Large-scale analysis of microarray data to identify molecular signatures of mouse pluripotent stem cells

Publicly-available microarray data constitutes a huge resource for researchers in biological science. A wealth of microarray data is available for the model organism – the mouse. Pluripotent embryonic stem (ES) cells are able to give rise to all of the adult tissues of the organism and, as such, are much-studied for their myriad applications in regenerative medicine. Fully differentiated, somatic cells can also be reprogrammed to pluripotency to give induced pluripotent stem cells (iPSCs). ES cells progress through a range of cellular states between ground state pluripotent stem cells, through the primed state ready for differentiation, to actual differentiation. Microarray data available in public, online repositories is annotated with several important fields, although this accompanying annotation often contains issues which can impact its usefulness to human and / or programmatic interpretation for downstream analysis. This thesis assembles and makes available to the research community the largest-to-date pluripotent mouse ES cell (mESC) microarray dataset and details the manual annotation of those samples for several key fields to allow further investigation of the pluripotent state in mESCs. Microarray samples from a given laboratory or experiment are known to be similar to each other due to batch effects. The same has been postulated about samples which use the same cell line. This work therefore precedes the investigation of transcriptional events in mESCs with an investigation into whether a sample's cell line or source laboratory is a greater contributor to the similarity between samples in this collected pluripotent mESC dataset using a method employing Random Submatrix Total Variability, and so named RaSToVa. Further, an extension of the same permutation and analysis method is developed to enable Discovery of Annotation-Linked Gene Expression Signatures (DALGES), and this is applied to the gathered data to provide the first large-scale analysis of transcriptional profiles and biological pathway activity of three commonly-used mESC cell lines and a selection of iPSC samples, seeking insight into potential biological differences that may result from these. This work then goes on to re-order the pluripotent mESC data by markers of known pluripotency states, from ground state pluripotency through primed pluripotency to earliest differentiation and analyses changes in gene expression and biological pathway activity across this spectrum, using differential expression and a window-scanning approach, seeking to recapitulate transcriptional patterns known to occur in mESCs, revealing the existence of putative “early” and “late” naïve pluripotent states and thereby identifying several lines of enquiry for in-laboratory investigation

Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer to fibroblasts

Background: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML), the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC) which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming. Results: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours. Conclusions: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection

PASI: A novel pathway method to identify delicate group effects

Pathway analysis is a common approach in diverse biomedical studies, yet the currently-available pathway tools do not typically support the increasingly popular personalized analyses. Another weakness of the currently-available pathway methods is their inability to handle challenging data with only modest group-based effects compared to natural individual variation. In an effort to address these issues, this study presents a novel pathway method PASI (Pathway Analysis for Sample-level Information) and demonstrates its performance on complex diseases with different levels of group-based differences in gene expression. PASI is freely available as an R package

Systematic evaluation of differential splicing tools for RNA-seq studies

Differential splicing (DS) is a post-transcriptional biological process with critical, wide-ranging effects on a plethora of cellular activities and disease processes. To date, a number of computational approaches have been developed to identify and quantify differentially spliced genes from RNA-seq data, but a comprehensive intercomparison and appraisal of these approaches is currently lacking. In this study, we systematically evaluated 10 DS analysis tools for consistency and reproducibility, precision, recall and false discovery rate, agreement upon reported differentially spliced genes and functional enrichment. The tools were selected to represent the three different methodological categories: exon-based (DEXSeq, edgeR, JunctionSeq, limma), isoform-based (cuffdiff2, DiffSplice) and event-based methods (dSpliceType, MAJIQ, rMATS, SUPPA). Overall, all the exon-based methods and two event-based methods (MAJIQ and rMATS) scored well on the selected measures. Of the 10 tools tested, the exon-based methods performed generally better than the isoform-based and event-based methods. However, overall, the different data analysis tools performed strikingly differently across different data sets or numbers of samples

In utero exposures to perfluoroalkyl substances and the human fetal liver metabolome in Scotland : a cross-sectional study

The Fetal Human study was funded by the UK Medical Research Council (MR/L010011/1) to PAF, PJO’S, JPI, DCH, and AD, by the Seventh Framework Programme of the European Union under Grant Agreement 212885, and by NHS Grampian Endowments grants (08/02, 09/12, 13/56, and 15/1/010) to PAF. The metabolomics and exposomics study was supported by the Swedish Research Council (to TH and MO; grants 2016-05176 and 2020-03674), Formas (to TH and MO; grant 2019-00869), Novo Nordisk Foundation (to TH; grant NNF20OC0063971), Research Council of Finland (to MO; grant 333981), the Inflammation in human early life: targeting impacts on life-course health consortium funded by the Horizon Europe Program of the European Union (Grant Agreement 101094099 to MO, TH, and PAF), and the framework of the European Partnership for the Assessment of Risks from Chemicals, and has received funding from the European Union's Horizon Europe research and innovation programme (grant agreement 101057014). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The authors would like to thank (1) the Fowler team members, NHS Grampian research nurses, and staff at the Pregnancy Counselling Service for their essential work in recruiting, collecting, and processing fetuses; and (2) the Centre for Genome Enabled Biology and Medicine, University of Aberdeen, for carrying out the RNA-Seq. The RNA-Seq data analysis was supported by use of the University of Aberdeen Maxwell High Performance Computer Cluster.Peer reviewedPublisher PD

Perfluoroalkyl substances are increased in patients with late-onset ulcerative colitis and induce intestinal barrier defects ex vivo in murine intestinal tissue

