Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes

Background: The HNF1A, HNF1B and HNF4A genes are part of an autoregulatory network in mammalian pancreas, liver, kidney and gut. The layout of this network appears to be similar in rodents and humans, but inactivation of HNF1A, HNF1B or HNF4A genes in animal models cause divergent phenotypes to those seen in man. We hypothesised that some differences may arise from variation in the expression profile of alternatively processed isoforms between species. Methodology/Principal Findings: We measured the expression of the major isoforms of the HNF1A, HNF1B and HNF4A genes in human and rodent pancreas, islet, liver and kidney by isoform-specific quantitative real-time PCR and compared their expression by the comparative Ct (??Ct) method. We found major changes in the expression profiles of the HNF genes between humans and rodents. The principal difference lies in the expression of the HNF1A gene, which exists as three isoforms in man, but as a single isoform only in rodents. More subtle changes were to the balance of HNF1B and HNF4A isoforms between species; the repressor isoform HNF1B(C) comprised only 6% in human islets compared with 24–26% in rodents (p = 0.006) whereas HNF4A9 comprised 22% of HNF4A expression in human pancreas but only 11% in rodents (p = 0.001). Conclusions/Significance: The differences we note in the isoform-specific expression of the human and rodent HNF1A, HNF1B and HNF4A genes may impact on the absolute activity of these genes, and therefore on the activity of the pancreatic transcription factor network as a whole. We conclude that alterations to expression of HNF isoforms may underlie some of the phenotypic variation caused by mutations in these genes.

Glasgow antipsychotic side-effects scale for Clozapine - development and validation of a clozapine-specific side-effects scale

OBJECTIVE: The authors developed and validated a clozapine-specific side-effects scale capable of eliciting the subjectively unpleasant side-effects of clozapine. METHODS: Questions from the original Glasgow Antipsychotic Side-effects Scale (GASS) were compared to a list of the most commonly reported clozapine side-effects and those with a significant subjective burden were included in the GASS for Clozapine (GASS-C). The original authors of the GASS and a group of mental health professionals from the UK and Ireland were enlisted to comment on the questions in the GASS-C based on their clinical experience. 110 clozapine outpatients from two sites completed the GASS-C, the original GASS and a repeat GASS-C. Statistical analyses were performed using SPSS for Windows version 19. RESULTS: The GASS-C was shown to have construct validity, in that Spearman's correlation coefficient was 0.816 (p<0.001) with the original GASS, whilst Cohen's kappa coefficient was >0.77 (p<0.001) for one question and >0.81 (p<0.001) for remaining relevant questions. GASS-C was also shown to have strong test-retest reliability, in that Cronbach's alpha coefficient was >0.907 (p<0.001), whilst Cohen's kappa coefficient was >0.81 (p<0.001) for 12 questions and >0.61 (p<0.001) for the remaining four questions. CONCLUSION: The GASS-C is a valid and reliable clinical tool to enable a systematic assessment of the subjectively unpleasant side-effects of clozapine. Future research should focus on how the scale can be utilised as a clinical tool to improve real-world outcomes such as adherence to clozapine therapy and quality of life.

Design and function of trilobite exoskeletons

The fossil arthropod Class Trilobita is characterised by the possession of a highly mineralised dorsal exoskeleton with an incurved marginal flange (doublure). This cuticle is usually the only part of the organism to be preserved. Despite the common occurrence of trilobites in Palaeozoic sediments, the original exoskeletal mineralogy has not been determined previously. Petrographic data involving over seventy trilobite species, ranging in age from Cambrian to Devonian, together with atomic absorption and stable isotope analyses, indicate a primary low-magnesian calcite composition. Trilobite cuticles exhibit a variety of preservational textures which are related to the different diagenetic realms through which they have passed. A greater knowledge of post-depositional processes and the specific features they produce, has enabled post-mortem artefacts to be distinguished from primary cuticular microstructures. Alterations of the cuticle can either enhance or destroy primary features, and their effects are best observed in thin-sections, both under transmitted light and cathodoluminescence. Well-preserved trilobites often retain primary microstructures such as laminations, canals, and tubercles. These have been examined in stained thin-sections and by scanning electron microscopy, from as wide a range of trilobites as possible. Construction of sensory field maps has shown that although the basic organisation of the exoskeleton is the same in all trilobites, the types of microstructures found, and their distribution is species-specific. The composition, microstructure, and architecture of the trilobite exoskeleton have also been studied from a biomechanical viewpoint. Total cuticle thickness, and the relative proportions of the different layers, together with the overall architecture all affected the mechanical properties of the exoskeleton.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.

