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دراسي و بحثي
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البحوث المنشورة
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البحث (1):
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عنوان البحث:
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Multiple
metabolomics of uropathogenic E. coli reveal different information content in
terms of metabolic potential compared to virulence factors.
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رابط إلى البحث:
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تاريخ النشر:
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07/09/2014
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موجز عن البحث:
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No single analytical method can cover the whole metabolome and the
choice of which platform to use may inadvertently introduce chemical
selectivity. In order to investigate this we analysed a collection of
uropathogenic Escherichia coli. The selected strains had previously undergone
extensive characterisation using classical microbiological methods for a
variety of metabolic tests and virulence factors. These bacteria were
analysed using Fourier transform infrared (FT-IR) spectroscopy; gas
chromatography mass spectrometry (GC-MS) after derivatisation of polar
non-volatile analytes; as well as reversed-phase liquid chromatography mass
spectrometry in both positive (LC-MS(+ve)) and negative (LC-MS(-ve))
electrospray ionisation modes. A comparison of the discriminatory ability of
these four methods with the metabolic test and virulence factors was made
using Procrustes transformations to ascertain which methods produce congruent
results. We found that FT-IR and LC-MS(-ve), but not LC-MS(+ve), were
comparable with each other and gave highly similar clustering compared with
the virulence factors tests. By contrast, FT-IR and LC-MS(-ve) were not
comparable to the metabolic tests, and we found that the GC-MS profiles were
significantly more congruent with the metabolic tests than the virulence
determinants. We conclude that metabolomics investigations may be biased to
the analytical platform that is used and reflects the chemistry employed by
the methods. We therefore consider that multiple platforms should be employed
where possible and that the analyst should consider that there is a danger of
false correlations between the analytical data and the biological
characteristics of interest if the full metabolome has not been measured.
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البحث ( 2 ):
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عنوان البحث:
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Metabolic Fingerprinting of Pseudomonas
putida DOT-T1E Strains: Understanding the Influence of Divalent Cations
in Adaptation Mechanisms Following Exposure to Toluene
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رابط إلى البحث:
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تاريخ النشر:
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26/04/2016
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موجز عن البحث:
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Pseudomonas putida strains can adapt and overcome the activity of
toxic organic solvents by the employment of several resistant mechanisms
including efflux pumps and modification to lipopolysaccharides (LPS) in their
membranes. Divalent cations such as magnesium and calcium play a crucial role
in the development of solvent tolerance in bacterial cells. Here, we have
used Fourier transform infrared (FT-IR) spectroscopy directly on cells
(metabolic fingerprinting) to monitor bacterial response to the absence and
presence of toluene, along with the influence of divalent cations present in
the growth media. Multivariate analysis of the data using principal
component-discriminant function analysis (PC-DFA) showed trends in scores
plots, illustrating phenotypic alterations related to the effect of Mg2+,
Ca2+ and toluene on cultures. Inspection of PC-DFA loadings plots
revealed that several IR spectral regions including lipids, proteins and
polysaccharides contribute to the separation in PC-DFA space, thereby
indicating large phenotypic response to toluene and these cations. Finally,
the saturated fatty acid ratio from the FT-IR spectra showed that upon
toluene exposure, the saturated fatty acid ratio was reduced, while it
increased in the presence of divalent cations. This study clearly
demonstrates that the combination of metabolic fingerprinting with
appropriate chemometric analysis can result in practicable knowledge on the
responses of important environmental bacteria to external stress from
pollutants such as highly toxic organic solvents, and indicates that these
changes are manifest in the bacterial cell membrane. Finally, we demonstrate
that divalent cations improve solvent tolerance in P. putida DOT‑T1E
strains.
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البحث (3):
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عنوان البحث:
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Metabolic analysis
of the response of Pseudomonas putida DOT-T1E strains to toluene
using Fourier transform infrared spectroscopy and gas chromatography
mass spectrometry
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here
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تاريخ النشر:
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21/06/2016
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موجز عن البحث:
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Introduction
An exceptionally interesting stress response of Pseudomonas
putida strains to toxic substances is the induction of efflux pumps
that remove toxic chemical substances from the bacterial cell out to the
external environment. To exploit these microorganisms to their full potential
a deeper understanding of the interactions between the bacteria and organic
solvents is required. Thus, this study focuses on investigation of metabolic
changes in P. putida upon exposure to toluene.
Objective
Investigate observable metabolic alterations during interactions of
three strains of P. putida(DOT-T1E, and its mutants DOT-T1E-PS28
and DOT-T1E-18) with the aromatic hydrocarbon toluene.
Methods
The growth profiles were measured by taking optical density (OD)
measurement at 660 nm (OD660) at various time points during incubation.
For fingerprinting analysis, Fourier-transform infrared (FT-IR) spectroscopy
was used to investigate any phenotypic changes resulting from exposure to
toluene. Metabolic profiling analysis was performed using gas
chromatography-mass spectrometry (GC–MS). Principal component—discriminant
function analysis (PC-DFA) was applied to the FT-IR data while multiblock
principal component analysis (MB-PCA) and N-way analysis of
variance (N-way ANOVA) were applied to the GC–MS data.
