Biology subjects

Steele, J. R.

Publications and source records attributed to Steele, J. R..

6 recordsLinked to original sources

bioRxiv2024

Analysis and visualization of quantitative proteomics data using FragPipe-Analyst

The FragPipe computational proteomics platform is gaining widespread popularity among the proteomics research community because of its fast processing speed and user-friendly graphical interface. Although FragPipe produces well-formatted output tables that are ready for analysis, there is still a need for an easy-to-use and user-friendly downstream statistical analysis and visualization tool. FragPipe-Analyst addresses this need by providing an R shiny web server to assist FragPipe users in conducting downstream analyses of the resulting quantitative proteomics data. It supports major quantification workflows including label-free quantification, tandem mass tags, and data-independent acquisition. FragPipe-Analyst offers a range of useful functionalities, such as various missing value imputation options, data quality control, unsupervised clustering, differential expression (DE) analysis using Limma, and gene ontology and pathway enrichment analysis using Enrichr. To support advanced analysis and customized visualizations, we also developed FragPipeAnalystR, an R package encompassing all FragPipe-Analyst functionalities that is extended to support site-specific analysis of post-translational modifications (PTMs). FragPipe-Analyst and FragPipeAnalystR are both open-source and freely available.

bioinformatics↗

bioRxiv2024

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates and citrate synthase activity but that these deficits can be offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggests that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

pathology↗

bioRxiv2023

Structure of a Rhs effector clade domain identifies new mechanistic insights into type VI secretion system toxin delivery.

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins directly into adjacent bacteria, often providing a competitive advantage. Rearrangement hotspot (Rhs) effectors are known T6SS cargo effectors, but the precise delivery and activation of these toxins is poorly defined. We present the structure of a novel T6SS Rhs effector (Tse15) from the multidrug resistant nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered y-cocoon Rhs domain with an N-terminal -helical clade domain and an unfolded C-terminal toxin domain located entirely inside the Rhs cage. We identify that Tse15 is cleaved into three domains, through two independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for the cleavage of the N-terminal clade domain. Proteomic analyses showed that the N-terminal clade and toxin domains, but not the y-strand rich Rhs cage, are delivered outside of the cell, suggesting a novel mechanism for Rhs toxin delivery and activation. Our findings suggest that this delivery mechanism requires an interaction between the N-terminal clade and toxin domains, with the clade domain acting as the internal chaperone to mediate tethering of the toxin to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggest this may be a common mechanism for T6SS toxin delivery.

microbiology↗

bioRxiv2023

Hypoxic injury triggers maladaptive repair in human kidney organoids

Acute kidney injury (AKI) is a common clinical disorder linked to high rates of illness and death. Ischemia is a leading cause of AKI, which can result in chronic kidney disease (CKD) through maladaptive repair marked by impaired epithelial regeneration, inflammation, and metabolic dysregulation. There are no targeted therapies for AKI or to prevent progression to CKD and insight into human disease mechanisms remains limited. Here we show that human kidney organoids recapitulate key molecular and metabolic signatures of AKI and maladaptive repair in response to hypoxic injury. Transcriptional, proteomic, and metabolomic profiling revealed tubular injury, cell death, cell cycle arrest and metabolic reprogramming in organoids exposed to hypoxia. Following return to normoxic conditions, injured organoids had increased signatures of TNF and NF-{kappa}B signalling pathways and S100A8/9, associated with maladaptive repair. Single cell RNA sequencing localized AKI and maladaptive repair markers including GDF15, MMP7, ICAM1, IL32, SPP1, C3 and CCN1 to injured tubules. Metabolic phenotypes linked to CKD were also evident, including dysregulated gluconeogenesis, altered amino acid metabolism and lipid peroxidation. iPSC-derived macrophages incorporated into organoids displayed a robust activation and inflammatory response to hypoxia. Spatial transcriptomics revealed a shift from a tissue resident-like to inflammatory macrophage states and localized effects on tubular injury and inflammation. This multi-omic analysis defines conserved mechanisms of human ischemic AKI and maladaptive repair, highlighting new opportunities to test therapeutics and model immune-mediated interactions.

systems biology↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2022

Systems-level investigation of mucopolysaccharidosis IIIA identifies deficient synaptic activity as a key driver of disease progression

Mucopolysaccharidoses are lysosomal storage diseases that collectively represent a major cause of lethal, treatment-refractory childhood dementias 1-7 Clinically-useful interventions are hampered due to an incomplete understanding of their neuropathological origins. Using the zebrafish sgsh model of mucopolysaccharidosis IIIA 8 (MPS IIIA, Sanfilippo syndrome A), we conducted several omics-based analyses, and developed and benchmarked a novel bioinformatic feature classification and ranking model for high-throughput datasets - ExIR - to prioritise important features in the progression of neurological manifestations of the disease. We find that the massive endolysosomal burden resulting from increased lysosomal storage of heparan sulfate and other secondarily accumulating substrates, such as sphingolipids, induces abnormal microtubule organisation and vesicle trafficking in neurons. This results in a gradual impairment of synaptic vesicle localisation at the presynaptic terminal and consequently impaired neuronal activity. Importantly, the endolysosomal phenotype in MPS IIIA zebrafish well-precedes the onset of neural pathology, though the larval MPS IIIA brain was found to be more susceptible to perturbation than wild type siblings. Collectively, these analyses demonstrate the presence of a progressive functional neurodegenerative phenotype underpinning neurological disease in MPS IIIA. Our findings provide direct mechanistic evidence linking the well-described lysosomal storage basis for MPS IIIA to its disproportionately severe neural clinical involvement, enabling development and refinement of future therapeutic interventions for this currently untreatable disorder. HighlightsO_LIMPS IIIA represents one of the most common causes of broadly fatal childhood dementia, but the mechanisms underlying disease progression are poorly understood. C_LIO_LIThe first systems-level analyses of disease state and progression in the CNS of an MPS IIIA animal model were performed. C_LIO_LIExperimental data-based Integrative Ranking (ExIR) was developed to provide unbiased prioritisation and classification of biological data as drivers, biomarkers and mediators of biological processes from high-throughput data at a systems level. C_LIO_LIApplication of ExIR to a transcriptomic and proteomic analyses of a zebrafish model of MPS IIIA implies progressive deficiencies in synaptic activity as a key driver of disease progression correlating with progressive neuronal endolysosomal burden and secondary storage diseases. C_LIO_LIA novel unifying explanation of pathobiology and progression of MPS IIIA facilitates identification of clinically targetable features and may be generalised to other neuronopathic storage disorders. C_LI

neuroscience↗