Microcavity array (MCA)-based biosensor chip for functional drug screening of 3D tissue models

Multicellular tumour spheroids that mimic a native cellular environment are widely used as model systems for drug testing. To study drug effects on three-dimensional cultures in real-time we designed and fabricated a novel type of sensor chip for fast, non-destructive impedance spectroscopy and extracellular recording. Precultured spheroids are trapped between four gold electrodes. Fifteen individual 100microm deep square microcavities with sizes from 200 to 400microm allow an optimised positioning during the measurement. Although apoptosis was induced in human melanoma spheroids by Camptothecin (CTT), treated cultures did not show disintegration but displayed increased impedance magnitudes compared to controls after 8h resulting from an altered morphology of the outer cells. Contractions in cardiomyocyte spheroids were monitored when the innovative chip was used for recording of extracellular potentials. The silicon-based electrode array is used as an acute test system for the monitoring of any kind of 3D cell cultures. Since no adherence of cells or labelling is necessary the multifunctional sensor chip provides a basis for improved drug development by high content screenings with reduced costs and assay times. Additional improvements for parallel testing of different substances on one chip are presented.     

Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering.

Cartilage cells are normally studied under atmospheric pressure conditions and without loading. However, since cartilage exists in a condition of reduced oxygen and intermittent hydrostatic pressure we hypothesized lower partial oxygen pressures (PO2) and different intermittent hydrostatic pressures (IHP) would increase articular chondrocyte proliferation and matrix production and to stabilize chondrocyte phenotype in vitro. Monolayers of adult bovine articular chondrocytes were cultured under 5% or 21% PO2 in combination with IHP (0.2 MPa amplitude, frequencies 5/5s = 0.1 Hz, 30/2 or 2/30 min on/off loading). We measured proliferation (3H-thymidine incorporation) and collagen secretion (protein-binding assay, collagen type II-ELISA and immunocytochemical staining of pericellular collagen types I, II and IX). Reduced PO2 stimulated proliferation and collagen type II and IX secretion of chondrocytes in comparison to 21% PO2. Additionally, collagen type I expression was delayed by low PO2, indicating a stabilization of the cell phenotype. IHP 5/5s and 30/2 min inhibited proliferation but increased collagen secretion (pericellular collagen type IX was decreased). IHP 30/2 min delayed first expression of collagen type I. In contrast, IHP 2/30 min increased proliferation, but lowered collagen expression. All stimulating or inhibiting effects of PO2 and IHP were additive and vice versa. Reduced PO2 and different settings of IHP increased proliferation, collagen secretion, and phenotype stability of chondrocytes. The oxygen- and IHP-induced effects were additive, suggesting that a combination of these parameters might be a useful tool in cartilage tissue engineering.     

Phytotoxicity of tin mine waste and accumulation of involved heavy metals in common buckwheat (Fagopyrum esculentum Moench)

Extraction and processing of cassiterite (SnO₂) left large tailings with high concentrations of tin, tungsten, molybdenum and lithium. Information on the phytotoxicity of mine waste is important with regard to ecological hazards. Exposure studies help to identify plants useful for the stabilization of waste tips and the phytomining of metals. A greenhouse study was performed using a dilution series of mine waste and four crops, a halophytic and a metallophytic species to derive dose response curves. Based on effective doses for growth reductions, sensitivity increased in the following order: maize > common buckwheat > quinoa > garden bean. Element analyses in different species and compartments of common buckwheat grown in a mixture of standard soil and 25% of the mine waste showed that only low levels of the metals were taken up and that transfer to seed tissues was negligible. As indicated by soil metal levels prior to and after the experiment, only lithium and arsenic proved to be plant available and reached high levels in green tissues while seed levels were low. The experiment confirmed differences in the uptake of metals with regard to elements and species. Common buckwheat is a suited candidate for cultivation on metal polluted soils.     

Thermal biology mediates responses of amphibians and reptiles to habitat modification

Human activities often replace native forests with warmer, modified habitats that represent novel thermal environments for biodiversity. Reducing biodiversity loss hinges upon identifying which species are most sensitive to the environmental conditions that result from habitat modification. Drawing on case studies and a meta‐analysis, we examined whether observed and modelled thermal traits, including heat tolerances, variation in body temperatures, and evaporative water loss, explained variation in sensitivity of ectotherms to habitat modification. Low heat tolerances of lizards and amphibians and high evaporative water loss of amphibians were associated with increased sensitivity to habitat modification, often explaining more variation than non‐thermal traits. Heat tolerances alone explained 24–66% (mean = 38%) of the variation in species responses, and these trends were largely consistent across geographic locations and spatial scales. As habitat modification alters local microclimates, the thermal biology of species will likely play a key role in the reassembly of terrestrial communities.     

Estimating product and energy substitution benefits in national‐scale mitigation analyses for Canada

The potential of forests and the forest sector to mitigate greenhouse gas (GHG) emissions is widely recognized, but challenging to quantify at a national scale. Mitigation benefits through the use of forest products are affected by product life cycles, which determine the duration of carbon storage in wood products and substitution benefits where emissions are avoided using wood products instead of other emissions‐intensive building products and energy fuels. Here we determined displacement factors for wood substitution in the built environment and bioenergy at the national level in Canada. For solid wood products, we compiled a basket of end‐use products and determined the reduction in emissions for two functionally equivalent products: a more wood‐intensive product vs. a less wood‐intensive one. Avoided emissions for end‐use products basket were weighted by Canadian consumption statistics to reflect national wood uses, and avoided emissions were further partitioned into displacement factors for sawnwood and panels. We also examined two bioenergy feedstock scenarios (constant supply and constrained supply) to estimate displacement factors for bioenergy using an optimized selection of bioenergy facilities which maximized avoided emissions from fossil fuels. Results demonstrated that the average displacement factors were found to be similar: product displacement factors were 0.54 tC displaced per tC of used for sawnwood and 0.45 tC tC^?1 for panels; energy displacement factors for the two feedstock scenarios were 0.47 tC tC^?1 for the constant supply and 0.89 tC tC^?1 for the constrained supply. However, there was a wide range of substitution impacts. The greatest avoided emissions occurred when wood was substituted for steel and concrete in buildings, and when bioenergy from heat facilities and/or combined heat and power facilities was substituted for energy from high‐emissions fossil fuels. We conclude that (1) national‐level substitution benefits need to be considered within a systems perspective on climate change mitigation to avoid the development of policies that deliver no net benefits to the atmosphere, (2) the use of long‐lived wood products in buildings to displace steel and concrete reduces GHG emissions, (3) the greatest bioenergy substitution benefits are achieved using a mix of facility types and capacities to displace emissions‐intensive fossil fuels.     

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, α-DsbA1 and α-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein α-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of α-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether α-DsbA1 is an oxidant or an isomerase based on functional activity. The purified α-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that α-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified α-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.