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| 2. |
- Encarnacao, Joao Crispim, Master, 1990-, et al.
(författare)
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A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells
- 2020
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Ingår i: FEBS Letters. - : WILEY. - 0014-5793 .- 1873-3468. ; 594:15, s. 2406-2420
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.
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| 3. |
- Encarnacao, Joao Crispim, et al.
(författare)
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Detecting ligand interactions in real time on living bacterial cells
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : SPRINGER. - 0175-7598 .- 1432-0614. ; 102:9, s. 4193-4201
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Tidskriftsartikel (refereegranskat)abstract
- Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracerA (R), originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.
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| 4. |
- Encarnacao, Joao Crispim, et al.
(författare)
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Impact of assay temperature on antibody binding characteristics in living cells : A case study
- 2017
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Ingår i: BIOMEDICAL REPORTS. - : Spandidos Publications. - 2049-9434 .- 2049-9442. ; 7:5, s. 400-406
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Tidskriftsartikel (refereegranskat)abstract
- Kinetic and thermodynamic studies of ligand-receptor interactions are essential for increasing the understanding of receptor activation mechanisms and drug behavior. The characterization of molecular interactions on living cells in real-time goes beyond most current binding assays, and provides valuable information about the dynamics and underlying mechanism of the molecules in a living system. The effect of temperature on interactions in cell-based assays is, however, rarely discussed. In the present study, the effect of temperature on binding of monoclonal antibodies, cetuximab and pertuzumab to specific receptors on living cancer cells was evaluated, and the affinity and kinetics of the interactions were estimated at selected key temperatures. Changes in the behavior of the interactions, particularly in the on- and off-rates were observed, leading to greatly extended time to reach the equilibrium at 21 degrees C compared with at 37 degrees C. However, the observed changes in kinetic characteristics were less than a factor of 10. It was concluded that it is possible to conduct real-time measurements with living cells at different temperatures, and demonstrated that influences of the ambient temperature on the interaction behavior are likely to be less than one order of magnitude.
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| 5. |
- Encarnação, João Crispim, Master, 1990-
(författare)
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Towards time-resolved molecular interaction assays in living bacteria
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rare and neglected diseases such as multidrug resistant (MDR) tuberculosis, malaria and trypanosomiasis are re-emerging in Europe. New strategies are needed to accelerate drug discovery to fight these pathogens. AEGIS is a Pan-European project that combines different technologies to accelerate the discovery of molecules suitable for drug development in selected neglected diseases. This thesis is part of the AEGIS research area that considers time in a multidisciplinary approach, combining biology, physics and mathematics to provide tools to characterize biological events for improving drug development and information about the target diseases and lead compounds.Real-time cell binding assays (RT-CBA) of receptor-ligand interactions are fundamental in basic research and drug discovery. However, this kind of assays are still rare on living cells, especially in the microbiology field. In this project, we apply the same high-precision assay type on bacterial systems and explored the interior of the cell with a time resolved assay.The effect of temperature was evaluated in the RT-CBA using LigandTracer to ensure that it was possible to use the technology in a range of temperatures suitable for bacteria. A method for attaching Gram positive and negative bacteria on the surface of a normal Petri dish, showing a high reproducibly and a high cellular viability after 16 h. With these two key steps, an RT-CBA fit for microbiology is available.Next, to answer biological questions, intracellular interactions were explored by expression and validation of intracellular proteins with fluorescent tags suitable for RT-CBAs. First, we used the subunit B from the Shiga toxin (STxB) as a model to understand different aspects about the internalization processes. RT-CBAs allowed to discovery new features of STxB binding and mechanism to deliver small molecules or small proteins into cancer cells. Then, for exploring intracellular interactions, insect cells were bioengineered for evaluating the ability of small molecules to internalize and bind to its target. Using Carbonic anhydrase II – sulfonamides as a model system, the molecular interaction in the cytoplasm could be measured using a quencher label approach. The development of this kind of novel RT-CBA tools provide new information about drug candidates for targets that are not properly expressed in bacterial cells.The assays in this project can make drug design more efficient. Furthermore, the evaluation of binding activity of the new compounds developed by AEGIS, focusing on rare/neglected diseases, in a biological environment has the potential to accelerate drug discovery for the targeted emerging diseases.
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| 7. |
- Rocklöv, Joacim, Professor, 1979-, et al.
(författare)
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Decision-support tools to build climate resilience against emerging infectious diseases in Europe and beyond
- 2023
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Ingår i: The Lancet Regional Health. - : Elsevier. - 2666-7762. ; 32
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Forskningsöversikt (refereegranskat)abstract
- Climate change is one of several drivers of recurrent outbreaks and geographical range expansion of infectious diseases in Europe. We propose a framework for the co-production of policy-relevant indicators and decision-support tools that track past, present, and future climate-induced disease risks across hazard, exposure, and vulnerability domains at the animal, human, and environmental interface. This entails the co-development of early warning and response systems and tools to assess the costs and benefits of climate change adaptation and mitigation measures across sectors, to increase health system resilience at regional and local levels and reveal novel policy entry points and opportunities. Our approach involves multi-level engagement, innovative methodologies, and novel data streams. We take advantage of intelligence generated locally and empirically to quantify effects in areas experiencing rapid urban transformation and heterogeneous climate-induced disease threats. Our goal is to reduce the knowledge-to-action gap by developing an integrated One Health—Climate Risk framework.
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