Biology subjects

Bradley, R.

Publications and source records attributed to Bradley, R..

10 recordsLinked to original sources

bioRxiv2025

Sub-cellular chemical mapping in bacteria using correlated cryogenic electron and mass spectrometry imaging

Electron cryomicroscopy (cryo-EM) allows high-resolution spatial visualization of biological specimens, however, it is challenging to chemically identify densities observed in cryo-EM. To overcome this, we combined cryo-EM with chemical imaging using focused ion beam secondary ion mass spectrometry (FIB-SIMS) for integrated spatio-chemical analysis of untagged specimens. We show that our correlative workflow permits sub-cellular localisation of molecules inside bacterial cells and is compatible with cryogenic light microscopy and FIB-milled lamellae of multicellular specimens. To highlight biological insights enabled by the workflow, we studied the uptake of Bisphenol-AF, a widespread chemical pollutant, by environmental bacteria, revealing the storage of these chemicals within cytosolic phase-separated aggregates in pollutant-exposed cells, where they cannot be removed by the bacterial efflux machinery despite its robust upregulation. These mechanistic insights were directly facilitated by the versatile cryo-EM-FIB-SIMS technique, showing that it is an effective avenue to map elemental and molecular signatures in near-native biological samples, which can be extended in the future for multiple applications in cell biology and imaging.

molecular biology↗

bioRxiv2025

Interspecies interactions drive bacterial proteome reorganisation and emergent metabolism

Species in microbial communities must respond to the presence of others to stave off resource competition or to capitalise on new resources that may become available due to additional metabolic activities. Such metabolic interactions manifest in growth effects such as competitive exclusion or cooperative growth boost. However, intra-cellular molecular changes that underpin these responses are very sparsely studied, preventing mechanistic insights into community function and dynamics. Here, we analyse meta-proteomics and metabolomic responses in 104 pairwise co-cultures of 15 diverse gut bacteria in a nutrient-rich medium. These co-cultures span a diversity of ecological interactions with competition, amensalism and exploitation being prominent, and with around one in five positive interactions. The species in co-cultures feature substantial molecular responses, with typically more than 60% of the proteome changing in response to at least one partner. Even closely related species and ortholog proteins show different expression profiles in response to the same partner indicating functional diversification at both protein and species level. We show that the magnitude of these responses is determined by genome size, species abundance, and pH. Small-molecule transport and carbon metabolism are among the most responsive processes indicating pervasive metabolic interactions. Using metabolomics, we identify likely cross-fed metabolites, emergent polyamine metabolism, and niche partitioning in amino acid utilisation. Overall, our study shows how bacteria respond to the presence of other species through extensive remodelling of their proteome and metabolome.

microbiology↗

bioRxiv2025

Impact of low-calorie sweeteners on gut bacteria is modulated by common xenobiotics

The gut microbiota is implicated in adverse effects associated with low-calorie sweeteners. Yet, the direct impact of sweeteners on gut bacteria remains largely uncharacterized. Here we report interactions between 25 phylogenetically diverse gut bacterial strains and 39 commercially used sweeteners. We tested these sweeteners individually and in combination with four commonly co-consumed compounds, viz., advantame, caffeine, vanillin, and duloxetine. Three quarters of the tested sweeteners individually impacted growth of at least one bacterial strain. Further, over 100 interactions were found between sweeteners and the four co-consumed compounds. Isosteviol, a commonly used sweetener, and duloxetine, an antidepressant, synergistically inhibited Roseburia intestinalis, a bacterium previously linked to glucose homeostasis, and Parabacteroides merdae, a prevalent commensal linked to healthy microbiota. Proteomic, metabolomic, and genetic analyses indicate altered small molecule transport underpinning this sweetener-drug synergy. The isosteviol-duloxetine combination also modulated metabolism of a synthetic gut bacterial community leading to increased toxicity to HeLa cells and altered secretion of inflammation modulatory cytokines IL-6 and IL-8 by Caco-2 cells. Together, our data bring forward the prevalence of interactions between low-calorie sweeteners and common xenobiotics.

microbiology↗

bioRxiv2024

RBM10 loss induces aberrant splicing of cytoskeletal and extracellular matrix mRNAs and promotes metastatic fitness.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse HrasG12V/Rbm1OKO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers. SUMMARYRNA splicing factor mutations are common in cancer but connecting phenotypes to specific misspliced genes has been challenging. We show that RBM10 loss leads to exon inclusions of transcripts regulating ECM-cytoskeletal interactions and RAC1-GTP activation, sufficient to promote metastatic fitness.

