Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens.

In Pseudomonas protegens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.

Gene flow and gene flux shape evolutionary patterns of variation in Drosophila subobscura

Gene flow (defined as allele exchange between populations) and gene flux (defined as allele exchange during meiosis in heterokaryotypic females) are important factors decreasing genetic differentiation between populations and inversions. Many chromosomal inversions are under strong selection and their role in recombination reduction enhances the maintenance of their genetic distinctness. Here we analyze levels and patterns of nucleotide diversity, selection and demographic history, using 37 individuals of Drosophila subobscura from Mount Parnes (Greece) and Barcelona (Spain). Our sampling focused on two frequent O-chromosome arrangements that differ by two overlapping inversions (OST and O3+4), which are differentially adapted to the environment as observed by their opposing latitudinal clines in inversion frequencies. The six analyzed genes (Pif1A, Abi, Sqd, Yrt, Atpa and Fmr1) were selected for their location across the O-chromosome and their implication in thermal adaptation. Despite the extensive gene flux detected outside the inverted region, significant genetic differentiation between both arrangements was found inside it. However, high levels of gene flow were detected for all six genes when comparing the same arrangement among populations. These results suggest that the adaptive value of inversions is maintained, regardless of the lack of genetic differentiation within arrangements from different populations, and thus favors the Local Adaptation hypothesis over the Coadapted Genome hypothesis as the basis of the selection acting on inversions in these populations.

Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: a case-control study in Spain.

Background: Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) gene mutation have been postulated as a possible cause of recurrent miscarriage (RM). There is a wide variation in the prevalence of MTHFR polymorphisms and homocysteine (Hcy) plasma levels among populations around the world. The present study was undertaken to investigate the possible association between hyperhomocysteinemia and its causative genetic or acquired factors and RM in Catalonia, a Mediterranean region in Spain. Methods: Sixty consecutive patients with ≥ 3 unexplained RM and 30 healthy control women having at least one child but no previous miscarriage were included. Plasma Hcy levels, MTHFR gene mutation, red blood cell (RBC) folate and vitamin B12 serum levels were measured in all subjects. Results: No significant differences were observed neither in plasma Hcy levels, RBC folate and vitamin B12 serum levels nor in the prevalence of homozygous and heterozygous MTHFR gene mutation between the two groups studied. Conclusions: In the present study RM is not associated with hyperhomocysteinemia, and/or the MTHFR gene mutation.

Integration and Implementation of Accounting Information Systems in Global Companies Case: The UPM-Kymmene Group

Tämän tutkielman tavoitteena on perehtyä globaalin yrityksen laskentatoimen tietojärjestelmien yhtenäistämiseen ja käyttöönottoon esimerkkinä UPM-Kymmene konsernin projekti. Tutkielmassa sovelletaan hermeneuttista tutkimusotetta. Teoreettisesti aihetta tarkastellaan globalisoitumisen ja laskentatoimen tietojärjestelmille asetettavien vaatimusten pohjalta, sekä järjestelmän muutosprosessin eri vaiheissa huomioon otettavien muuttujien perusteella. Yhtenäisen laskentatoimen tietojärjestelmän tuomat edut globaalille yritykselle ovat ilmeisiä. Ennen yhtenäisen projektin kehittelyä on olennaista tutkia lähtökohdat projektin onnistumiselle ja suunnitella projektin eri vaiheet huolella. Tutkielmassa havaitaan myös, että globaalissa yrityksessä tulee huomioida eri yrityskulttuurit sekä tulosyksiköiden erilaiset toimintatavat. Johtopäätöksenä todetaan, että sekä yritysjohdon että tulosyksiköiden sitoutuneisuus projektiin ja yhtenäiset tavoitteet ovat oleellisia projektin onnistumisen kannalta.

Localization and expression of EDS5H a homologue of the SA transporter EDS5.

BACKGROUND: An important signal transduction pathway in plant defence depends on the accumulation of salicylic acid (SA). SA is produced in chloroplasts and the multidrug and toxin extrusion transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5; At4g39030) is necessary for the accumulation of SA after pathogen and abiotic stress. EDS5 is localized at the chloroplast and functions in transporting SA from the chloroplast to the cytoplasm. EDS5 has a homologue called EDS5H (EDS5 HOMOLOGUE; At2g21340) but its relationship to EDS5 has not been described and its function is not known. RESULTS: EDS5H exhibits about 72% similarity and 59% identity to EDS5. In contrast to EDS5 that is induced after pathogen inoculation, EDS5H was constitutively expressed in all green tissues, independently of pathogen infection. Both transporters are located at the envelope of the chloroplast, the compartment of SA biosynthesis. EDS5H is not involved with the accumulation of SA after inoculation with a pathogen or exposure to UV stress. A phylogenetic analysis supports the hypothesis that EDS5H may be an H(+)/organic acid antiporter like EDS5. CONCLUSIONS: The data based on genetic and molecular studies indicate that EDS5H despite its homology to EDS5 does not contribute to pathogen-induced SA accumulation like EDS5. EDS5H most likely transports related substances such as for example phenolic acids, but unlikely SA.

