Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein’s stability remains a challenge. This is largely due to a shortage of atomically detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM β-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and <i>in vivo</i> measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design