cancer biology↗

bioRxiv2023

Laser Desorption Rapid Evaporative Ionisation Mass Spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionisation technique for mass spectrometry, laser desorption rapid evaporative ionisation mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100 % accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas Quinolone Signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were significantly reduced in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCEThis work demonstrates that a high-throughput ambient ionisation mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and markedly reduces production of the virulence factors pyocyanin and elastase in this bacterium. This approach has the potential to be widely applicable to the characterisation of the stress responses of bacteria.

microbiology↗

bioRxiv2023

Integrative single-cell characterization of frugivory adaptations in the bat kidney and pancreas

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we used integrative single-cell sequencing on insectivorous and frugivorous bat kidneys and pancreases and identified key cell population, gene expression and regulatory element differences associated with frugivorous adaptation that also relate to human disease, particularly diabetes. We found an increase in collecting duct cells and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the frugivore kidney. In the frugivorous pancreas, we observed an increase in endocrine and a decrease in exocrine cells and differences in genes and regulatory elements involved in insulin regulation. Combined, our work provides novel insights into frugivorous adaptation that also could be leveraged for therapeutic purposes.

genomics↗

bioRxiv2023

Diverging Parkinson's Disease Pathology between GBAN370S ,LRRK2G2019S and engineered SNCAA53T iPSC-derived Dopaminergic Neurons

Multiple neurodegenerative disorders, including Parkinsons disease (PD) and Alzheimers disease-associated dementia (ADAD), are linked with dopaminergic (DA) neuron death and a resulting reduction in dopamine levels in the brain. DA neuron degeneration and the risk of developing PD is connected to genetic mutations affiliated with lysosomal function and protein degradation. Accessible human cellular models for PD-relevant genetic mutations are needed to investigate mechanisms of DA cell death and define points of therapeutic intervention. Human induced pluripotent stem cell (iPSC)-derived midbrain DA neurons offer a developmentally and physiologically relevant in vitro model for investigating PD pathogenic mechanisms across genetic backgrounds. In this study, we generated DA neurons using iPSCs from two clinically diagnosed PD patients, one harboring an inherited GBAN370S mutation and the other a mutation in LRRK2G2019S and compared pathophysiology against DA neurons from genetically engineered SNCAA53T iPSCs and its isogenic apparently healthy normal (AHN) iPSCs. Our results present a novel phenotype for GBAN370S and LRRK2G2019S derived DA neurons, showing that they produced and released significantly more dopamine compared to the AHN and SNCAA53T mutant DA neurons. All mutant DA neurons developed deficient glucocerebrosidase (GCase) activity, increased mitochondrial stress, aberrant neuronal activity patterns, and increased -synuclein accumulation. Together these data suggest potentially divergent origins of PD pathogenesis in GBAN370S and LRRK2G2019S DA neurons. In addition, compound screening confirmed that GCase modulators can rescue enzyme activity and impact neural activity across all DA mutant neurons, to varying degrees. These data demonstrate unique in vitro phenotypes associated with PD and suggest a diversity of underlying mechanisms across different genetic backgrounds. Together, the cell lines used in this study present a valuable tool for new therapeutic discovery.

neuroscience↗

bioRxiv2018

The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSP (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.

cell biology↗

bioRxiv2018

The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

Somatic mutations in the genes encoding components of the spliceosome occur frequently in human neoplasms, including myeloid dysplasias and leukemias and less often in solid tumors. One of the affected factors, U2AF1, is involved in splice site selection, and the most common change, S34F, alters a conserved nucleic acid binding domain, recognition of the 3-splice site, and alternative splicing of many mRNAs. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1, showing that it binds mature mRNA in the cytoplasm and negatively regulates mRNA translation. This splicing-independent role of U2AF1 is altered by the S34F mutation, and polysomal profiling indicates the mutation affects translation of hundreds of mRNA. One functional consequence is increased synthesis of the secreted chemokine interleukin 8 which contributes to metastasis, inflammation, and cancer progression in mice and humans.

cell biology↗

bioRxiv2018

Impaired hematopoiesis and leukemia development in mice with a \"knock-in\" allele of U2af1(S34F)

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent \"knock-in\" alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-Ethyl-N-Nitrosourea (ENU). Overall, three of sixteen ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss-or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzfl. However, the engineered U2af1(S34F) missense mutation reverted to wild type (WT) in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.\n\nSIGNIFICANCE STATEMENTSomatic mutations in four splicing factor genes (U2AF1, SRSF2, SF3B1, and ZRSR2) are found in MDS and MDS-related AML, blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized, in part due to the paucity of disease-relevant models. Here, we report the establishment of mouse models to study the most common U2AF1 mutation, U2af1(S34F). Production of the mutant protein specifically in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice.

cancer biology↗