Evidence that the cold- and menthol-sensing functions of the human TRPM8 channel evolved separately.

Transient receptor potential melastatin 8 (TRPM8) is a temperature- and menthol-sensitive ion channel that contributes to diverse physiological roles, including cold sensing and pain perception. Clinical trials targeting TRPM8 have faced repeated setbacks predominantly due to the knowledge gap in unraveling the molecular underpinnings governing polymodal activation. A better understanding of the molecular foundations between the TRPM8 activation modes may aid the development of mode-specific, thermal-neutral therapies. Ancestral sequence reconstruction was used to explore the origins of TRPM8 activation modes. By resurrecting key TRPM8 nodes along the human evolutionary trajectory, we gained valuable insights into the trafficking, stability, and function of these ancestral forms. Notably, this approach unveiled the differential emergence of cold and menthol sensitivity over evolutionary time, providing a fresh perspective on complex polymodal behavior. These studies provide a paradigm for understanding polymodal behavior in TRPM8 and other proteins with the potential to enhance our understanding of sensory receptor biology and pave the way for innovative therapeutic interventions.

A novel induced pluripotent stem cell model of Schwann cell differentiation reveals NF2 - related gene regulatory networks of the extracellular matrix.

Schwann cells are vital to development and maintenance of the peripheral nervous system and their dysfunction has been implicated in a range of neurological and neoplastic disorders, including NF2 -related schwannomatosis. We developed a novel human induced pluripotent stem cell (hiPSC) model to study Schwann cell differentiation in health and disease. We performed transcriptomic, immunofluorescence, and morphological analysis of hiPSC derived Schwann cell precursors (SPCs) and terminally differentiated Schwann cells (SCs) representing distinct stages of development. To validate our findings, we performed integrated, cross-species analyses across multiple external datasets at bulk and single cell resolution. Our hiPSC model of Schwann cell development shared overlapping gene expression signatures with human amniotic mesenchymal stem cell (hAMSCs) derived SCs and in vivo mouse models, but also revealed unique features that may reflect species-specific aspects of Schwann cell biology. Moreover, we identified gene co-expression modules that are dynamically regulated during hiPSC to SC differentiation associated with ear and neural development, cell fate determination, the NF2 gene, and extracellular matrix (ECM) organization. By cross-referencing results between multiple datasets, we identified new genes potentially associated with NF2 expression. Our hiPSC model further provides a tractable platform for studying Schwann cell development in the context of human disease.

Long-term Outcomes After Interrupted Aortic Arch Repair.

BACKGROUND: Interrupted aortic arch (IAA) is associated with left ventricular outflow tract obstruction (LVOTO) and DiGeorge syndrome. High-risk infantile surgery is required to address IAA, with limited data available on long-term outcomes. We used the Pediatric Cardiac Care Consortium, a multicenter US-based registry for pediatric cardiac interventions, to assess long-term outcomes after IAA repair by patient characteristics and surgical approach. METHODS: This is a retrospective cohort study of patients undergoing IAA repair between 1982 and 2003. Kaplan-Meier plots and Cox proportional hazards regression were used to examine associations with postdischarge deaths tracked by matching with the US National Death Index. RESULTS: Of 390 patients meeting inclusion criteria, 309 (79.2%) survived to discharge. During a median follow-up of 23.6 years, 30-year survival reached 80.7% for patients surviving hospital discharge after initial IAA repair. Adjusted analysis revealed higher risk of death for type B vs type A (adjusted hazard ratio [aHR], 3.32; 95% CI, 1.48-7.44), staged repair (aHR, 2.50; 95% CI, 1.14-5.50), and LVOTO interventions during initial hospitalization (aHR, 4.12; 95% CI, 1.83-9.27) but not for LVOTO without need for interventions or presence of DiGeorge syndrome. There was a trend toward improved in-hospital and long-term survival over time during the study period. CONCLUSIONS: Staged repair, type B IAA, and need for LVOTO intervention during initial hospitalization for repair are associated with high risk of death out to 30 years. Survival outcomes are improving, but further efforts need to minimize staged approach and risks associated with LVOTO relief procedures.

Competition between inside-out unfolding and pathogenic aggregation in an amyloid-forming ß-propeller.

Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

Development of eighteen novel microsatellite loci for Masked Bobwhite, Colinus virginianus ridgwayi.

BACKGROUND: Masked Bobwhite (Colinus virginianus ridgwayi) is a critically-endangered New World quail species endemic to Sonoran Desert grasslands of North America. It suffered severe population declines during the nineteenth and twentieth centuries, with its persistence now reliant upon a captive breeding program that requires careful genetic management to maintain extant genetic diversity. Although nuclear microsatellite DNA markers existed for the closely related Northern Bobwhite (C. virginianus), none were available for Masked Bobwhite to inform necessary management decisions. METHODS AND RESULTS: Paired-end Illumina© sequencing was conducted to screen the Masked Bobwhite genome for microsatellite loci. We identified 18 loci exhibiting high polymorphism and limited deviations from genetic equilibrium expectations. These loci were amplified in 78 individuals. Familial relationships were reconstructed via sibship methods and compared to manually-curated pedigree data. Thirteen of fifteen full-sibling groups in the pedigree were exactly reconstructed (86.6%). Three other full-sibling groups partially matched pedigree relationships with high statistical confidence, and likely represented pedigree inaccuracies. Four additional full-sibling pairs were identified with low statistical confidence and likely resulted from analytical artifacts. CONCLUSIONS: The novel microsatellite loci accurately reconstructed parent-offspring and sibling relationships. These loci will be useful for guiding genetic management decisions and identifying pedigree inaccuracies in the captive breeding program.

Delivery of recombinant SARS-CoV-2 envelope protein into human cells

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.