Biology subjects

Cooke, J.

Publications and source records attributed to Cooke, J..

7 recordsLinked to original sources

bioRxiv2025

Simulated metabolic profiles reveal biases in pathway analysis methods

Initially developed for transcriptomics data, pathway analysis (PA) methods can introduce biases when applied to metabolomics data, especially if input parameters are not chosen with care. This is particularly true for exometabolomics data, where exported metabolites may be far from internal disruptions in the organism. Experimentally evaluating PA methods is practically impossible when the samples "true" metabolic disruption is unknown. Using in silico metabolic modelling, we simulated metabolic profiles for entire pathway knockouts, providing both a known disruption site as well as a simulated metabolic profile for use in PA methods. PA should be able to detect the known disrupted pathway among the significantly enriched pathways for that profile. Through network-level statistics, visualisation, and graph-based metrics, we show that even when a given pathway is completely blocked, it may not be detected as significantly enriched with PA. This work highlights how some metabolomics data may not be suited to typical PA methods, and serves as a benchmark for analysing, improving and developing new PA tools.

bioinformatics↗

bioRxiv2024

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

Autologous photoreceptor cell replacement therapy shows great promise for treating patients with multiple forms of inherited retinal degenerative blindness. Specifically, in disorders such as retinitis pigmentosa and Stargardts disease, selective death of photoreceptor cells results in irreversible blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of photoreceptor cells required for these therapies. However, in addition to photoreceptor cells, retinal organoids also contain many other retinal cell types. Therefore, approaches for isolating fate committed photoreceptors from dissociated retinal organoids are desirable to produce photoreceptor cell replacement therapies. In this work, we present a partial dissociation strategy, which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. Given that this technique can be performed with only standard plasticware and cGMP compliant reagents, it is an ideal candidate for use in the preparation of clinical cell therapies.

bioengineering↗

bioRxiv2024

PathIntegrate: Multivariate modelling approaches for pathway-based multi-omics data integration

As terabytes of multi-omics data are being generated, there is an ever-increasing need for methods facilitating the integration and interpretation of such data. Current multi-omics integration methods typically output lists, clusters, or subnetworks of molecules related to an outcome. Even with expert domain knowledge, discerning the biological processes involved is a time-consuming activity. Here we propose PathIntegrate, a method for integrating multi-omics datasets based on pathways, designed to exploit knowledge of biological systems and thus provide interpretable models for such studies. PathIntegrate employs single-sample pathway analysis to transform multi-omics datasets from the molecular to the pathway-level, and applies a predictive single-view or multi-view model to integrate the data. Model outputs include multi-omics pathways ranked by their contribution to the outcome prediction, the contribution of each omics layer, and the importance of each molecule in a pathway. Using semi-synthetic data we demonstrate the benefit of grouping molecules into pathways to detect signals in low signal-to-noise scenarios, as well as the ability of PathIntegrate to precisely identify important pathways at low effect sizes. Finally, using COPD and COVID-19 data we showcase how PathIntegrate enables convenient integration and interpretation of complex high-dimensional multi-omics datasets. The PathIntegrate Python package is available at https://github.com/cwieder/PathIntegrate. Author summaryOmics data, which provides a readout of the levels of molecules such as genes, proteins, and metabolites in a sample, is frequently generated to study biological processes and perturbations within an organism. Combining multiple omics data types can provide a more comprehensive understanding of the underlying biology, making it possible to piece together how different molecules interact. There exist many software packages designed to integrate multi-omics data, but interpreting the resulting outputs remains a challenge. Placing molecules into the context of biological pathways enables us to better understand their collective functions and understand how they may contribute to the condition under study. We have developed PathIntegrate, a pathway-based multi-omics integration tool which helps integrate and interpret multi-omics data in a single step using machine learning. By integrating data at the pathway rather than the molecular level, the relationships between molecules in pathways can be strengthened and more readily identified. PathIntegrate is demonstrated on Chronic Obstructive Pulmonary Disease and COVID-19 metabolomics, proteomics, and transcriptomics datasets, showcasing its ability to efficiently extract perturbed multi-omics pathways from large-scale datasets.

