Biology subjects

Higginbotham, J. N.

Publications and source records attributed to Higginbotham, J. N..

3 recordsLinked to original sources

bioRxiv2025

Catch and release of sialoglycoRNAs enables sequencing-based profiling across cellular and extracellular material

Glycosylated RNAs (glycoRNAs) represent a recently discovered class of small RNAs, but their systematic characterization has been limited by reliance on metabolic chemical reporters and high RNA input requirements. Here we present rPAL sequencing (rPAL-seq), a sensitive and selective platform for de novo discovery of sialoglycoRNAs. rPAL-seq combines enhanced periodate oxidation of sialic acids with a capture-release workflow and optimized library construction using poly(A) extension coupled with template-switching reverse transcription. The method enabled reproducible profiling from less than 100 ng of input RNA, corresponding to less than 2% of the material required by previous approaches. When applied across 13 human cell lines, rPAL-seq identified lineage-associated glycoRNA patterns alongside a conserved core dominated by uridine-rich snRNAs and snoRNAs, with modification signatures implicating glycosylation on acp3U or related uridine-based modifications. Extending to extracellular vesicles and non-vesicular nanoparticles, rPAL-seq revealed secreted glycoRNA profiles distinct from those of the cellular fraction. rPAL-seq provides a robust, scalable strategy for glycoRNA profiling, opening new avenues for studying this emerging biopolymer.

molecular biology↗

bioRxiv2025

Unbiased recording of clonal potency reveals species-specific regulation of mammalian intestine

The mammalian intestine regenerates rapidly after damage, yet the clonal dynamics and species-specific regulation of different populations remain poorly understood. Here we used synthetic or naturally occurring DNA alterations to reconstruct clonal histories of the mouse and human intestinal epithelium at single-cell resolution. In mice, we uncovered the clonal architecture of different cell types and their roles in regeneration, supporting a hierarchical regenerative response model. We identified a rare embryonic precursor population that persisted in the adult and was crucial for regeneration after irradiation. This population was marked by Tob2, which is required for nuclear transport of Ascl2. A parallel clonal analysis of 65 human colonic biopsies revealed secretory lineage bias and an age-associated decline in clonal diversity in the distal colon. Unlike highly proliferative murine Lgr5+ stem cells, human LGR5+ cells were found largely quiescent, revealing species-specific difference in clonal potency, and suggesting a distinct regulation of intestinal stemness.

genomics↗

bioRxiv2023

Detection of EGFR and its Activity State in Plasma CD63-EVs from Glioblastoma Patients: Rapid Profiling using an Anion Exchange Membrane Sensor

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/L and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

cancer biology↗