Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries

The selection of cytochrome P450 enzymes from large variant libraries, and the subsequent use of these enzymes in preparative scale biotransformations, remains a formidable challenge due to the complexities of the associated electron transport systems. Here, a powerful approach for the generation and screening of P450cam libraries for new function is presented that is both flexible and robust. A targeted library was generated wherein only the P450cam active-site amino acids Y96 and F98 were fully randomized and biotransformations, using a novel P450cam whole-cell system, were screened by GC–MS for the hydroxylation of diphenylmethane. One in 50 of the reactions screened, including 16 different variants, produced 4-hydroxydiphenylmethane with up to 92% conversion observed in the case of the Y96A variant. These results demonstrate a primary example of the screening of P450cam libraries in a format that is compatible with extension to preparative scale reactions.

The IKBALS project: multi-modal reasoning in legal knowledge based systems

In attempting to build intelligent litigation support tools, we have moved beyond first generation, production rule legal expert systems. Our work integrates rule based and case based reasoning with intelligent information retrieval. When using the case based reasoning methodology, or in our case the specialisation of case based retrieval, we need to be aware of how to retrieve relevant experience. Our research, in the legal domain, specifies an approach to the retrieval problem which relies heavily on an extended object oriented/rule based system architecture that is supplemented with causal background information. We use a distributed agent architecture to help support the reasoning process of lawyers. Our approach to integrating rule based reasoning, case based reasoning and case based retrieval is contrasted to the CABARET and PROLEXS architectures which rely on a centralised blackboard architecture. We discuss in detail how our various cooperating agents interact, and provide examples of the system at work. The IKBALS system uses a specialised induction algorithm to induce rules from cases. These rules are then used as indices during the case based retrieval process. Because we aim to build legal support tools which can be modified to suit various domains rather than single purpose legal expert systems, we focus on principles behind developing legal knowledge based systems. The original domain chosen was theAccident Compensation Act 1989 (Victoria, Australia), which relates to the provision of benefits for employees injured at work. For various reasons, which are indicated in the paper, we changed our domain to that ofCredit Act 1984 (Victoria, Australia). This Act regulates the provision of loans by financial institutions. The rule based part of our system which provides advice on the Credit Act has been commercially developed in conjunction with a legal firm. We indicate how this work has lead to the development of a methodology for constructing rule based legal knowledge based systems. We explain the process of integrating this existing commercial rule based system with the case base reasoning and retrieval architecture.

Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry

Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears.Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum. © 2011 Elsevier Ltd.

Identification and characterization of flowering genes in kiwifruit : sequence conservation and role in kiwifruit flower development

Background Flower development in kiwifruit (Actinidia spp.) is initiated in the first growing season, when undifferentiated primordia are established in latent shoot buds. These primordia can differentiate into flowers in the second growing season, after the winter dormancy period and upon accumulation of adequate winter chilling. Kiwifruit is an important horticultural crop, yet little is known about the molecular regulation of flower development. Results To study kiwifruit flower development, nine MADS-box genes were identified and functionally characterized. Protein sequence alignment, phenotypes obtained upon overexpression in Arabidopsis and expression patterns suggest that the identified genes are required for floral meristem and floral organ specification. Their role during budbreak and flower development was studied. A spontaneous kiwifruit mutant was utilized to correlate the extended expression domains of these flowering genes with abnormal floral development. Conclusions This study provides a description of flower development in kiwifruit at the molecular level. It has identified markers for flower development, and candidates for manipulation of kiwifruit growth, phase change and time of flowering. The expression in normal and aberrant flowers provided a model for kiwifruit flower development.

Identification of Mendel's white flower character

Background The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. Methodology/Principal Findings We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. Conclusions/Significance We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

Background Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes.

