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.

Mechanical thromboembolectomy for acute ischemic stroke: comparison of the catch thrombectomy device and the Merci Retriever in vivo

BACKGROUND AND PURPOSE: The purpose of the study was to compare efficacy and potential complications of 2 commercially available devices for mechanical thromboembolectomy. METHODS: Devices were tested in an established animal model allowing the use of routine angiography catheters and thrombectomy devices. Radio-opaque thrombi were used for visualization of thrombus-device interaction during angiography. The Merci Retrieval System and the Catch Thromboembolectomy System were assessed each in 10 vessel occlusions. For every occluded vessel up to 5 retrieval attempts were performed. RESULTS: Sufficient recanalization was achieved with the Merci Retriever in 90% of occlusions, and with the Catch device recanalization was achieved in 70% of occlusions. Recanalization at the first attempt occurred significantly more often with the Merci Retriever compared to the Catch device (OR, 21; 95% CI, 1.78-248.11). Consequently, significantly more attempts (P=0.02) had to be performed with the Catch device; therefore, time to recanalization was longer. Thrombus fragmentations during retrieval were caused more often by the Catch device compared to the Merci Retriever (OR, 15.6; 95% CI, 1.73-140.84), resulting in a higher distal embolization rate. During retrieval both devices lost thrombotic material at the tip of the guide catheter, which was then aspirated in most cases. CONCLUSIONS: Both distal devices are effective for thromboembolectomy. To avoid loss of thrombotic material and distal embolization, the use of large luminal balloon guide catheters and aspiration during retrieval seems to be mandatory. The design of the Merci Retriever appears to be more efficient during thrombus mobilization and retrieval with less fragmentation compared to the Catch Thromboembolectomy System.

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

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.

Sensory and conceptual representations in memory: Motor images which cannot be imaged

The paper argues for a distinction between sensory-and conceptual-information storage in the human information-processing system. Conceptual information is characterized as meaningful and symbolic, while sensory information may exist in modality-bound form. Furthermore, it is assumed that sensory information does not contribute to conscious remembering and can be used only in data-driven process reptitions, which can be accompanied by a kind of vague or intuitive feeling. Accordingly, pure top-down and willingly controlled processing, such as free recall, should not have any access to sensory data. Empirical results from different research areas and from two experiments conducted by the authors are presented in this article to support these theoretical distinctions. The experiments were designed to separate a sensory-motor and a conceptual component in memory for two-digit numbers and two-letter items, when parts of the numbers or items were imaged or drawn on a tablet. The results of free recall and recognition are discussed in a theoretical framework which distinguishes sensory and conceptual information in memory.

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

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to > 1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p's molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: A comparison of tricyclo-DNA with LNA

The nuclear antisense properties of a series of tricyclo(tc)-DNA oligonucleotide 9-15mers, targeted against the 3' and 5' splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence and dose dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by a factor of at least 4-5. A tc-oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2‘M concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction of CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc-15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc- and LNA-oligonucleotides. The obtained results confirm the power of tricyclo-DNA for nuclear antisense applications. Morover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.