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Large technology and software engineering programmes, such as enterprise system pro- grammes, are increasingly implemented through a mixture of customer and specialist third-party resources. These multi-partner working environments can be thought of as a complex social system, which oftentimes experience various forms of conflict. This can be due to competing objectives and priorities of the various organizations, along with incompatibilities of team members within the work-based social network of the implementation programme. If not brought under control, conflict can lead to complex emergent behaviours and dynamics within the wider social network, which can severely impact the likelihood of successful programme implementation of these software-intensive systems. Using social network analysis and thematic coding analysis within a case study, we show that the project management of complex software-intensive implementations requires considerable focus on control and communication across the programme-wide social network of team members, which we represent as a cybernetic system. A conceptual framework has been developed that extends extant literature around conflict in teams by framing the individual projects and the overall programme-wide implementation as cybernetic systems. The conceptual framework illustrates how a cybernetics approach to conflict within enterprise system implementations, can provide new insights into how conflict develops within project teams. Finally, we argue that the cybernetic approach allows us to develop project management interventions to mitigate the risk of conflict development, or control and regulate conflict once it has developed. We conclude by setting the agenda for future research on how conflict can be controlled within the implementation of software-intensive systems, such as enterprise systems.

Background: Heritability explains 45-82% of muscle mass and strength variation, yet polygenic models for muscle phenotypes in older women are scarce. Therefore, the objective of the present study was to (1) assess if total genotype predisposition score (GPSTOTAL) for a set of polymorphisms differed between older women with low and high muscle mass, and (2) utilise a data-driven GPS (GPSDD) to predict the variance in muscle size and strength-related phenotypes. Methods: In three-hundred 60- to 91-year-old Caucasian women (70.7 ± 5.7 years), skeletal muscle mass, biceps brachii thickness, vastus lateralis anatomical cross-sectional area (VLACSA), hand grip strength (HGS), and elbow flexion (MVCEF) and knee extension (MVCKE) maximum voluntary contraction were measured. Participants were classified as having low muscle mass if the skeletal muscle index (SMI) < 6.76 kg/m2 or relative skeletal muscle mass (%SMMr) < 22.1%. Genotyping was completed for 24 single-nucleotide polymorphisms (SNPs). GPSTOTAL was calculated from 23 SNPs and compared between the low and high muscle mass groups. A GPSDD was performed to identify the association of SNPs with other skeletal muscle phenotypes. Results: There was no significant difference in GPSTOTAL between low and high muscle mass groups, irrespective of classification based on SMI or %SMMr. The GPSDD model, using 23 selected SNPs, revealed that 13 SNPs were associated with at least one skeletal muscle phenotype: HIF1A rs11549465 was associated with four phenotypes and, in descending number of phenotype associations, ACE rs4341 with three; PTK2 rs7460 and CNTFR rs2070802 with two; and MTHFR rs17421511, ACVR1B rs10783485, CNTF rs1800169, MTHFR rs1801131, MTHFR rs1537516, TRHR rs7832552, MSTN rs1805086, COL1A1 rs1800012, and FTO rs9939609 with one phenotype. The GPSDD with age included as a predictor variable explained 1.7% variance of biceps brachii thickness, 12.5% of VLACSA, 19.0% of HGS, 8.2% of MVCEF, and 9.6% of MVCKE. Conclusions: In older women, GPSTOTAL did not differ between low and high muscle mass groups. However, GPSDD was associated with muscle size and strength phenotypes. Further advancement of polygenic models to understand skeletal muscle function during ageing might become useful in targeting interventions towards older adults most likely to lose physical independence. ispartof: GENES vol:13 issue:6 ispartof: location:Switzerland status: published

War and asymmetrical conflicts are becoming increasingly prevalent in the modern world. Due to improvements in conflict medicine, survivable injuries are now more severe than they once were. Therefore, it is now more important than ever that there exist scientific and engineering methods for replicating wartime injuries in the context of the laboratory. We have developed one such method: a shock tube platform for testing ex vivo samples of the porcine respiratory system. Using this platform, we can, to some extent, simulate the pathophysiological consequences of blast lung. This is a condition commonly present in victims of explosive blasts, both those due to typical armaments and Improvised Explosive Devices (IEDs). Presented here are the results of experiments conducted using porcine bronchiole tissue as ex vivo organ cultures. Data presented show epithelial damage, consistent with known trauma-induced cell injury that can lead to acute respiratory distress syndrome (ARDS).

