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The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical β-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124−/− embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124- regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors. [ABSTRACT FROM AUTHOR]

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7–8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery. [ABSTRACT FROM AUTHOR]

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self‐renewal properties is essential for long‐term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19–21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations. [ABSTRACT FROM AUTHOR]

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency. [Display omitted] • scRNA-seq of developing human distal lung mesenchyme identified cellular heterogeneity • RSPO2+ mesenchymal cells lie adjacent to LGR5+ epithelial bud tip progenitors • Blocking RSPO2/LGR5 in vitro reduced WNT signaling and led to airway differentiation • RSPO2+ mesenchyme provides a niche for bud tips in co-cultures Hein et al. present the scRNA-seq of the developing human distal lung, showing transcriptionally distinct populations of mesenchyme. Spatial profiling showed that RSPO2+ mesenchymal cells are physically adjacent to LGR5+ epithelial bud tips. Organoid experiments revealed that RSPO2+ cells create a high WNT signaling environment, supporting bud tip identity and multipotency. [ABSTRACT FROM AUTHOR]

R5339 --> 731.8 --> Background: Small intestinal bicarbonate transport, is critical for epithelial protection, digestion, and absorption. Membrane trafficking and/or recycling of transporters is a critical means to modulate their function, however, the cellular mechanisms that drive these processes in the duodenum remain unclear. The inositol 1,4,5‐trisphosphate (IP3) receptor binding protein released with IP3 (IRBIT) is an intracellular protein that has been shown to regulate localization of bicarbonate transporters outside of the intestine, yet its role in regulating duodenal bicarbonate secretion is unknown. Aim: To examine duodenal expression and function of IRBIT in regulating bicarbonate transport. Methods: Duodenal endoscopic biopsies from subjects without gross or histological evidence of disease were used directly or cultured using traditional apical‐in or flipped apical‐out enteroids. mRNA expression was evaluated using qPCR and re‐analysis of a duodenal sc‐RNAseq dataset. Protein expression and localization was determined by Western blot and confocal immunofluorescence, respectively. In vivo bicarbonate secretion was measured in anesthetized mice by duodenal perfusion and back‐titration. Results: IRBIT is highly expressed in human duodenal biopsies, with protein densitometric analysis showing ≥3.5‐fold higher expression than Calu‐3 airway submucosal gland cells or HEK293 kidney‐derived cells (n=1 each). Analysis of duodenal sc‐RNAseq data (1,625 cells) showed ACHYL1 in crypts and villi. Immunofluorescence staining of human duodenal biopsies (n=3) showed IRBIT staining along the crypt‐villus axis, primarily localizing in E‐cadherin positive epithelial cells, but also present in the lamina propria. In patient‐derived duodenal enteroids, IRBIT was expressed in both crypt‐like (undifferentiated) and villus‐like (differentiated) enteroids (n=3 each), with greater IRBIT mRNA and protein expression in the latter, a pattern similar to SLC26A3 and SLC26A6 chloride/bicarbonate exchangers (n=3 each). In sc‐RNAseq analysis, AHCYL1 and SLC26A3 were co‐expressed in 38% of all villus cells, compared to only 5% co‐expressing AHCYL1 and SLC26A6. CFTR‐independent duodenal bicarbonate secretion stimulated by linaclotide (10‐7 M) was significantly inhibited by phospholipase C inhibition (U7312, 10‐6 M; 91±11%, n=3), which we showed disrupts IRBIT membrane localization, or SLC26A3 inhibition (DRAinh‐A250, 10‐5 M; 68±8%, n=10). Linaclotide stimulation of apical‐out enteroids increased both IRBIT (n=3) and SLC26A3 (n=12) membrane trafficking. Summary and Conclusions: IRBIT is highly expressed in the duodenum and appears to functionally interact with SLC26A3. IRBIT may be an attractive target to modulate small intestinal pH in diseases like cystic fibrosis, where impaired bicarbonate secretion contributes to intestinal disease and malnutrition. [ABSTRACT FROM AUTHOR]