Results
The growth profiles demonstrated the effect of toluene on bacterial
cultures and the results suggest that the mutant P. putida DOT-T1E−18
was more sensitive (significantly affected) to toluene compared to the other
two strains. PC-DFA on FT-IR data demonstrated the differentiation between
different conditions of toluene on bacterial cells, which indicated
phenotypic changes associated with the presence of the solvent within the
cell. Fifteen metabolites associated with this phenotypic change, in P.
putida due to exposure to solvent, were from central metabolic
pathways. Investigation of MB-PCA loading plots and N-way ANOVA
for condition | strain × time blocking (dosage of toluene)
suggested ornithine as the most significant compound that increased upon
solvent exposure.
Conclusion
The combination of metabolic fingerprinting and profiling with
suitable multivariate analysis revealed some interesting leads for
understanding the mechanism of Pseudomonas strains response
to organic solvent exposure.
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البحث (4):
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عنوان البحث:
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Metabolomics Analysis
Reveals the Participation of Efflux Pumps and Ornithine in the Respons of Pseudomonas putida DOT-T1E Cells to Challenge with Propranolol
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here
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تاريخ النشر:
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22/06/2016
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موجز عن البحث:
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Efflux
pumps are critically important membrane components that play a crucial role
in strain tolerance in Pseudomonas putida to antibiotics and
aromatic hydrocarbons that result in these toxicants being expelled from the
bacteria. Here, the effect of propranolol on P. putida was
examined by sudden addition of 0.2, 0.4 and 0.6 mg mL-1 of this
β-blocker to several strains ofP. putida, including the
wild type DOT-T1E and the efflux pump knockout mutants DOT-T1E-PS28 and
DOT-T1E-18. Bacterial viability measurements reveal that the efflux pump
TtgABC plays a more important role than the TtgGHI pump in strain tolerance
to propranolol. Mid-infrared (MIR) spectroscopy was then used as a rapid,
high-throughput screening tool to investigate any phenotypic changes
resulting from exposure to varying levels of propranolol. Multivariate
statistical analysis of these MIR data revealed gradient trends in resultant
ordination scores plots, which were related to the concentration of
propranolol. MIR illustrated phenotypic changes associated with the presence
of this drug within the cell that could be assigned to significant changes
that occurred within the bacterial protein components. To complement this
phenotypic fingerprinting approach metabolic profiling was performed using
gas chromatography mass spectrometry (GC-MS) to identify metabolites of
interest during the growth of bacteria following toxic perturbation with the
same concentration levels of propranolol. Metabolic profiling revealed that
ornithine, which was only produced by P. putidacells
in the presence of propranolol, presents itself as a major metabolic feature
that has important functions in propranolol stress tolerance mechanisms
within this highly significant and environmentally relevant species of
bacteria.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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IRDG infrared & Raman Discussion Group
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تاريخ الإنعقاد:
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17/09/2013
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مكان الإنعقاد:
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Glasgow. UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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The effect of abiotic stress on Pseudomonas putida
using FT-IR spectroscopy
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ملخص المشاركة:
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Efflux pumps are probably the most important mechanisms that play a
crucial role in solvent tolerance in Pseudomonas putida DOT-T1E, as they
expel antibiotics and aromatic hydrocarbons. Three pumps are involved in solvent
tolerance: TtgABC, TtgDEF and TtgGHI. The sudden addition of 0.05% (vol/vol)
toluene or 0.2% propranolol (a human -blocker) to P. putida DOT-T1E, P.
putida DOT-T1E-PS28 (lacking TtgGHI pump) and P. putida DOT-T1E-18 (lacking
the TtgABC pump) grown in LB medium, in the presence or absence of toluene
via gas phase, had a clear effect on bacterial survival. In this study the
effect of a particular concentration of toxic solvent or drug on the
bacterial cell was examined. Once a solvent is toxic to a cell, it is
hypothesised that this toxic substance have a phenotypic effect on the cells
even at very low concentration, so infrared (IR) spectroscopy can be used to
investigate whether the phenotype of an organism has changed by exposing it
to different levels of toxic solvents. This would allow us to establish the
foundation for MS-based metabolomics experiments that could be used to study
metabolites at particular time points following toxic perturbation. The
collected data from gas chromatography-mass spectrometry (GC-MS) and liquid
chromatography-mass spectrometry (LC-MS) will then be analysed by
chemometrics techniques in order to interpret the results and elucidate the
mode of action and the role of specific efflux pumps.
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المؤتمر (2):
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عنوان المؤتمر:
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Metabomeeting 2015
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تاريخ الإنعقاد:
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07/12/2015
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مكان الإنعقاد:
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Cambridge, UK
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طبيعة المشاركة:
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Poster presentation
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عنوان المشاركة:
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A Metabolomics Study of the Effect of Toluene on
Pseudomonas Putida DOT-T1E Strains
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ملخص المشاركة:
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Organic solvents such as toluene, benzene, and xylenes with a logP
between 1.5 and 3.5 are highly toxic for microorganism. Bacteria have the
ability to adapt the activity of toxic organic solvent by the employment of
various resistant mechanisms such as efflux pump which remove the toxic
substances from the bacterial cell before they reach their target. Here,
metabolomics approaches were employed in order to elucidate some metabolites
changes during interactions of Pseudomonas putida (DOT-T1E, DOT-T1E-PS28 and
DOT-T1E-18) strains with toluene. Fourier-transform infrared (FT-IR)
spectroscopy and gas chromatography-mass spectrometry (GC-MS) were used to
generate metabolic fingerprinting and profiling in order to elucidate solvent
adaptation mechanisms in P. putida strains. Observed metabolome alterations
can support postulated adaption mechanisms, or may prove to be indicative of
novel adaption mechanisms for which there is little evidence up to date.
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