Biology subjects

Flannery, S.

Publications and source records attributed to Flannery, S..

3 recordsLinked to original sources

bioRxiv2025

Isolation of functional supramolecular attack particles (SMAPs)

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are [~]130-150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100-110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca2+-stabilized SMAPs (F3) trigger caspase-3-dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance StatementCell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo. These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

immunology↗

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗

bioRxiv2020

Effects of population, latitude, and individual tree to leaf variation in oaks: An empirical study and simulation of sampling strategies in bur oak (Quercus macrocarpa, Fagaceae)

PREMISELeaf shape and size figure strongly in trees adaptation to their environments. Oaks are notoriously variable in leaf morphology. Our study examines the degree to which within-tree, among-tree, and among-site variation contribute to latitudinal variation in leaf shape and size of bur oak (Quercus macrocarpa: Fagaceae), one of North Americas most geographically widespread oak species. METHODSSamples were collected from four sites each at northern, central, and southern latitudes of the bur oak range. Ten leaf size traits were measured, and variance in these traits and eight ratios based on these traits was partitioned into tree, population, and latitude components. We then parameterized a series of leaf collection simulations using empirical covariance among leaves on trees and trees at sites. We used the simulations to assess the efficiency of different collecting strategies for estimating among-population differences in leaf shape and size. KEY RESULTSLeaf size measurements were highly responsive to latitude. Site contributed more than tree to total variation in leaf morphology. Simulations suggest that power to detect among-site variance in leaf shape and size can be estimated most efficiently with increases in either leaves per tree (10-11 leaves from each of 5 trees) or trees per site (5 leaves from each of 10+ trees). CONCLUSIONSOur study demonstrates the utility of simulating sampling and controlling for variance in sampling for leaf morphology, whether the questions being addressed are ecological, evolutionary, or taxonomic. Simulation code is provided as an R package (traitsPopSim) to help researchers plan morphological sampling strategies.

plant biology↗