Karl Burgess

Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency

There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-invasive tools with which to address the stem cell niche.

Toward Global Metabolomics Analysis with Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: Improved Metabolite Identification by Retention Time Prediction

Metabolomics is an emerging field of postgenomic biology concerned with comprehensive analysis of small molecules in biological systems. However, difficulties associated with the identification of detected metabolites currently limit its application. Here we demonstrate that a retention time prediction model can improve metabolite identification on a hydrophilic interaction chromatography (HILIC)-high-resolution mass spectrometry metabolomics platform. A quantitative structure retention relationship (QSRR) model, incorporating six physicochemical variables in a multiple-linear regression based on 120 authentic standard metabolites, shows good predictive ability for retention times of a range of metabolites (cross-validated R(2) = 0.82 and mean squared error = 0.14). The predicted retention times improved metabolite identification by removing 40% of the false identifications that occurred with identification by accurate mass alone. The importance of this procedure was demonstrated by putative identification of 690 metabolites in extracts of the protozoan parasite Trypanosoma brucei, thus allowing identified metabolites to be mapped onto an organism-wide metabolic network, providing opportunities for future studies of cellular metabolism from a global systems biology perspective.

IDEOM: An Excel interface for analysis of LC-MS based metabolomics data

SUMMARY The application of emerging metabolomics technologies to the comprehensive investigation of cellular biochemistry has been limited by bottlenecks in data processing, particularly noise filtering and metabolite identification. IDEOM provides a user-friendly data processing application that automates filtering and identification of metabolite peaks, paying particular attention to common sources of noise and false identifications generated by liquid chromatography-mass spectrometry (LC-MS) platforms. Building on advanced processing tools such as mzMatch and XCMS, it allows users to run a comprehensive pipeline for data analysis and visualization from a graphical user interface within Microsoft Excel, a familiar program for most biological scientists. AVAILABILITY AND IMPLEMENTATION IDEOM is provided free of charge at http://mzmatch.sourceforge.net/ideom.html, as a macro-enabled spreadsheet (.xlsb). Implementation requires Microsoft Excel (2007 or later). R is also required for full functionality. CONTACT michael.barrett@glasgow.ac.uk SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Stable Isotope-Assisted Metabolomics for Network-Wide Metabolic Pathway Elucidation

The combination of high-resolution LC–MS-based untargeted metabolomics with stable isotope tracing provides a global overview of the cellular fate of precursor metabolites. This methodology enables detection of putative metabolites from biological samples and simultaneous quantification of the pattern and extent of isotope labeling. Labeling of Trypanosoma brucei cell cultures with 50% uniformly 13C-labeled glucose demonstrated incorporation of glucose-derived carbon into 187 of 588 putatively identified metabolites in diverse pathways including carbohydrate, nucleotide, lipid, and amino acid metabolism. Labeling patterns confirmed the metabolic pathways responsible for the biosynthesis of many detected metabolites, and labeling was detected in unexpected metabolites, including two higher sugar phosphates annotated as octulose phosphate and nonulose phosphate. This untargeted approach to stable isotope tracing facilitates the biochemical analysis of known pathways and yields rapid identification of previously unexplored areas of metabolism.

Nanotopographical Effects on Mesenchymal Stem Cell Morphology and Phenotype

There is a rapidly growing body of literature on the effects of topography and critically, nanotopography on cell adhesion, apoptosis and differentiation. Understanding the effects of nanotopography on cell adhesion and morphology and the consequences of cell shape changes in the nucleus, and consequently, gene expression offers new approaches to the elucidation and potential control of stem cell differentiation. In the current study we have used molecular approaches in combination with immunohistology and transcript analysis to understand the role of nanotopography on mesenchymal stem cell morphology and phenotype. Results demonstrate large changes in cell adhesion, nucleus and lamin morphologies in response to the different nanotopographies. Furthermore, these changes relate to alterations in packing of chromosome territories within the interphase nucleus. This, in turn, leads to changes in transcription factor activity and functional (phenotypical) signalling including cell metabolism. Nanotopography provides a useful, non‐invasive tool for studying cellular mechanotransduction, gene and protein expression patterns, through effects on cell morphology. The different nanotopographies examined, result in different morphological changes in the cyto‐ and nucleo‐skeleton. We propose that both indirect (biochemical) and direct (mechanical) signalling are important in these early stages of regulating stem cell fate as a consequence of altered metabolic changes and altered phenotype. The current studies provide new insight on cell–surface interactions and enhance our understanding of the modulation of stem cell differentiation with significant potential application in regenerative medicine. J. Cell. Biochem. 115: 380–390, 2014.

Untargeted Metabolomics Reveals a Lack Of Synergy between Nifurtimox and Eflornithine against Trypanosoma brucei

A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Nanotopographical Induction of Osteogenesis through Adhesion, Bone Morphogenic Protein Cosignaling, and Regulation of MicroRNAs

It is emerging that nanotopographical information can be used to induce osteogenesis from mesenchymal stromal cells from the bone marrow, and it is hoped that this nanoscale bioactivity can be utilized to engineer next generation implants. However, the osteogenic mechanism of surfaces is currently poorly understood. In this report, we investigate mechanism and implicate bone morphogenic protein (BMP) in up-regulation of RUNX2 and show that RUNX2 and its regulatory miRNAs are BMP sensitive. Our data demonstrate that osteogenic nanotopography promotes colocalization of integrins and BMP2 receptors in order to enhance osteogenic activity and that vitronectin is important in this interface. This provides insight that topographical regulation of adhesion can have effects on signaling cascades outside of cytoskeletal signaling and that adhesions can have roles in augmenting BMP signaling.

Pyrimidine Salvage in Trypanosoma Brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidines

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes.

Pathos: A web facility that uses metabolic maps to display experimental changes in metabolites identified by mass spectrometry

This work describes a freely available web-based facility which can be used to analyse raw or processed mass spectrometric data from metabolomics experiments and display the metabolites identified – and changes in their experimental abundance – in the context of the metabolic pathways in which they occur. The facility, Pathos (http://motif.gla.ac.uk/Pathos/), employs Java servlets and is underpinned by a relational database populated from the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Input files can contain either raw m/z values from experiments conducted in different modes, or KEGG or MetaCyc IDs assigned by the user on the basis of the m/z values and other criteria. The textual output lists the KEGG pathways on an XHTML page according to the number of metabolites or potential metabolites that they contain. Filtering by organism is also available. For metabolic pathways of interest, the user is able to retrieve a pathway map with identified metabolites highlighted. A particular feature of Pathos is its ability to process relative quantification data for metabolites identified under different experimental conditions, and to present this in an easily comprehensible manner. Results are colour-coded according to the degree of experimental change, and bar charts of the results can be generated interactively from either the text listings or the pathway maps. The visual presentation of the output from Pathos is designed to allow the rapid identification of metabolic areas of potential interest, after which particular results may be examined in detail. Copyright