Neutrophil extracellular traps impair regeneration.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.

Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation.

The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.

Cell-Cycle-Dependent ERK Signaling Dynamics Direct Fate Specification in the Mammalian Preimplantation Embryo.

Despite the noisy nature of single cells, multicellular organisms robustly generate different cell types from one zygote. This process involves dynamic cross regulation between signaling and gene expression that is difficult to capture with fixed-cell approaches. To study signaling dynamics and fate specification during preimplantation development, we generated a transgenic mouse expressing the ERK kinase translocation reporter and measured ERK activity in single cells of live embryos. Our results show primarily active ERK in both the inner cell mass and trophectoderm cells due to fibroblast growth factor (FGF) signaling. Strikingly, a subset of mitotic events results in a short pulse of ERK inactivity in both daughter cells that correlates with elevated endpoint NANOG levels. Moreover, endogenous tagging of Nanog in embryonic stem cells reveals that ERK inhibition promotes enhanced stabilization of NANOG protein after mitosis. Our data show that cell cycle, signaling, and differentiation are coordinated during preimplantation development.

A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia.

Erwinia amylovora is a plant pathogen from the Erwiniaceae family and a causative agent of the devastating agricultural disease fire blight. Here we characterize eight lytic bacteriophages of E. amylovora that we isolated from the Wasatch front (Utah, United States) that are highly similar to vB_EamM_Ea35-70 which was isolated in Ontario, Canada. With the genome size ranging from 271 to 275 kb, this is a novel jumbo family of bacteriophages. These jumbo bacteriophages were further characterized through genomic and proteomic comparison, mass spectrometry, host range and burst size. Their proteomes are highly unstudied, with over 200 putative proteins with no known homologs. The production of 27 of these putative proteins was confirmed by mass spectrometry analysis. These bacteriophages appear to be most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity, however, we were only able to detect infection of Erwinia and the closely related strains of Pantoea.