Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.

Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier.

BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.

Applying a multi-criteria genetic algorithm framework for brownfield reuse optimization: improving redevelopment options based on stakeholder preferences.

The reuse of underused or abandoned contaminated land, so-called brownfields, is increasingly seen as an important means for reducing the consumption of land and natural resources. Many existing decision support systems are not appropriate because they focus mainly on economic aspects, while neglecting sustainability issues. To fill this gap, we present a framework for spatially explicit, integrated planning and assessment of brownfield redevelopment options. A multi-criteria genetic algorithm allows us to determine optimal land use configurations with respect to assessment criteria and given constraints on the composition of land use classes, according to, e.g., stakeholder preferences. Assessment criteria include sustainability indicators as well as economic aspects, including remediation costs and land value. The framework is applied to a case study of a former military site near Potsdam, Germany. Emphasis is placed on the trade-off between possibly conflicting objectives (e.g., economic goals versus the need for sustainable development in the regional context of the brownfield site), which may represent different perspectives of involved stakeholders. The economic analysis reveals the trade-off between the increase in land value due to reuse and the costs for remediation required to make reuse possible. We identify various reuse options, which perform similarly well although they exhibit different land use patterns. High-cost high-value options dominated by residential land use and low-cost low-value options with less sensitive land use types may perform equally well economically. The results of the integrated analysis show that the quantitative integration of sustainability may change optimal land use patterns considerably.

Integrated planning and spatial evaluation of megasite remediation and reuse options.

Redevelopment of large contaminated brownfields (megasites) is often hampered by a lack of communication and harmonization among diverse stakeholders with potentially conflicting interests. Decision support is required to provide integrative yet transparent evaluation of often complex spatial information to stakeholders with different areas of expertise. It is considered crucial for successful redevelopment to identify a shared vision of how the respective contaminated site could be remediated and redeveloped. We describe a framework of assessment methods and models that analyzes and visualizes site- and land use-specific spatial information at the screening level, with the aim to support the derivation of recommendable land use layouts and to initiate further and more detailed planning. The framework integrates a GIS-based identification of areas to be remediated, an estimation of associated clean-up costs, a spatially explicit market value appraisal, and an assessment of the planned future land use's contribution to sustainable urban and regional development. Case study results show that derived options are potentially favorable in both a sustainability and an economic sense and that iterative re-planning is facilitated by the evaluation and visualization of economic, ecological and socio-economic aspects. The framework supports an efficient early judgment about whether and how abandoned land may be assigned a sustainable and marketable land use.

Extracellular iron biomineralization by photoautotrophic iron-oxidizing bacteria.

Iron oxidation at neutral pH by the phototrophic anaerobic iron-oxidizing bacterium Rhodobacter sp. strain SW2 leads to the formation of iron-rich minerals. These minerals consist mainly of nano-goethite (alpha-FeOOH), which precipitates exclusively outside cells, mostly on polymer fibers emerging from the cells. Scanning transmission X-ray microscopy analyses performed at the C K-edge suggest that these fibers are composed of a mixture of lipids and polysaccharides or of lipopolysaccharides. The iron and the organic carbon contents of these fibers are linearly correlated at the 25-nm scale, which in addition to their texture suggests that these fibers act as a template for mineral precipitation, followed by limited crystal growth. Moreover, we evidence a gradient of the iron oxidation state along the mineralized fibers at the submicrometer scale. Fe minerals on these fibers contain a higher proportion of Fe(III) at cell contact, and the proportion of Fe(II) increases at a distance from the cells. All together, these results demonstrate the primordial role of organic polymers in iron biomineralization and provide first evidence for the existence of a redox gradient around these nonencrusting, Fe-oxidizing bacteria.