Phylogenetic distribution of traits associated with plant colonization in Escherichia coli.

Plants are increasingly considered as secondary reservoirs for commensal and pathogenic Escherichia coli strains, but the ecological and functional factors involved in this association are not clear. To address this question, we undertook a comparative approach combining phenotypic and phylogenetic analyses of E. coli isolates from crops and mammalian hosts. Phenotypic profiling revealed significant differences according to the source of isolation. Notably, isolates from plants displayed higher biofilm and extracellular matrix production and higher frequency of utilization of sucrose and the aromatic compound p-hydroxyphenylacetic acid. However, when compared with mammalian-associated strains, they reached lower growth yields on many C-sources commonly used by E. coli. Strikingly, we observed a strong association between phenotypes and E. coli phylogenetic groups. Strains belonging to phylogroup B1 were more likely to harbour traits indicative of a higher ability to colonize plants, whereas phylogroup A and B2 isolates displayed phenotypes linked to an animal-associated lifestyle. This work provides clear indications that E. coli phylogroups are specifically affected by niche-specific selective pressures, and provides an explanation on why E. coli population structures vary in natural environments, implying that different lineages in E. coli have substantially different transmission ecology.

Does prolonged chewing reduce food intake? Fletcherism revisited.

Horace Fletcher (1849-1919) spread his doctrine to chew each mouthful thoroughly in order to prevent gaining weight. We sought to test this idea by manipulating chewing instructions whilst using electromyography to monitor chewing behaviour. Comparing 35 with 10 chews per mouthful, we showed that higher chewing counts reduced food intake despite increasing chewing speed, and despite doubling meal duration for achieving a subjective reference point for feeling 'comfortably full'. Although limited by a low sample size, our preliminary findings confirm Mr. Fletcher's doctrine, and provide a basis for further research in this area. Outcomes and implications are discussed.

Ulcerative colitis and irritable bowel patients exhibit distinct abnormalities of the gut microbiota.

BACKGROUND: Previous studies suggest a link between gut microbiota and the development of ulcerative colitis (UC) and irritable bowel syndrome (IBS). Our aim was to investigate any quantitative differences in faecal bacterial compositions in UC and IBS patients compared to healthy controls, and to identify individual bacterial species that contribute to these differences. METHODS: Faecal microbiota of 13 UC patients, 11 IBS patients and 22 healthy volunteers were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE) using universal and Bacteroides specific primers. The data obtained were normalized using in-house developed statistical method and interrogated by multivariate approaches. The differentiated bands were excised and identified by sequencing the V3 region of the 16S rRNA genes. RESULTS: Band profiles revealed that number of predominant faecal bacteria were significantly different between UC, IBS and control group (p < 10-4). By assessing the mean band numbers in UC (37 ± 5) and IBS (39 ± 6), compared to the controls (45 ± 3), a significant decrease in bacterial species is suggested (p = 0.01). There were no significant differences between IBS and UC. Biodiversity of the bacterial species was significantly lower in UC (µ = 2.94, σ = 0.29) and IBS patients (µ = 2.90, σ = 0.38) than controls (µ = 3.25, σ = 0.16; p = 0.01). Moreover, similarity indices revealed greater biological variability of predominant bacteria in UC and IBS compared to the controls (median Dice coefficients 76.1% (IQR 70.9 - 83.1), 73.8% (IQR 67.0 - 77.5) and 82.9% (IQR 79.1 - 86.7) respectively). DNA sequencing of discriminating bands suggest that the presence of Bacteroides vulgatus, B. ovatus, B. uniformis, and Parabacteroides sp. in healthy volunteers distinguishes them from IBS and UC patients. DGGE profiles of Bacteroides species revealed a decrease of Bacteroides community in UC relative to IBS and controls. CONCLUSION: Molecular profiling of faecal bacteria revealed abnormalities of intestinal microbiota in UC and IBS patients, while different patterns of Bacteroides species loss in particular, were associated with UC and IBS.

PCR-denaturing gradient gel electrophoresis of complex microbial communities: a two-step approach to address the effect of gel-to-gel variation and allow valid comparisons across a large dataset.

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.

Gene expression profile of primary prostate epithelial and stromal cells in response to sulforaphane or iberin exposure.

