Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Purpose The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Methods Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Results Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. Conclusions This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

The WR content of IC10 - first detection of WC9 stars in a low metallicity environment?

We present deep, narrow-hand photometry of the Local Group starburst galaxy IC10. Our dedicated photometric system provides detection of 13 new Wolf-Rayet (WR) stars and allows spectral subtypes to be assigned. Three of these new stars appear to be WC9 subtypes. If confirmed, these would be the very first WC9 stars ever detected in a low metallicity environment, hence putting strong new constraints on the formation and evolution models of massive stars. Eight of the new WR stars are of the WC subtype, which does not significantly modify the anomalously high WC/WN ratio in IC10. However it is likely that a number of Wolf-Rayet stars of the WNE and WC spectral subtypes are still to be discovered in the heart of the galaxy.

Critical factors affecting management of site operatives and personal in confined construction site

The aim of this paper is to identify and classify the numerous managerial issues encountered in the management of personnel in confined site construction. For the purpose of this research, a confined construction site is defined as a site where permanent works fit the site footprint, extending to levels above and/or below ground level, leaving spatial restrictions for other operations (e.g. plant and material movements, materials storage and temporary accommodation etc.) and require effective resource co-ordination beyond normal on-site management input. A literature review and analysis, case studies incorporating interviews and focus groups along with a questionnaire survey were used in order to gain a comprehensive insight into the issues in the management of personnel in a confined construction site environment. The following are the top five leading issues highlighted in the management of personnel in confined site construction; (1) Accidents due to an untidy site, (2) One contractor holding up another because of the lack of space, (3) A risk to personnel because of vehicular traffic on-site, (4) Difficult to facilitate several contractors at one work location, and (5) Numerous personnel working within the one space. In today’s modern environment, spatial restrictions are quickly becoming the norm in the industry. Therefore, the management of personnel on-site becomes progressively more difficult with the decrease in available space on-site. Where such environments exist, acknowledging the numerous issues highlighted above, aids site management in the supervision and co-ordination of personnel on-site, thus reducing accidents, increasing productivity and increase profit margins, in spatially restricted environments. As on-site management professionals successfully identify, acknowledge and counteract the numerous issues illustrated, the successful management of personnel on a confined construction site is achievable. By identifying the numerous issues, on-site management can proactively mitigate such issues through adopting counteractive measures and through successful identification of the traits identified.

Two-photon double ionization of He

We determine generalized cross sections for two-photon double ionization of He in the photon energy region between 40.7 and 47 eV where absorption of two photons can lead to non-sequential double ionization only. The present cross sections, obtained in R-matrix Floquet theory, agree with cross sections obtained from time-dependent calculations. By examining the ratio of two-photon double ionization to two-photon single ionization, we demonstrate that core excitation effects at an intensity of 10(13) W cm(-2) are relatively unimportant at 45 eV, but that they are significant at other photon energies.

C∗-Segal algebras with order unit

We introduce the notion of a (noncommutative) C *-Segal algebra as a Banach algebra (A, {norm of matrix}{dot operator}{norm of matrix} A) which is a dense ideal in a C *-algebra (C, {norm of matrix}{dot operator}{norm of matrix} C), where {norm of matrix}{dot operator}{norm of matrix} A is strictly stronger than {norm of matrix}{dot operator}{norm of matrix} C onA. Several basic properties are investigated and, with the aid of the theory of multiplier modules, the structure of C *-Segal algebras with order unit is determined.

