Biology subjects

Bear, C. E.

Publications and source records attributed to Bear, C. E..

4 recordsLinked to original sources

bioRxiv2023

Collagen Tubular Airway-on-Chip for Extended Epithelial Culture andInvestigation of Ventilation Dynamics

The lower respiratory tract is a hierarchical network of compliant tubular structures that are made from extracellular matrix proteins with a wall lined by an epithelium. While microfluidic airway-on-a-chip models incorporate the effects of shear and stretch on the epithelium, week-long air-liquid-interface (ALI) culture remains limited to static conditions. The circular cross-section and substrate compliance associated with intact airways have yet to be recapitulated to allow studies of epithelial injuries under physiological and ventilation conditions. To overcome these limitations, we present a collagen tube-based airway model. Sustaining a functional human bronchial epithelium during two-week perfusion is accomplished by continuously supplying warm, humid air at the apical side and culture medium at the basal side. The model faithfully recapitulates human airways in size, composition, and mechanical microenvironment, allowing for the first time dynamic studies of elastocapillary phenomena associated with regular breathing as well as mechanical ventilation, along with the impact on epithelial cells. Findings reveal the epithelium to become increasingly damaged when subjected to repetitive collapse and reopening as opposed to overdistension and suggest expiratory flow resistance to reduce atelectasis. We expect the model to find broad potential applications in organ-on-a-chip applications for various tubular tissues.

bioengineering↗

bioRxiv2022

Viscoelastic Notch Signaling Hydrogel Induces Liver Bile Duct Organoid Growth and Morphogenesis

Cholangiocyte organoids can be used to model liver biliary disease; however, both a defined matrix in which to emulate cholangiocyte self-assembly and the mechano-transduction pathways involved therein remain elusive. We designed a series of defined viscoelastic hyaluronan hydrogels in which to culture primary cholangiocytes and found that by mimicking the stress relaxation rate of liver tissue, we could induce cholangiocyte organoid growth and significantly increase expression of Yes-associated protein (YAP) target genes. Strikingly, inhibition of matrix metalloproteinases (MMPs) did not significantly affect organoid growth in 3D culture, suggesting that mechanical remodeling of the viscoelastic microenvironment - and not MMP-mediated degradation - is key to cholangiocyte organoid growth. By immobilizing jagged1 to the hyaluronan, stress relaxing hydrogel, self-assembled bile duct structures formed in organoid culture, indicating the synergistic effects of Notch signaling and viscoelasticity. By uncovering critical roles of hydrogel viscoelasticity, YAP signaling and Notch activation, we controlled cholangiocyte organogenesis, thereby paving the way for their use in disease modeling and/or transplantation.

bioengineering↗

bioRxiv2021

High-throughput functional analysis of CFTR and other apically localized channels in iPSC derived intestinal organoids

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. In these proof-of-concept studies, we show how this fluorescence-based assay of apical membrane potential can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This multiparameter phenotypic platform promises to expand CF therapy discovery to include strategies to target multiple determinants of epithelial fluid transport.

molecular biology↗

bioRxiv2018

Comprehensive mapping of Cystic Fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking predicts corrector binding site

BackgroundCystic Fibrosis (CF) is caused by mutations in the CFTR gene, of which over 2000 have been reported to date. Mutations have yet to be analyzed in aggregate to assess their distribution across the tertiary structure of the CFTR protein, an approach that could provide valuable insights into the structure-function relationship of CFTR. In addition, the binding site of Class I correctors (VX-809, VX-661, C18) is not well understood.\n\nMethodsExonic CFTR mutations and mutant allele frequencies described in three curated databases (ABCMdb, CFTR1 and CFTR2, comprising >130,000 data points) were mapped to two different structural models: a homology model of full-length CFTR protein in the open-channel state, and a cryo-electron microscopy core-structure of CFTR in the closed-channel state. Immunoblotting confirmed the approximate binding site of Class I correctors, and molecular docking generated binding poses for their complex with the cryo-electron microscopy structure.\n\nResultsResidue positions of six high-frequency mutant CFTR alleles were found to spatially co-localize in CFTR protein, and a significant cluster was identified at the NBD1:ICL4 interdomain interface. Further, Class I correctors VX-809, VX-661 and C18 were shown to act via a similar mechanism in vitro, and a putative multi-domain corrector binding site near residues F374-L375 was predicted in silico.\n\nConclusionsOur results confirm the significance of interdomain interfaces as susceptible to disruptive mutation, and identify a putative corrector binding site. The structural pharmacogenomics approach of mapping mutation databases to protein models shows promise for facilitating drug discovery and personalized medicine for monogenetic diseases.

biochemistry↗