Mucus is responsible for controlling transport and barrier function in biological systems, and its properties can be significantly affected by compositional and environmental changes. In this study, the impacts of pH and cacl 2 were examined on the solution-to-gel transition of mucin, the primary structural component of mucus. Microscale structural changes were correlated with macroscale viscoelastic behavior as a function of pH and calcium addition using rheology, dynamic light scattering, zeta potential, surface tension, and ftiR spectroscopic characterization. Mucin solutions transitioned from solution to gel behavior between pH 4-5 and correspondingly displayed a more than tenfold increase in viscoelastic moduli. Addition of CaCl 2 increased the sol-gel transition pH value to ca. 6, with a twofold increase in loss moduli at low frequencies and tenfold increase in storage modulus. changing the ionic conditions-specifically [H + ] and [ca 2+ ]-modulated the sol-gel transition pH, isoelectric point, and viscoelastic properties due to reversible conformational changes with mucin forming a network structure via non-covalent cross-links between mucin chains. Mucin is a polyelectrolyte glycoprotein found in mucus, the structured complex fluid found in all types of organisms from bacteria to humans. In vertebrates, mucus is produced by mucus membranes and can be found lining the eyelids, mouth, and nose, as well as gastrointestinal, respiratory, and genital tracts. Since the 1970s 1 , animal mucus and mucin solutions have been well studied, especially the roles mucus plays in drug delivery 2-6 , disorders like cystic fibrosis 7,8 , and its protective biological functions 9-12. Mucin glycoproteins are present in mucus at concentrations of 1-5%, along with electrolytes (ca. 1%), lipids (1-2%), other proteins (1-2%), and water (90-95%) 13. Despite being present in low concentrations, mucin glycoproteins are primarily responsible for the protective and lubricative functions of mucus within the body. Solubilized mucin behaves as a complex fluid with changing viscoelastic and structural properties in response to its environmental conditions. Mucin glycoproteins are responsible for the majority of the physical properties of mucus, as mucin conformation, intra-and inter-strand bonding, and microstructure changes in response to environmental factors such as pH 6,7,12,14 , temperature 2,15 , and ion content 1,14,16. All these factors affect the reversible supramolecular bonding between functional groups within the protein strands, which modifies the microstructure as well as the mechanical and transport properties of the mucin network. In aqueous solutions, biopolymers like mucin form networks that can change dynamically due to non-covalent crosslinks, including physical entanglements and supramolecular bonding. Mucins resemble a bottle brush polymer with a protein backbone and oligosaccharide (carbohydrate) side chains arranged radially from the backbone. The protein backbone of the mucin biopolymer has areas that are dense w...

Background Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the three most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. Methods We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176,645 cells as a reference for an in-depth comparison to 4,416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. Results We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. Conclusion Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar “lineage of origin”.

Understanding the cellular origins of childhood brain tumors is key for discovering novel tumor-specific therapeutic targets. Previous strategies mapping cellular origins typically involved comparing human tumors to murine embryonal tissues, a potentially imperfect approach due to spatio-temporal gene expression differences between species. Here we use an unprecedented single-nucleus atlas of the developing human cerebellum (Sepp, Leiss, et al) and extensive bulk and single-cell transcriptome tumor data to map their cellular origins with focus on three most common pediatric brain tumors - pilocytic astrocytoma, ependymoma, and medulloblastoma. Using custom bioinformatics approaches, we postulate the astroglial and glial lineages as the origins for posterior fossa ependymomas and radiation-induced gliomas (secondary tumors after medulloblastoma treatment), respectively. Moreover, we confirm that SHH, Group3 and Group4 medulloblastomas stem from granule cell/unipolar brush cell lineages, whereas we propose pilocytic astrocytoma to originate from the oligodendrocyte lineage. We also identify genes shared between the cerebellar lineage of origin and corresponding tumors, and genes that are tumor specific; both gene sets represent promising therapeutic targets. As a common feature among most cerebellar tumors, we observed compositional heterogeneity in terms of similarity to normal cells, suggesting that tumors arise from or differentiate into multiple points along the cerebellar "lineage of origin".

