Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, Fasciolidae)

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. Ln addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic. (C) 2001 Wiley-Liss, Inc.

Development of the nervous system in Dugesia tigrina during regeneration after fission and decapitation

In the present study of Dugesia tigrina the development of the nervous system is followed and compared during regeneration after fission and after decapitation. Immunocytochemistry was used, with antisera raised against the biogenic amine, 5-hydroxytryptamine (5-HT) and the two neuropeptides, neuropeptide F (NPF), and FMRF amide. The results indicate that two processes are involved in the formation of the new cerebral ganglion. First, new processes sprouting from the original main longitudinal nerve cords bend transversely, indicating the position of the developing horseshoe-shaped anterior cerebral commissure. Then new nerve cells in front of the commissure differentiate from neoblasts and their growth cones fasciculate with the fibres from the old main longitudinal nerve cords. In the cerebral ganglion, 5-HT-IR cells appear before NPF-IR cells, in contrast to the pharynx where NPF-IR cells differentiate before the 5-HT-IR cells. In the peripheral nervous system, NPF-IR fibres and cells appear at a very early stage and dominate the whole regeneration process. A role for the PNS in early pattern formation is suggested.

Strategic development in exact calculation: Group and individual differences in four achievement subtypes

This longitudinal study sought to identify developmental changes
in strategy use between 5 and 7 years of age when solving exact
calculation problems. Four mathematics and reading achievement
subtypes were examined at four time points. Five strategies were
considered: finger counting, verbal counting, delayed retrieval,
automatic retrieval, and derived fact retrieval. Results provided
unique insights into children’s strategic development in exact calculation
at this early stage. Group analysis revealed relationships
between mathematical and/or reading difficulties and strategy
choice, shift, and adaptiveness. Use of derived fact retrieval by
7 years of age distinguished children with mathematical difficulties
from other achievement subtypes. Analysis of individual differences
revealed marked heterogeneity within all subtypes,
suggesting (inter alia) no marked qualitative distinction between
our two mathematical difficulty subtypes.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

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.

The 1863 Melbourne Shakespeare War: Barry Sullivan, Charles and Ellen Kean, and the Play of Cultural Usurpation on the Australian Stage

This essay investigates an intricate drama of cultural identity in performances of Shakespeare on the nineteenth-century Melbourne stage. It considers the rivalry between Charles and Ellen Kean and their competitor, Barry Sullivan, for the two-month period in 1863 during which their Australian tours overlapped. This Melbourne Shakespeare war was anticipated,augmented, and richly documented in Melbourne’s papers: The Age, The Argus and Melbourne Punch. This essay pursues two seams of inquiry. The first is an investigation of the discourses of cultural and aesthetic value laced through the language of reviews of their Shakespearean roles.The essay identifies how reviewers register affective engagement with the performers in these roles, and suggests how the roles themselves reflected, by accident or design, the terms of the dispute. The second is concerned with the national identity of the actors. Kean, although born in Waterford, Ireland, had held the post of Queen Victoria’s Master of the Revels and identified himself as English. Sullivan, although born in Birmingham, was of Cork parentage and was identified as Irish by both his supporters and his detractors. This essay tracks the development of the actors’ national and artistic identities established prior to Melbourne and ask how they played out on in the context of the particularities of Australian reception. It shows that, in this instance, these actors were implicated in complex debates over national authority and cultural ownership.

Immunomodulatory molecules of Fasciola hepatica:Candidates for both vaccine and immunotherapeutic development

The liver fluke, Fasciola hepatica, causes fascioliasis in domestic animals (sheep, cattle), a global disease that is also an important infection of humans. As soon as the parasite invades the gut wall its interaction with various host immune cells (e.g. dendritic cells, macrophages and mast cells) is complex. The parasite secretes a myriad of molecules that direct the immune response towards a favourable non-protective Th2-mediate/regulatory environment. These immunomodulatory molecules, such as cathepsin L peptidase (FhCL1), are under development as the first generation of fluke vaccines. However, this peptidase and other molecules, such as peroxiredoxin (FhPrx) and helminth defence molecule (FhHDM-1), exhibit various immunomodulatory properties that could be harnessed to help treat immune-related conditions in humans and animals.

Addressing potentially inappropriate prescribing in older patients: development and pilot study of an intervention in primary care (the OPTI-SCRIPT study)

Background: Potentially inappropriate prescribing (PIP) in older people is common in primary care and can result in increased morbidity, adverse drug events, hospitalizations and mortality. The prevalence of PIP in Ireland is estimated at 36% with an associated expenditure of over [euro sign]45 million in 2007. The aim of this paper is to describe the application of the Medical Research Council (MRC) framework to the development of an intervention to decrease PIP in Irish primary care.

Methods: The MRC framework for the design and evaluation of complex interventions guided the development of the study intervention. In the development stage, literature was reviewed and combined with information obtained from experts in the field using a consensus based methodology and patient cases to define the main components of the intervention. In the pilot stage, five GPs tested the proposed intervention. Qualitative interviews were conducted with the GPs to inform the development and implementation of the intervention for the main randomised controlled trial.

