Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 104–106 M–1 and stoichiometries, n1, of 2–13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 103–105 M–1 and stoichiometries, n2, of 16–30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern–Volmer quenching constant, KSV.

On the flexibility of attention to race

Research on the flexibility of race-based processing offers divergent results. Some studies find that race affects processing in an obligatory fashion. Other studies suggest dramatic flexibility. The current study attempts to clarify this divergence by examining a process that may mediate flexibility in race-based processing: the engagement of visual attention. In this study, White participants completed an exogenous cuing task designed to measure attention to White and Black faces. Participants in the control condition showed a pronounced bias to attend to Black faces. Critically, participants in a goal condition were asked to process a feature of the stimulus that was unrelated to race. The induction of this goal eliminated differential attention to Black faces, suggesting that attentional engagement responds flexibly to top-down goals, rather than obligatorily to bottom-up racial cues.

Flexibility of representational states in working memory

The relationship between working memory (WM) and attention is a highly interdependent one, with evidence that attention determines the state in which items in WM are retained. Through focusing of attention, an item might be held in a more prioritized state, commonly termed as the focus of attention (FOA). The remaining items, although still retrievable, are considered to be in a different representational state. One means to bring an item into the FOA is to use retrospective cues (‘retro-cues’) which direct attention to one of the objects retained in WM. Alternatively, an item can enter a privileged state once attention is directed towards it through bottom-up influences (e.g. recency effect) or by performing an action on one of the retained items (‘incidental’ cueing). In all these cases, the item in the FOA is recalled with better accuracy compared to the other items in WM. Far less is known about the nature of the other items in WM and whether they can be flexibly manipulated in and out of the FOA. We present data from three types of experiments as well as transcranial magnetic stimulation to early visual cortex to manipulate the item inside FOA. Taken together, our results suggest that the context in which items are retained in WM matters. When an item remains behaviourally relevant, despite not being inside the FOA, re-focusing attention upon it can increase its recall precision. This suggests that a non-FOA item can be held in a state in which it can be later retrieved. However, if an item is rendered behaviourally unimportant because it is very unlikely to be probed, it cannot be brought back into the FOA, nor recalled with high precision. Under such conditions, some information appears to be irretrievably lost from WM. These findings, obtained from several different methods, demonstrate quite considerable flexibility with which items in WM can be represented depending upon context. They have important consequences for emerging state-dependent models of WM.

Peak residential electricity demand and social practices: Deriving flexibility and greenhouse gas intensities from time use and locational data

Peak residential electricity demand takes place when people conduct simultaneous activities at specific times of the day. Social practices generate patterns of demand and can help understand why, where, with whom and when energy services are used at peak time. The aim of this work is to make use of recent UK time use and locational data to better understand: (i) how a set of component indices on synchronisation, variation, sharing and mobility indicate flexibility to shift demand; and (ii) the links between people’s activities and peaks in greenhouse gases’ intensities. The analysis is based on a recent UK time use dataset, providing 1 minute interval data from GPS devices and 10 minute data from diaries and questionnaires for 175 data days comprising 153 respondents. Findings show how greenhouse gases’ intensities and flexibility to shift activities vary throughout the day. Morning peaks are characterised by high levels of synchronisation, shared activities and occupancy, with low variation of activities. Evening peaks feature low synchronisation, and high spatial mobility variation of activities. From a network operator perspective, the results indicate that periods with lower flexibility may be prone to more significant local network loads due to the synchronization of electricity-demanding activities.

Genomic signals of migration and continuity in Britain before the Anglo-Saxons

The purported migrations that have formed the peoples of Britain have been the focus of generations of scholarly controversy. However, this has not benefited from direct analyses of ancient genomes. Here we report nine ancient genomes (~1 x) of individuals from northern Britain: seven from a Roman era York cemetery, bookended by earlier Iron-Age and later Anglo-Saxon burials. Six of the Roman genomes show affinity with modern British Celtic populations, particularly Welsh, but significantly diverge from populations from Yorkshire and other eastern English samples. They also show similarity with the earlier Iron-Age genome, suggesting population continuity, but differ from the later Anglo-Saxon genome. This pattern concords with profound impact of migrations in the Anglo-Saxon period. Strikingly, one Roman skeleton shows a clear signal of exogenous origin, with affinities pointing towards the Middle East, confirming the cosmopolitan character of the Empire, even at its northernmost fringes.

Increasing thermal plant flexibility in a high renewables power system

Thermal generation is a vital component of mature and reliable electricity markets. As the share of renewable electricity in such markets grows, so too do the challenges associated with its variability. Proposed solutions to these challenges typically focus on alternatives to primary generation, such as energy storage, demand side management, or increased interconnection. Less attention is given to the demands placed on conventional thermal generation or its potential for increased flexibility. However, for the foreseeable future, conventional plants will have to operate alongside new renewables and have an essential role in accommodating increasing supply-side variability. This paper explores the role that conventional generation has to play in managing variability through the sub-system case study of Northern Ireland, identifying the significance of specific plant characteristics for reliable system operation. Particular attention is given to the challenges of wind ramping and the need to avoid excessive wind curtailment. Potential for conflict is identified with the role for conventional plant in addressing these two challenges. Market specific strategies for using the existing fleet of generation to reduce the impact of renewable resource variability are proposed, and wider lessons from the approach taken are identified.

Genomic evidence for the Pleistocene and recent population history of Native Americans

How and when the Americas were populated remains contentious. Using ancient and modern genome-wide data, we found that the ancestors of all present-day Native Americans, including Athabascans and Amerindians, entered the Americas as a single migration wave from Siberia no earlier than 23 thousand years ago (ka) and after no more than an 8000-year isolation period in Beringia. After their arrival to the Americas, ancestral Native Americans diversified into two basal genetic branches around 13 ka, one that is now dispersed across North and South America and the other restricted to North America. Subsequent gene flow resulted in some Native Americans sharing ancestry with present-day East Asians (including Siberians) and, more distantly, Australo-Melanesians. Putative “Paleoamerican” relict populations, including the historical Mexican Pericúes and South American Fuego-Patagonians, are not directly related to modern Australo-Melanesians as suggested by the Paleoamerican Model.

Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line.

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Estrogen and thyroid hormone receptor interactions: physiological flexibility by molecular specificity

The influence of thyroid hormone on estrogen actions has been demonstrated both in vivo and in vitro. In transient transfection assays, the effects of liganded thyroid hormone receptors (TR) on transcriptional facilitation by estrogens bound to estrogen receptors (ER) display specificity according to the following: 1) ER isoform, 2) TR isoform, 3) the promoter through which transcriptional facilitation occurs, and 4) cell type. Some of these molecular phenomena may be related to thyroid hormone signaling of seasonal limitations upon reproduction. The various combinations of these molecular interactions provide multiple and flexible opportunities for relations between two major hormonal systems important for neuroendocrine feedbacks and reproductive behaviors.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Estrogens and thyroid hormone: Non-genomic effects are coupled to transcription.

Estrogens and thyroid hormones are regulators of important diverse physiological processes such as reproduction, thermogenesis, neural development, neural differentiation and cardiovascular functions. Both are ligands for receptors in the nuclear receptor superfamily, which act as ligand-dependent transcription factors, regulating transcription. However, estrogens and thyroid hormones also rapidly (within minutes or seconds) activate kinase cascades and calcium increases, presumably initiated at the cell membrane. We discuss the relevance of both modes of hormone action, including the membrane estrogen receptor, to physiology, with particular reference to lordosis behavior. We first showed that estrogen restricted to the membrane can, in fact, lead to subsequent increases in transcription from a consensus estrogen response element-based reporter in the neuroblastoma cell line, SK-N-BE(2)C. Using a novel hormonal paradigm, we also showed that the activation of protein kinase A, protein kinase C, mitogen activated protein kinase and increases in calcium were important in the ability of the membrane-limited estrogen to potentiate transcription. We discuss the source of calcium important in transcriptional potentiation. Since estrogens and thyroid hormones have common effects on neuroprotection, cognition and mood, we also hypothesized that crosstalk could occur between the rapid actions of thyroid hormones and the genomic actions of estrogens. In neural cells, we showed that triiodothyronine acting rapidly via MAPK can increase transcription by the nuclear estrogen receptor ERa from a consensus estrogen response element, possibly by the phosphorylation of the ERa. Novel mechanisms that link signals initiated by hormones from the membrane to the nucleus are physiologically relevant and can achieve neuroendocrine integration

Non-genomic actions of estrogens and their interaction with genomic actions in the brain

Ligands for the nuclear receptor superfamily have at least two mechanisms of action: (a) classical transcriptional regulation of target genes (genomic mechanisms); and (b) non-genomic actions, which are initiated at the cell membrane, which could also impact transcription. Though transcriptional mechanisms are increasingly well understood, membrane-initiated actions of these ligands are incompletely understood. This has led to considerable debate over the physiological relevance of membrane-initiated actions of hormones versus genomic actions of hormones, with genomic actions predominating in the endocrine field. There is good evidence that the membrane-limited actions of hormones, particularly estrogens, involve the rapid activation of kinases and the release of calcium and that these are linked to physiologically relevant scenarios in the brain. We show evidence in this review, that membrane actions of estrogens, which activate these rapid signaling cascades, can also potentiate nuclear transcription in both the central nervous system and in non-neuronal cell lines. We present a theoretical scenario which can be used to understand this phenomenon. These signaling cascades may occur in parallel or in series but subsequently, converge at the modification of transcriptionally relevant molecules such as nuclear receptors and/or coactivators. In addition, other non-cognate hormones or neurotransmitters may also activate cascades to crosstalk with estrogen receptor-mediated transcription, though the relevance of this is less clear. The idea that coupling between membrane-initiated and genomic actions of hormones is a novel idea in neuroendocrinology and provides us with a unified view of hormone action in the central nervous system.