Fetal Tracheal Occlusion Increases Lung Basal Cells via Increased Yap Signaling.

Fetal endoscopic tracheal occlusion (FETO) is an emerging surgical therapy for congenital diaphragmatic hernia (CDH). Ovine and rabbit data suggested altered lung epithelial cell populations after tracheal occlusion (TO) with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA sequencing (mRNA-seq) data and used quantitative image analysis in fetal rabbit lung TO, which had increased basal cells and reduced ciliated cells after TO. In a fetal mouse TO model, flow cytometry showed increased basal cells, and immunohistochemistry demonstrated basal cell extension to subpleural airways. Nuclear Yap, a known regulator of basal cell fate, was increased in TO lung, and Yap ablation on the lung epithelium abrogated TO-mediated basal cell expansion. mRNA-seq of TO lung showed increased activity of downstream Yap genes. Human lung specimens with congenital and fetal tracheal occlusion had clusters of subpleural basal cells that were not present in the control. TO increases lung epithelial cell nuclear Yap, leading to basal cell expansion.

Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366).

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389-402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233-425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233-425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233-425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.