Hierarchical Bayesian models in ecology: Reconstructing species interaction networks from non-homogeneous species abundance data

The relationships among organisms and their surroundings can be of immense complexity. To describe and understand an ecosystem as a tangled bank, multiple ways of interaction and their effects have to be considered, such as predation, competition, mutualism and facilitation. Understanding the resulting interaction networks is a challenge in changing environments, e.g. to predict knock-on effects of invasive species and to understand how climate change impacts biodiversity. The elucidation of complex ecological systems with their interactions will benefit enormously from the development of new machine learning tools that aim to infer the structure of interaction networks from field data. In the present study, we propose a novel Bayesian regression and multiple changepoint model (BRAM) for reconstructing species interaction networks from observed species distributions. The model has been devised to allow robust inference in the presence of spatial autocorrelation and distributional heterogeneity. We have evaluated the model on simulated data that combines a trophic niche model with a stochastic population model on a 2-dimensional lattice, and we have compared the performance of our model with L1-penalized sparse regression (LASSO) and non-linear Bayesian networks with the BDe scoring scheme. In addition, we have applied our method to plant ground coverage data from the western shore of the Outer Hebrides with the objective to infer the ecological interactions. (C) 2012 Elsevier B.V. All rights reserved.

Equidiscrimination contours around an achromatic colour are convex but not balanced

A model based on the postreceptor channels followed by a Minkowski norm (Minkowski model) is widely used to fit experimental data on colour discrimination. This model predicts that contours of equal discrimination in colour space are convex and balanced (symmetrical). We have tested these predictions in an experiment. Two new statistical tests have been developed to analyse convexity and balancedness of experimental curves. Using these tests we have found that while they are in line with the convexity prediction, our experimental contours strongly testify against balancedness. It follows that the Minkowski model is, generally, inappropriate to model colour discrimination data. © 2002 Elsevier Science (USA).

Bending It Like Beckham: How to Visually Fool the Goalkeeper

Background: As bending free-kicks becomes the norm in modern day soccer, implications for goalkeepers have largely been ignored. Although it has been reported that poor sensitivity to visual acceleration makes it harder for expert goalkeepers to perceptually judge where the curved free-kicks will cross the goal line, it is unknown how this affects the goalkeeper's actual movements.

Methodology/Principal Findings: Here, an in-depth analysis of goalkeepers' hand movements in immersive, interactive virtual reality shows that they do not fully account for spin-induced lateral ball acceleration. Hand movements were found to be biased in the direction of initial ball heading, and for curved free-kicks this resulted in biases in a direction opposite to those necessary to save the free-kick. These movement errors result in less time to cover a now greater distance to stop the ball entering the goal. These and other details of the interceptive behaviour are explained using a simple mathematical model which shows how the goalkeeper controls his movements online with respect to the ball's current heading direction. Furthermore our results and model suggest how visual landmarks, such as the goalposts in this instance, may constrain the extent of the movement biases.

Conclusions: While it has previously been shown that humans can internalize the effects of gravitational acceleration, these results show that it is much more difficult for goalkeepers to account for spin-induced visual acceleration, which varies from situation to situation. The limited sensitivity of the human visual system for detecting acceleration, suggests that curved free-kicks are an important goal-scoring opportunity in the game of soccer.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis

The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

Fasciola hepatica:The therapeutic potential of a worm secretome

The success of helminth parasites is partly related to their ability to modulate host immune responses towards an anti-inflammatory/regulatory phenotype. This ability resides with the molecules contained in the secretome of various helminths that have been shown to interact with host immune cells and influence their function. Consequently, there exists a unique opportunity to exploit these molecules for the prophylactic and therapeutic treatment of human pro- and auto-inflammatory disorders (for example septic shock, transplant rejection and autoimmune disease). In this review, we describe the mechanisms used by the trematode parasite, Fasciola hepatica, to modulate the immune responses of its host and discuss the potent immune-modulatory effects of three individual molecules within the secretome; namely cathepsin L1, peroxiredoxin and helminth defence molecule. With a focus on the requirements from industry, we discuss the strategies by which these molecules may be clinically developed to control human immune responses in a way that is conducive to the prevention of immune-mediated diseases.

Introduction

The scholarly interest in norm implementation in the area of minority rights protection emerged during the period of the European accession of post-communist societies, which were characterised by persistent cleavage along ethnic lines. These studies have analysed democratic transition of ethnically segmented societies and developed into a broad field that focuses its attention on inter-ethnic relations, the role of history, interstate cooperation and the impact of international organisations on post-communist states' treatment of domestic minorities. All these are usually conducted from a vantage point of the European conditionality research. In our introduction to the special issue we sketch a number of reasons why it is encouraging and indeed, desirable to move beyond the perusal of conditionality into the domain of implementation research.