BACKGROUND: Congenital portosystemic shunt (cPSS) is one of the most common congenital disorders diagnosed in dogs. Hepatic encephalopathy (HE) is a frequent complication in dogs with a cPSS and is a major cause of morbidity and mortality. Despite HE been a major cause of morbidity in dogs with a cPSS, little is known about the cellular changes that occur in the central nervous system of dogs with a cPSS.OBJECTIVES: The objective of this study was to characterise the histological changes in the cerebral cortex and cerebellum of dogs with cPSS with particular emphasis on astrocyte morphology.METHODS: Eight dogs with a confirmed cPSS were included in the study.RESULTS: Six dogs had substantial numbers of Alzheimer type II astrocytes and all cases had increased immunoreactivity for glial fibrillary acidic protein in the cerebral cortex, even if there were minimal other morphological changes.CONCLUSIONS: This study demonstrates that dogs with a cPSS have marked cellular changes in the cerebral cortex and cerebellum. The cellular changes that occur in the cerebral cortex and cerebellum of dogs with spontaneously arising HE are similar to changes which occur in humans with HE, further validating dogs with a cPSS as a good model for human HE.

Drawing on the central themes of open innovation of the knowledge inflows and outflows for a firm’s innovative achievement, this research investigates the application of external knowledge (i.e. competitor intelligence) for product innovation. We posit that open-mindedness and inter-functional coordination mediate the relationship between competitor intelligence and product innovation. We further explore the joint moderating effect of the dynamic emerging market environment on the effects of open-mindedness and inter-functional coordination on product innovation. Survey data were collected from 284 executives from Chinese IT SMEs. Analysis methods of Confirmatory Factor Analysis (CFA) and Structural Equation Modelling (SEM) are applied. Results showed that competitor intelligence has a positive and direct effect on product innovation, and their relationships can be further strengthened by the mediators—open-mindedness and inter-functional coordination. In testing the interaction with the dynamic emerging market, we found that the external environment interacts positively with open-mindedness, but negatively with inter-functional coordination on the effect on product innovation. This research enriches our understanding of external knowledge inflow for open innovation application. It contextualises the positive impact of being an open-minded and inter-functional coordinated on a firm’s product innovation. Managerial implications and future research avenues are discussed

Aims: Renovascular hypertension is a rare cause of paediatric hypertension. It is however, a potentially treatable cause particularly when caused by renal artery stenosis (RAS). Materials and Methods: We present the case of an 11-month-old girl presenting with cardiac dysfunction. She was found to be hypertensive with a systolic blood pressure >180mmHg. DMSA demonstrated a small right kidney and a divided renal function of 6% on the right and 94% on the left. Spectral analysis demonstrated abnormal waveforms suggestive of RAS of the left kidney. Results: Angioplasty with a cutting balloon was successful. Blood pressure measurements, renal function and left ventricular function improved. Conclusion: RAS can be successfully treated with cutting balloon angioplasty after failure of convention balloon angioplasty to relieve the narrowing. In our case, there was an immediate successful angiographic result that on mid-term follow-up demonstrated significant improvement in clinical and biochemical outcomes and cessation of all anti-hypertensive medication.

Objective Despite poor clinic communication and staff treatment being reported by donors, high rates of overall satisfaction are still reported in surveys. This study will evaluate the importance of communication and interaction between donors and fertility clinic staff in gamete donor care. Methods We report on 120 egg and sperm donors’ responses to a UK-wide online satisfaction survey. The survey focused on donors’ interactions with fertility clinic staff pre-, during, and post- donation. Basic cross-tabulation was performed on the data using online survey software. Textual data was read and extracts identified, which illustrated and expanded on the findings from the numerical data. Diagrammatic modeling was also utilised to analyse the textual data, with particular focus to relationships between the donors and clinic staff, the main activities within the gamete donation process, and how these activities may affect donor satisfaction with the gamete donation process. Results Donors expressed concern for the infertile couple and the resulting child; conveyed frustration at not receiving information on the expenses they could claim; felt lost in the system regarding the ease of making clinic appointments, and once made they were routinely not seen on time for these appointments. Donors also negatively commented on aftercare, the location and condition of the donation room, and information on contraception. In addition, Ovarian Hyperstimulation Syndrome was frequently reported, with these egg donors believing that clinic staff were not concerned with their physical or emotional well-being, but were instead disproportionately focused on extracting the eggs. Conclusions The multifaceted notion of donors highlights the complexity inherent to the gamete donation process, which comprises various aspects of uncertainty in the donation system, and ambiguity in the donation process. Categorising donors as Altruist, Customer, and Patient, conveys the particular importance of staff communication and treatment in donor care. These categories are not mutually exclusive however, in that an individual donor may experience more than one of these perspectives during the course of their gamete donation journey. Finally, there were a number of exemplar cases, where donors reported high satisfaction throughout, and these correlated with them being given a single point of contact at the clinic. Subject to resource constraints, we suggest that this practice should be implemented throughout clinics in the UK, so that donors have access to dedicated clinic staff who not only support them emotionally and physically throughout the gamete donation process, but also ensure that communication is open, clear, timely, and consistent.

Robert Rosen's (M,R) system is an abstract biological network architecture that is allegedly both irreducible to sub-models of its component states and non-computable on a Turing machine. (M,R) stands as an obstacle to both reductionist and mechanistic presentations of systems biology, principally due to its self-referential structure. If (M,R) has the properties claimed for it, computational systems biology will not be possible, or at best will be a science of approximate simulations rather than accurate models. Several attempts have been made, at both empirical and theoretical levels, to disprove this assertion by instantiating (M,R) in software architectures. So far, these efforts have been inconclusive. In this paper, we attempt to demonstrate why - by showing how both finite state machine and stream X-machine formal architectures fail to capture the self-referential requirements of (M,R). We then show that a solution may be found in communicating X-machines, which remove self-reference using parallel computation, and then synthesise such machine architectures with object-orientation to create a formal basis for future software instantiations of (M,R) systems.

Computational modelling and simulation is increasingly being used to complement traditional wet-lab techniques when investigating the mechanistic behaviours of complex biological systems. In order to ensure computational models are fit for purpose, it is essential that the abstracted view of biology captured in the computational model, is clearly and unambiguously defined within a conceptual model of the biological domain (a domain model), that acts to accurately represent the biological system and to document the functional requirements for the resultant computational model. We present a domain model of the IL-1 stimulated NF-κB signalling pathway, which unambiguously defines the spatial, temporal and stochastic requirements for our future computational model. Through the development of this model, we observe that, in isolation, UML is not sufficient for the purpose of creating a domain model, and that a number of descriptive and multivariate statistical techniques provide complementary perspectives, in particular when modelling the heterogeneity of dynamics at the single-cell level. We believe this approach of using UML to define the structure and interactions within a complex system, along with statistics to define the stochastic and dynamic nature of complex systems, is crucial for ensuring that conceptual models of complex dynamical biosystems, which are developed using UML, are fit for purpose, and unambiguously define the functional requirements for the resultant computational model.

Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal.

Robert Rosen's (M,R) system is an abstract biological network architecture that is allegedly non-computable on a Turing machine. If (M,R) is truly non-computable, there are serious implications for the modelling of large biological networks in computer software. A body of work has now accumulated addressing Rosen's claim concerning (M,R) by attempting to instantiate it in various software systems. However, a conclusive refutation has remained elusive, principally since none of the attempts to date have unambiguously avoided the critique that they have altered the properties of (M,R) in the coding process, producing merely approximate simulations of (M,R) rather than true computational models. In this paper, we use the Unified Modelling Language (UML), a diagrammatic notation standard, to express (M,R) as a system of objects having attributes, functions and relations. We believe that this instantiates (M,R) in such a way than none of the original properties of the system are corrupted in the process. Crucially, we demonstrate that (M,R) as classically represented in the relational biology literature is implicitly a UML communication diagram. Furthermore, since UML is formally compatible with object-oriented computing languages, instantiation of (M,R) in UML strongly implies its computability in object-oriented coding languages.

Toll-like receptors (TLRs) recognize specific pathogen–associated molecular patterns and initiate innate immune responses through signaling pathways that depend on the adaptor proteins MyD88 (myeloid differentiation marker 88) or TRIF (TIR domain–containing adaptor protein–inducing interferon-β). TLR4, in particular, uses both adaptor proteins to activate the transcription factor nuclear factor κB (nF-κB); however, the specificity and redundancy of these two pathways remain to be elucidated. We developed a mathematical model to show how each pathway encodes distinct dynamical features of NF-κB activity and makes distinct contributions to the high variability observed in single-cell measurements. The assembly of a macromolecular signaling platform around MyD88 associated with receptors at the cell surface determined the timing of initial responses to generate a reliable, digital NF-κB signal. In contrast, ligand-induced receptor internalization into endosomes produced noisy, delayed, yet sustained NF-κB signals through TRIF. With iterative mathematical model development, we predicted the molecular mechanisms by which the MyD88- and TRIF-mediated pathways provide ligand concentration–dependent signaling dynamics that transmit information about the pathogen threat.

Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.

In this review article, we discuss the current state of computational modelling of the nuclear factor-kappa B (NF-ΚB) signalling pathway. NF-ΚB is a transcription factor, which is ubiquitous within cells and controls a number of immune responses, including inflammation and apoptosis. The NF-ΚB signalling pathway is tightly regulated, commencing with activation at the cell membrane, signal transduction through various components within the cytoplasm, translocation of NF-ΚB into the nucleus and, finally, the transcription of various genes relating to the innate and adaptive immune responses. There have been a number of computational (mathematical) models developed of the signalling pathway over the past decade. This review describes how these approaches have helped advance our understanding of NF-ΚB control.

Predicting efficacy and optimal drug delivery strategies for small molecule and biological therapeutics is challenging due to the complex interactions between diverse cell types in different tissues that determine disease outcome. Here we present a new methodology to simulate inflammatory disease manifestation and test potential intervention strategies in silico using agent-based computational models. Simulations created using this methodology have explicit spatial and temporal representations, and capture the heterogeneous and stochastic cellular behaviours that lead to emergence of pathology or disease resolution. To demonstrate this methodology we have simulated the prototypic murine T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. In the simulation immune cell dynamics, neuronal damage and tissue specific pathology emerge, closely resembling behaviour found in the murine model. Using the calibrated simulation we have analysed how changes in the timing and efficacy of T cell receptor signalling inhibition leads to either disease exacerbation or resolution. The technology described is a powerful new method to understand cellular behaviours in complex inflammatory disease, permits rational design of drug interventional strategies and has provided new insights into the role of TCR signalling in autoimmune disease progression.

A PhD thesis is a project with an established goal and a deadline. As such, the tools, strategies and insight of professional project management can be used effectively to improve both research success and personal well-being.

Background Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. Results Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death. Conclusions Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

The presence of cytokines, prostaglandins and lipocortin-1 was investigated in terminally affected mice in two models of scrapie. There was marked induction of glial interleukin-1β, tumour necrosis factor α, prostaglandin E2, prostaglandin F2α and lipocortin-1 immunoreactivity in those areas of the brain showing the characteristic vacuolation of scrapie. A comparison of these staining patterns with those of GFAP and F4/80 showed that their expression occurred predominantly in astrocytes. It is possible that cytokines play a significant role in the pathogenesis of neurodegeneration in scrapie.

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.

Accumulation of β-sheet-rich peptide (Aβ) is strongly associated with Alzheimer's disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aβ species are potent neurotoxins, however the molecular mechanism responsible for Aβ toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aβ 1-40 and Aβ 1-42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aβ toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aβ receptors in N2a cells, triggering a multi factorial toxicity. © 2011 Manzoni et al.

Background The pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) is associated with the accumulation of aggregated forms of the α-synuclein (αSN) protein. An early event in the neuropathology of PD and DLB is the loss of synapses and a corresponding reduction in the level of synaptic proteins. However, the molecular mechanisms involved in synapse damage in these diseases are poorly understood. In this study the process of synapse damage was investigated by measuring the amount of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission, in cultured neurons incubated with αSN, or with amyloid-β (Aβ) peptides that are thought to trigger synapse degeneration in Alzheimer's disease. Results We report that the addition of recombinant human αSN reduced the amount of synaptophysin in cultured cortical and hippocampal neurons indicative of synapse damage. αSN also reduced synaptic vesicle recycling, as measured by the uptake of the fluorescent dye FM1-43. These effects of αSN on synapses were modified by interactions with other proteins. Thus, the addition of βSN reduced the effects of αSN on synapses. In contrast, the addition of amyloid-β (Aβ)1-42 exacerbated the effects of αSN on synaptic vesicle recycling and synapse damage. Similarly, the addition of αSN increased synapse damage induced by Aβ1-42. However, this effect of αSN was selective as it did not affect synapse damage induced by the prion-derived peptide PrP82-146. Conclusions These results are consistent with the hypothesis that oligomers of αSN trigger synapse damage in the brains of Parkinson's disease patients. Moreover, they suggest that the effect of αSN on synapses may be influenced by interactions with other peptides produced within the brain.

