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.

Characterising transiting exoplanets by way of differential photometry

This paper describes a simple activity for plotting and characterising the light curve from an exoplanet transit event by way of differential photometry analysis. Using free digital imaging software, participants analyse a series of telescope images with the goal of calculating various exoplanet parameters, including its size, orbital radius and habitability. The activity has been designed for a high-school or undergraduate university level and introduces fundamental concepts in astrophysics and an understanding of the basis for exoplanetary science, the transit method and digital photometry.

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.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17β-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17β-estradiol was withdrawn. Stimulation of invasiveness with 17β-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.

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.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

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.

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.