1. No category

MERLION Metabolomics
Workshop Singapore 2014
19 – 21 November 2014 University Town, NUS, Singapore
Developing Metabolomics Platform Technologies through
Singapore-French Research Alliance
Workshop Programme
And Abstract Book
Organised by:
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
CONTENT
Content Page ...............................................................................................1
Welcome Message ......................................................................................2
Organisers’ Welcome ..................................................................................3
Organising Committee ................................................................................4
NUS Environmental Research Institute .................................................... 5
MetaboHUB .................................................................................................6
Institut Français de Singapour – French Embassy ................................... 7
Keynote Speakers........................................................................................8
Programme ................................................................................................13
Young Researcher Award .........................................................................21
Abstracts for Main Session.......................................................................22
Abstracts for Young Researchers Session ...............................................55
Agilent Sponsored Talk .............................................................................73
Technical Site Visit ....................................................................................74
Sponsors’ profiles ......................................................................................76
Location Map of University Town ...........................................................79
Notes ..........................................................................................................81
1
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Welcome Message
from the Organising Committee Chairperson
As the Chairperson, it is my pleasure to welcome
you to the Merlion Metabolomics Workshop
Singapore 2014.
This is the first 3-day workshop jointly organized by
Singapore and France, providing first-hand insights
and experiences revolving around 3 broad themes
of metabolomics – Human Health, Food and
Science Technology and Environmental. Through
this workshop, we hope to encourage technical exchanges and catalyze the
development of research collaboration in the field of metabolomics
between Singapore and France. Apart from Singapore and France, we have
participants from Australia, China, Japan, Hong Kong, South Korea, India,
Ireland, Thailand, Malaysia, Czech Republic, Italy and United Kingdom. This
workshop is truly an epitome of Singapore’s identity, a cosmopolitan melting
pot, where east meets west. We hope to encourage the transfer of
knowledge and technologies developed in Europe to Asia and vice versa.
To end, I would like to express my personal gratitude to faculty and staff at
the National University of Singapore, Metabohub and the Institut Français
de Singapour – French Embassy, industry partners and sponsors for their
work and dedication. Also to participants, thank you for helping us make this
Merlion Metabolomics Workshop Singapore 2014 a success.
Sincerely,
Chairperson, MMWS 2014
Associate Professor, Department of Biological Sciences, NUS
Deputy Director, NUS-NERI
Director, Graduate Program, NTU-SCELSE
2
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Organisers’ Welcome
Dear Participants,
We would like to extend a warm welcome to everyone attending the 2014
Merlion Metabolomics Workshop Singapore!
We have received an overwhelming response from the scientific community.
We would like to thank every invited speakers and participants for attending
this workshop. Your enthusiastic participation ensures the very success of
this workshop.
This Merlion Metabolomics workshop has an exciting three days programme
addressing areas of Human Health, Food and Science Technology and
Environmental Metabolomics. The goal of this workshop is to establish an
open channel of communication, networking and to encourage mutual
sharing of ongoing research results in the field of metabolomics between
Singapore and France. At the end of each day, we have sessions for young
researchers to share their research findings.
We hope that this will be an enjoyable and interactive event for all. Enjoy
your stay in Singapore!
Best wishes to all delegates,
Organising Committee
MMWS2014
3
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Organising Committee
Organising Committee
Associate Professor Sanjay SWARUP, National University of Singapore
Professor Choon Nam ONG, National University of Singapore
Professor Sam Fong Yau LI, National University of Singapore
Associate Professor Eric Chun Yong CHAN, National University of Singapore
Dr. Fabien JOURDAN, French National Research Institute for Agricultural
Research
Ms. Caroline SAUTOT, French National Research Institute for Agricultural
Research Transfert
Mr. Geoffry SMITH, International Life Sciences Institute Southeast Asia
Mr. Florent BEAU, Institut Français de Singapour – French Embassy
Dr. Adeline MARTIN, Institut Français de Singapour – French Embassy
Dr. Peter KEW, National University of Singapore
Ms. Elaine TAY, National University of Singapore
Ms. Eileen Mei Hui TAN, National University of Singapore
Scientific Committee
Associate Professor Sanjay SWARUP, National University of Singapore
Professor Dominique ROLIN, University of Bordeaux
Dr. Fabien JOURDAN, French National Research Institute for Agricultural
Research
Professor Sam Fong Yau LI, National University of Singapore
Mr. Geoffry SMITH, International Life Sciences Institute Southeast Asia
4
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
About NUS Environmental Research
Institute (NERI)
The NUS Environmental Research Institute (NERI) aims to be a leading global
institute for interdisciplinary research, education and expertise in
environmental science and technology in Asia. NERI coordinates and
facilitates cross-faculty research and educational activities to conduct
cutting-edge research addressing critical environmental issues. NERI
proactively engages strategic partners within NUS' global networks, with
government agencies, and industry.