Strategies for Effective Management of Health and Safety in Confined Site Construction

The overall aim of this research is to identify and catalogue the numerous managerial strategies for effective management of health and safety on a confined, urban, construction site. A mixed methods methodology was adopted using interviews and focus group discussions on three selected case studies of confined construction sites. In addition to these, a questionnaire survey was used based on the findings from the interviews and the focus group discussions. The top five key strategies include (1) Employ safe system of work plans to mitigate personnel health and safety issues; (2) Inform personnel, before starting on-site, of the potential issues using site inductions; (3) Effective communication among site personnel; (4) Draft and implement an effective design site layout prior to starting on-site; and (5) Use of banksman (traffic co-ordinator) to segregate personnel from vehicular traffic. The construction sector is one of the leading industries in accident causation and with the continued development and regeneration of our urban centres, confined site construction is quickly becoming the norm - an environment which only fuels accident creation within the construction sector. This research aids on-site management that requires direction and assistance in the identification and implementation of key strategies for the management of health and safety, particularly in confined construction site environments.

Hypercyclic operators on countably dimensional spaces

According to Grivaux, the group GL(X) of invertible linear operators on a separable infinite dimensional Banach space X acts transitively on the set s (X) of countable dense linearly independent subsets of X. As a consequence, each A? s (X) is an orbit of a hypercyclic operator on X. Furthermore, every countably dimensional normed space supports a hypercyclic operator. Recently Albanese extended this result to Fréchet spaces supporting a continuous norm. We show that for a separable infinite dimensional Fréchet space X, GL(X) acts transitively on s (X) if and only if X possesses a continuous norm. We also prove that every countably dimensional metrizable locally convex space supports a hypercyclic operator.

Behavior, pain perception, and the extremely low-birth weight survivor

This article explores the literature concerning responses to pain of both premature and term-born newborn infants, the evidence for short-term and long-term effects of pain, and behavioral sequelae in individuals who have experienced repeated early pain in neonatal life as they mature. There is no doubt that pain causes stress in babies and this in turn may adversely affect long-term neurodevelopmental outcome. Although there are methods for assessing dimensions of acute reactivity to pain in an experimental setting, there are no very good measures available at the present time that can be used clinically. In the clinical setting repeated or chronic pain is more likely the norm rather than infrequent discrete noxious stimuli of the sort that can be readily studied. The wind-up phenomenon suggests that, exposed to a cascade of procedures as happens with clustering of care in the clinical setting in an attempt to provide periods of rest for stressed babies, an infant may in fact perceive procedures that are not normally viewed as noxious, as pain. Pain exposure during lifesaving intensive medical care of ELBW neonates may also affect subsequent reactivity to pain in the neonatal period, but behavioral differences are probably not likely to be clinically significant in the long term. Prolonged and repeated untreated pain in the newborn period, however, may produce a relatively permanent shift in basal autonomic arousal related to prior NICU pain experience, which may have long-term sequelae. In the long run, the most significant clinical effects of early pain exposure may be on neurodevelopment, contributing to later attention, learning, and behavior problems in these vulnerable children. Although there is considerable evidence to support a variety of adverse effects of early pain, there is less information about the long-term effects of opiates and benzodiazepines on the developing central nervous system. Current evidence reviewed suggests that judicious use of morphine for adjustment to mechanical ventilation may ameliorate the altered autonomic response. It may be very important, however, to distinguish stress from pain. Animal evidence suggests that the neonatal brain is affected differently when exposed to morphine administered in the absence of pain than in the presence of pain. Pain control may be important for many reasons but overuse of morphine or benzodiazepines may have undesirable long-term effects. This is a rapidly evolving area of knowledge of clear relevance to clinical management likely to affect long-term outcomes of high-risk children.