RESUMO
The "Cell Painting" technology utilizes multiplexed fluorescent staining of various cell organelles, to produce high-content microscopy images of cells for multidimensional phenotype assessment. The phenotypic profiles extracted from those images can be analyzed upon perturbations with biologically active molecules to annotate the mode of action or biological activity by comparison with reference profiles of already known mechanisms of action, ultimately enabling the determination of on-target and off-target effects. This approach is already described in various human cell cultures, the most commonly used being the U2OS cell line, yet allows broad applications in additional areas of chemical-biological research. Here we describe for the first time the application and adaptation of Cell Painting to an insect cell line, the Sf9 cells from Spodoptera frugiperda. By adjusting image acquisition and analysis models, specific phenotypic profiles were obtained in a dose-dependent manner for 20 reference compounds, including representatives for the most relevant insecticidal modes of action categories (nerve & muscle, respiration and growth & development). Through a dimensionality-reduction method, both calculations of phenotypic half maximal inhibition concentration (IC50) values as well as similarity analysis of the obtained profiles by hierarchical clustering were performed. By Cell Painting effects on the phenotype could be obtained at higher sensitivity than in other assay formats, such as cytotoxicity assessments. More importantly, these analyses provide insight into mechanistic determinants of biological activity. Compounds with similar modes of action showed a high degree of proximity in a hierarchical clustering analysis while being distinct from actives with an unrelated mode of action. In essence, we provide strong evidence on the impact of Cell Painting mechanistic understanding of insecticides with regards to determinants of efficacy and safety utilizing an insect cell model system.
Assuntos
Inseticidas , Spodoptera , Animais , Spodoptera/efeitos dos fármacos , Spodoptera/citologia , Inseticidas/farmacologia , Inseticidas/toxicidade , Células Sf9RESUMO
Mitochondria are central hubs of cellular metabolism and are tightly connected to signaling pathways. The dynamic plasticity of mitochondria to fuse, divide, and contact other organelles to flux metabolites is central to their function. To ensure bona fide functionality and signaling interconnectivity, diverse molecular mechanisms evolved. An ancient and long-overlooked mechanism is the generation of mitochondrial-derived vesicles (MDVs) that shuttle selected mitochondrial cargoes to target organelles. Just recently, we gained significant insight into the mechanisms and functions of MDV transport, ranging from their role in mitochondrial quality control to immune signaling, thus demonstrating unexpected and diverse physiological aspects of MDV transport. This review highlights the origin of MDVs, their biogenesis, and their cargo selection, with a specific focus on the contribution of MDV transport to signaling across cell and organ barriers. Additionally, the implications of MDVs in peroxisome biogenesis, neurodegeneration, metabolism, aging, and cancer are discussed.
Assuntos
Mitocôndrias , Peroxissomos , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Transporte BiológicoRESUMO
Automated disassembly is increasingly in focus for Recycling, Re-use, and Remanufacturing (Re-X) activities. Trends in digitalization, in particular digital twin (DT) technologies and the digital product passport, as well as recently proposed European legislation such as the Net Zero and the Critical materials Acts will accelerate digitalization of product documentation and factory processes. In this contribution we look beyond these activities by discussing digital information for stakeholders at the Re-X segment of the value-chain. Furthermore, we present an approach to automated product disassembly based on different levels of available product information. The challenges for automated disassembly and the subsequent requirements on modeling of disassembly processes and product states for electronic waste are examined. The authors use a top-down (e.g., review of existing standards and process definitions) methodology to define an initial data model for disassembly processes. An additional bottom-up approach, whereby 5 exemplary electronics products were manually disassembled, was employed to analyze the efficacy of the initial data model and to offer improvements. This paper reports on our suggested informal data models for automatic electronics disassembly and the associated robotic skills.