The current key research tracks are:
•
•
•
•
Environmental Surveillance and Treatment
Environmental and Human Health
Green Chemistry and Sustainable Energy
Impacts of Climate Change on the Environment
For more information, visit http://www.nus.edu.sg/neri/.
FOR A SUSTAINABLE FUTURE
5
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
About Metabohub
MetaboHUB is the French infrastructure of metabolomics and fluxomics that provides tools and
services to academic research teams and industrial partners in the fields of health, nutrition,
agriculture, environment and biotechnology.
MetaboHUB infrastructure gathers the equipment, means, competences, and critical mass to
respond to the specific requirements of current and future metabolomics: multi-tools
requirement, large-scale analysis programs, standardization of data and databases establishment,
multicompetences requirements and systems biology approach.
MetaboHUB provides a large variety of metabolomics services and expertise to your researcher.
The main services consist in analyzing of many biological matrices using the tools of
metabolomics and fluxomics. Our laboratories have the expertise and knowledge of sample
preparation and data acquisition from a wide range of biological matrices (plants,
microorganisms, human cells, animal tissues and biofluids). These services include data
acquisition by mass spectrometry (GC, LC), NMR (liquid and HR-MAS) and LC-NMR, NMR/MS
hyphenation and processing, annotation, visualization and exploration of large data set. A team
of bioinformaticians and biostatisticians is supporting the achievement of these remaining tasks.
The advanced services cores offers collaborations on a project-basis in specific areas of
metabolism from targeted metabolites analysis including primary, secondary metabolites, lipids,
xenobiotics, drugs, etc. to non-targeted metabolomics including lipidomics. MetaboHUB hosts a
comprehensive park of analytical tools and owns a strong expertise for the quantification of a
wide range of metabolites.
MetaboHUB is committed to transfer its technical knowledge and communicate on
breakthroughs to the broad scientific community (from classroom to lab bench). MetaboHUB
partners already propose a wide range of personalized training sessions to the scientific
community upon request (e.g. in metabolomics and fluxomics; in data treatment and
biostatistics; in NMR profiling, database management, data curation).
MetaboHUB staff members are already committed to knowledge dissemination through the
“Réseau Français de Métabolomique et Fluxomique” (‘RFMF’, French Metabolomics and
Fluxomics Network) and through "Réseau Français des Lipidomistes" (RFL). Various events are
being organized by MetaboHUB staff through RFMF and MetaboHUB is largely involved in the
organization of several national and international congresses.
For more information, visit: http://www.metabohub.fr/en/
Contact: [email protected]
ANR-11-INBS-0010
6
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
About Institut Français de Singapour
– French Embassy
The Institut Français de Singapour (IFS) of the
French Embassy is a vector that develops
cooperation between France and Singapore in the
areas of culture, science and technology, and
higher education.
It promotes the French language, educational opportunities, creativity and
French innovation and research. It thus has a far reaching mandate and is a
center of expertise and advice.
Always at the forefront of current issues, the Institut Français de Singapour’s
priority is a focus on innovation, making it a factor of attractiveness for
French expertise. Among the numerous programs of the IFS, the PHC
Merlion program actively supports research development.