BACKGROUND: Broccoli consumption has been associated with a reduced risk of prostate cancer. Isothiocyanates (ITCs) derived from glucosinolates that accumulate in broccoli are dietary compounds that may mediate these health effects. Sulforaphane (SF, 4-methylsulphinylbutyl ITC) derives from heading broccoli (calabrese) and iberin (IB, 3-methylsulphinypropyl ITC) from sprouting broccoli. While there are many studies regarding the biological activity of SF, mainly undertaken with cancerous cells, there are few studies associated with IB. METHODS: Primary epithelial and stromal cells were derived from benign prostatic hyperplasia tissue. Affymetrix U133 Plus 2.0 whole genome arrays were used to compare global gene expression between these cells, and to quantify changes in gene expression following exposure to physiologically appropriate concentrations of SF and IB. Ontology and pathway analyses were used to interpret results. Changes in expression of a subset of genes were confirmed by real-time RT-PCR. RESULTS: Global gene expression profiling identified epithelial and stromal-specific gene expression profiles. SF induced more changes in epithelial cells, whereas IB was more effective in stromal cells. Although IB and SF induced different changes in gene expression in both epithelial and stromal cells, these were associated with similar pathways, such as cell cycle and detoxification. Both ITCs increased expression of PLAGL1, a tumor suppressor gene, in stromal cells and suppressed expression of the putative tumor promoting genes IFITM1, CSPG2, and VIM in epithelial cells. CONCLUSION: These data suggest that IB and SF both alter genes associated with cancer prevention, and IB should be investigated further as a potential chemopreventative agent. Prostate 69: 1411-1421, 2009. (c) 2009 Wiley-Liss, Inc.

Multivariate techniques and their application in nutrition: a metabolomics case study.

The post-genomic technologies are generating vast quantities of data but many nutritional scientists are not trained or equipped to analyse it. In high-resolution NMR spectra of urine, for example, the number and complexity of spectral features mean that computational techniques are required to interrogate and display the data in a manner intelligible to the researcher. In addition, there are often multiple underlying biological factors influencing the data and it is difficult to pinpoint which are having the most significant effect. This is especially true in nutritional studies, where small variations in diet can trigger multiple changes in gene expression and metabolite concentration. One class of computational tools that are useful for analysing this highly multivariate data include the well-known 'whole spectrum' methods of principal component analysis and partial least squares. In this work, we present a nutritional case study in which NMR data generated from a human dietary Cu intervention study is analysed using multivariate methods and the advantages and disadvantages of each technique are discussed. It is concluded that an alternative approach, called feature subset selection, will be important in this type of work; here we have used a genetic algorithm to identify the small peaks (arising from metabolites of low concentration) that have been altered significantly following a dietary intervention.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins in dry-cured hams: data registration and multivariate analysis across multiple gels.

This study investigates whether dry-cured hams from two European countries can be distinguished using SDS-PAGE. Thirty-seven commercial hams (19 Spanish, 18 French) were used in the study. Four protein fractions were extracted from each sample, with sufficient material prepared to allow each fraction to be analysed in triplicate lanes. The complete extraction process was carried out in duplicate. The 24 specimens originating from each ham sample were randomly allocated to different lane positions and gels, as were at least two reference lanes (for reference proteins). In total, 118 gels were prepared. Mathematical routines were developed using a matrix language to process the gel image files. Procedures were written to carry out 'within-gel' image correction, lane extraction and normalization, 'between-gel' data registration and linear discriminant analysis (LDA) of each fraction's data to establish whether the provenance could be systematically distinguished. The between-gel registration was carried out using a genetic algorithm (GA). Feature selection was also performed using a GA, to pass subsets of features to the LDA routine. Cross-validated classification success rates were 84, 91, 81 and 85%, respectively, for the four fractions. We conclude that SDS-PAGE can be conducted in a sufficiently quantitative manner and can potentially verify the provenance of regional speciality dry-cured hams.

FTIR spectroscopy and multivariate analysis can distinguish the geographic origin of extra virgin olive oils.

This work investigates whether Fourier transform infrared spectroscopy (FTIR), in combination with multivariate analysis, can distinguish extra virgin olive oils from different producing countries. Duplicate spectra were collected from 60 oils from four European countries. Two approaches to data analysis were used as follows: first, the "whole spectrum" method of partial least squares (PLS) followed by distance-based linear discriminant analysis (LDA) applied to the PLS scores, and second, a genetic algorithm (GA) for variate selection from the raw data, followed by LDA applied to the selected subset. The PLS-LDA approach produced a cross-validation success rate of 96%, whereas the GA-LDA approach achieved a 100% cross-validation success rate, from subsets comprising only eight variates. Neither the selected variate nor the whole spectrum approach was able to offer insight into the origin of the discrimination in biochemical terms. However, FTIR analysis is rapid, and this work shows that it has the required discriminatory power to potentially offer a "black box" method of screening oils to verify their country of origin.