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now.

Divergence with gene flow and fine-scale phylogeographical structure in the wedge-billed woodcreeper, Glyphorynchus spirurus, a Neotropical rainforest bird.

Determining the relative roles of vicariance and selection in restricting gene flow between populations is of central importance to the evolutionary process of population divergence and speciation. Here we use molecular and morphological data to contrast the effect of isolation (by mountains and geographical distance) with that of ecological factors (altitudinal gradients) in promoting differentiation in the wedge-billed woodcreeper, Glyphorynchus spirurus, a tropical forest bird, in Ecuador. Tarsus length and beak size increased relative to body size with altitude on both sides of the Andes, and were correlated with the amount of moss on tree trunks, suggesting the role of selection in driving adaptive divergence. In contrast, molecular data revealed a considerable degree of admixture along these altitudinal gradients, suggesting that adaptive divergence in morphological traits has occurred in the presence of gene flow. As suggested by mitochondrial DNA sequence data, the Andes act as a barrier to gene flow between ancient subspecific lineages. Genome-wide amplified fragment length polymorphism markers reflected more recent patterns of gene flow and revealed fine-scale patterns of population differentiation that were not detectable with mitochondrial DNA, including the differentiation of isolated coastal populations west of the Andes. Our results support the predominant role of geographical isolation in driving genetic differentiation in G. spirurus, yet suggest the role of selection in driving parallel morphological divergence along ecological gradients.

Prototypical Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Financial integration and TARGET2-Securities – A stakeholder approach

The purpose of this study was to analyze how different stakeholders, such as the European Central Bank, the European Commission, the European Central Securities Depositories Association, the Bank of International Settlements and the International Monetary Fund, have influenced the European financial integration in the area of securities clearing and settlement since the introduction of the single currency. The results show that the contribution differs from stakeholder to another and can take the form of standards and recommendations, legislation, research and publications, risk analysis etc. The Eurosystem has been particularly active in this area by proving and planning central bank services, such as TARGET2, CCMB2 and T2S. Along the way the development has and continues to face challenges that need to be solved by European authorities. Coordination is a key factor for the future as we have seen during the recent financial turmoil.

Information integration and its impacts on logistics performance - Using information technology and systems to facilitate information integration

Fast changing environment sets pressure on firms to share large amount of information with their customers and suppliers. The terms information integration and information sharing are essential for facilitating a smooth flow of information throughout the supply chain, and the terms are used interchangeably in research literature. By integrating and sharing information, firms want to improve their logistics performance. Firms share information with their suppliers and customers by using traditional communication methods (telephone, fax, Email, written and face-to-face contacts) and by using advanced or modern communication methods such as electronic data interchange (EDI), enterprise resource planning (ERP), web-based procurement systems, electronic trading systems and web portals. Adopting new ways of using IT is one important resource for staying competitive on the rapidly changing market (Saeed et al. 2005, 387), and an information system that provides people the information they need for performing their work, will support company performance (Boddy et al. 2005, 26). The purpose of this research has been to test and understand the relationship between information integration with key suppliers and/or customers and a firm’s logistics performance, especially when information technology (IT) and information systems (IS) are used for integrating information. Quantitative and qualitative research methods have been used to perform the research. Special attention has been paid to the scope, level and direction of information integration (Van Donk & van der Vaart 2005a). In addition, the four elements of integration (Jahre & Fabbe-Costes 2008) are closely tied to the frame of reference. The elements are integration of flows, integration of processes and activities, integration of information technologies and systems and integration of actors. The study found that information integration has a low positive relationship to operational performance and a medium positive relationship to strategic performance. The potential performance improvements found in this study vary from efficiency, delivery and quality improvements (operational) to profit, profitability or customer satisfaction improvements (strategic). The results indicate that although information integration has an impact on a firm’s logistics performance, all performance improvements have not been achieved. This study also found that the use of IT and IS have a mediocre positive relationship to information integration. Almost all case companies agreed on that the use of IT and IS could facilitate information integration and improve their logistics performance. The case companies felt that an implementation of a web portal or a data bank would benefit them - enhance their performance and increase information integration.

MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.