Characterisation and correction of signal fluctuations in successive acquisitions of microarray images

<p>Abstract</p> <p>Background</p> <p>There are many sources of variation in dual labelled microarray experiments, including data acquisition and image processing. The final interpretation of experiments strongly relies on the accuracy of the measurement of the signal intensity. For low intensity spots in particular, accurately estimating gene expression variations remains a challenge as signal measurement is, in this case, highly subject to fluctuations.</p> <p>Results</p> <p>To evaluate the fluctuations in the fluorescence intensities of spots, we used series of successive scans, at the same settings, of whole genome arrays. We measured the decrease in fluorescence and we evaluated the influence of different parameters (PMT gain, resolution and chemistry of the slide) on the signal variability, at the level of the array as a whole and by intensity interval. Moreover, we assessed the effect of averaging scans on the fluctuations. We found that the extent of photo-bleaching was low and we established that 1) the fluorescence fluctuation is linked to the resolution e.g. it depends on the number of pixels in the spot 2) the fluorescence fluctuation increases as the scanner voltage increases and, moreover, is higher for the red as opposed to the green fluorescence which can introduce bias in the analysis 3) the signal variability is linked to the intensity level, it is higher for low intensities 4) the heterogeneity of the spots and the variability of the signal and the intensity ratios decrease when two or three scans are averaged.</p> <p>Conclusion</p> <p>Protocols consisting of two scans, one at low and one at high PMT gains, or multiple scans (ten scans) can introduce bias or be difficult to implement. We found that averaging two, or at most three, acquisitions of microarrays scanned at moderate photomultiplier settings (PMT gain) is sufficient to significantly improve the accuracy (quality) of the data and particularly those for spots having low intensities and we propose this as a general approach. For averaging and precise image alignment at sub-pixel levels we have made a program freely available on our web-site <url>http://bioinfome.cgm.cnrs-gif.fr</url> to facilitate implementation of this approach.</p

Semiochemical-based alternatives to synthetic toxicant insecticides for pollen beetle management

There is an urgent need to develop sustainable pest management systems to protect arable crops in order to replace the current over-reliance on synthetic insecticides. Semiochemicals are insect- or plant-derived chemicals that are used by organisms as information signals. Integrated pest management tools are currently in development that utilise semiochemicals to manipulate the behaviour of pest insects and their natural enemies to provide effective control of pests within the crop. These innovative tools usually require fewer inputs and can involve multiple elements therefore reducing the likelihood of resistance developing compared with use of synthetic toxicants. We review here the life cycle of the pollen beetle Brassicogethes aeneus (previously known as Meligethes aeneus) which is a pest insect of oilseed rape (Brassica napus) and describe the current knowledge of any behaviour mediated by semiochemicals in this species. We discuss the behavioural processes where semiochemical-based control approaches may be appropriate and consider how these approaches could be integrated into an integrated pest management strategy for this important arable crop

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Do lambs perceive regular human stroking as pleasant? Behavior and heart rate variability analyses

Stroking by humans is beneficial to the human-animal relationship and improves welfare in many species that express intraspecific allogrooming, but very few studies have looked at species like sheep that do not express such contact except around parturition. This study investigated the way lambs perceive regular human tactile contact using behavioral and physiological responses. Twenty-four lambs were reared and bucket-fed in groups of four. All were stroked daily by their familiar caregiver. At 8 weeks of age, the lambs were individually tested in their home pen but in a 1×1m open-barred pen after a 15h period of habituation to physical separation from peers while remaining in visual and auditory contact. Half of the lambs received stroking by their caregiver for 8min and half were exposed to their caregiver’s immobile presence. Heart rate and heart rate variability were recorded and analyzed by 2-min slots over the same interval based on three measures: mean heart rate value (HR), root mean square of successive differences (RMSSD) and standard deviation of all intervals measured between consecutive sinus beats (SDNN). Behavioral responses (ear postures of the lamb and time spent in contact with the familiar caregiver, on the knees of the familiar caregiver, and moving) were recorded throughout the test. Lamb HR decreased continuously while in the presence of their caregiver. Lambs being stroked showed slower HR and higher RMSSD which reflected positive emotional states compared to lambs left unstroked. All behavioral variables were highly correlated with the main component axis of the PCA analyses: the more the animals stayed in contact with their caregiver, the less they moved and the more their ears were hanging. This first component clearly differentiates lambs being stroked or not. Behavioral and physiological observations support the hypothesis that gentle physical contact with the caregiver is perceived positively by lambs

Science and society in education

This booklet is for teachers who want to expand their teaching approaches to include socio-scientific issues which enrich and give meaning to core scientific principles. It is meant to enhance young people’s curiosity about the social and scientific world and raise important questions about issues which affect their lives. We call this approach Socio-Scientific Inquiry-Based Learning, or ‘SSIBL’ for short. Chapters 1 and 2 present an introduction to the theoretical background of SSIBL. In chapter 3, SSIBL will be approached from a classroom perspective, providing a simplified version of the framework and showing teaching examples

Redundant and Specific Roles of the ARGONAUTE Proteins AGO1 and ZLL in Development and Small RNA-Directed Gene Silencing

The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5′-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function

Human performance and strategies while solving an aircraft routing and sequencing problem: an experimental approach

As airport resources are stretched to meet increasing demand for services, effective use of ground infrastructure is increasingly critical for ensuring operational efficiency. Work in operations research has produced algorithms providing airport tower controllers with guidance on optimal timings and sequences for flight arrivals, departures, and ground movement. While such decision support systems have the potential to improve operational efficiency, they may also affect users’ mental workload, situation awareness, and task performance. This work sought to identify performance outcomes and strategies employed by human decision makers during an experimental airport ground movement control task with the goal of identifying opportunities for enhancing user-centered tower control decision support systems. To address this challenge, thirty novice participants solved a set of vehicle routing problems presented in the format of a game representing the airport ground movement task practiced by runway controllers. The games varied across two independent variables, network map layout (representing task complexity) and gameplay objective (representing task flexibility), and verbal protocol, visual protocol, task performance, workload, and task duration were collected as dependent variables. A logistic regression analysis revealed that gameplay objective and task duration significantly affected the likelihood of a participant identifying the optimal solution to a game, with the likelihood of an optimal solution increasing with longer task duration and in the less flexible objective condition. In addition, workload appeared unaffected by either independent variable, but verbal protocols and visual observations indicated that high-performing participants demonstrated a greater degree of planning and situation awareness. Through identifying human behavior during optimization problem solving, the work of tower control can be better understood, which, in turn, provides insights for developing decision support systems for ground movement management

An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells

INTRODUCTION: About 70% of breast cancers express oestrogen receptor alpha (ESR1/ERalpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERalpha-positive tumour cells, ERalpha-positive cells in normal breast tissue rarely proliferate. Because ERalpha expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERalpha-negative. Currently only tumour cell lines are available to model ERalpha-positive disease. To create an ERalpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERalpha in combination with other relevant breast cancer genes. METHODS: Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. RESULTS: Microarray analysis of ERalpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERalpha, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/SCID mice results in the formation of ERalpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. CONCLUSION: We have created a genetically defined model of ERalpha-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasi

Perceiving What Is Reachable Depends on Motor Representations: Evidence from a Transcranial Magnetic Stimulation Study

Background: Visually determining what is reachable in peripersonal space requires information about the egocentric location of objects but also information about the possibilities of action with the body, which are context dependent. The aim of the present study was to test the role of motor representations in the visual perception of peripersonal space. Methodology: Seven healthy participants underwent a TMS study while performing a right-left decision (control) task or perceptually judging whether a visual target was reachable or not with their right hand. An actual grasping movement task was also included. Single pulse TMS was delivered 80 % of the trials on the left motor and premotor cortex and on a control site (the temporo-occipital area), at 90 % of the resting motor threshold and at different SOA conditions (50ms, 100ms, 200ms or 300ms). Principal Findings: Results showed a facilitation effect of the TMS on reaction times in all tasks, whatever the site stimulated and until 200ms after stimulus presentation. However, the facilitation effect was on average 34ms lower when stimulating the motor cortex in the perceptual judgement task, especially for stimuli located at the boundary of peripersonal space. Conclusion: This study provides the first evidence that brain motor area participate in the visual determination of what is reachable. We discuss how motor representations may feed the perceptual system with information about possibl

Angiopoietin 2 Alters Pancreatic Vascularization in Diabetic Conditions

Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1-10 twice/week. In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1-10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. this work highligghted an important role of Ang2 in pancreatic vascular defects induced by hyperglycemia