GeneMCL in microarray analysis

Accurately and reliably identifying the actual number of clusters present with a dataset of gene expression profiles, when no additional information on cluster structure is available, is a problem addressed by few algorithms. GeneMCL transforms microarray analysis data into a graph consisting of nodes connected by edges, where the nodes represent genes, and the edges represent the similarity in expression of those genes, as given by a proximity measurement. This measurement is taken to be the Pearson correlation coefficient combined with a local non-linear rescaling step. The resulting graph is input to the Markov Cluster (MCL) algorithm, which is an elegant, deterministic, non-specific and scalable method, which models stochastic flow through the graph. The algorithm is inherently affected by any cluster structure present, and rapidly decomposes a graph into cohesive clusters. The potential of the GeneMCL algorithm is demonstrated with a 5730 gene subset (IGS) of the Van't Veer breast cancer database, for which the clusterings are shown to reflect underlying biological mechanisms. (c) 2005 Elsevier Ltd. All rights reserved.

Tissue engineering a fetal membrane

The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype.

Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erbalpha pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erbalpha target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erbalpha pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo11C-acetate and PET-CT scanning to show that colonic acetate crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression. Furthermore, we demonstrate through 13C high-resolution magic-angle-spinning that 13C acetate from fermentation of 13C-labelled carbohydrate in the colon increases hypothalamic 13C acetate above baseline levels. Hypothalamic 13C acetate regionally increases the 13C labelling of the glutamate–glutamine and GABA neuroglial cycles, with hypothalamic 13C lactate reaching higher levels than the ‘remaining brain’. These observations suggest that acetate has a direct role in central appetite regulation.

Cell cycle profiles and expression of p21CIP1 and P27KIP1 during myocyte development

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

Use of global gene expression patterns in mechanistic studies of oestrogen action in MCF7 human breast cancer cells

Over the years, the MCF7 human breast cancer cell line has provided a model system for the study of cellular and molecular mechanisms in oestrogen regulation of cell proliferation and in progression to oestrogen and antioestrogen independent growth. Global gene expression profiling has shown that oestrogen action in MCF7 cells involves the coordinated regulation of hundreds of genes across a wide range of functional groupings and that more genes are down regulated than upregulated. Adaptation to long-term oestrogen deprivation, which results in loss of oestrogen-responsive growth, involves alterations to gene patterns not only at early time points (0-4 weeks) but continuing through to later times (20-55 weeks), and even involves alterations to patterns of oestrogen-regulated gene expression. Only 48% of the genes which were regulated >= 2-fold by oestradiol in oestrogen-responsive cells retained this responsiveness after long-term oestrogen deprivation but other genes developed de novo oestrogen regulation. Long-term exposure to fulvestrant, which resulted in loss of growth inhibition by the antioestrogen, resulted in some very large fold changes in gene expression up to 10,000-fold. Comparison of gene profiles produced by environmental chemicals with oestrogenic properties showed that each ligand gave its own unique expression profile which suggests that environmental oestrogens entering the human breast may give rise to a more complex web of interference in cell function than simply mimicking oestrogen action at inappropriate times. (C) 2009 Elsevier Ltd. All rights reserved.

A molecular and structural characterization of senescing Arabidopsis siliques and comparison of transcriptional profiles with senescing petals and leaves

Senescence of plant organs is a genetically controlled process that regulates cell death to facilitate nutrient recovery and recycling, and frequently precedes, or is concomitant with, ripening of reproductive structures. In Arabidopsis thaliana, the seeds are contained within a silique, which is itself a photosynthetic organ in the early stages of development and undergoes a programme of senescence prior to dehiscence. A transcriptional analysis of the silique wall was undertaken to identify changes in gene expression during senescence and to correlate these events with ultrastructural changes. The study revealed that the most highly up-regulated genes in senescing silique wall tissues encoded seed storage proteins, and the significance of this finding is discussed. Global transcription profiles of senescing siliques were compared with those from senescing Arabidopsis leaf or petal tissues using microarray datasets and metabolic pathway analysis software (MapMan). In all three tissues, members of NAC and WRKY transcription factor families were up-regulated, but components of the shikimate and cell-wall biosynthetic pathways were down-regulated during senescence. Expression of genes encoding ethylene biosynthesis and action showed more similarity between senescing siliques and petals than between senescing siliques and leaves. Genes involved in autophagy were highly expressed in the late stages of death of all plant tissues studied, but not always during the preceding remobilization phase of senescence. Analyses showed that, during senescence, silique wall tissues exhibited more transcriptional features in common with petals than with leaves. The shared and distinct regulatory events associated with senescence in the three organs are evaluated and discussed.

N-nitroso compound exposure-associated transcriptomic profiles are indicative of an increased risk for colorectal cancer

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

Ethnic differences in beta-cell function, dietary intake and expression of the metabolic syndrome among UK adults of south Asian, black African-Caribbean and white-European origin at high risk of metabolic syndrome

A cross-sectional analysis of ethnic differences in dietary intake, insulin sensitivity and beta-cell function, using the intravenous glucose tolerance test (IVGTT), was conducted on 497 healthy adult participants of the ‘Reading, Imperial, Surrey, Cambridge, and Kings’ (RISCK) study. Insulin sensitivity (Si) was significantly lower in African-Caribbean (AC) and South Asian (SA) participants [IVGTT-Si; AC: 2.13 vs SA: 2.25 vs white-European (WE): 2.84 (×10−4 mL µU min)2, p < 0.001]. AC participants had a higher prevalence of anti-hypertensive therapy (AC: 19.7% vs SA: 7.5%), the most cardioprotective lipid profile [total:high-density lipoprotein (HDL); AC: 3.52 vs SA: 4.08 vs WE: 3.83, p = 0.03] and more pronounced hyperinsulinaemia [IVGTT–acute insulin response (AIR)] [AC: 575 vs SA: 428 vs WE: 344 mL/µU/min)2, p = 0.002], specifically in female participants. Intake of saturated fat and carbohydrate was lower and higher in AC (10.9% and 50.4%) and SA (11.1% and 52.3%), respectively, compared to WE (13.6% and 43.8%, p < 0.001). Insulin resistance in ACs is characterised by ‘normal’ lipid profiles but high rates of hypertension and pronounced hyperinsulinaemia.

Identification of novel cholesteatoma-related gene expression signatures using full-genome microarrays

This study demonstrates that the expression profile of cholesteatoma is similar to a metastatic tumour and chronically inflamed tissue. Based on the investigated profiles we present novel protein-protein interaction and signal transduction networks, which include cholesteatoma-regulated transcripts and may be of great value for drug targeting and therapy development.