Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Developing and Deploying an XML-based Learning Content Management System at the FernUniversität Hagen

This paper is a report about the FuXML project carried out at the FernUniversität Hagen. FuXML is a Learning Content Management System (LCMS) aimed at providing a practical and efficient solution for the issues attributed to authoring, maintenance, production and distribution of online and offline distance learning material. The paper presents the environment for which the system was conceived and describes the technical realisation. We discuss the reasons for specific implementation decisions and also address the integration of the system within the organisational and technical infrastructure of the university.

Matching Software and Context in Open Learning Scenarios

We describe the use of log file analysis to investigate whether the use of CSCL applications corresponds to its didactical purposes. Exemplarily we examine the use of the web-based system CommSy as software support for project-oriented university courses. We present two findings: (1) We suggest measures to shape the context of CSCL applications and support their initial and continuous use. (2) We show how log files can be used to analyze how, when and by whom a CSCL system is used and thus help to validate further empirical findings. However, log file analyses can only be interpreted reasonably when additional data concerning the context of use is available.

Bemerkungen zu kunsthistorischen Informationssystemen; vornehmlich aus der Sicht der Informatik

Der Einsatz von Informationssystemen hat in der Kunstgeschichte sowie auch in anderen Bereichen der Geisteswissenschaften eine lange Tradition. Gesteuert wurde dieser Einsatz in vielen Fällen davon, was zuständige Wissenschaftler als Erfordernisse der Informatik ansahen. Betrachtet man die Informatik etwas genauer, so lässt sich feststellen, dass Prinzipien, die zunächst abstrakt und grundlegend erscheinen, häufig eine ganz bestimmte technische Situation widerspiegeln und außerhalb eines recht eng definierten Rahmens nicht angewandt werden können. Dies ist vor allem der Fall in Bezug auf Informationssysteme und die Logik der Informationsbeschaffung. Innerhalb dieses Bereiches hat sich der technische Aspekt in den letzten Jahren extrem gewandelt. Es wird deshalb dafür plädiert, dass technische Erfordernisse (welche leicht missverstanden werden können und sich schnell wandeln) nicht als primäre Richtlinien für die Herstellung von kunstgeschichtlichen Informationssystemen dienen sollten, sondern die Erfordernisse für den Einsatz solcher Informationssysteme selbst, die in ihrer Anwendung deutlich werden. Wenn die Informatik als Referenzpunkt für die Herstellung solcher Systeme gelten soll, sollte sie anhand präziser technischer Literatur definiert werden und nicht anhand von Reflexionen über die Bedeutung einiger weniger technischer Begriffe, die leicht missverstanden werden können.

Extraktion und kartographische Visualisierung von Informationen aus Weblogs

Beim Information Retrieval ist in Anbetracht der Informationsflut entscheidend, relevante Informationen zu finden. Ein vielversprechender Ansatz liegt im Semantischen Web, wobei dem System die Bedeutung von Informationen ontologiebasiert beigebracht wird. Sucht der Benutzer nach Stichworten, werden ihm anhand der Ontologie verwandte Begriffe angezeigt und er kann mittels Mensch-Maschine-Interaktion seine relevanten Informationen extrahieren. Um eine solche Interaktion zu fördern, werden die Ergebnisse visuell aufgearbeitet. Dabei liegt der Mehrwert darin, dass der Benutzer anstelle von Tausenden von Suchresultaten in einer fast endlosen Liste, ein kartographisch visualisiertes Suchresultat geliefert bekommt. Dabei hilft die Visualisierung, unvorhergesehene Beziehungen zu entdecken und zu erforschen.