published, reports on the use of enzymes to achieve oxyfunctionalisation on a synthetically relevant scale are fewer. Here, we describe the scale-up of the conversion of 4-ethylphenol to (R)-1-(4′-hydroxyphenyl)ethanol using the flavin-dependent enzyme vanillyl alcohol oxidase. The process was optimised by testing different reaction media and substrate and enzyme concentrations and by performing it under an oxygen atmosphere. Under optimised reaction conditions, 4.10 g (R)-1-(4′-hydroxyphenyl)ethanol at 97% ee was obtained from 10 g 4-ethylphenol (isolated yield 36%). These results highlight some of the challenges that can be encountered during scale-up of an enzymatic oxyfunctionalisation process to a synthetically relevant scale and will be of use for the development of enzymatic processes for the synthesis of industrially relevant compounds. (Figure presented.).</p

A Survey of Genomic Traces Reveals a Common Sequencing Error, RNA Editing, and DNA Editing

While it is widely held that an organism's genomic information should remain constant, several protein families are known to modify it. Members of the AID/APOBEC protein family can deaminate DNA. Similarly, members of the ADAR family can deaminate RNA. Characterizing the scope of these events is challenging. Here we use large genomic data sets, such as the two billion sequences in the NCBI Trace Archive, to look for clusters of mismatches of the same type, which are a hallmark of editing events caused by APOBEC3 and ADAR. We align 603,249,815 traces from the NCBI trace archive to their reference genomes. In clusters of mismatches of increasing size, at least one systematic sequencing error dominates the results (G-to-A). It is still present in mismatches with 99% accuracy and only vanishes in mismatches at 99.99% accuracy or higher. The error appears to have entered into about 1% of the HapMap, possibly affecting other users that rely on this resource. Further investigation, using stringent quality thresholds, uncovers thousands of mismatch clusters with no apparent defects in their chromatograms. These traces provide the first reported candidates of endogenous DNA editing in human, further elucidating RNA editing in human and mouse and also revealing, for the first time, extensive RNA editing in Xenopus tropicalis. We show that the NCBI Trace Archive provides a valuable resource for the investigation of the phenomena of DNA and RNA editing, as well as setting the stage for a comprehensive mapping of editing events in large-scale genomic datasets

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

A Baseline for the Multivariate Comparison of Resting-State Networks

As the size of functional and structural MRI datasets expands, it becomes increasingly important to establish a baseline from which diagnostic relevance may be determined, a processing strategy that efficiently prepares data for analysis, and a statistical approach that identifies important effects in a manner that is both robust and reproducible. In this paper, we introduce a multivariate analytic approach that optimizes sensitivity and reduces unnecessary testing. We demonstrate the utility of this mega-analytic approach by identifying the effects of age and gender on the resting-state networks (RSNs) of 603 healthy adolescents and adults (mean age: 23.4 years, range: 12–71 years). Data were collected on the same scanner, preprocessed using an automated analysis pipeline based in SPM, and studied using group independent component analysis. RSNs were identified and evaluated in terms of three primary outcome measures: time course spectral power, spatial map intensity, and functional network connectivity. Results revealed robust effects of age on all three outcome measures, largely indicating decreases in network coherence and connectivity with increasing age. Gender effects were of smaller magnitude but suggested stronger intra-network connectivity in females and more inter-network connectivity in males, particularly with regard to sensorimotor networks. These findings, along with the analysis approach and statistical framework described here, provide a useful baseline for future investigations of brain networks in health and disease

Agribusiness Sheep Updates - 2004 part 2

Precision Pastures Using Species Diversity to Improve Pasture Performance Anyou Liu and Clinton Revell, Department of Agriculture, Western Australia New Annual Pasture Legumes for Sheep Graziers Phil Nichols, Angelo Loi, Brad Nutt and Darryl McClements Department of Agriculture Western Australia Pastures from Space – Can Satellite Estimates of Pasture Growth Rate be used to Increase Farm Profit? Lucy Anderton, Stephen Gherardi and Chris Oldham Department of Agriculture Western Australia Summer-active Perennial Grasses for Profitable Sheep Production Paul Sanford and John Gladman, Department of Agriculture, Western Australia Pastures From Space – Validation Of Predictions Of Pasture Growth Rates DONALD, G.E.A, EDIRISINGHE, A.A, HENRY, D.A.A, MATA, G.A, GHERARDI, S.G.B, OLDHAM, C.M.B, GITTINS, S.P.B AND SMITH, R. C. G.C ACSIRO, Livestock Industries, PMB 5, Wembley, WA, 6913. BDepartment of Agriculture Western Australia, Bentley, WA, 6983. C Department of Land Information Western Australia, Floreat, WA, 6214. Production and Management of Biserrula Pasture - Managing the Risk of Photosensitivity Dr Clinton Revell and Roy Butler, Department of Agriculture Western Australia Meat Quality of Sheep Grazed on a Saltbush-based Pasture Kelly Pearce1,2, David Masters1, David Pethick2, 1 CSIRO LIVESTOCK INDUSTRIES, WEMBLEY, WA 2 SCHOOL OF VETERINARY AND BIOMEDICAL SCIENCE, MURDOCH UNIVERSITY, MURDOCH, WA Precision Sheep Lifetime Wool – Carryover Effects on Subsequent Reproduction of the Ewe Flock Chris Oldham, Department of Agriculture Western Australia Andrew Thompson, Primary Industries Research Victoria (PIRVic), Dept of Primary Industries, Hamilton, Vic Ewe Productivity Trials - a Linked Analysis Ken Hart, Johan Greeff, Department of Agriculture Western Australia, Beth Paganoni, School of Animal Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia. Grain Finishing Systems For Prime Lambs Rachel Kirby, Matt Ryan, Kira Buttler, Department of Agriculture, Western Australia The Effects of Nutrition and Genotype on the Growth and Development, Muscle Biochemistry and Consumer Response to Lamb Meat David Pethick, Department of Veterinary Science, Murdoch University, WA, Roger Heggarty and David Hopkins, New South Wales Agriculture ‘Lifetime Wool’ - Effects of Nutrition During Pregnancy and Lactation on Mortality of Progeny to Hogget Shearing Samantha Giles, Beth Paganoni and Tom Plaisted, Department of Agriculture Western Australia, Mark Ferguson and Darren Gordon, Primary Industries Research Victoria (PIRVic), Dept of Primary Industries, Hamilton, Vic Lifetime Wool - Target Liveweights for the Ewe Flock J. Young, Farming Systems Analysis Service, Kojonup, C. Oldham, Department of Agriculture Western Australia, A. Thompson, Primary Industries Research Victoria (PIRVic), Hamilton, VIC Lifetime Wool - Effects of Nutrition During Pregnancy and Lactation on the Growth and Wool Production of their Progeny at Hogget Shearing B. Paganoni, University of Western Australia, Nedlands WA, C. Oldham, Department of Agriculture Western Australia, M. Ferguson, A. Thompson, Primary Industries Research Victoria (PIRVic), Hamilton, VIC RFID Technology – Esperance Experiences Sandra Brown, Department of Agriculture Western Australia The Role of Radio Frequency Identification (RFID) Technology in Prime Lamb Production - a Case Study. Ian McFarland, Department of Agriculture, Western Australia. John Archer, Producer, Narrogin, Western Australia Win with Twins from Merinos John Milton, Rob Davidson, Graeme Martin and David Lindsay The University of Western Australia Precision Sheep Need Precision Wool Harvesters Jonathan England, Castle Carrock Merinos, Kingston SE, South Australia Business EBVs and Indexes – Genetic Tools for your Toolbox Sandra Brown, Department of Agriculture Western Australia Green Feed Budget Paddock Calculator Mandy Curnow, Department of Agriculture Western Australia Minimising the Impact of Drought - Evaluating Flock Recovery Options using the ImPack Model Karina P. Wood, Ashley K. White, B. Lloyd Davies, Paul M. Carberry, NSW Department of Primary Industries (NSW DPI), Lifetime Wool - Modifying GrazFeed® for WA Mike Hyder, Department of Agriculture Western Australia , Mike Freer, CSIRO Plant Industry, Canberra, A.C.T. , Andrew van Burgel, and Kazue Tanaka, Department of Agriculture Western Australia Profile Calculator – A Way to Manage Fibre Diameter Throughout the Year to Maximise Returns Andrew Peterson, Department of Agriculture, Western Australia Pasture Watch - a Farmer Friendly Tool for Downloading and Analysing Pastures from Space Data Roger Wiese,Fairport Technologies International, South Perth, WA, Stephen Gherardi, BDepartment of Agriculture Western Australia, Gonzalo Mata, CCSIRO, Livestock Industries, Wembley, Western Australia, and Chris Oldham, Department of Agriculture Western Australia Sy Sheep Cropping Systems An Analysis of a Cropping System Containing Sheep in a Low Rainfall Livestock System. Evan Burt, Amanda Miller, Anne Bennett, Department of Agriculture, Western Australia Lucerne-based Pasture for the Central Wheatbelt – is it Good Economics? Felicity FluggeA, Amir AbadiA,B and Perry DollingA,B,A CRC for Plant-based Management of Dryland Salinity: BDept. of Agriculture, WA Sheep and Biserrula can Control Annual Ryegrass Dean Thomas, John Milton, Mike Ewing and David Lindsay, The University of WA, Clinton Revell, Department of Agriculture, Western Australia Sustainable Management Pasture Utilisation, Fleece Weight and Weaning Rate are Integral to the Profitability of Dohnes and SAMMs. Emma Kopke,Department of Agriculture Western Australia, John Young, Farming Systems Analysis Service Environmental Impact of Sheep Confinement Feeding Systems E A Dowling and E K Crossley, Department of Agriculture, Western Australia Smart Grazing Management for Production and Environmental Outcomes Dr Brien E (Ben) Norton, Centre for the Management of Arid Environments, Curtin University of Technology, WA Common Causes of Plant Poisoning in the Eastern Wheatbelt of Western Australia. Roy Butler, Department of Agriculture, Western Australia Selecting Sheep for Resistance to Worms and Production Trait Responses John Karlsson, Johan Greeff, Department of Agriculture, Western Australia, Geoff Pollott, Imperial College, London UK Production and Water Use of Lucerne and French Serradella in Four Soil Types, Diana Fedorenko1,4, Darryl McClements2,4 and Robert Beard3,4, 12Department of Agriculture, Western Australia; 3Farmer, Meckering; 4CRC for Plant-based Management of Dryland Salinity. Worm Burdens in Sheep at Slaughter Brown Besier, Department of Agriculture Western Australia, Una Ryan, Caroline Bath, Murdoch Universit

Exercise and Type 2 Diabetes: The American College of Sports Medicine and the American Diabetes Association: joint position statement

Although physical activity (PA) is a key element in the prevention and management of type 2 diabetes, many with this chronic disease do not become or remain regularly active. High-quality studies establishing the importance of exercise and fitness in diabetes were lacking until recently, but it is now well established that participation in regular PA improves blood glucose control and can prevent or delay type 2 diabetes, along with positively affecting lipids, blood pressure, cardiovascular events, mortality, and quality of life. Structured interventions combining PA and modest weight loss have been shown to lower type 2 diabetes risk by up to 58% in high-risk populations. Most benefits of PA on diabetes management are realized through acute and chronic improvements in insulin action, accomplished with both aerobic and resistance training. The benefits of physical training are discussed, along with recommendations for varying activities, PA-associated blood glucose management, diabetes prevention, gestational diabetes mellitus, and safe and effective practices for PA with diabetes-related complications

SUDOSCAN: A Simple, Rapid, and Objective Method with Potential for Screening for Diabetic Peripheral Neuropathy.

Clinical methods of detecting diabetic peripheral neuropathy (DPN) are not objective and reproducible. We therefore evaluated if SUDOSCAN, a new method developed to provide a quick, non-invasive and quantitative assessment of sudomotor function can reliably screen for DPN. 70 subjects (45 with type 1 diabetes and 25 healthy volunteers [HV]) underwent detailed assessments including clinical, neurophysiological and 5 standard cardiovascular reflex tests (CARTs). Using the American Academy of Neurology criteria subjects were classified into DPN and No-DPN groups. Based on CARTs subjects were also divided into CAN, subclinical-CAN and no-CAN. Sudomotor function was assessed with measurement of hand and foot Electrochemical Skin Conductance (ESC) and calculation of the CAN risk score. Foot ESC (μS) was significantly lower in subjects with DPN [n = 24; 53.5(25.1)] compared to the No-DPN [77.0(7.9)] and HV [77.1(14.3)] groups (ANCOVA p<0.001). Sensitivity and specificity of foot ESC for classifying DPN were 87.5% and 76.2%, respectively. The area under the ROC curve (AUC) was 0.85. Subjects with CAN had significantly lower foot [55.0(28.2)] and hand [53.5(19.6)] ESC compared to No-CAN [foot ESC, 72.1(12.2); hand ESC 64.9(14.4)] and HV groups (ANCOVA p<0.001 and 0.001, respectively). ROC analysis of CAN risk score to correctly classify CAN revealed a sensitivity of 65.0% and specificity of 80.0%. AUC was 0.75. Both foot and hand ESC demonstrated strong correlation with individual parameters and composite scores of nerve conduction and CAN. SUDOSCAN, a non-invasive and quick test, could be used as an objective screening test for DPN in busy diabetic clinics, insuring adherence to current recommendation of annual assessments for all diabetic patients that remains unfulfilled