Mucus is a complex fluid that maintains moisture and simultaneously acts as a barrier and facilitates transport of select materials between the body and adjacent fluids. While mucus is comprised primarily of water, it is highly heterogeneous containing the biopolymer mucin along with lipids, salts, DNA, proteins, and cells. Complex mechanisms control the reversible network formation observed in mucus and mucin solutions. Some isolated relationships between biopolymer network structure, pH and ionic strength, and rheology have been identified; however, a complete understanding of the interplay in these mechanisms is lacking. In this effort, rheology of native mucus and mucin solutions was examined as a function of pH and salt concentration. Bulk rheology of native and artificial lung mucus confirmed that native mucus displays a solid-like behavior at low strain values. Mucus displays this solid-like gel behavior at pH values ca. 4 and below, and displays solution behavior at higher pH values. Ion concentration also plays an important role with divalent cations (Ca2+) reducing the viscosity of gelled mucin and increasing the viscosity of solution-state mucin. In addition to rheological characterization, mucin-mucin and mucin-solute interactions and resultant changes in microstructure were also studied. Morphological and chemical changes at the nano-scale were correlated to the micro-structural changes observed with rheology. Dynamic light scattering showed mucin polymer particle size heterogeneity as well as a significant increase in particle size at pH values ca. 4 and below — where the solutions display a gel behavior. Using zeta potential a decrease in dispersive forces was observed that allows for polymer-polymer interaction and particle aggregation at low pH values. Atomic force micrography showed spheroid-like aggregates between adjacent mucin particles under acidic conditions. The ability to understand and control the reversible association of network structures in polymer and biopolymer systems, such as mucin, through supramolecular interactions has fundamental impacts in the field of polymer science and engineering. There is also significant potential to advance applications involving novel hydrogel materials, such as disease treatments and drug delivery.

Medulloblastoma (MB) is a highly malignant childhood brain tumor comprising a collection of molecularly and clinically distinct entities. Mutations in chromatin-modifying genes distribute across subgroups and account for nearly half of all MBs. However, the molecular consequences of chromatin modifier alterations remain poorly understood. Herein, we characterize the chromatin landscape of fifty-two primary MBs representing each MB subgroup using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for six histone marks and perform multi-layered integration with somatic genetics, genome-wide DNA methylation, and transcriptomic datasets extracted from the same tumors. Analysis of differential chromatin states across MB subgroups identified significant enrichment of a bivalent enhancer state (EnhBiv) in Group 4 MB. Group 4-specific EnhBivs mapped to promoters of key neurodevelopmental genes and transcription factors and was associated with increased DNA methylation and reduced expression. In accordance with higher EnhBivs coverage in Group 4 MB, we found that KDM2B, a H3K4me3 and H3K36me2 demethylase, is highly expressed in Group 4 MBs and patient-derived xenografts (PDXs). KDM2B knockout mediated by CRISPR targeting suppressed growth of both Group 3 MB cell lines in vitro and Group 4 MB PDXs in vivo but had no effect on control SHH-MB cells, suggesting that KDM2B is a novel selective dependency in MB. Knockout of KDM2B in MB cells induced profound upregulation of PRC2 targets, genes regulated by bivalent histone marks, as well as genes involved in neuronal differentiation. Further biochemical and molecular studies demonstrated that KDM2B interacts with PRC2 complex subunits and occupies gene promoters associated with Group 4-specific EnhBiv chromatin. In summary, our comprehensive characterization of the MB chromatin landscape in a large cohort of primary tumors provides novel insights into the epigenetic basis of MB and implicates KDM2B as a novel Group 4 MB dependency that should be prioritized as a therapeutic target in this subgroup.