Abstract
Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation ...followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.
The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative ...complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.
TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces ...the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with "knob"-mutations, and the variable region of the light chain (VL) fused to CH3 with matching "holes". The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent "in between" these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.
We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and ...subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is ...quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
Highlights • The role of mGlu5 in modulating consequences of chronic psychosocial stress using the CSC paradigm was investigated. • Genetic ablation and pharmacological blockade of mGlu5 decreased ...the vulnerability to CSC-induced alterations. • CSC exposure dysregulated hippocampal mGlu5 binding sites in wildtype mice. • mGlu5 seems to be involved in chronic psychosocial stress-induced affective and somatic consequences.
Individualized Internet marketing represents a paradigm shift in how customers are engaged with products and services through the World Wide Web. Internet marketing has become ubiquitous through the ...influence of technological advancements such as the use of location-based services and geolocation. Disaggregate methods of marketing segmentation are recent developments that largely replace aggregate methods such as geodemographic profiling. Individualized marketing relies on data collected via the interactions of users with the Internet to develop custom user profiles and marketing strategies. Spatial data collected through Internet marketing are orders of magnitude larger in extent and complexity than data obtained through previous methods. The advent of Big Data reflects the influence of technology on social interaction patterns. The spatial data potential of these patterns poses new opportunities and challenges for Geographic Information Science. Internet modification is framed as a cyber-spatial extension of theories about the production of space. Individualized marketing effectively steers consumer behavior by reinforcing confidence in personal preferences and cultivating brand loyalty. This thesis explores the inherent spatial ramifications of these consumer reactions to Internet marketing and their implications for future investments.
The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative ...complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 mu g/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1:50. Reproducibility assays showed a variation below 25% at dilutions less than 1:1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 mu g/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.
A major bottleneck delaying the further commercialization of thin-film solar cells based on hybrid organohalide lead perovskites is interface loss in state-of-the-art devices. We present a generic ...interface architecture that combines solution-processed, reliable, and cost-efficient hole-transporting materials without compromising efficiency, stability, or scalability of perovskite solar cells. Tantalum-doped tungsten oxide (Ta-WOₓ)/conjugated polymer multilayers offer a surprisingly small interface barrier and form quasi-ohmic contacts universally with various scalable conjugated polymers. In a simple device with regular planar architecture and a self-assembled monolayer, Ta-WOₓ–doped interface–based perovskite solar cells achieve maximum efficiencies of 21.2% and offer more than 1000 hours of light stability. By eliminating additional ionic dopants, these findings open up the entire class of organics as scalable hole-transporting materials for perovskite solar cells.
Organic solar cells are promising in terms of full‐solution‐processing which enables low‐cost and large‐scale fabrication. While single‐junction solar cells have seen a boost in power conversion ...efficiency (PCE), multi‐junction solar cells are promising to further enhance the PCE. In all‐solution‐processed multi‐junction solar cells, interfacial losses are often encountered between hole‐transporting layer (HTL) and the active layers and therefore greatly limit the application of newly developed high‐performance donor and acceptor materials in multi‐junction solar cells. Here, the authors report on a systematic study of interface losses in both single‐junction and multi‐junction solar cells based on representative polymer donors and HTLs using electron spectroscopy and time‐of‐flight secondary ion mass spectrometry. It is found that a facile mixed HTL containing poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and MoO
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nanoparticles successfully overcomes the interfacial losses in both single‐ and multi‐junction solar cells based on various active layers by reducing interface protonation, promoting better energy‐level alignment, and forming a dense and smooth layer. Solution‐processed single‐junction solar cells are demonstrated to reach the same performance as with evaporated MoO
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(over 7%). Multi‐junction solar cells with polymers containing nitrogen atoms as the first layer and the mixed PEDOT:PSS and MoO
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nanoparticles as hole extraction layer reach fill factor (FF) of over 60%, and PCE of over 8%, while the identical stack with pristine PEDOT:PSS or MoO
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nanoparticles show FF smaller than 50% and PCE less than 5%.
The interface losses in both solution‐processed inverted single‐ and multi‐junction solar cells using representative polymer donors and hole‐transporting layers (HTLs) are systematically investigated. A facile mixed HTL containing poly(3,4‐ethylenedioxythiophene) polystyrene sulfonate and MoO
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nanoparticles successfully overcomes the interfacial losses by reducing interface protonation, promoting better energy‐level alignment, and forming a dense and smooth layer.
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