The PHC Merlion Program is a joint Franco-Singaporean collaboration, and
managed by the IFS in partnership with Singaporean institutions. The aim is
to encourage and support new scientific development in research between
French and Singaporean laboratories, through funding the scientists’ trip
exchanges. The program takes the form of an annual call for proposals, and
since its inception in 2006, has funded more than 150 bilateral projects, one
of which is the MERLION Metabolomics Workshop Singapore 2014!
More information and the calendar of cultural and scientific events may be
found on our website. In addition, do not hesitate to subscribe to our
newsletter.
http://www.institutfrancais.sg/
7
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Keynote Speakers
Professor Choon Nam ONG
School of Public Health, National University of
Singapore (NUS)
Director of the NUS Environmental Research Institute,
Singapore
His current research interests include evaluation of
Biomarkers using metabolomics platform and
Mechanistic-based investigations of chronic disease
and their prevention. He has published over 300 peerreviewed articles with close to 15,000 citations. He sits on the editorial board of
several scientific journals and currently an Associate Editor of Environmental
Research.
Professor Dominique ROLIN
Metabohub – Bordeaux
University of Bordeaux, France
D. Rolin is a professor at University of Bordeaux. He
has recognized expertise in the fields of NMR
spectroscopy, biology, plant and fruit metabolism,
metabolomics and fluxomics (M&F). He is heading
MetaboHUB and also CGFB a federation of
technological platforms dedicated to the study of living organisms at University of
Bordeaux. In 2002, he has set up and led the Metabolome Facility of Bordeaux
(BMP), which can be considered as a pioneer in plant metabolomics in France. D.
Rolin is also deeply involved in the promotion of M&F in France as co-founder
(2005), treasurer (2007-2010) and President (2010-2015) of the RFMF.
8
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Professor Royston GOODACRE
University of Manchester, United Kingdom
Educated in University of Bristol, UK where he was
awarded his BSc in Microbiology and his PhD in
analytical methods applied to microbiological
problems. After which he did a three year PDRA in
University of Wales, Aberystwyth, UK and then four
years as a Wellcome Trust Fellow where he
investigated chemometrics and artificial neural networks for the analysis of
spectroscopic data. Following this he was appointed a Lecturer in Microbiology.
He moved in Feb 2003 to take up the position of Reader in Analytical Science in
the School of Chemistry, The University of Manchester, UK. His current position
since Aug 2005 is as Professor of Biological Chemistry. His research focuses on
developing MS-based metabolomics along with new chemometric approaches
that he applies to help understand how organisms respond to external
perturbations. His group also develops Raman spectroscopy for chemical imaging
and surface enhanced Raman scattering for trace detection of (bio)chemicals. He
is founder and Editor-in-Chief of Metabolomics, on the Editorial Advisory Boards
of Analyst and Journal of Analytical and Applied Pyrolysis.
Associate Professor Robert TRENGOVE
Separation Science and Metabolomics Laboratory
Murdoch University, Australia
Rob Trengove has pioneered the development of
MS-based metabolomics techniques for more than
20 years, collaborating with Australian and
International researchers and industry. He currently
leads a team of more than 15 researchers working
on a diverse range of topics including HIV/AIDs, iron disorders, desalination,
microalgae lipidomics, fungal and bacterial metabolomics. Rob Trengove has
published more than 60 high-impact journal articles and has ongoing commercial
contract research arrangements with major industry players in the petrochemical,
clinical and animal health pharmaceutical sector.
9
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Professor Huiru Tang
Wuhan Institute of Physics and Mathematics, Chinese
Academy of Sciences, China
Prof. TANG received his bachelor’s degree in 1986
from Shannxi University of Science and Technology
and doctor’s degree in 1994 from University of
London. His research interests include metabolomic
analytical methods, interaction between host
metabolic and intestinal flora, pathophysiological metabonomic studies. He has
published more than 130 papers with more than 2000 citations.
Professor Jean Charles Portais
Metabolic Biochemistry at the University of Toulouse,
France
JC PORTAIS is Professor of Metabolic Biochemistry at
the University of Toulouse (France) and has recognized
expertise in the fields of metabolism, metabolomics
and fluxomics. He is heading the research group
MetaSys at LISBP, which aims at the comprehensive,
system-level understanding of metabolic adaptation
and its role in the behavior of organisms, with applications in biotechnology and
human health. Prof. JC Portais is also heading MetaToul, the Metabolomics &
Fluxomics Center of Toulouse, one of the very first Metabolomics centers in
France. He is also deeply involved in the promotion of M&F in France as cofounder and President (2005-2010) of the RFMF.
10
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Professor Eiichiro Fukusaki
Division of Science and Biotechnology, Graduate
School of Engineering, Osaka University, Japan
Eiichiro Fukusaki entered a private company, Nitto
Denko Co, after receiving master degree from Osaka
University on 1985. He received PhD from Osaka
University on 1993 through his company work. After
ten years company experience, he returned back to
Osaka University as an associate professor. On 2007
he has been assigned as a full professor in Osaka University. His current research
interests are focusing on development and application of metabolomics
technology. He has published over 200 original papers and 50 patents.
Dr Christophe Junot
French Alternative Energies and Atomic Energy
Commission, France
C. Junot is a doctor of Pharmacy. After having
gained a PhD in Analytical Chemistry in 2000 (Pierre
et Marie Curie University, Paris 6), he joined
GlaxoSmithKline laboratories and developed
experience in the field of pharmacokinetics and
metabolism applied to drug discovery for 2 years.
From 2002, he has been a group leader at the Life Science Division of CEA
(Commissariat à l’Energie Atomique) where he develops mass spectrometry based
analytical methodologies for metabolome analysis. C. Junot has been appointed as
head of the Laboratory for Drug Metabolism Studies since September 2010 and he
is member of the board of the French Metabolomics and Fluxomics Network. C.
Junot is the deputy coordinator of the French MetaboHUB platform and also in
charge of the coordination of analytical chemistry developments within this
platform.
11
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Dr Fabien Jourdan
MetaboHub-Toulouse
French National Research Institute for Agricultural
Research, France
Fabien Jourdan is a research scientist at INRA (the
French National Research Institute for Agricultural
Research). He is developing bioinformatics methods
to study genome-scale metabolic networks. These
approaches are mainly based on graph models. The
aim is, based on experimental data like metabolomics ones, to retrieve parts of
the organism metabolism affected by genetic or environmental perturbations.
Fabien Jourdan is leading the development of MetExplore web server
(www.metexplore.fr) which is gathering most of these algorithms and allows
visualization of metabolic networks. MetExplore development is supported by the
French National infrastructure for metabolomics and fluxomics, MetaboHub..
12
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Scientific Programme
Date (Day)
Time
Event
19 Nov 2014
(Wednesday)
A1
A2
Speakers
Day 1
8.30 - 9.00 a.m.
Registration and Breakfast
9.00 - 9.10 a.m.
Opening address
by National University of
Singapore
Assoc Prof Sanjay Swarup
Chairman
Merlion Metabolomics
Workshop
9.10 - 9.20 a.m.
Welcome address
by Invited Guest
National University of
Singapore
Prof Andrew Wee
Vice President (University
and Global Relations)
National University of
Singapore
9.20 - 9.30 a.m.
Speech
by Guest-of-Honor
Embassy of France to
Singapore
His Excellency
Mr Benjamin Dubertret
Ambassador of France to
Singapore
9.30 - 10.00 a.m.
Opening Speaker
(Singapore)
Introducing metabolomics in
Singapore
Prof Ong Choon Nam
NUS Environmental
Research Institute National
University of Singapore
10.00 - 10.30 a.m.
Opening Speaker (France)
MetaboHUB and RFMF: Two
tools at the service of
metabolomics and fluxomics
in France
Prof Dominique Rolin
Metabohub - Bordeaux
University of Bordeaux,
France
10.30 - 10.50 a.m.
Tea break
13
MERLION METABOLOMICS WORKSHOP SINGAPORE
19 Nov 2014
(Wednesday)
Abstract No.
2014
Day 1
Session 1 - Food Science and Technology Metabolomics
Chaired by: Dr Fabien Jourdan and Dr Naweed Naqvi
10.50 – 11.20
a.m.
Keynote Speaker
Metabolomics the way
forward
Prof Royston Goodacre
Manchester Institute of
Biotechnology
University of Manchester,
United Kingdom
A4
11.20 – 11.50
a.m.
Keynote Speaker
Metabolic network
modelling and Food
Toxicology
Dr Fabien Jourdan
MetaboHub-Toulouse
French National Research
Institute for Agricultural
Research, France
A5
11.50 – 12.10
p.m.
Untargeted metabolomic
approaches and data mining
tools for marker discovery in
nutrition
Dr Estelle Pujos-Guillot
French National Research
Institute for Agricultural
Research, France
A6
12.10 – 12.30
p.m.
Lipidomic analysis of palm oil
variability
Dr Laetitia Fouillen
French National Center for
Scientific Research, France
12.30 - 12.50
p.m.
Novel Metabolites Involved
in Pathogen-Host Interaction
and Disease Control
Dr Naweed Naqvi
Temasek Life Sciences
Laboratory
National University of
Singapore
A3
A7
12.50 - 1.30
p.m.
Lunch
14
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
19 Nov 2014
(Wednesday)
Day 1
Abstract No.
Session 2 – Human Health Metabolomics
Chaired by: Dr Christophe Junot and Assoc Prof Chan Chun Yong, Eric
1.30 – 2.00 p.m.
Keynote Speaker
Mass spectrometry based
metabolomics and lipidomics
for the study rare diseases
Dr Christophe Junot
French Alternative Energies
and Atomic Energy
Commission, France
2.00 – 2.20 p.m.
Lipidomics: a key tool for
human health
Dr Justine Bertrand Michel
French National Institute of
Health and Medical
Research, France
2.20 – 2.40 p.m.
Urinary metabolomics
revealed arsenic exposurerelated metabolic alteration:
a proof-of-concept study in a
Chinese male cohort
Prof Zhang Jie
Institute of Urban
Environment
Chinese Academy of
Sciences, China
A11
2.40 – 3.00 p.m.
Noninvasive urinary
metabonomic diagnosis of
human bladder cancer
A12
3.00 – 3.20 p.m.
Natural variation of
lipidomes
A8
A9
A10
3.20 - 3.40 p.m.
Assoc Prof Chan Chun Yong,
Eric
Department of Pharmacy
National University of
Singapore, Singapore
Assoc Prof Marcus Wenk
Department of Biochemistry
and Department of
Biological Sciences
National University of
Singapore, Singapore
Tea break
Session 3 - Young Researchers Session
Y1-Y8
3.40 - 5.40 p.m
5.40 – 6.00 p.m.
6.00 – 8.00 p.m.
8 oral presentations (15 min
each) - Human Health
Presenters:
1. Dr Mrinal Kumar Das
2. Dr Cui Liang
3. Dr Liu Feng
4. Ms Yin Xuejiao
5. Mr Erhan Simsek
6. Ms Anna Karen
Carrasco Laserna
7. Dr Kenjiro Kami
8. Dr Robert L Davidson
Chaired By:
Dr Sastia Prama Putri
Department of
Biotechnology
Graduate School of
Engineering
Osaka University, Japan
Agilent Sponsored Talk
Dr Oliver Jones
Two-dimensional liquid
School of Applied Sciences,
chromatography: a new
RMIT University, Australia
technique for metabolomics?
Welcome Cocktail Reception
15
MERLION METABOLOMICS WORKSHOP SINGAPORE
20 Nov 2014
(Thursday)
2014
Day 2
Session 4 – Food Science and Technology Metabolomics
Chaired by: Prof Royston Goodacre and Dr Blandine Comte
9.00 – 9.30 a.m.
Keynote Speaker
Application of metabolic
fingerprinting for food
quality assessments
Prof Eiichiro Fukusaki
Department of
Biotechnology
Graduate School of
Engineering
Osaka University, Japan
9.30 – 9.50 a.m.
Metabolomics and systems
biology in the context of
food toxicology
Dr Daniel Zalko
French National Research
Institute for Agricultural
Research, France
9.50 – 10.10 a.m.
Use of metabolomics for
gene function discovery in a
dwarf rice mutant
A16
10.10 – 10.30 a.m.
Nutritional metabolomics:
an integrative
understanding of metabolic
disease development
Dr Blandine Comte
French National Research
Institute for Agricultural
Research, France
A17
10.30 - 10.50 a.m.
A totally new way to
measure the germination of
grain using metabolomics
Dr Oliver Jones
School of Applied Sciences,
RMIT University, Australia
A13
A14
A15
10.50 - 11.10 a.m.
Prof Prakash Kumar
Department of Biological
Sciences
National University of
Singapore, Singapore
Tea break
16
MERLION METABOLOMICS WORKSHOP SINGAPORE
20 Nov 2014
(Thursday)
Day 2
Abstract No.
Session 5 – Human Health Metabolomics
Chaired by: Prof Ong Choon Nam and Dr Etienne Thevenot
2014
A18
11.10 - 11.40 a.m.
Keynote Speaker
Metabolomic Aspects of
Symbiosis and
Transgenomic Interactions
In Mammals
A19
11.40 – 12.00 p.m.
Metabolomic applications in Dr Ho Ying Swan
Bioprocessing Technology
bioprocessing and clinical
Institute, A*STAR, Singapore
research
12.00 a.m. – 12.20
p.m.
Metabolomics driven the
discovery of functional
small molecules towards a
diversity of biological
innovations
Prof Lu Haitao
Chongqing University
Innovative Drug and
Research Centre, China
12.20 – 12.40 p.m.
Urine, metabolite profiling,
its applications
Dr Ong Eng Shi
Singapore University of
Technology and Design,
Singapore
12.40 – 1.00 p.m.
Biostatistics for biomarker
discovery and phenotype
prediction
Dr Etienne Thevenot
French Alternative Energies
and Atomic Energy
Commission
1.00 – 1.20 p.m.
Metabolomics-A great tool
for generating hypothesis
Dr Wang Yulan
Wuhan Institute of Physics
and Mathematics Chinese
Academy of Sciences, China
A20
A21
A22
A23
1.20 - 2.10 p.m.
Prof Tang Huiru
Wuhan Institute of Physics
and Mathematics Chinese
Academy of Sciences, China
Lunch
17
MERLION METABOLOMICS WORKSHOP SINGAPORE
20 Nov 2014
(Thursday)
Day 2
Abstract No.
Session 6- Environmental Metabolomics
Chaired by: Assoc Prof Sanjay Swarup and Dr Cyril Jousse
A24
A25
2014
2.10 – 2.40 p.m.
Keynote Speaker
Model systems for the
metabolomics study of
environmental exposure
Prof Robert Trengove
Separation Science and
Metabolomics Laboratory
Murdoch University,
Australia
2.40 – 3.00 p.m.
Environmental metabolomics
to understand resource
partitioning strategies in algae
Assoc Prof Sanjay Swarup
NUS Environmental
Research Institute
National University of
Singapore, Singapore
A26
3.00 – 3.20 p.m.
A27
3.20 – 3.40 p.m.
Non targeted metabolomics
for assessment of
environmental exposure to
contaminants and their
biological effects: towards
exposomics
Dr Laurent Debrauwer
MetaboHub-Toulouse
French National Institute
for Agronomy Research,
France
A study on Honeybee losses.
First insight into
environmental interactions.
Dr Cyril Jousse
University of Blaise Pascal,
France
3.40 - 4.00 p.m.
Tea Break
Session 7 - Young researchers session
Y9-16
4.00 - 6.00 p.m.
7.30 - 9.30 p.m.
8 oral presentations
(15 min each)
Presenters:
Environmental
• Mr Umashankar
Shivshankar
• Ms Gao Yan
• Ms Zhang Wenlin
• Dr Amit Rai
• Dr Saravan Periasamy
Food Science & Technology
• Ms Nay Min Min
Thaw Saw
• Dr Aw Wanping
• Dr Rajesh N. Patkar
Chaired By:
Dr Blandine Comte
French National Research
Institute for Agricultural
Research, France
Cocktail Reception and Dinner Banquet
18
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
21 Nov 2014
(Friday)
Day 3
Abstract No.
Session 8 - Environmental Metabolomics
Chaired by: Prof Sam Li Fong Yau and Prof Robert Trengove
A28
A29
A30
A31
A32
9.00 – 9.30 a.m.
Keynote Speaker
Comprehensive
investigations of cellular
metabolic networks using
13C-fluxomics and
applications to systems
microbiology and
biotechnology
Prof Jean Charles Portais
MetaboHub-Toulouse
University of Toulouse,
France
9.30 – 9.50 a.m.
Response of cloud
microorganisms to
atmospheric stresses: the
case study of cold shock
Dr Anne-Marie Delort
University of Blaise Pascal,
France
9.50 – 10.10 a.m.
Analysis of leachates from
gasification of solid wastes
by metabolomics
techniques
Prof Sam Li Fong Yau
NUS Environmental
Research Institute
National University of
Singapore, Singapore
10.10 – 10.30 a.m.
Methods for dealing with
batch effects in
metabolome data
Dr Rohan Benjamin Hugh
Williams
Singapore Centre for
Environmental Life Sciences
Engineering
National University of
Singapore, Singapore
10.30 - 10.50 a.m.
Metabolomics-driven
strain improvement of
higher alcohols in
Escherichia coli
10.50 – 11.10 a.m.
11.10 a.m. - 12.30
p.m.
12.30 - 12.35 p.m.
12.35 - 12.40 p.m.
Dr Sastia Prama Putri
Department of
Biotechnology
Graduate School of
Engineering
Osaka University, Japan
Tea break
Panel Discussion on Metabolomics Research Directions
Closing Remarks
(France)
Closing Remarks
(Singapore)
19
Dr Fabien Jourdan
MetaboHub-Toulouse
French National Research
Institute for Agricultural
Research, France
Assoc Prof Sanjay Swarup
NUS Environmental
Research Institute National
University of Singapore,
Singapore
MERLION METABOLOMICS WORKSHOP SINGAPORE
12.40 - 1.30 p.m.
2014
Lunch
Site 1: Agilent Factory in
Yishun Industrial Estate
1.30 - 4.30 p.m.
Technical Site visit
Site 2: NUS Environmental
Research Institute (NERI)
Laboratory at T-Lab and
CREATE.
Singapore and French Discussion (By Invitation Only)
Workshop End
In Summary
Item
No. of Speakers
Duration
Keynotes
9
25 mins presentation and 5 mins Q&A
Oral Presentations
23
15 mins presentation and 5 mins Q&A
Young researchers
presentations
16
10 mins presentation and 5 mins Q&A
20
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Young Researcher Award
The “Young Researchers Award” recognizes and celebrates innovation in
research as presented during the Merlion Metabolomics Workshop Young
Researchers’ session. Selected members of the Workshop’s Scientific
Committee have graciously agreed to serve as judges for the Award and
their decisions are final.
Two prizes shall be presented in 2014. Each prizewinner will receive a CRC
Press book titled: “Mass Spectrometry-Based Metabolomics: A Practical
Guide” (RRP USD140).
Mass Spectrometry-Based Metabolomics: A Practical
Guide is a simple, step-by-step reference for profiling
metabolites in a target organism. The book
summarizes all steps in metabolomics research, from
experimental design to sample preparation, analytical
procedures, and data analysis. Case studies are
presented for easy understanding of the metabolomics
workflow and its practical applications in different
research fields. The book includes an in-house library
and built-in software so that those new to the field can
begin to analyze real data samples. In addition to
being an excellent introductory text, the book also
contains the latest advancements in this emerging
field and can thus be a useful reference for metabolomics specialists.
For more information: http://www.crcpress.com/product/isbn/9781482223767
Many thanks to Professor Eiichiro Fukusaki, Dr Sastia Putri And CRC Press
for their sponsorship of this award.