bioinformatics↗

bioRxiv2023

Genome scale metabolic network modelling for metabolic profile predictions

Metabolic profiling (metabolomics) aims at measuring small molecules (metabolites) in complex samples like blood or urine for human health studies. While biomarker-based assessment often relies on a single molecule, metabolic profiling combines several metabolites to create a more complex and more specific fingerprint of the disease. However, in contrast to genomics, there is no unique metabolomics setup able to measure the entire metabolome. This challenge leads to tedious and resource consuming preliminary studies to be able to design the right metabolomics experiment. In that context, computer assisted metabolic profiling can be of strong added value to design more quickly and efficiently metabolomics studies. We propose a constraint-based modelling approach which predicts in silico profiles of metabolites that are more likely to be differentially abundant under a given metabolic perturbation (e.g. due to a genetic disease), using flux simulation. In genome-scale metabolic networks, the fluxes of exchange reactions, also known as the flow of metabolites through their external transport reactions, can be simulated and compared between control and disease conditions in order to calculate changes in metabolite import and export. These import/export flux differences would be expected to induce changes in circulating biofluid levels of those metabolites, which can then be interpreted as potential biomarkers or metabolites of interest. In this study, we present SAMBA (SAMpling Biomarker Analysis), an approach which simulates fluxes in exchange reactions following a metabolic perturbation using random sampling, compares the simulated flux distributions between the baseline and modulated conditions, and ranks predicted differentially exchanged metabolites as potential biomarkers for the perturbation. We show that there is a good fit between simulated metabolic exchange profiles and experimental differential metabolites detected in plasma, such as patient data from the disease database OMIM, and metabolic trait-SNP associations found in mGWAS studies. These biomarker recommendations can provide insight into the underlying mechanism or metabolic pathway perturbation lying behind observed metabolite differential abundances, and suggest new metabolites as potential avenues for further experimental analyses. Author summaryAssociating diseases and other metabolic disruptions with physiological markers is key for diagnostic and personalised medicine. These markers can be metabolites - small molecules involved in every living beings metabolism, and can be measured in biofluids such as blood or urine using metabolic profiling (metabolomics). Nevertheless, this experimental metabolomics design needs to be tailor made for each disease to ensure that most relevant metabolites will be detected. The selection of metabolites to analyse for future experiments can be time-consuming and expensive. In this paper, we build upon an existing computational method for simulating metabolite changes in a human model. This provides a prediction of the change in biofluid abundance of every known metabolite involved in human metabolism in a potentially large number of metabolic situations. The newly introduced method produces a change score and a rank for each metabolite in each condition. We show the strong potential of the approach by comparing predictions with experimental results.

systems biology↗

bioRxiv2021

Pathway analysis in metabolomics: pitfalls and best practice for the use of over-representation analysis

Over-representation analysis (ORA) is one of the commonest pathway analysis approaches used for the functional interpretation of metabolomics datasets. Despite the widespread use of ORA in metabolomics, the community lacks guidelines detailing its best-practice use. Many factors have a pronounced impact on the results, but to date their effects have received little systematic attention in the field. We developed in-silico simulations using five publicly available datasets and illustrated that changes in parameters, such as the background set, differential metabolite selection methods, and pathway database choice, could all lead to profoundly different ORA results. The use of a non-assay-specific background set, for example, resulted in large numbers of false-positive pathways. Pathway database choice, evaluated using three of the most popular metabolic pathway databases: KEGG, Reactome, and BioCyc, led to vastly different results in both the number and function of significantly enriched pathways. Metabolomics data specific factors, such as reliability of compound identification and assay chemical bias also impacted ORA results. Simulated metabolite misidentification rates as low as 4% resulted in both gain of false-positive pathways and loss of truly significant pathways across all datasets. Our results have several practical implications for ORA users, as well as those using alternative pathway analysis methods. We offer a set of recommendations for the use of ORA in metabolomics, alongside a set of minimal reporting guidelines, as a first step towards the standardisation of pathway analysis in metabolomics. Author summaryMetabolomics is a rapidly growing field of study involving the profiling of small molecules within an organism. It allows researchers to understand the effects of biological status (such as health or disease) on cellular biochemistry, and has wide-ranging applications, from biomarker discovery and personalised medicine in healthcare to crop protection and food security in agriculture. Pathway analysis helps to understand which biological pathways, representing collections of molecules performing a particular function, are involved in response to a disease phenotype, or drug treatment, for example. Over-representation analysis (ORA) is perhaps the most common pathway analysis method used in the metabolomics community. However, ORA can give drastically different results depending on the input data and parameters used. In this work, we have established the effects of these factors on ORA results using computational simulations applied to five real-world datasets. Based on our results, we offer the research community a set of best-practice recommendations applicable not only to ORA but also to other pathway analysis methods to help ensure the reliability and reproducibility of results.