An investigation of the stress-buffering effects of social support in the occupational stress process as a function of team identification

This study explored how the social context influences the stress-buffering effects of social support on employee adjustment. It was anticipated that the positive relationship between support from colleagues and employee adjustment would be more marked for those strongly identifying with their work team. Furthermore, as part of a three-way interactive effect, it was predicted that high identification would increase the efficacy of coworker support as a buffer of two role stressors (role overload and role ambiguity). One hundred and 55 employees recruited from first-year psychology courses enrolled at two Australian universities were surveyed. Hierarchical multiple regression analyses revealed that the negative main effect of role ambiguity on job satisfaction was significant for those employees with low levels of team identification, whereas high team identifiers were buffered from the deleterious effect of role ambiguity on job satisfaction. There also was a significant interaction between coworker support and team identification. The positive effect of coworker support on job satisfaction was significant for high team identifiers, whereas coworker support was not a source of satisfaction for those employees with low levels of team identification. A three-way interaction emerged among the focal variables in the prediction of psychological well-being, suggesting that the combined benefits of coworker support and team identification under conditions of high demand may be limited and are more likely to be observed when demands are low.

Multi-shot person re-identification via relational stein divergence

Person re-identification is particularly challenging due to significant appearance changes across separate camera views. In order to re-identify people, a representative human signature should effectively handle differences in illumination, pose and camera parameters. While general appearance-based methods are modelled in Euclidean spaces, it has been argued that some applications in image and video analysis are better modelled via non-Euclidean manifold geometry. To this end, recent approaches represent images as covariance matrices, and interpret such matrices as points on Riemannian manifolds. As direct classification on such manifolds can be difficult, in this paper we propose to represent each manifold point as a vector of similarities to class representers, via a recently introduced form of Bregman matrix divergence known as the Stein divergence. This is followed by using a discriminative mapping of similarity vectors for final classification. The use of similarity vectors is in contrast to the traditional approach of embedding manifolds into tangent spaces, which can suffer from representing the manifold structure inaccurately. Comparative evaluations on benchmark ETHZ and iLIDS datasets for the person re-identification task show that the proposed approach obtains better performance than recent techniques such as Histogram Plus Epitome, Partial Least Squares, and Symmetry-Driven Accumulation of Local Features.

Structural identification of hindered amine light stabilisers in coil coatings using electrospray ionisation tandem mass spectrometry

Hindered amine light stabilisers (HALS) are the most effective antioxidants currently available for polymer systems in post-production, in-service applications, yet the mechanism of their action is still not fully understood. Structural characterisation of HALS in polymer matrices, particularly the identification of structural modifications brought about by oxidative conditions, is critical to aid mechanistic understanding of the prophylactic effects of these molecules. In this work, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was applied to the analysis of a suite of commercially available 2,2,6,6-tetramethylpiperidine-based HALS. Fragmentation mechanisms for the \[M + H](+) ions are proposed, which provide a rationale for the product ions observed in the MS/MS and MS(3) mass spectra of N-H, N-CH(3), N-C(O)CH(3) and N-OR containing HALS (where R is an alkyl substituent). A common product ion at m/z 123 was identified for the group of antioxidants containing N-H, N-CH3 or N-C(0)CH3 functionality, and this product ion was employed in precursor ion scans on a triple quadrupole mass spectrometer to identify the HALS species present in a crude extract from of a polyester-based coil coating. Using MS/MS, two degradation products were unambiguously identified. This technique provides a simple and selective approach to monitoring HALS structures within complex matrices. Copyright (C) 2010 John Wiley & Sons, Ltd.

Identification of double bond position in lipids : From GC to OzID

Recent developments in mass spectrometry and chromatography provide new possibilities for the identification and in some instances quantification of a wide range of lipids in complex matrices. These advances in analytical technologies have provided a tantalizing glimpse of the true structural diversity of lipids in nature and have reinvigorated interest in the role of lipids in biology. While technological advances have been impressive, difficulties in the ready identification of sites of unsaturation (i.e., double bond position) within these molecules presents a significant impediment to understanding lipid biochemistry. This is of particular importance given the growing body of literature suggesting that the presence of naturally occurring lipid double bond isomers can have a significant influence, both positive and negative, on the development of pathologies such as cancer, cardiovascular disease and type 2 diabetes. This article provides a critical review of the Current suite of analytical approaches to the challenge of identification of the position of carbon-carbon double bonds in intact lipids. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Identification and Validity of Accelerometer Cut-Points for Toddlers

The purpose of this study was to derive ActiGraph cut-points for sedentary (SED), light-intensity physical activity (LPA), and moderate-to-vigorous physical activity (MVPA) in toddlers and evaluate their validity in an independent sample. The predictive validity of established preschool cut-points were also evaluated and compared. Twenty-two toddlers (mean age = 2.1 years ± 0.4 years) wore an ActiGraph accelerometer during a videotaped 20-min play period. Videos were subsequently coded for physical activity (PA) intensity using the modified Children's Activity Rating Scale (CARS). Receiver operating characteristic (ROC) curve analyses were conducted to determine cut-points. Predictive validity was assessed in an independent sample of 18 toddlers (mean age = 2.3 ± 0.4 years). From the ROC curve analyses, the 15-s count ranges corresponding to SED, LPA, and MVPA were 0–48, 49–418, and >418 counts/15 s, respectively. Classification accuracy was fair for the SED threshold (ROC-AUC = 0.74, 95% confidence interval = 0.71–0.76) and excellent for MVPA threshold (ROC-AUC = 0.90, 95% confidence interval = 0.88–0.92). In the cross-validation sample, the toddler cut-point and established preschool cut-points significantly overestimated time spent in SED and underestimated time in spent in LPA. For MVPA, mean differences between observed and predicted values for the toddler and Pate cut-points were not significantly different from zero. In summary, the ActiGraph accelerometer can provide useful group-level estimates of MVPA in toddlers. The results support the use of the Pate cut-point of 420 counts/15 s for MVPA.

Methodology for developing real-time motorway traffic risk identification models using individual-vehicle data

Most of existing motorway traffic safety studies using disaggregate traffic flow data aim at developing models for identifying real-time traffic risks by comparing pre-crash and non-crash conditions. One of serious shortcomings in those studies is that non-crash conditions are arbitrarily selected and hence, not representative, i.e. selected non-crash data might not be the right data comparable with pre-crash data; the non-crash/pre-crash ratio is arbitrarily decided and neglects the abundance of non-crash over pre-crash conditions; etc. Here, we present a methodology for developing a real-time MotorwaY Traffic Risk Identification Model (MyTRIM) using individual vehicle data, meteorological data, and crash data. Non-crash data are clustered into groups called traffic regimes. Thereafter, pre-crash data are classified into regimes to match with relevant non-crash data. Among totally eight traffic regimes obtained, four highly risky regimes were identified; three regime-based Risk Identification Models (RIM) with sufficient pre-crash data were developed. MyTRIM memorizes the latest risk evolution identified by RIM to predict near future risks. Traffic practitioners can decide MyTRIM’s memory size based on the trade-off between detection and false alarm rates. Decreasing the memory size from 5 to 1 precipitates the increase of detection rate from 65.0% to 100.0% and of false alarm rate from 0.21% to 3.68%. Moreover, critical factors in differentiating pre-crash and non-crash conditions are recognized and usable for developing preventive measures. MyTRIM can be used by practitioners in real-time as an independent tool to make online decision or integrated with existing traffic management systems.

Two-photon fluorescence spectroscopy for identification of healthy and malignant biological tissues

Two-photon fluorescence spectroscopy has been performed on rat skeletal muscles to investigate the effect of fixation processes on the micro-environments of the endogenous fluorophors in rat skeletal muscles. The two-photon fluorescence spectra measured for different fixation periods show a differential among those samples that were fixed in water, formalin and methanol, respectively. The results imply that two-photon fluorescence spectroscopy can be a potential technique for identification of healthy and malignant biological tissues.

A novel identification method for ultra trace detection of biomolecules using functionalised Surface Enhanced Raman Spectroscopy (SERS)

This thesis developed a new method for measuring extremely low amounts of organic and biological molecules, using Surface enhanced Raman Spectroscopy. This method has many potential applications, e.g. medical diagnosis, public health, food provenance, antidoping, forensics and homeland security. The method development used caffeine as the small molecule example, and erythropoietin (EPO) as the large molecule. This method is much more sensitive and specific than currently used methods; rapid, simple and cost effective. The method can be used to detect target molecules in beverages and biological fluids without the usual preparation steps.