Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.     

Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster

The deoxyhexose sugar fucose has an important fine-tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP-fucose, which is generated from GDP-mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP-mannose dehydratase (GMD; EC 4.2.1.47) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd-1 and gmd-2) and one GER-encoding gene (ger-1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.     

Automated measurement and quantification of heterotrophic bacteria in water samples based on the MPN method

Quantification of heterotrophic bacteria is a widely used measure for water analysis. Especially in terms of drinking water analysis, testing for microorganisms is strictly regulated by the European Drinking Water Directive, including quality criteria and detection limits. The quantification procedure presented in this study is based on the most probable number (MPN) method, which was adapted to comply with the need for a quick and easy screening tool for different kinds of water samples as well as varying microbial loads. Replacing tubes with 24-well titer plates for cultivation of bacteria drastically reduces the amount of culture media and also simplifies incubation. Automated photometric measurement of turbidity instead of visual evaluation of bacterial growth avoids misinterpretation by operators. Definition of a threshold ensures definite and user-independent determination of microbial growth. Calculation of the MPN itself is done using a program provided by the US Food and Drug Administration (FDA). For evaluation of the method, real water samples of different origins as well as pure cultures of bacteria were analyzed in parallel with the conventional plating methods. Thus, the procedure described requires less preparation time, reduces costs and ensures both stable and reliable results for water samples.