Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

A molecular cell atlas of the human lung from single-cell RNA sequencing.

Although single-cell RNA sequencing studies have begun to provide compendia of cell expression profiles1-9, it has been difficult to systematically identify and localize all molecular cell types in individual organs to create a full molecular cell atlas. Here, using droplet- and plate-based single-cell RNA sequencing of approximately 75,000 human cells across all lung tissue compartments and circulating blood, combined with a multi-pronged cell annotation approach, we create an extensive cell atlas of the human lung. We define the gene expression profiles and anatomical locations of 58 cell populations in the human lung, including 41 out of 45 previously known cell types and 14 previously unknown ones. This comprehensive molecular atlas identifies the biochemical functions of lung cells and the transcription factors and markers for making and monitoring them; defines the cell targets of circulating hormones and predicts local signalling interactions and immune cell homing; and identifies cell types that are directly affected by lung disease genes and respiratory viruses. By comparing human and mouse data, we identified 17 molecular cell types that have been gained or lost during lung evolution and others with substantially altered expression profiles, revealing extensive plasticity of cell types and cell-type-specific gene expression during organ evolution including expression switches between cell types. This atlas provides the molecular foundation for investigating how lung cell identities, functions and interactions are achieved in development and tissue engineering and altered in disease and evolution.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/ß-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.

Extended access to cocaine self-administration results in reduced glutamate function within the medial prefrontal cortex.

Previous studies have shown that brief access to cocaine yields an increase in D2 receptor binding in the medial prefrontal cortex (mPFC), but that extended access to cocaine results in normalized binding of D2 receptors (i.e. the D2 binding returned to control levels). Extended-access conditions have also been shown to produce increased expression of the NR2 subunit of the N-Methyl-D-aspartate receptor in the mPFC. These results implicate disrupted glutamate and dopamine function within this area. Therefore, in the present study, we monitored glutamate and dopamine content within the mPFC during, or 24 hours after, cocaine self-administration in animals that experienced various amounts of exposure to the drug. Naïve subjects showed decreased glutamate and increased dopamine levels within the mPFC during cocaine self-administration. Exposure to seven 1-hour daily cocaine self-administration sessions did not alter the response to self-administered cocaine, but resulted in decreased basal dopamine levels. While exposure to 17 1-hour sessions also resulted in reduced basal dopamine levels, these animals showed increased dopaminergic, but completely diminished glutamatergic, response to self-administered cocaine. Finally, exposure to 17 cocaine self-administration sessions, the last 10 of which being 6-hour sessions, resulted in diminished glutamatergic response to self-administered cocaine and reduced basal glutamate levels within the mPFC while normalizing (i.e. causing a return to control levels) both the dopaminergic response to self-administered cocaine as well as basal dopamine levels within this area. These data demonstrate directly that the transition to escalated cocaine use involves progressive changes in dopamine and glutamate function within the mPFC.

Successful management of scalp avulsion with full-thickness graft.

Scalp avulsion injuries are one of the life-threatening traumatic injuries. Rapid management in the emergency department is vital to the successful rescue of an avulsed scalp. There are many replantation methods to treat scalp avulsion, so the best aesthetic and functional results are achieved. Skin grafting, free flaps, microvascular surgery, and hyperbaric oxygen are all suitable for reconstructive plans. We report a rare case of a scalp avulsion injury in a female due to an entanglement of a headscarf in a motorized machine resulting in defects and tissue loss of the hair-bearing skin, nasal area, forehead, left ear, and bilateral eyelids and eyebrows. Preoperative management included early blood transfusion, intravenous fluids, and wound compression after rapid physical examination. Reconstructive surgery was performed using a full-thickness skin graft and the outcomes were pristine. There are no clear guidelines to determine which reconstructive method is superior to another in each condition. Our case demonstrates that relatively primitive methods like skin grafting can give greater results if done with circumspection.

Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells.

Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat alveolar type 1 (AT1) cells, which mediate gas exchange, and AT2 cells, which secrete surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling, and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: Maintaining Wnt signaling prevents transdifferentiation, whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting "bulk" AT2s as progenitors. Thus, individual AT2 stem cells reside in single-cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface and is coopted in cancer.