Complex metaprobe production and characterisation for metabolic studies in relation to bowel health

Introduction - Colorectal cancer is the third most common cancer in the UK and the second most common cause of cancer mortality. There is increasing evidence that the presence of a systemic inflammatory response plays an important role in predicting survival in patients with colorectal cancer. The Glasgow Prognostic Score includes a measure of systemic inflammatory response and has been used successfully as a predictor of survival rate in cancer patients. Butyric acid is one of the short chain fatty acids (SCFA) produced by colonic bacteria through fermentation of dietary non-digestible carbohydrate (NDC). This simple organic acid is known to have anti-inflammatory and possibly direct anti-neoplastic effects. The inaccessibility of the human colon means that little is known of the in vivo rate of butyrate production, its metabolic fate or the foodstuffs that may maximise its production. Butyrate may be produced in greater quantities from other SCFA such as acetate and lactate rather than by direct production by saccharolytic bacteria. Specialist organisms may be responsible for producing butyrate through conversion of other SCFA which are produced by fermentation in the colon. The distal site of butyrate production may be a very important feature and the implication that slowly fermentable NDC, such as resistant starch, may be significant. Previous Work - The European Union project EUROSTARCH (www.eurostarch.org) investigated the way in which different starchy foods are digested and metabolised within the body. The results of this project have informed the development of low GI foods. Results have also increased our knowledge of NDC fermentation and promise to lead to improvements in human bowel health. We pioneered the measurement of starch digestion and fermentation during this project. What are Metaprobes? The term metaprobe is used to describe stable isotope labelled tracers. Complex metaprobes are produced by biological synthesis e.g. the introduction of 13C enriched CO2 gas during the production of crops. Simple metaprobes are produced by chemical synthesis e.g. urea. We can produce complex metaprobes by labelling staple foods such as cereals using the stable isotope 13C incorporated from enriched 13CO2 during anthesis of the crops. Complex metaprobes are unique tools with which to study human metabolism in health and disease and they can be used in several ways. The labelled cereals allow in vivo measurement of butyrate production from dietary NDC and to permit its production to be monitored. Hypothesis - 30% of cancer deaths in the UK are bowel related. Butyric acid produced naturally through fermentation of dietary carbohydrates, is a potent and natural anti-inflammatory agent which can protect against cancer. We urgently need to develop methods to quantify butyrate production in vivo. Aims - Three aims are recognised: optimising the preparation of complex metaprobes of barley and wheat grain; optimising yield and 13C-enrichment of whole grain and its starch and protein fractions; using complex metaprobes to compare SCFA production in vivo. Plan of Work - This project will include a review of complex metaprobe production for human nutrition and health studies in relation to bowel health. The practical work will include production and characterisation of complex metaprobes (13C-labelled cereals) and their application to study butyrate production in a pilot study on volunteer human subjects. I have successfully grown cereal crops with different varieties of barley and wheat in glasshouse conditions. During the anthesis phase, the plants were placed in growth cabinets at 18oC with 18 hours of light in every 24 hours. The plants were labelled using 13C enriched CO2 gas and are incubated for a further 24 hours and returned to the glasshouse. Examples of the harvest are Wheat var Paragon produced 1.7 Kg labelled grain at 0.278 ape 13C enrichment and Barley var Cellar produced 3.4 Kg labelled grain at 0.198 ape 13C enrichment. Some of the 13C labelled grain has already been used for human nutrition studies. The 13C-enrichment of starch glucose has been measured by liquid chromatography isotope ratio mass spectrometry (LC-IRMS) analysis. These cereal grains can be incorporated into common foodstuffs for in vivo studies of the production of butyrate and other SCFA. Volunteers were fed two meals made from 13C-enriched barley grain: porridge and whole grain barley. Whole grains being rich in resistant starch. Samples of urine will be used to measure butyrate production. Breath samples will be used to measure oxidation of the 13C-enriched barley, which is a global measure of digestion and fermentation. Laboratory procedures such as ultrafiltration and solid phase extraction will be applied to urine samples. These sample preparation procedures will be followed by SCFA analysis by GC-IRMS. Future Direction - Future clinical trials will be considered with Prof Donald C McMillan, University Dept of Surgery, Glasgow Royal Infirmary as a means of identifying the role of butyrate in colonic cancer. The current screening process recognises three phases: early stage diagnosis; polyp development; diagnosis and treatment of advance stage colonic cancer. We wish to study the role of butyrate and its production using our newly developed complex metaprobe tools

Explaining participation in the informal economy a purchaser perspective

Purpose Participation in the informal economy has been predominantly explained from a supply-side perspective by evaluating the rationales for people working in this sphere. Recognising that many transactions in the informal economy are often instigated by customers, exemplified by purchasers asking “how much for cash?”, the aim of this paper is to explain the informal economy from a demand-side perspective by evaluating citizens‟ rationales for making purchases in the informal economy. Here, we test three potential explanations for acquiring goods and services in the informal economy, grounded in rational economic actor, social actor and formal economy imperfections theoretical perspectives. Methodology To do this, a 2013 Eurobarometer survey, involving 27,563 face-to-face interviews conducted in 28 European Union member states is reported. Findings The finding is that all three rationales apply but the weight given to each varies across populations. A multinomial logit regression analysis then pinpoints the specific groups variously using the informal economy to obtain a lower price, for social or redistributive rationales, or due to the failures of the formal economy in terms of the availability, speed and quality of provision. Practical Implications The outcome is to reveal that the policy approach of changing the cost/benefit ratios confronting purchasers will only be effective for those purchasers citing a lower price as their prime rationale. Different policy measures will be required for those making informal economy purchases due to the shortcomings of the formal economy, and for social ends. These policy measures are then discussed. Originality/value The value and originality of this paper is that it explains participation in the informal economy from a purchaser, rather than the predominant supplier, perspective

First Cultivation and Characterization of Mycobacterium ulcerans from the Environment

Mycobacterium ulcerans infection, or Buruli ulcer, is the third most common mycobacteriosis of humans worldwide, after tuberculosis and leprosy. Buruli ulcer is a neglected, devastating, necrotizing disease, sometimes producing massive, disfiguring ulcers, with huge social impact. Buruli ulcer occurs predominantly in impoverished, humid, tropical, rural areas of Africa, where the incidence has been increasing, surpassing tuberculosis and leprosy in some regions. Besides being a disease of the poor, Buruli ulcer is a poverty-promoting chronic infectious disease. There is strong evidence that M. ulcerans is not transmitted person to person but is an environmental pathogen transmitted to humans from its aquatic niches. However, until now M. ulcerans has not been isolated in pure culture from environmental sources. This manuscript describes the first isolation, to our knowledge, of M. ulcerans in pure culture from an environmental source. This strain, which is highly virulent for mice, has microbiological features typical of African strains of M. ulcerans and was isolated from an aquatic insect from a Buruli ulcer–endemic area in Benin, West Africa. Our findings support the concept that M. ulcerans is a pathogen of humans with an aquatic environmental niche and will have positive consequences for the control of this neglected and socially important tropical disease

Eight common genetic variants associated with serum dheas levels suggest a key role in ageing mechanisms

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant circulating steroid secreted by adrenal glands-yet its function is unknown. Its serum concentration declines significantly with increasing age, which has led to speculation that a relative DHEAS deficiency may contribute to the development of common age-related diseases or diminished longevity. We conducted a meta-analysis of genome-wide association data with 14,846 individuals and identified eight independent common SNPs associated with serum DHEAS concentrations. Genes at or near the identified loci include ZKSCAN5 (rs11761528; p = 3.15×10-36), SULT2A1 (rs2637125; p = 2.61×10-19), ARPC1A (rs740160; p = 1.56×10-16), TRIM4 (rs17277546; p = 4.50×10-11), BMF (rs7181230; p = 5.44×10-11), HHEX (rs2497306; p = 4.64×10-9), BCL2L11 (rs6738028; p = 1.72×10-8), and CYP2C9 (rs2185570; p = 2.29×10-8). These genes are associated with type 2 diabetes, lymphoma, actin filament assembly, drug and xenobiotic metabolism, and zinc finger proteins. Several SNPs were associated with changes in gene expression levels, and the related genes are connected to biological pathways linking DHEAS with ageing. This study provides much needed insight into the function of DHEAS

Extent, causes, and consequences of small RNA expression variation in human adipose tissue.

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population

Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes.

Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whereas cis regulatory patterns of gene expression have been extensively explored, the identification of trans regulatory effects in humans has attracted less attention. Here we show that the type 2 diabetes and high-density lipoprotein cholesterol-associated cis-acting expression quantitative trait locus (eQTL) of the maternally expressed transcription factor KLF14 acts as a master trans regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly correlated with concurrently measured metabolic traits, and a subset of the trans-regulated genes harbor variants directly associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk and offers a potential model for other complex traits

Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement Nu 241500 (BuruliVac), from Fundacao Calouste Gulbenkian and from Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). JBG, TGM and AGF had a personal grant from the Portuguese Science and Technology Foundation (FCT) (SFRH/BD/33573/2009, SFRH/BD/41598/2007 and SFRH/BPD/68547/2010, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.This work was supported by a Canadian Institute of Health Research (CIHR) team grant awarded to E.G., A.T., M.C.V. and M.L. (TEC-128093) and the CIHR funded Epigeneome Mapping Centre at McGill University (EP1-120608) awarded to T.P. and M.L., and the Swedish Research Council, Knut and Alice Wallenberg Foundation and the Torsten Söderberg Foundation awarded to L.R. F.A. holds studentship from The Research Institute of the McGill University Health Center (MUHC). F.G. is a recipient of a research fellowship award from the Heart and Stroke Foundation of Canada. A.T. is the director of a Research Chair in Bariatric and Metabolic Surgery. M.C.V. is the recipient of the Canada Research Chair in Genomics Applied to Nutrition and Health (Tier 1). E.G. and T.P. are recipients of a Canada Research Chair Tier 2 award. The MuTHER Study was funded by a programme grant from the Wellcome Trust (081917/Z/07/Z) and core funding for the Wellcome Trust Centre for Human Genetics (090532). TwinsUK was funded by the Wellcome Trust; European Community's Seventh Framework Programme (FP7/2007-2013). The study also receives support from the National Institute for Health Research (NIHR)-funded BioResource, Clinical Research Facility and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London. T.D.S. is a holder of an ERC Advanced Principal Investigator award. SNP genotyping was performed by The Wellcome Trust Sanger Institute and National Eye Institute via NIH/CIDR. Finally, we thank the NIH Roadmap Epigenomics Consortium and the Mapping Centers (http://nihroadmap.nih.gov/epigenomics/) for the production of publicly available reference epigenomes. Specifically, we thank the mapping centre at MGH/BROAD for generation of human adipose reference epigenomes used in this study.This is the final version. It was first published by NPG at http://www.nature.com/ncomms/2015/150529/ncomms8211/full/ncomms8211.html#abstrac

The Architecture of Gene Regulatory Variation across Multiple Human Tissues: The MuTHER Study

While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis—MCTA) permits immediate replication of eQTLs using co-twins (93%–98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%–20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits