PathwAX: a web server for network crosstalk based pathway annotation

Pathway annotation of gene lists is often used to functionally analyse biomolecular data such as gene expression in order to establish which processes are activated in a given experiment. Databases such as KEGG or GO represent collections of how genes are known to be organized in pathways, and the challenge is to compare a given gene list with the known pathways such that all true relations are identified. Most tools apply statistical measures to the gene overlap between the gene list and pathway. It is however problematic to avoid false negatives and false positives when only using the gene overlap. The pathwAX web server (http://pathwAX.sbc.su.se/) applies a different approach which is based on network crosstalk. It uses the comprehensive network FunCoup to analyse network crosstalk between a query gene list and KEGG pathways. PathwAX runs the BinoX algorithm, which employs Monte-Carlo sampling of randomized networks and estimates a binomial distribution, for estimating the statistical significance of the crosstalk. This results in substantially higher accuracy than gene overlap methods. The system was optimized for speed and allows interactive web usage. We illustrate the usage and output of pathwAX

A novel method for crosstalk analysis of biological networks: improving accuracy of pathway annotation

Analyzing gene expression patterns is a mainstay to gain functional insights of biological systems. A plethora of tools exist to identify significant enrichment of pathways for a set of differentially expressed genes. Most tools analyze gene overlap between gene sets and are therefore severely hampered by the current state of pathway annotation, yet at the same time they run a high risk of false assignments. A way to improve both true positive and false positive rates (FPRs) is to use a functional association network and instead look for enrichment of network connections between gene sets. We present a new network crosstalk analysis method BinoX that determines the statistical significance of network link enrichment or depletion between gene sets, using the binomial distribution. This is a much more appropriate statistical model than previous methods have employed, and as a result BinoX yields substantially better true positive and FPRs than was possible before. A number of benchmarks were performed to assess the accuracy of BinoX and competing methods. We demonstrate examples of how BinoX finds many biologically meaningful pathway annotations for gene sets from cancer and other diseases, which are not found by other methods. BinoX is available at http://sonnhammer.org/BinoX

Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer

Economic vs. juristic thinking in Carl Menger’s principles of economics

Austrian Economic School is increasingly being treated as one of the most significant and ever more influential bodies of ideas affecting the contemporary economic science. It is well known that Carl Menger, the founder of the Austrian School, was a lawyer by education. The paper is devoted to the degree to which his legal education influenced his economic theorizing. After a close examination of the Menger’s conceptual apparatus and the epistemological ramifications of the key notions underlying his thinking, the paper concludes that the juristic background of the far-reaching theoretical contributions contained in Menger’s Principles was truly decisive. That holds true despite the fact that Menger only exceptionally utilized the juristic vocabulary. The key ingredients of his theoretical creation? such as the individual autonomy, the coincidence of the wills in concluding a contract the effective protection of property and making transactions based on the individual motivation to further one’s own interests to the utmost? are clearly inspired and in many ways influenced by private law, particularly the doctrines underlying the Roman Law and the Austrian Civil Code

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken

Investigating 3R in vivo approaches for bio-distribution and efficacy evaluation of nucleic acid nanocarriers: studies on peptide-mimicking ionizable lipid

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.Drug Delivery Technolog

The increasing threat to European forests from the invasive foliar pine pathogen, Lecanosticta acicola

European forests are threatened by increasing numbers of invasive pests and pathogens. Over the past century, Lecanosticta acicola, a foliar pathogen predominantly of Pinus spp., has expanded its range globally, and is increasing in impact. Lecanosticta acicola causes brown spot needle blight, resulting in premature defoliation, reduced growth, and mortality in some hosts. Originating from southern regions of North American, it devastated forests in the USA's southern states in the early twentieth century, and in 1942 was discovered in Spain.Derived from Euphresco project 'Brownspotrisk,' this study aimed to establish the current distribution of Lecanosticta species, and assess the risks of L. acicola to European forests. Pathogen reports from the literature, and new/ unpublished survey data were combined into an open-access geo-database (http://www.portaloff orestpathology.com), and used to visualise the pathogen's range, infer its climatic tolerance, and update its host range. Lecanosticta species have now been recorded in 44 countries, mostly in the northern hemisphere. The type species, L. acicola, has increased its range in recent years, and is present in 24 out of the 26 European countries where data were available. Other species of Lecanosticta are largely restricted to Mexico and Central America, and recently Colombia.The geo-database records demonstrate that L. acicola tolerates a wide range of climates across the northern hemisphere, and indicate its potential to colonise Pinus spp. forests across large swathes of the Europe. Pre-liminary analyses suggest L. acicola could affect 62% of global Pinus species area by the end of this century, under climate change predictions.Although its host range appears slightly narrower than the similar Dothistroma species, Lecanosticta species were recorded on 70 host taxa, mostly Pinus spp., but including, Cedrus and Picea spp. Twenty-three, including species of critical ecological, environmental and economic significance in Europe, are highly susceptible to L. acicola, suffering heavy defoliation and sometimes mortality. Variation in apparent susceptibility between reports could reflect variation between regions in the hosts' genetic make-up, but could also reflect the signif-icant variation in L. acicola populations and lineages found across Europe. This study served to highlight sig-nificant gaps in our understanding of the pathogen's behaviour.Lecanosticta acicola has recently been downgraded from an A1 quarantine pest to a regulated non quarantine pathogen, and is now widely distributed across Europe. With a need to consider disease management, this study also explored global BSNB strategies, and used Case Studies to summarise the tactics employed to date in Europe

Targeted Disruption of the PME-1 Gene Causes Loss of Demethylated PP2A and Perinatal Lethality in Mice

Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/-) tissues, which also displayed alterations in phosphoproteome content.These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo