DNA-based self-assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA-based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6-helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3'-ends and azides on their 5'-ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so-called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.
Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.
Geminiviruses are transmitted by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Gennadius) is the most efficient vector of Subgroup III geminiviruses. An isolate of Abutilon mosaic virus (AbMV), a bipartite geminivirus, was not detectable in the DNA extract from insects by Southern blot analysis, nor was the isolate transmissible by the B-biotype of B. tabaci, although the virus DNA was amplified (by PCR) from some insects. In contrast, Sida golden mosaic virus (SiGMV-Co), a closely related geminivirus, was acquired and transmitted by B. tabaci to various host plants. The coat protein of AbMV was replaced with that of SiGMV-Co. The resulting chimeric AbMV was acquired and transmitted to various host plants by B. tabaci, indicating that the coat protein plays an essential role in the transmission process by B. tabaci.
Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein -glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial culture after a multistep purification process. This glycosylation-optimized factor H showed full complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.
Geminiviruses are transmitted in a circulative manner by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Genn.) is the vector for members of the genus Begomovirus. The closely related bipartite Central American begomoviruses Abutilon mosaic virus (AbMV), Sida golden mosaic virus originating from Costa Rica (SiGMV-CR), and Sida golden mosaic virus originating from Honduras (SiGMV-Hoyv) were used to study transmission by their insect vector. The AbMV isolate is defective in transmission, whereas the two Sida-infecting viruses are readily transmitted by B. tabaci. These three viruses are able to form pseudorecombinant viruses by exchange of genomic components. The pseudorecombinant virus SiGMV-Hoyv A/AbMV B was transmissible, whereas the reciprocal pseudorecombinant virus AbMV A/SiGMV-Hoyv B was not transmitted, indicating that DNA B is not involved in the transmission defect. However, the uptake of the pseudorecombinant virus AbMV A/SiGMV-Hoyv B was much better than AbMV itself, indicating that DNA B or DNA B gene products enhance uptake of viral DNA. Exchange of AbMV coat protein with that of SiGMV-CR resulted in a transmissible chimeric AbMV. Mutagenesis of the AbMV coat protein showed that the exchange of two amino acids, at positions 124 and 149, was sufficient to obtain a whitefly-transmissible AbMV mutant. However, when amino acid 174 was altered in addition to amino acids 124 and 149 AbMV was readily transmitted by B. tabaci. From this we conclude that it is not a concise motif, such as the amino acid triplet, aspartate-alanine-glycine (DAG), involved in aphid transmission of potyviruses, that determines transmissibility of begomoviruses by B. tabaci. Instead it is the composition of the coat protein domain from amino acid 123 to 149, as a minimal transmission domain, with the contribution of amino acids 149 to 174 for efficient transmission.
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