Background Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105–132 of the murine prion protein (MoPrP105-132) in neuroblastoma cells and primary cortical neurons. Results Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1) and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2) and cyclo-oxygenase-1 (COX-1), enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1. Conclusion As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

In the present study the accumulation of protease resistant prion protein (PrPres) in scrapie-infected neuroblastoma cells (ScN2a cells) was shown to be dependent on culture conditions. The highest levels of PrPres were found in slow growing cells. Further increases in PrPres accumulation were observed in ScN2a cells treated with retinoic acid, a compound that is associated with neuronal differentiation. The effects of retinoic acid were dose-dependent with a maximal effect at 200 ng/ml. A similar increase in PrPres was observed in another prion-infected cell line, scrapie-mouse brain (SMB) cells, treated with retinoic acid while retinoic acid increased the amount of PrPC in non-infected cells. Other drugs reported to cause neuronal differentiation, such as phorbol esters, did not increase the PrPres content of ScN2a cells. The survival of retinoic acid-treated ScN2a cells co-cultured with microglia was significantly reduced when compared to untreated ScN2a cells and an inverse correlation was demonstrated between the PrPres content of cells and their survival when co-cultured with microglia. The production of interleukin-6 by microglia cultured with retinoic acid-treated ScN2a cells was significantly higher than that of microglia cultured with untreated ScN2a cells.

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.

Epidemiological studies have shown that the risk of developing Alzheimer's disease is reduced by the chronic use of classical non-steroidal anti-inflammatory drugs (NSAIDs), drugs that inhibit the cyclo-oxygenase (COX) enzymes that convert arachidonic acid to prostaglandins. In the present study, human SH-SY5Y neuroblastoma cells or murine primary cortical neurones treated with NSAIDs were protected against the otherwise toxic effects of amyloid-beta1-42. COX-1 selective inhibitors provided greater protection than did COX-2 selective inhibitors or lipoxygenase inhibitors, suggesting that activation of COX-1 is required for amyloid-beta1-42-induced neurotoxicity. Although the production of neuronal prostaglandin E2 in response to amyloid-beta1-42 was reduced by the presence of COX-1 inhibitors, no neurotoxic effects of prostaglandin E2, or any other prostaglandin, were observed.

The appearance of immunoreactive interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α, prostaglandin (PG) E 2 and lipocortin-1 in the central nervous system was investigated during the development of lesions in a 301V/VM murine scrapie model. Focal PrP Sc deposition was present after 30 days of the 115–120 day incubation period; this immunoreactivity increased in intensity and distribution thereafter. Staining for IL-1β and TNFα in perivascular macrophages, and PGE 2 immunoreactivity in astrocytes, was detected in those areas showing PrP Sc deposition from 60 days. Increased GFAP and F4/80 immunoreactivity, indicating activation of astrocytes and microglia, was also evident in these areas from 60 days. Glial cytokine and lipocortin immunoreactivity was detected after 90 days, in the absence of clinical signs. The disease-induced cytokine, PG and lipocortin immunoreactivity occurred only in those brain areas showing PrP Sc deposition, glial activation and, in later stages, vacuolation. These findings support the concept that PrP Sc deposition induces glial cytokine production. These glial cytokines may contribute to the development of the pathological lesions in scrapie. © 1997 Elsevier Science B.V. All rights reserved.

Background The transmissible spongiform encephalopathies, otherwise known as prion diseases, occur following the conversion of the cellular prion protein (PrPC) to an alternatively folded, disease-associated isoform (PrPSc). Recent studies suggest that this conversion occurs via a cholesterol-sensitive process, as cholesterol synthesis inhibitors reduced the formation of PrPSc and delayed the clinical phase of scrapie infection. Since polyunsaturated fatty acids also reduced cellular cholesterol levels we tested their effects on PrPSc formation in three prion-infected neuronal cell lines (ScGT1, ScN2a and SMB cells). Results We report that treatment with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or the cholesterol synthesis inhibitor simvastatin reduced the amounts of free cholesterol in membrane extracts from prion-infected neuronal cells. Simvastatin reduced cholesterol production while DHA and EPA promoted the conversion of free cholesterol to cholesterol esters. Crucially, while simvastatin reduced PrPSc formation, both DHA and EPA significantly increased the amounts of PrPSc in these cells. Unlike simvastatin, the effects of DHA and EPA on PrPSc content were not reversed by stimulation of cholesterol synthesis with mevalonate. Treatment of ScGT1 cells with DHA and EPA also increased activation of cytoplasmic phospholipase A2 and prostaglandin E2 production. Finally, treatment of neuronal cells with DHA and EPA increased the amounts of PrPC expressed at the cell surface and significantly increased the half-life of biotinylated PrPC. Conclusion We report that although treatment with DHA or EPA significantly reduced the free cholesterol content of prion-infected cells they significantly increased PrPSc formation in three neuronal cell lines. DHA or EPA treatment of infected cells increased activation of phospholipase A2, a key enzyme in PrPSc formation, and altered the trafficking of PrPC. PrPC expression at the cell surface, a putative site for the PrPSc formation, was significantly increased, and the rate at which PrPC was degraded was reduced. Cholesterol depletion is seen as a potential therapeutic strategy for prion diseases. However, these results indicate that a greater understanding of the precise relationship between membrane cholesterol distribution, PrPC trafficking, cell activation and PrPSc formation is required before cholesterol manipulation can be considered as a prion therapeutic.

Background The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrPC) to an alternatively folded isoform (PrPSc). The accumulation of PrPSc within the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined. Results We report the novel observation that prion infection altered the membrane composition and significantly increased total cholesterol levels in two neuronal cell lines (ScGT1 and ScN2a cells). There was a significant correlation between the concentration of free cholesterol in ScGT1 cells and the amounts of PrPSc. This increase was entirely a result of increased amounts of free cholesterol, as prion infection reduced the amounts of cholesterol esters in cells. These effects were reproduced in primary cortical neurons by the addition of partially purified PrPSc, but not by PrPC. Crucially, the effects of prion infection were not a result of increased cholesterol synthesis. Stimulating cholesterol synthesis via the addition of mevalonate, or adding exogenous cholesterol, had the opposite effect to prion infection on the cholesterol balance. It did not affect the amounts of free cholesterol within neurons; rather, it significantly increased the amounts of cholesterol esters. Immunoprecipitation studies have shown that cytoplasmic phospholipase A2 (cPLA2) co-precipitated with PrPSc in ScGT1 cells. Furthermore, prion infection greatly increased both the phosphorylation of cPLA2 and prostaglandin E2 production. Conclusion Prion infection, or the addition of PrPSc, increased the free cholesterol content of cells, a process that could not be replicated by the stimulation of cholesterol synthesis. The presence of PrPSc increased solubilisation of free cholesterol in cell membranes and affected their function. It increased activation of the PLA2 pathway, previously implicated in PrPSc formation and in PrPSc-mediated neurotoxicity. These observations suggest that the neuropathogenesis of prion diseases results from PrPSc altering cholesterol-sensitive processes. Furthermore, they raise the possibility that disturbances in membrane cholesterol are major triggering events in neurodegenerative diseases.

Background The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ1–42 were examined. Results The addition of Aβ1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E2. Conclusion Pre-treatment with ginkgolides A or B protects neurons against Aβ1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E2, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.

Background An early event in the neuropathology of prion and Alzheimer's diseases is the loss of synapses and a corresponding reduction in the level of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission. The molecular mechanisms involved in synapse degeneration in these diseases are poorly understood. In this study the process of synapse degeneration was investigated by measuring the synaptophysin content of cultured neurones incubated with the prion derived peptide (PrP82-146) or with Aβ1-42, a peptide thought to trigger pathogenesis in Alzheimer's disease. A pharmacological approach was used to screen cell signalling pathways involved in synapse degeneration. Results Pre-treatment with phospholipase A2 inhibitors (AACOCF3, MAFP and aristolochic acids) protected against synapse degeneration in cultured cortical and hippocampal neurones incubated with PrP82-146 or Aβ1-42. Synapse degeneration was also observed following the addition of a specific phospholipase A2 activating peptide (PLAP) and the addition of PrP82-146 or Aβ1-42 activated cytoplasmic phospholipase A2 within synapses. Activation of phospholipase A2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B, Hexa-PAF and CV6029) protected against synapse degeneration induced by PrP82-146, Aβ1-42 and PLAP. PAF facilitated the production of prostaglandin E2, which also caused synapse degeneration and pre-treatment with the prostanoid E receptor antagonist AH13205 protected against PrP82-146, Aβ1-42 and PAF induced synapse degeneration. Conclusions Our results are consistent with the hypothesis that PrP82-146 and Aβ1-42trigger abnormal activation of cytoplasmic phospholipase A2 resident within synapses, resulting in elevated levels of PAF and prostaglandin E2that cause synapse degeneration. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse degeneration seen during Alzheimer's or prion diseases.