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.

Geschichte und Trends im IR

Diese Seminararbeit wurde im Rahmen des Seminars Angewandtes Information Retrievalgeschrieben und beschäftigt sich mit der Geschichte der Daten, der Internetgeschichte und dem untrennbar dazugehörenden Information Retrieval, welches als Wiedergewinnung vonbereits zur Verfügung stehenden Daten gesehen werden kann.In einem ersten Teil befasst sich diese Arbeit mit der Geschichte der menschlichen Daten-sammlung und Speicherung. Hier wird die Geschichte von den Anfängen der Datensammlungbis hin zur heutigen digitalen Zeit durchlaufen.Im zweiten Teil werden die Evolution und Funktionsweise verschiedener Systeme vorgestellt,wobei eine Trennung vorgenommen wird in die Geschichte des Information Retrieval, alsAntwort auf die Datenmengen, welche durch die Evolution hervorgebracht wurde, und dannwird auf heutige Trends des Information Retrievals eingegangen. Hierbei werde ich nocheinmal die grundlegenden Probleme der Informationssuche aufzeigen und die aktuellen For-schungen in diesem Gebiet erwähnen.Danach wird ein Fazit gezogen und kritisch Stellung bezüglich der aktuellen Trends einge-nommen. In meinem Schlusswort gebe ich meine Vision einer Suchmaschine wieder, wiediese in Zukunft aussehen könnte

Pilot Project: Use of Wikis to Supplement Student Orientation

Background: During the orientation process, new students are often inundated with manuals, maps, and other materials essential to their success as students. The experience can leave students feeling overwhelmed, unable to sift through the substantial amount of information that has been given to them. Wikis, in contrast, are well-suited for facilitating userinteraction with vast amounts of diverse information. [See PDF for complete abstract]

A day in the life of PubMed: analysis of a typical day's query log.

OBJECTIVE: To characterize PubMed usage over a typical day and compare it to previous studies of user behavior on Web search engines. DESIGN: We performed a lexical and semantic analysis of 2,689,166 queries issued on PubMed over 24 consecutive hours on a typical day. MEASUREMENTS: We measured the number of queries, number of distinct users, queries per user, terms per query, common terms, Boolean operator use, common phrases, result set size, MeSH categories, used semantic measurements to group queries into sessions, and studied the addition and removal of terms from consecutive queries to gauge search strategies. RESULTS: The size of the result sets from a sample of queries showed a bimodal distribution, with peaks at approximately 3 and 100 results, suggesting that a large group of queries was tightly focused and another was broad. Like Web search engine sessions, most PubMed sessions consisted of a single query. However, PubMed queries contained more terms. CONCLUSION: PubMed's usage profile should be considered when educating users, building user interfaces, and developing future biomedical information retrieval systems.

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity