Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins

Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL_(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTβR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.     

Living on the edge: The use of fruit-feeding butterflies to evaluate edge effect on subtropical assemblages

This study evaluated how the edge effect influences the structuration of fruit-feeding butterfly assemblages in swamp forest fragments of the subtropical Atlantic Forest, Southern Brazil. Sampling was carried out twice in 10 fragments using baited traps placed in sampling units both at the forest edge and 50 m within the forest interior, with the habitats being defined by a set of environmental variables. Richness and abundance were higher for edge habitats with an effect of temperature depending on humidity and luminosity. The subfamily/tribe composition of fruit-feeding butterflies was segregated between edge and interior and was predicted by wind speed and the interaction between humidity and luminosity. Fifty meters within the forest interior is not sufficient to cause homogenization of butterfly composition between the edge and interior of swamp forest fragments, indicating distinct assemblages in each habitat. The interior harboured forest-loving butterfly groups while the edge harboured generalist sun-loving and common butterflies associated with disturbed areas, suggesting resistance to the effects of habitat fragmentation. We highlight the importance of using fruit-feeding butterfly groups, instead of species, to evaluate edge effects. We also suggest that a heterogeneous matrix with native habitats and distinct semi-natural land-use systems be maintained to manage subtropical areas by increasing connectivity within the landscape. Considering the impacts that the Atlantic Forest suffers, increased knowledge of modifications caused at small and regional scales is crucial for the maintenance of ecological processes and represents a tool for conservation planning and environmental agendas.     

Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species during Hypoxia Adaptation of Aspergillus fumigatus

Aspergillus fumigatus faces abrupt changes in oxygen concentrations at the site of infection. An increasing number of studies has demonstrated that elevated production of intracellular reactive oxygen species (ROS) under low oxygen conditions plays a regulatory role in modulating cellular responses for adaptation to hypoxia. To learn more about this process in A. fumigatus, intracellular ROS production during hypoxia has been determined. The results confirm increased amounts of intracellular ROS in A. fumigatus exposed to decreased oxygen levels. Moreover, nuclear accumulation of the major oxidative stress regulator AfYap1 is observed after low oxygen cultivation. For further analysis, iodoTMT labeling of redox‐sensitive cysteine residues is applied to identify proteins that are reversibly oxidized. This analysis reveals that proteins with important roles in maintaining redox balance and protein folding, such as the thioredoxin Asp f 29 and the disulfide‐isomerase PdiA, undergo substantial thiol modification under hypoxia. The data also show that the mitochondrial respiratory complex IV assembly protein Coa6 is significantly oxidized by hypoxic ROS. Deletion of the corresponding gene results in a complete absence of hypoxic growth, indicating the importance of complex IV during adaptation of A. fumigatus to oxygen‐limiting conditions.