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1.  Age and Microenvironment Outweigh Genetic Influence on the Zucker Rat Microbiome 
PLoS ONE  2014;9(9):e100916.
Animal models are invaluable tools which allow us to investigate the microbiome-host dialogue. However, experimental design introduces biases in the data that we collect, also potentially leading to biased conclusions. With obesity at pandemic levels animal models of this disease have been developed; we investigated the role of experimental design on one such rodent model. We used 454 pyrosequencing to profile the faecal bacteria of obese (n = 6) and lean (homozygous n = 6; heterozygous n = 6) Zucker rats over a 10 week period, maintained in mixed-genotype cages, to further understand the relationships between the composition of the intestinal bacteria and age, obesity progression, genetic background and cage environment. Phylogenetic and taxon-based univariate and multivariate analyses (non-metric multidimensional scaling, principal component analysis) showed that age was the most significant source of variation in the composition of the faecal microbiota. Second to this, cage environment was found to clearly impact the composition of the faecal microbiota, with samples from animals from within the same cage showing high community structure concordance, but large differences seen between cages. Importantly, the genetically induced obese phenotype was not found to impact the faecal bacterial profiles. These findings demonstrate that the age and local environmental cage variables were driving the composition of the faecal bacteria and were more deterministically important than the host genotype. These findings have major implications for understanding the significance of functional metagenomic data in experimental studies and beg the question; what is being measured in animal experiments in which different strains are housed separately, nature or nurture?
doi:10.1371/journal.pone.0100916
PMCID: PMC4169429  PMID: 25232735
2.  Investigation of chronic alcohol consumption in rodents via UHPLC-MS based metabolite profiling 
Journal of chromatography. A  2012;1259:128-137.
Alcohol consumption in man, when seen in its extreme form of alcoholism, is a complex and socially disruptive disorder that can result in significant levels of liver injury. Here the rodent “intragastric feeding model” was used together with UHPLC-TOFMS analysis to determine changes in global metabolite profiles for plasma and urine from alcohol treated rats and mice compared to control animals. Multivariate statistical analysis (using principal components analysis, PCA) revealed robust differences between profiles from control and alcohol-treated animals from both species. A large number of metabolites were seen to differ between control and alcohol-treated animals, for both biofluids.
doi:10.1016/j.chroma.2012.02.053
PMCID: PMC3387526  PMID: 22446076
Alcohol; Metabolite profiling; Ultra Performance Liquid Chromatography (UPLC); Time of Flight mass spectrometry (TOF-MS); metabonomics; metabolomics
3.  Role of nitric oxide in regulating stomatal apertures 
Plant Signaling & Behavior  2009;4(5):467-469.
During stomatal closure, nitric oxide (NO) operates as one of the key intermediates in the complex, abscisic acid (ABA)-mediated, guard cell signaling network that regulates this process. However, data concerning the role of NO in stomatal closure that occurs in turgid vs. dehydrated plants is limited. The data presented demonstrate that, while there is a requirement for NO during the ABA-induced stomatal closure of turgid leaves, such a requirement does not exist for ABA-enhanced stomatal closure observed to occur during conditions of rapid dehydration. The data also indicate that the ABA signaling pathway must be both functional and to some degree activated for guard cell NO signaling to occur. These observations are in line with the idea that the effects of NO in guard cells are mediated via a Ca2+-dependent rather than a Ca2+-independent ABA signaling pathway. It appears that there is a role for NO in the fine tuning of the stomatal apertures of turgid leaves that occurs in response to fluctuations in the prevailing environment.
PMCID: PMC2676769  PMID: 19816112
abscisic acid; nitrate reductase; nitric oxide; stomatal apertures; guard cells
4.  Hydrogen sulfide effects on stomatal apertures 
Plant Signaling & Behavior  2011;6(10):1444-1446.
Hydrogen sulfide (H2S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H2S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H2S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H2S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.
doi:10.4161/psb.6.10.17104
PMCID: PMC3256366  PMID: 21904118
abscisic acid; GYY4137; hydrogen sulfide; nitric oxide; stomatal aperture
5.  Does the Mass Spectrometer Define the Marker? A Comparison of Global Metabolite Profiling Data Generated Simultaneously via UPLC-MS on Two Different Mass Spectrometers 
Analytical chemistry  2010;82(19):8226-8234.
By coupling a single UPLC separation to two different types of mass spectrometer an unbiased comparison of the metabolite profiles produced by each instrument for a set of rat urine samples was obtained. The flow from the UPLC column was split equally and both streams of eluent were simultaneously directed to the inlets of the two mass spectrometers. Mass spectrometry on the eluent was undertaken using a triple quadrupole linear ion trap and a hybrid quadrupole time of flight mass spectrometer using both positive and negative ESI. Data from both mass spectrometers were subjected to multivariate statistical analysis, after applying the same data extraction software, and showed the same general pattern of correlation between the samples using both unsupervised and supervised methods of statistical analysis. Based on orthogonal partial least square discriminant analysis models a number of ions were recognized as “responsible” for the separation of the animal groups. From the peaks detected, and denoted as significant by the statistical analysis a number of ions were found to be unique to one dataset or the other, a result which may have consequences for biomarker discovery and inter-laboratory comparisons. The software package used for data analysis also had an effect on the outcome of the statistical analysis.
doi:10.1021/ac1016612
PMCID: PMC2951889  PMID: 20828141
UPLC-MS; QTOF; QTRAP; metabolomics; metabonomics; urine metabolite profiling; multivariate analysis
6.  MicroRNAs: their discovery, biogenesis, function and potential use as biomarkers in non-invasive prenatal diagnostics 
MiRNAs are a widespread class of small non-coding RNAs that have the ability to silence gene expression through sequence complementarity to their targets. We describe their initial discovery in the nematode C.elegans and review what is currently known about their biogenesis. The regulation of expression and processing of miRNAs, and the mechanisms through which miRNAs locate their correct targets are not yet fully understood. MiRNAs are involved in a multitude of developmental and pathological processes leading to an explosion of research in disparate subject areas. In this review we draw attention to placentally expressed miRNAs that can be detected in the maternal plasma; and we discuss their potential use as biomarkers in non-invasive prenatal diagnostics.
PMCID: PMC3166153  PMID: 21915364
Small non-coding RNAs; miRNAs; circulating nucleic acids; maternal plasma biomarkers; prenatal diagnostics
7.  The relationship of proline content and metabolism on the productivity of maize plants 
Plant Signaling & Behavior  2011;6(2):251-257.
The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots.
doi:10.4161/psb.6.2.14336
PMCID: PMC3121986  PMID: 21415600
maize; maize silk; plant productivity; pollen; proline; proline dehydrogenase; Δ1-pyrroline-5-carboxylate synthetase; Zea mays L.
8.  Site and Strain-Specific Variation in Gut Microbiota Profiles and Metabolism in Experimental Mice 
PLoS ONE  2010;5(1):e8584.
Background
The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.
Methodology/Principal Findings
The GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial –specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.
Conclusion/Significance
Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions.
doi:10.1371/journal.pone.0008584
PMCID: PMC2798964  PMID: 20052418
9.  Nitric oxide and nitrite are likely mediators of pollen interactions 
Plant Signaling & Behavior  2009;4(5):416-418.
The ability of plants to produce nitric oxide (NO) is now well recognised. In plants, NO is involved in the control of organ development and in regulating some of their physiological functions. We have recently shown that pollen generates NO in a constitutive manner and have measured both intra- and extracellular production of this radical. Furthermore, we have shown that nitrite accumulates in the media surrounding the pollen and have suggested that the generation of these signaling molecules may be important for the normal interaction between the pollen grain and the stigma on which it alights. However, pollen grains inevitably come into contact with other tissues, including those of animals and it is likely that the NO produced will influence the behavior of the cells associated with these tissues. Such non-animal-derived, NO-mediated effects on mammalian cells may not be restricted to pollen and plant debris and fungal spore-derived NO may elicit similar effects.
PMCID: PMC2676752  PMID: 19816116
allergy; fungal spores; nitric oxide; nitrite; pollen
10.  Cold- and light-induced changes in the transcriptome of wheat leading to phase transition from vegetative to reproductive growth 
BMC Plant Biology  2009;9:55.
Background
For plants to flower at the appropriate time, they must be able to perceive and respond to various internal and external cues. Wheat is generally a long-day plant that will go through phase transition from vegetative to floral growth as days are lengthening in spring and early summer. In addition to this response to day-length, wheat cultivars may be classified as either winter or spring varieties depending on whether they require to be exposed to an extended period of cold in order to become competent to flower. Using a growth regime to mimic the conditions that occur during a typical winter in Britain, and a microarray approach to determine changes in gene expression over time, we have surveyed the genes of the major pathways involved in floral transition. We have paid particular attention to wheat orthologues and functional equivalents of genes involved in the phase transition in Arabidopsis. We also surveyed all the MADS-box genes that could be identified as such on the Affymetrix genechip wheat genome array.
Results
We observed novel responses of several genes thought to be of major importance in vernalisation-induced phase transition, and identified several MADS-box genes that might play an important role in the onset of flowering. In addition, we saw responses in genes of the Gibberellin pathway that would indicate that this pathway also has some role to play in phase transition.
Conclusion
Phase transition in wheat is more complex than previously reported, and there is evidence that day-length has an influence on genes that were once thought to respond exclusively to an extended period of cold.
doi:10.1186/1471-2229-9-55
PMCID: PMC2685395  PMID: 19432994
11.  Markedly different gene expression in wheat grown with organic or inorganic fertilizer 
Nitrogen is the major determinant of crop yield and quality and the precise management of nitrogen fertilizer is an important issue for farmers and environmentalists. Despite this, little is known at the level of gene expression about the response of field crops to different amounts and forms of nitrogen fertilizer. Here we use expressed sequence tag (EST)-based wheat microarrays in combination with the oldest continuously running agricultural experiment in the world to show that gene expression is significantly influenced by the amount and form of nitrogenous fertilizer. In the Broadbalk winter wheat experiment at Rothamsted in the United Kingdom and at three other diverse test sites, we show that specific genes have surprisingly different expression levels in the grain endosperm when nitrogen is supplied either in an organic or an inorganic form. Many of the genes showing differential expression are known to participate in nitrogen metabolism and storage protein synthesis. However, others are of unknown function and therefore represent new leads for future investigation. Our observations show that specific gene expression is diagnostic for use of organic sources of nitrogen fertilizer and may therefore have useful applications in defining the differences between organically and conventionally grown wheat.
doi:10.1098/rspb.2005.3161
PMCID: PMC1559878  PMID: 16191595
transcriptomics; microarrays; organic farming; wheat; nitrogen

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