Background Environmental factors are strongly implicated in late-onset of inflammatory bowel disease. Here, we investigate whether high levels of perfluoroalkyl substances are associated with (1) late-onset inflammatory bowel disease, and (2) disturbances of the bile acid pool. We further explore the effect of the specific perfluoroalkyl substance perfluorooctanoic acid on intestinal barrier function in murine tissue. Methods Serum levels of perfluoroalkyl substances and bile acids were assessed by ultra-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer in matched samples from patients with ulcerative colitis (n = 20) and Crohn's disease (n = 20) diagnosed at the age of >= 55 years. Age and sex-matched blood donors (n = 20), were used as healthy controls. Ex vivo Ussing chamber experiments were performed to assess the effect of perfluorooctanoic acid on ileal and colonic murine tissue (n = 9). Results The total amount of perfluoroalkyl substances was significantly increased in patients with ulcerative colitis compared to healthy controls and patients with Crohn's disease (p Discussion Our results demonstrate that perfluoroalkyl substances levels are increased in patients with late-onset ulcerative colitis and may contribute to the disease by inducing a dysfunctional intestinal barrier.</p

Exposure to environmental contaminants is associated with altered hepatic lipid metabolism in non-alcoholic fatty liver disease

Background & aims: Recent experimental models and epidemiological studies suggest that specific environmental contaminants (ECs) contribute to the initiation and pathology of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms linking EC exposure with NAFLD remain poorly understood and there is no data on their impact on the human liver metabolome. Herein, we hypothesized that exposure to ECs, particularly perfluorinated alkyl substances (PFAS), impacts liver metabolism, specifically bile acid metabolism. Methods: In a well-characterized human NAFLD cohort of 105 individuals, we investigated the effects of EC exposure on liver metabolism. We characterized the liver (via biopsy) and circulating metabolomes using 4 mass spectrometry-based analytical platforms, and measured PFAS and other ECs in serum. We subsequently compared these results with an exposure study in a PPARa-humanized mouse model. Results: PFAS exposure appears associated with perturbation of key hepatic metabolic pathways previously found altered in NAFLD, particularly those related to bile acid and lipid metabolism. We identified stronger associations between the liver metabolome, chemical exposure and NAFLD-associated clinical variables (liver fat content, HOMA-IR), in females than males. Specifically, we observed PFAS-associated upregulation of bile acids, triacylglycerols and ceramides, and association between chemical exposure and dysregulated glucose metabolism in females. The murine exposure study further corroborated our findings, vis-a-vis a sex-specific association between PFAS exposure and NAFLD-associated lipid changes. Conclusions: Females may be more sensitive to the harmful impacts of PFAS. Lipid-related changes subsequent to PFAS exposure may be secondary to the interplay between PFAS and bile acid metabolism. Lay summary: There is increasing evidence that specific environmental contaminants, such as perfluorinated alkyl substances (PFAS), contribute to the progression of non-alcoholic fatty liver disease (NAFLD). However, it is poorly understood how these chemicals impact human liver metabolism. Here we show that human exposure to PFAS impacts metabolic processes associated with NAFLD, and that the effect is different in females and males. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.Peer reviewe

Systems biology approaches to study lipidomes in health and disease

Lipids have many important biological roles, such as energy storage sources, structural components of plasma membranes and as intermediates in metabolic and signaling pathways. Lipid metabolism is under tight homeostatic control, exhibiting spatial and dynamic complexity at multiple levels. Consequently, lipid-related disturbances play important roles in the pathogenesis of most of the common diseases. Lipidomics, defined as the study of lipidomes in biological systems, has emerged as a rapidly-growing field. Due to the chemical and functional diversity of lipids, the application of a systems biology approach is essential if one is to address lipid functionality at different physiological levels. In parallel with analytical advances to measure lipids in biological matrices, the field of computational lipidomics has been rapidly advancing, enabling modeling of lipidomes in their pathway, spatial and dynamic contexts. This review focuses on recent progress in systems biology approaches to study lipids in health and disease, with specific emphasis on methodological advances and biomedical applications.</div

Metabolic signatures across the full spectrum of non-alcoholic fatty liver disease

Background & AimsNon-alcoholic fatty liver disease (NAFLD) is a progressive liver disease with potentially severe complications including cirrhosis and hepatocellular carcinoma. Previously, we have identified circulating lipid signatures associating with liver fat content and non-alcoholic steatohepatitis (NASH). Here, we develop a metabolomic map across the NAFLD spectrum, defining interconnected metabolic signatures of steatosis (non-alcoholic fatty liver, NASH, and fibrosis).MethodsWe performed mass spectrometry analysis of molecular lipids and polar metabolites in serum samples from the European NAFLD Registry patients (n = 627), representing the full spectrum of NAFLD. Using various univariate, multivariate, and machine learning statistical approaches, we interrogated metabolites across 3 clinical perspectives: steatosis, NASH, and fibrosis.ResultsFollowing generation of the NAFLD metabolic network, we identify 15 metabolites unique to steatosis, 18 to NASH, and 15 to fibrosis, with 27 common to all. We identified that progression from F2 to F3 fibrosis coincides with a key pathophysiological transition point in disease natural history, with n = 73 metabolites altered.ConclusionsAnalysis of circulating metabolites provides important insights into the metabolic changes during NAFLD progression, revealing metabolic signatures across the NAFLD spectrum and features that are specific to NAFL, NASH, and fibrosis. The F2–F3 transition marks a critical metabolic transition point in NAFLD pathogenesis, with the data pointing to the pathophysiological importance of metabolic stress and specifically oxidative stress.</p