Biology subjects

McKay, E. C.

Publications and source records attributed to McKay, E. C..

3 recordsLinked to original sources

bioRxiv2026

Spectronaut-nf: A Nextflow Pipeline for Parallel Processing of DIA Data with Spectronaut

SummaryContemporary proteomics methods can now generate large-scale DIA datasets of thousands of files that demand substantial computational resources for efficient analysis. Spectronaut is a widely used platform for DIA data processing; however, large-scale searches are often constrained by computational performance and long execution times when run on single workstations. Here, we present Spectronaut-nf, a Nextflow-based pipeline that enables scalable and parallelized execution of Spectronaut analyses across high-performance computing (HPC) environments. The workflow divides directDIA analysis into modular stages, including spectral library generation, DIA searching, and merging results, allowing efficient distribution of tasks across multiple compute nodes. Benchmarking using 72 diaPASEF raw files using typical hardware demonstrated that Spectronaut-nf completed searches in 23.77 hours, compared with 39.09 hours on a Windows workstation and 67.04 hours on a single-node Linux HPC setup. Stress testing with 1,037 diaPASEF raw files further demonstrated the scalability and robustness of the workflow for large proteomics datasets. Across platforms, protein and peptide identifications remained consistent, with only minimal variability attributable to platform-specific differences. Overall, Spectronaut-nf provides a flexible, scalable, and efficient framework for high-throughput DIA proteomics analysis in HPC environments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/741433v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4a7107org.highwire.dtl.DTLVardef@14287d8org.highwire.dtl.DTLVardef@e484e9org.highwire.dtl.DTLVardef@d21c07_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

NLRP3 is a thermosensor that is negatively regulated by high temperature

Inflammation is an essential response to infection and injury, but unregulated inflammation is damaging and must be limited by negative feedback signalling. Inflammasome signalling drives local inflammation and systemic responses like fever. However, our understanding of how inflammasome signalling is negatively regulated is limited. NLRP3 is activated by a vast number of stimuli and senses perturbations of cytoplasmic homeostasis. As temperature is a fundamental environmental stressor, we hypothesised that NLRP3 inflammasome signalling would be sensitive to increased temperatures and so we investigated the effects of high temperatures on NLRP3 in macrophages. Short-term incubation at high fever range temperatures significantly inhibits NLRP3 activation, while secretion of the inflammasome-independent cytokines TNF and IL-6 are much less affected. High temperature blocks NLRP3 inflammasome formation in a transcription-independent manner, and NLRP3 is highly sensitive to temperature-mediated inhibition relative to the NLRC4, AIM2, and NLRP1 inflammasomes. Using cellular assays and molecular simulations we show that the effect of high temperature on NLRP3 is protein intrinsic. NLRP3 activation is associated with a decrease in the thermal stability of the protein and multiscale molecular dynamics simulations identified a peptide in the C-terminal of the FISNA domain (COFI) that is highly flexible and undergoes a significant conformational shift at high temperature. Cellular assays demonstrate that the COFI regulates NLRP3 stability and is required for activation. Furthermore, mice exposed to high temperature display attenuated inflammatory cytokine production upon in vivo LPS challenge. Our studies thus reveal that high temperatures associated with fever limit NLRP3 activity and identify a novel role for NLRP3 as a protein thermosensor.

immunology↗

bioRxiv2025

Induced pluripotent stem cell-derived macrophages as a model for human inflammasome signaling

Macrophage models are a mainstay of inflammasome research, however current human in vitro macrophage models have significant limitations. Here we generate induced pluripotent stem cell (iPSC)-derived macrophages (iMacs) to study inflammasome signaling and benchmark them with human monocyte-derived macrophages (HMDMs). We confirm that iMacs express high levels of macrophage markers and are highly phagocytic. Whole cell proteomics analysis shows that iMacs express many inflammasome sensors and related proteins, and in functional assays iMacs respond to multiple inflammasome stimuli. The NLRP3 inflammasome is strongly activated in iMacs and we find that nigericin alone activates NLRP3. The non-canonical inflammasome does not require a priming step in iMacs as caspase-4 is constitutively expressed. High levels of NAIP/NLRC4 inflammasome activation are also observed in response to needle toxin. Finally, unlike HMDMs, iMacs activate NLRP1. Therefore, we demonstrate that iMacs are a physiologically relevant and highly tractable model to study human inflammasome signaling and regulation. MotivationiPSC-derived macrophages (iMacs) are functionally, transcriptionally, and phenotypically similar to primary human macrophages. iMacs therefore offer new opportunities to study inflammasome activity in a human macrophage model, but to date they have not been widely used. In this study, we describe a protocol to differentiate and characterize iMacs. We then describe how to activate a range of different inflammasomes within these cells and assess the inflammasome response by measuring pyroptosis, cytokine release, ASC speck formation, and processing of inflammasome-related proteins. We also benchmark iMac responses with the current gold standard primary human monocyte derived macrophage model.

immunology↗