bioinformatics↗

bioRxiv2020

Alternative splicing of GluN1 gates glycine-primed internalization of NMDA receptors

N-methyl-D-aspartate receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Gating of the NMDARs requires binding of four co-agonist molecules, but the receptors can signal non-ionotropically through binding of glycine, alone, to its cognate site on GluN1. A consequence of this signalling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit is not a singular molecular species in the CNS, rather there are 8 alternatively spliced isoforms of this subunit produced by including or excluding the N1 and the C1, C2 or C2 polypeptide cassettes. Whether alternative splicing affects glycine priming signalling is unknown. Here, using recombinant NMDARs expressed heterologously we discovered that glycine priming of NMDARs critically depends on alternative splicing: the four splice isoforms lacking the N1 cassette, encoded in exon 5, are primed by glycine whereas glycine priming is blocked in the four splice variants containing the N1 cassette. On the other hand, the C-terminal cassettes - C1, C2 or C2 - had no effect on glycine priming signalling. Nor was glycine priming affected by the GluN2 subunit in the receptor. In wild-type mice we found that glycine primed internalization of synaptic NMDARs in CA1 hippocampal pyramidal neurons. With mice we engineered such that GluN1 obligatorily contained the N1 cassette, glycine did not prime synaptic NMDARs in pyramidal neurons. In contrast to pyramidal neurons, we discovered that in wild-type mice, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling non-ionotropic signalling of NMDAR by glycine and the consequential cell surface dynamics of the receptors.

neuroscience↗

bioRxiv2019

Assessing the specificity of the relationship between brain alpha oscillations and tonic pain

Recent research has pointed to alpha brain oscillations as a potential clinical biomarker for sensitivity to pain. In particular, researchers claimed that the slowing of individual alpha frequency (IAF) could be an objective marker of pain during prolonged nociceptive stimulation. However, prolonged pain has been also associated with increased IAF. To date, there is insufficient evidence to conclude that IAF can be a neural marker of pain.\n\nThe current study aimed at elucidating the conflicting findings by assessing the specificity of the relationship between brain alpha oscillations and tonic pain. We recorded electroencephalography (EEG) on healthy volunteers during exposure to consecutive 5-minute sessions of painful hot water immersion, innocuous warm water immersion and an aversive, non-painful auditory stimulus, matched by unpleasantness to the painful condition. Participants rated stimulus unpleasantness throughout each condition. We also asked participants to sit still with eyes closed and eyes open right before and after the three experimental conditions in order to obtain a robust estimate of baseline alpha. Our findings revealed both increased and decreased IAF during tonic hot stimulation depending on the alpha range targeted (7-13 Hz vs. 8-10 Hz). In addition, they provide mild evidence for a negative relationship between IAF and the unpleasantness of the experience. Finally, we could not identify a difference between IAF during tonic hot temperature and during tonic auditory experience. Altogether, these findings emphasize a twofold frequency pattern (increase vs. decrease) for IAF during tonic thermal pain, thus indicating the need for robust methodological scrutiny of IAF as a neural marker of pain.

neuroscience↗