Technology sourcing versus technology exploitation:An analysis of US foreign direct investment flows

The traditional paradigm of foreign direct investment (FDI) suggests that FDI is undertaken principally to exploit some firm-specific advantage in a foreign country which provides a locational advantage to the investor. However, recent theoretical work suggests a model of FDI in which the motivation is not to exploit existing technological advantages in a foreign country, but to access such technology and transfer it from the host economy to the investing multinational corporation via spillover effects. This paper tests the technology sourcing versus technology exploiting hypotheses for a panel of sectoral FDI flows between the United States and major OECD nations over a 15 year period. The research makes use of Patel and Vega's (Research Policy, 28, 145-55, 1999) taxonomy of sectors which are likely a priori to exhibit technology sourcing and exploiting behaviour respectively. While there is evidence that FDI flows into the United States are attracted to R and D intensive sectors, very little support is found for the technology sourcing hypothesis either for inward or outward FDI flows. The results suggest that, in aggregate, firm-specific 'ownership' effects remain powerful determinants of FDI flows.

Double-blind, placebo-controlled, randomized study of eicosapentaenoic acid diester in patients with cancer cachexia

Purpose: Eicosapentaenoic acid (EPA) has been proposed to have specific anticachectic effects. This trial compared EPA diethyl ester with placebo in cachectic cancer patients for effects on weight and lean body mass. Patients and Methods: Five hundred eighteen weight-losing patients with advanced gastrointestinal or lung cancer were studied in a multicenter, double-blind, placebo controlled trial. Patients were randomly assigned to receive a novel preparation of pure EPA at a dose of 2 g or 4 g daily or placebo (2g EPA, n = 175; 4 g EPA, n = 172; placebo, n = 171). Patients were assessed at 4 weeks and 8 weeks. Results: The groups were well balanced at baseline. Mean weight loss at baseline was 18% (n = 518). Over the 8-week treatment period, both intention-to-treat analysis and per protocol analysis revealed no statistically significant improvements in survival, weight, or other nutritional variables. There was, however, a trend in favor of EPA with analysis of the primary end point, weight, at 8 weeks showing a borderline, nonsignificant treatment effect (P = .066). Relative to placebo, mean weight increased by 1.2 kg with 2 g EPA (95% CI, 0 kg to 2.3 kg) and by 0.3 kg with 4g EPA (-0.9 kg to 1.5 kg). Conclusion: The results indicate no statistically significant benefit from single agent EPA in the treatment of cancer cachexia. Future studies should concentrate on other agents or combination regimens. © 2006 by American Society of Clinical Oncology.

Phenotypic and genotypic analysis of intestinal spirochacies

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.

Surface properties of enterococcus faecalis in relation to infective endocarditis

The effect of growth conditions on both the appearance and the antigenic profile of cells of Enterococcus faecalis was investigated using electron micrographs of ruthenium red stained and sectioned cells and SDS-PAGE and blotting techniques respectively. Three specific antigens of molecular weights 73, 40 and 37 kdaltons were of particular interest being expressed most strongly after growth in serum. This medium was deemed to most closely mimic jn vjvo growth conditions reflecting an environment similar to that which the microorganisms would encounter during bacteraemia, preceding the colonisation of the endocardium and the development of infective endocarditis. The 40 and 37 kdalton antigens were shown by immunoqold labelling to be exposed on the surface of the cells although they did not appear to be connected with the fimbriae shown to exist on some of the E. faecalis cells examined by negative staining. The 73, 40 and 37 kdalton antigens were crudely purified using sarkosyl and ammonium sulphate precipitation, and used as the basis of a serodiagnostic test for E. faecalis endocarditis using an ELISA system. This was tested in a blind trial and the success rates were 94% for positives, 90% for negatives with endocarditis caused by other organisms and 80% for E. faecalis infections other than endocarditis. The binding of E.faecalis cells to the serum proteins fibronectin and albumin was investigated using 125I labelled proteins, followed by Scatchard analysis. This showed that· E.faecalis cells do loosely bind large amounts of both of these proteins, thus surely affecting the way in which the host's immune system perceives the cells. The E.faecalis receptor for fibronectin was partially characterised and appeared to involve protein and/or carbohydrate containing components. but did not involve LTA or the 40 and 37 kdalton species specific antigens.

Evaluation of biodegradable polymer microspheres and an anionic dendrimer for the potential delivery of antisense oligonucleotides

Antisense oligonucleotides (AODNs) can selectively inhibit individual gene expression by binding specifically to rnRNA. The over-expression of the epidermal growth factor receptor (EGFR) has been observed in human breast and glioblastoma tumours and therefore AODNs designed to target the EGFR would be a logical approach to treat such tumours. However, poor pharmacokinetic/pharmacodynamic and cellular uptake properties of AODNs have limited their potential to become successful therapeutic agents. Biodegradable polymeric poly (lactide-co-glycolide) (P(LA-GA)) and dendrimer delivery systems may allow us to overcome these problems. The use of combination therapy of AODNs and cytotoxic agents such as 5-fluorouracil (5-FU) in biodegradable polymeric formulations may further improve therapeutic efficacy. AODN and 5-FU were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations (double emulsion method) and release profiles determined in vitro. The release rates (biphasic) of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Sustained release over 35 days was observed in both types of formulation. Naked and microsphere-loaded AODN and 5-FU (in separate formulations) were tested on an A431 vulval carcinoma cell line. Combining naked or encapsulated drugs produced a greater reduction in viable cell number as compared with either agent alone. However, controls and Western blotting indicated that non-sequence specific cytotoxic effects were responsible for the differences in viable cell number. The uptake properties of an anionic dendrimer based on a pentaerythritol structure covalently linked to AODNs (targeting the EGFR) have been characterised. The cellular uptake of AODN linked to the dendrimer was up to 3.5-fold higher in A431 cells as compared to naked AODN. Mechanistic studies suggested that receptor-mediated and adsorptive (binding protein-mediated) endocytosis were the predominant uptake mechanisms for the dendrimer-AODN. RNase H cleavage assay suggested that the dendrimer-AODN was able to bind and cleave the target site. A reduction of 20%, 28% and 45% in EGFR expression was observed with 0.05μM, 0.1μM and 0.5μM dendrimer-AODN treatments respectively with a reduction in viable cell number. These results indicated that the dendrimer delivery system may reduce viable cell number by an antisense specific mechanism.

Observation of a finger or an object movement primes imitative responses differentially

Behavioural advantages for imitation of human movements over movements instructed by other visual stimuli are attributed to an ‘action observation-execution matching’ (AOEM) mechanism. Here, we demonstrate that priming/exogenous cueing with a videotaped finger movement stimulus (S1) produces specific congruency effects in reaction times (RTs) of imitative responses to a target movement (S2) at defined stimulus onset asynchronies (SOAs). When contrasted with a moving object at an SOA of 533 ms, only a human movement is capable of inducing an effect reminiscent of ‘inhibition of return’ (IOR), i.e. a significant advantage for imitation of a subsequent incongruent as compared to a congruent movement. When responses are primed by a finger movement at SOAs of 533 and 1,200 ms, inhibition of congruent or facilitation of incongruent responses, respectively, is stronger as compared to priming by a moving object. This pattern does not depend on whether S2 presents a finger movement or a moving object, thus effects cannot be attributed to visual similarity between S1 and S2. We propose that, whereas both priming by a finger movement and a moving object induces processes of spatial orienting, solely observation of a human movement activates AOEM. Thus, S1 immediately elicits an imitative response tendency. As an overt imitation of S1 is inadequate in the present setting, the response is inhibited which, in turn, modulates congruency effects.

Attentional inhibition has social-emotional consequences for unfamiliar faces

Visual attention studies often rely on response time measures to show the impact of attentional facilitation and inhibition. Here we extend the investigation of the effects of attention on behavior and show that prior attentional states associated with unfamiliar faces can influence subsequent social-emotional judgments about those faces. Participants were shown pairs of face images and were asked to withhold a response if a transparent stop-signal cue appeared over one of the faces. This served to associate the cued face with an inhibitory state. Later, when asked to make social-emotional choices about these face pairs, participants chose uncued faces more often than cued faces as "more trustworthy" and chose cued faces more often than uncued faces as "less trustworthy." For perceptual choices, there was no effect of how the question was framed (which face is "on a lighter background" vs. "on a darker background"). These results suggest that attentional inhibition can be associated with socially relevant stimuli, such as faces, and can have specific, deleterious effects on social-emotional judgments.

Chemical specificity in REDOX-responsive materials:the diverse effects of different Reactive Oxygen Species (ROS) on polysulfide nanoparticles

REDOX responsive (nano)materials typically exhibit chemical changes in response to the presence and concentration of oxidants/reductants. Due to the complexity of biological environments, it is critical to ascertain whether the chemical response may depend on the chemical details of the stimulus, in addition to its REDOX potential, and whether chemically different responses can determine a different overall performance of the material. Here, we have used oxidation-sensitive materials, although these considerations can be extended also to reducible ones. In particular, we have used poly(propylene sulfide) (PPS) nanoparticles coated with a PEGylated emulsifier (Pluronic F127); inter alia, we here present also an improved preparative method. The nanoparticles were exposed to two Reactive Oxygen Species (ROS) typically encountered in inflammatory reactions, hydrogen peroxide (H2O2) and hypochlorite (ClO−); their response was evaluated with a variety of techniques, including diffusion NMR spectroscopy that allowed to separately characterize the chemically different colloidal species produced. The two oxidants triggered a different chemical response: H2O2 converted sulfides to sulfoxides, while ClO− partially oxidized them further to sulfones. The different chemistry correlated to a different material response: H2O2 increased the polarity of the nanoparticles, causing them to swell in water and to release the surface PEGylated emulsifier; the uncoated oxidized particles still exhibited very low toxicity. On the contrary, ClO− rapidly converted the nanoparticles into water-soluble, depolymerized fragments with a significantly higher toxicity. The take-home message is that it is more correct to discuss ‘smart’ materials in terms of an environmentally specific response to (REDOX) stimuli. Far from being a problem, this could open the way to more sophisticated and precisely targeted applications.

Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction

BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling.