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PhD Discussions: Ainhoa Ferret and Marta Badia
Friday, January 26 @ 10:00 am–11:00 am
3D bioengineered liver for the study of acute and chronic hepatic damage
Ainhoa Ferret, Biosensors for bioengineering group
The liver, a vital organ, faces acute and chronic insults that disrupt its normal function. Understanding the mechanisms underlying acute and chronic liver damage is crucial for developing effective treatments. Traditional liver models face several limitations. As a result, 3D models have emerged as a more physiologically cellular microenvironment for investigating disease progression, identifying potential therapeutic targets, and developing new drugs. We developed a 3D liver using human hepatocytes, HSCs, and monocytes. The cells were encapsulated in a mixture of GelMA and CMCMA, and LAP as a photo-initiator. The 3D livers were kept in culture for up to 30 days in serum-free medium. They were challenged with acetaminophen and LPS (APAP-LPS), known hepatotoxic compounds, to recreate the pathophysiological phenotype of liver damage in vitro. Extensive liver damage characterized by hepatic stellate cell (HSC) activation and proliferation was observed upon challenge with APAP-LPS. In vivo, these cells exhibited the myofibroblast phenotype typical of activated HSCs. Additionally, impaired gene expression of hepatocyte functionality markers was observed. The transition from monocytes to proinflammatory cytokine-releasing macrophages measured the inflammation level. Notably, dexamethasone demonstrated potent beneficial effects, reducing hepatocyte damage, inhibiting HSC activation, and decreasing collagen production. These results were observed in both acute (high APAP-LPS concentration/3 days) and chronic (low APAP-LPS concentration/30 days) models. The 3D model presented here demonstrates its value as a versatile platform for drug screening in both acute and chronic liver damage scenarios. Its ability to reproduce critical features of liver pathophysiology, including hepatocyte functionality impairment, HSC activation, and inflammation, makes it a valuable tool for studying liver diseases and evaluating potential therapeutic interventions. Furthermore, the adaptability of this model for high-throughput screening provides an opportunity to accelerate the drug discovery process and improve patient outcomes in liver damage-related conditions.
Disclosures
Conflict of interest: This study is supported by Grifols.
What makes a prion behave like a prion? Lessons from deep mutagenesis
Marta Badia, Protein phase transitions in health and disease group
Prions are proteins capable of promoting conformational changes of other protein isoforms. When prion proteins switch from a soluble (non-prion) state to a misfolded (prion) state, they can bind to each other forming small nuclei that can rapidly incorporate other monomers and form amyloid-like aggregates. Subtle differences in the sequence of prionic proteins are enough to impair the recruitment of monomers into these small nuclei, creating a barrier for the nucleation of aggregates. Learning how this barrier is established (and overcome) is fundamental to explain prion nucleation and to understand cross-species prion infection.
Yeast prions serve as a good and tractable model to study amyloid formation and protein aggregation. Sup35 is one of the most intensively studied yeast prions and its N-terminal domain is sufficient for prion nucleation and the maintenance of its prionic state. However, the mechanisms by which Sup35 starts nucleating amyloid aggregates and the features that prevent this nucleation still need to be elucidated.
Using deep mutagenesis, we built a library encompassing all single amino acid changes in the QN-rich region (aa 2-40) of the Sup35 N-terminal domain. We then employed a massively parallel approach that combines high-throughput sequencing with a selection assay that is able to measure Sup35 nucleation in yeast cells.
By systematically quantifying the effect of hundreds of mutants in the QN-rich domain of Sup35 we determined the compatibility of each mutation with an effective Sup35 nucleation. Thanks to this dataset, we identified a nine-residue segment (residues 17-25) crucial for this process. On the other hand, our comprehensive dataset also uncovers mutants that increase Sup35 nucleation, gaining mechanistic insights on the nucleation of this model system and how prion species barriers can be overcome.
