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Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures. 相似文献
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Michael E. Joesten Moira E. Royston Marlene Jimenez Axel Wadewitz Diana Melian Richard A. Lockshin 《Journal of insect physiology》1982,28(7):589-599
Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO2 trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO3 and small amounts of Na+, Ca2+, Mg2+, PO4?3 and some proteins. Haemolymph levels of K+ decrease, and those of Ca2+ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion. 相似文献
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John Draper Amanda J. Lloyd Royston Goodacre Manfred Beckmann 《Metabolomics : Official journal of the Metabolomic Society》2013,9(1):4-29
Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design. 相似文献
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Stina K. Lien Håvard Sletta Trond E. Ellingsen Svein Valla Elon Correa Royston Goodacre Kai Vernstad Sven Even Finborud Borgos Per Bruheim 《Metabolomics : Official journal of the Metabolomic Society》2013,9(2):403-417
Metabolic profiling of Pseudomonas fluorescens SBW25 and various mutants derived thereof was performed to explore how the bacterium adapt to changes in carbon source and upon induction of alginate synthesis. The experiments were performed at steady-state conditions in nitrogen-limited chemostats using either fructose or glycerol as carbon source. Carbon source consumption was up-regulated in the alginate producing mutant with inactivated anti-sigma factor MucA. The mucA- mutants (also non-alginate producing mucA- control strains) had a higher dry weight yield on carbon source implying a change in carbon and energy metabolism due to the inactivation of the anti-sigma factor MucA. Both LC–MS/MS and GC–MS methods were used for quantitative metabolic profiling, and major reorganization of primary metabolite pools in both an alginate producing and a carbon source dependent manner was observed. Generally, larger changes were observed among the phosphorylated glycolytic metabolites, the pentose phosphate pathway metabolites and the nucleotide pool than among amino acids and citric acid cycle compounds. The most significant observation at the metabolite level was the significantly reduced energy charge of the mucA- mutants (both alginate producing and non-producing control strains) compared to the wild type strain. This reduction was caused more by a strong increase in the AMP pool than changes in the ATP and ADP pools. The alginate-producing mucA- mutant had a slightly increased GTP pool, while the GDP and GMP pools were strongly increased compared to non-producing mucA- strains and to the wild type. Thus, whilst changes in the adenosine phosphate nucleotide pool are attributed to the mucA inactivation, adjustments in the guanosine phosphate nucleotide pool are consequences of the GTP-dependent alginate production induced by the mucA inactivation. This metabolic profiling study provides new insight into carbon and energy metabolism of the alginate producer P. fluorescens. 相似文献
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Bajhaiya Amit K. Dean Andrew P. Driver Thomas Trivedi Drupad K. Rattray Nicholas J. W. Allwood J. William Goodacre Royston Pittman Jon K. 《Metabolomics : Official journal of the Metabolomic Society》2016,12(1):1-14
Metabolomics - Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require... 相似文献
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George G. Harrigan Tyamagondlu V. Venkatesh Mark Leibman Jonathan Blankenship Timothy Perez Steven Halls Alexander W. Chassy Oliver Fiehn Yun Xu Royston Goodacre 《Metabolomics : Official journal of the Metabolomic Society》2016,12(5):82
Introduction
Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM).Objectives
Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible.Methods
Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography–time of flight-mass spectrometry (GC–TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis.Results
Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices.Conclusion
The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.10.
Proposed minimum reporting standards for chemical analysis 总被引:4,自引:0,他引:4
Lloyd W. Sumner Alexander Amberg Dave Barrett Michael H. Beale Richard Beger Clare A. Daykin Teresa W.-M. Fan Oliver Fiehn Royston Goodacre Julian L. Griffin Thomas Hankemeier Nigel Hardy James Harnly Richard Higashi Joachim Kopka Andrew N. Lane John C. Lindon Philip Marriott Andrew W. Nicholls Michael D. Reily John J. Thaden Mark R. Viant 《Metabolomics : Official journal of the Metabolomic Society》2007,3(3):211-221
There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale
metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context
for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly,
the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics
experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes
the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation,
experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently
focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques
in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line
discussion forum at or . Further, community input related to this document can also be provided via this electronic forum.
The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration
Sponsor: Metabolomics Society http://www.metabolomicssociety.org/
Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/
http://msi-workgroups.sourceforge.net/chemical-analysis/
Version: Revision: 5.1
Date: 09 January, 2007 相似文献