Results: The literature review identified PIP criteria for inclusion in the study and two initial intervention components - academic detailing and medicines review supported by therapeutic treatment algorithms. Through patient case studies and a focus group with a group of 8 GPs, these components were refined and a third component of the intervention identified - patient information leaflets. The intervention was tested in a pilot study. In total, eight medicine reviews were conducted across five GP practices. These reviews addressed ten instances of PIP, nine of which were addressed in the form of either a dose reduction or a discontinuation of a targeted medication. Qualitative interviews highlighted that GPs were receptive to the intervention but patient preference and time needed both to prepare for and conduct the medicines review, emerged as potential barriers. Findings from the pilot study allowed further refinement to produce the finalised intervention of academic detailing with a pharmacist, medicines review with web-based therapeutic treatment algorithms and tailored patient information leaflets.

Conclusions: The MRC framework was used in the development of the OPTI-SCRIPT intervention to decrease the level of PIP in primary care in Ireland. Its application ensured that the intervention was developed using the best available evidence, was acceptable to GPs and feasible to deliver in the clinical setting. The effectiveness of this intervention is currently being tested in a pragmatic cluster randomised controlled trial.

Trial registration: Current controlled trials ISRCTN41694007.© 2013 Clyne et al.; licensee BioMed Central Ltd.

Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a ß-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis-Menten kinetics in both directions, with Km values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s(-1) for the conversion of dihydroxyacetone phosphate and 1900 s(-1) for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. ß-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.

Fasciola hepatica in the rat:immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.

Development of small and medium-sized enterprise horizontal innovation networks: UK agri-food sector study

Within the management literature, there is an emergent discourse on horizontal collaboration among small and medium-sized enterprises (SMEs), whereby individual rivalries are overcome by the need for more resources and innovation, leading to increased competitiveness through joint product development. In particular, a number of these horizontal collaborations between SMEs have occurred within the agri-food sector. As a consequence, this article aims to explore the longitudinal development of horizontal innovation networks within an artisan bakers’ network as part of the UK SME agri-food sector. An interpretivist research approach was used, whereby the development and evolution of an artisan bakers’ horizontal network was studied over a 27-month period. The findings, as summarised in conceptual models which draw upon knowledge-based open innovation and social network constructs, illustrate that a complex three-stage life cycle development occurred within the bakers’ horizontal network.

Seasonal and gestation stage associated differences in aflatoxin exposure in pregnant Gambian women

Objective: Aflatoxin is known to cross the placental barrier and exposures in utero could influence genomic programming, foetal growth and development, resulting in long-term health effects. We aimed to determine aflatoxin exposure in Gambian women at two stages of pregnancy and during the rainy and dry seasons.

Methods: We examined aflatoxin exposure in pregnant Gambian women at early (<16 weeks) and later (16 weeks onward) stages of pregnancy and at different times of the year, during the rainy (June to October 2009) or dry (November to May 2010) season, using aflatoxin–albumin adducts (AF-alb).

Results: Mean AF-alb was higher during the dry season than in the rainy season, in both early and later pregnancy although the difference was strongest in later pregnancy. There was a modest increase in AF-alb in later than early pregnancy (geometric mean 41.8 vs. 34.5 pg/mg, P < 0.05), but this was restricted to the dry season when exposures were generally higher.

Conclusions: The study confirmed that Gambian pregnant women were exposed to aflatoxin throughout the pregnancy, with higher levels in the dry season. There was some evidence in the dry season that women in later pregnancy had higher AF-alb levels than those in earlier pregnancy. Further research on the effects of exposure to this potent mutagen and carcinogen throughout pregnancy, including the epigenetic modification of foetal gene expression and impact on pre- and post-natal growth and development, are merited.

Virtual metrology enabled early stage prediction for enhanced control of multi-stage fabrication processes

Semiconductor fabrication involves several sequential processing steps with the result that critical production variables are often affected by a superposition of affects over multiple steps. In this paper a Virtual Metrology (VM) system for early stage measurement of such variables is presented; the VM system seeks to express the contribution to the output variability that is due to a defined observable part of the production line. The outputs of the processed system may be used for process monitoring and control purposes. A second contribution of this work is the introduction of Elastic Nets, a regularization and variable selection technique for the modelling of highly-correlated datasets, as a technique for the development of VM models. Elastic Nets and the proposed VM system are illustrated using real data from a multi-stage etch process used in the fabrication of disk drive read/write heads. © 2013 IEEE.

Retirement Transition Migration: Implications for Rural Development

Recent literature suggests that the increasingly blurred relationship between paid employment and retirement facilitates a retirement transition period, a life course stage which may involve a change of residence. The role of pre-retirement mobility in the repopulation of rural areas has, however, received relatively little academic scrutiny from UK geographers. This article draws upon findings from a two-year study conducted in three UK case study areas. It examines the extent of pre-retirement age (aged 50-64) migration into rural communities and the impacts this type of movement has upon economic activity, social and community engagement and service provision. It is argued that while this under-researched group offers significant potential to support the social and economic sustainability of rural communities (at least in the short and medium term), there are notable regional variations which are likely to have important long term implications for rural communities as this cohort ages in situ.

RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.