Kartezio: Evolutionary Design of Explainable Pipelines for Biomedical Image Analysis

An unresolved issue in contemporary biomedicine is the overwhelming number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. Crucially however, these frameworks require large human-annotated datasets for training and the resulting models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming based computational strategy that generates transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets, a feature which confers tremendous flexibility, speed, and functionality to this approach. We also deployed Kartezio to solve semantic and instance segmentation problems in four real-world Use Cases, and showcase its utility in imaging contexts ranging from high-resolution microscopy to clinical pathology. By successfully implementing Kartezio on a portfolio of images ranging from subcellular structures to tumoral tissue, we demonstrated the flexibility, robustness and practical utility of this fully explicable evolutionary designer for semantic and instance segmentation.Comment: 36 pages, 6 main Figures. The Extended Data Movie is available at the following link: https://www.youtube.com/watch?v=r74gdzb6hdA. The source code is available on Github: https://github.com/KevinCortacero/Kartezi

Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host

Building a community to engineer synthetic cells and organelles from the bottom-up

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Bottom-up Assembly of Functional Extracellular Vesicles – Implications for Synthetic Biology and Biomedical Applications

Formation of lipid-based compartments is a distinguishing feature of eukaryotic life forms. These compartments play a crucial role in orchestrating independent and self-contained metabolic, signalling or synthesis processes. Moreover, cellderived lipid compartments, like extracellular vesicles (EVs), have been shown to be essential for intercellular signalling and are involved in a wide variety of disease states. Although attaining a holistic understanding of EV-based communication is a compelling goal, the extensive molecular and structural complexity of these vesicles as well as a lack of reliable EV isolation techniques, have impaired detailed mechanistic insights. Inspired by bottom-up synthetic biology principles, the central goal of my interdisciplinary research was the development of a bio-inspired EV model system, which serves as a platform to study EV-based intercellular signalling and empowers novel EV-inspired therapeutics. In this thesis, I present two major methodologies developed for the controlled high-throughput assembly of synthetic vesicles. First, I describe a droplet-based microfluidic approach for the production of giant unilamellar vesicles (GUVs) with wellcontrolled biophysical and biochemical properties. I report on systematic investigations of GUV interactions with living cells and present concepts on how fine-tuning of the vesicles surface characteristics can be applied for targeted cellular delivery of macromolecular cargos. Moreover, I show how these vesicles can be reconceptualised as synthetic organelles, functioning within living cells and providing them with synthetic functionalities. Based on these fundamental characterizations, in the second part of my thesis, I present a complementary and quantitative approach for the sequential bottom-up assembly of fully synthetic EVs (fsEVs). To exemplify the application of fsEVs for new therapeutic concepts, I show that they exert analogous functionalities to naturally occurring wound healing EVs. Furthermore, by combining the fsEV technology with whole-transcriptome analysis, I systematically decode the synergistic functionalities between individual EV components. This approach enabled me to perform an analytical dissection of the associated EV signalling processes mediated by tetraspanin proteins. Bioinspired and biocompatible synthetic compartments with precisely controllable biophysical and biochemical properties are desirable tools for a wide range of living and synthetic cells research. This study makes it tempting to view EV-like compartments in a broader perspective. For example, they have great application potential as on-demand drug delivery systems, paving the way for hitherto impossible approaches towards administration of advanced cargos such as microparticles, viruses or synthetic organelles. Moreover, I anticipate that the highly controlled assembly of fsEVs will provide a robust framework for innovative therapeutic applications of bottom-up assembled synthetic biological modules and will additionally allow for new insights into fundamental EVrelated principles that govern cellular communication

Membranes on the move: The functional role of the extracellular vesicle membrane for contact‐dependent cellular signalling

Abstract Extracellular vesicles (EVs), lipid‐enclosed structures released by virtually all life forms, have gained significant attention due to their role in intercellular and interorganismal communication. Despite their recognized importance in disease processes and therapeutic applications, fundamental questions about their primary function remain. Here, we propose a different perspective on the primary function of EVs, arguing that they serve as essential elements providing membrane area for long‐distance, contact‐dependent cellular communication based on protein‐protein interaction. While EVs have been recognized as carriers of genetic information, additional unique advantages that they could provide for cellular communication remain unclear. Here, we introduce the concept that the substantial membrane area provided by EVs allows for membrane contact‐dependent interactions that could be central to their function. This membrane area enables the lateral diffusion and sorting of membrane ligands like proteins, polysaccharides or lipids in two dimensions, promoting avidity‐driven effects and assembly of co‐stimulatory architectures at the EV‐cell interface. The concept of vesicle‐induced receptor sequestration (VIRS), for example, describes how EVs confine and focus receptors at the EV contact site, promoting a dense local concentration of receptors into signalosomes. This process can increase the signalling strength of EV‐presented ligands by 10‐1000‐fold compared to their soluble counterparts. The speculations in this perspective advance our understanding of EV‐biology and have critical implications for EV‐based applications and therapeutics. We suggest a shift in perspective from viewing EVs merely as transporters of relevant nucleic acids and proteins to considering their unique biophysical properties as presentation platforms for long‐distance, contact‐dependent signalling. We therefore highlight the functional role of the EV membrane rather than their content. We further discuss how this signalling mechanism might be exploited by virus‐transformed or cancer cells to enhance immune‐evasive mechanisms