Biology subjects

Crowley, T.

Publications and source records attributed to Crowley, T..

5 recordsLinked to original sources

bioRxiv2026

Interrogating the Mechanisms of Cas9-mediated Allele Conversion

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

molecular biology↗

bioRxiv2025

The dietary emulsifier polysorbate-80 induces lipid accumulation and cell death in intestinal epithelial cells via ferroptosis

Chronic inflammatory and metabolic diseases are major global health issues increasingly linked to dietary factors. Consumption of dietary emulsifiers like polysorbate-80 (p80), common in ultra-processed foods and pharmaceuticals, has raised concerns about gut health. RNA sequencing on intestinal epithelial cells (IECs) exposed to p80 revealed increased expression of ferroptosis-associated genes and disruption of lipid metabolism pathways further demonstrated by mitochondrial dysfunction, including altered membrane potential and architecture, and accumulation of reactive oxygen species, iron, lipid peroxidation, and lipid droplet formation. Lipidomic profiling identified significant alterations in triglyceride species and elevated pro-ferroptotic polyunsaturated fatty acids. These data indicate that p80 disrupts lipid homeostasis in IECs and triggers ferroptotic cell death, mechanisms potentially contributing to the increased incidence of chronic conditions like inflammatory bowel disease and metabolic syndrome. The study highlights critical implications for public health, emphasizing the need for reassessment of emulsifier safety standards while balancing needs with consumer safety.

cell biology↗

bioRxiv2025

IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

IFN-{gamma} is secreted by multiple lymphoid subsets in response to antigen stimulation and can reprogram and prime macrophages epigenetically and transcriptionally to increase responses to inflammatory stimuli such as LPS, IL-1{beta} or TNF-. IFN-{gamma}-driven M1-like inflammatory macrophage states are shared across human immune-mediated inflammatory diseases (IMIDs) and while IFN-{gamma} is nonredundant for defense to intracellular pathogens it is unclear if this is also the case in IMIDs. To identify additional secreted ligands which could prime and induce M1-like macrophages we screened >600 human proteins in human primary macrophages. Using complementary functional genomics approaches, we discovered that IL-32{beta} induced an M1-like inflammatory state in non-primed and IFN-{gamma}-primed macrophages. IL-32{beta} induced signaling, transcriptional, tolerance, cross-tolerance, and inflammatory responses in macrophages which were MyD88, IRAK1 and Myddosome-dependent. These responses to host IL-32{beta} were similar to yet distinct from, those induced by microbial LPS. IL-32 protein was elevated in serum from patients with severe COVID-19 and IL-32{beta} together with IFN-{gamma} were expressed by T cells and induced a macrophage transcriptional response which was shared by monocytes and macrophages in mild and severe COVID-19.

immunology↗

bioRxiv2017

Differential expression analysis of log-ratio transformed counts: benchmarking methods for RNA-Seq data

BackgroundCount data generated by next-generation sequencing assays do not measure absolute transcript abundances. Instead, the data are constrained to an arbitrary \"library size\" by the sequencing depth of the assay, and typically must be normalized prior to statistical analysis. The constrained nature of these data means one could alternatively use a log-ratio transformation in lieu of normalization, as often done when testing for differential abundance (DA) of operational taxonomic units (OTUs) in 16S rRNA data. Therefore, we benchmark how well the ALDEx2 package, a transformation-based DA tool, detects differential expression in high-throughput RNA-sequencing data (RNA-Seq), compared to conventional RNA-Seq differential expression methods.\n\nResultsTo evaluate the performance of log-ratio transformation-based tools, we apply the ALDEx2 package to two simulated, and one real, RNA-Seq data sets. The latter was previously used to benchmark dozens of conventional RNA-Seq differential expression methods, enabling us to directly compare transformation-based approaches. We show that ALDEx2, widely used in meta-genomics research, identifies differentially expressed genes (and transcripts) from RNA-Seq data with high precision and, given sufficient sample sizes, high recall too (regardless of the alignment and quantification procedure used). Although we show that the choice in log-ratio transformation can affect performance, ALDEx2 has high precision (i.e., few false positives) across all transformations. Finally, we present a novel, iterative log-ratio transformation (now implemented in ALDEx2) that further improves performance in simulations.\n\nConclusionsOur results suggest that log-ratio transformation-based methods can work to measure differential expression from RNA-Seq data, provided that certain assumptions are met. Moreover, these methods have high precision (i.e., few false positives) in simulations and perform as good as, or better than, than conventional methods on real data. With previously demonstrated applicability to 16S rRNA data, ALDEx2 can work as a single tool for data from multiple sequencing modalities.

bioinformatics↗

bioRxiv2017

propr: An R-package for Identifying Proportionally Abundant Features Using Compositional Data Analysis

In the life sciences, many assays measure only the relative abundances of components for each sample. These data, called compositional data, require special handling in order to avoid misleading conclusions. For example, in the case of correlation, treating relative data like absolute data can lead to the discovery of falsely positive associations. Recently, researchers have proposed proportionality as a valid alternative to correlation for calculating pairwise association in relative data. Although the question of how to best measure proportionality remains open, we present here a computationally efficient R package that implements two proposed measures of proportionality. In an effort to advance the understanding and application of proportionality analysis, we review the mathematics behind proportionality, demonstrate its application to genomic data, and discuss some ongoing challenges in the analysis of relative abundance data.

bioinformatics↗