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

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

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV‐based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV‐based communication and how protein‐based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle‐induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV‐membranes. By bottom‐up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV‐like vesicles, increases their signalling potential by more than 100‐fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV‐presented proteins. By this the authors propose vesicle‐triggered local clustering of membrane receptors as the principle structural mechanism underlying EV‐based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter‐protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV‐based intercellular signalling

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells

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.BN/Marileen Dogterom La