21
MERLION METABOLOMICS WORKSHOP SINGAPORE
Abstracts for Main Session
22
2014
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A1. Introducing metabolomics in Singapore
Eric Chan Chun Yong 1, Sam Li Fong Yau 2, 5, Sanjay Swarup3 ,5 and Choon Nam Ong 4, 5 *
1
Department of Pharmacy
Department of Chemistry
3
Department of Biological Sciences
4
School of Public Health
5
NUS Environmental Research Institute (NERI)
National University of Singapore
*E-mail: [email protected] (corresponding author)
2
Abstract
Metabolomics is a burgeoning ‘omics’ field that leverages upon the established analytical
chemistry technologies and bioinformatics tools for the study of small metabolites. In Singapore,
a core group of scientists have been applying metabolomics during the past decade developing
suitable technologies for metabolomics investigations and concurrently using this technology to
solve real-world problems such as the discovery of novel metabolic signatures for the diagnosis of
diseases, elucidation of effect of host-gut microbiota interactions on drug disposition, diet and
disease, plant and microbial metabolomics, metabolic changes of exposure to environmental
contaminants, and identification of biomarkers for early disease prevention and treatment. The
team also working closely with researchers locally and internationally and the analytical
platforms used including Nuclear Magnetic Resonance (NMR) and different Mass Spectrometry
(HPLC-MS and GC-MS). The team also engaged commercial and industrial sectors to develop
advanced technologies for metabolomic investigations. In this paper the authors will update
some of their recent works on environmental and biomedical related metabolomics. The
challenges and future of this field in Singapore will also be discussed.
23
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A2. MetaboHUB and RFMF: two tools at the service of metabolomics and
fluxomics in France
D. Rolin1*, F. Bellvert2, J. Bertrand-Michel2, C. Canlet2, R. Cole3, S. Colombié1, C. Deborde1,
L. Debrauwer3, M. Ferrara4, L. Fouillen1, F. Jourdan2, C. Jousse4, C. Junot3, A. Moing1, JC.
Portais2, E. Pujos-Guillot4, C. Sautot5, E. Thevenot3
1
MetaboHUB Bordeaux, INRA, CNRS, Université Bordeaux, F-33140 Villenave d'Ornon, France
MetaboHUB Toulouse, CNRS, INRA, INSA, INSERM, Université Paul Sabatier, F-31000 Toulouse,
France
3
MetaboHUB Saclay-Paris, CEA, University Paris VI, F-91190 Gif-sur-Yvette, France
4
MetaboHUB Clermont-Ferrand, INRA, Université Blaise Pascal, CNRS, F-63122 Saint-GenèsChampanelle, France
5
INRA Transfert, F-44300 Nantes, France
*E-mail: [email protected] (corresponding author)
2
Abstract
MetaboHUB and RFMF are two tools developed in France to serve the scientific
community in Metabolomics and Fluxomics (M&F). The French Metabolomics and Fluxomics
Network (RFMF, http://bit.ly/fr_metabolomics) is a scientific society which contributes to the
development of M&F and more broadly to the overall analysis of metabolism in France through
various actions:
• Foster relationships between French researchers working in M&F (public and private sectors);
• Promote and structure education and training in M&F;
• Organize and support the organization of conferences in France and abroad;
• Encourage the participation of young researchers to conferences through travel grants;
• Allocate funds mission or prizes for accomplished work in M&F.
In 2013, RFMF and the international Metabolomics Society formed an international
affiliation to promote metabolomics.
MetaboHUB project (http://www.metabohub.fr/en/) aims at creating a national
infrastructure that will place France among the European leaders for advanced research services
in M&F. MetaboHUB provides equipment and services to academic research and industrial
partners in nutrition, health, agriculture, environment and biotechnology. MetaboHUB is the
merging of 4 existing facilities (Bordeaux, Paris-Saclay, Toulouse and Clermont-Ferrand) into a
unique infrastructure sharing common regulations and complementary M&F tools. Besides
providing state-of-the-art services and support to national and international projects, a major
ambition of MetaboHUB is to develop innovative tools and methods to address critical biological
questions. MetaboHUB will provide custom solutions for (i) high-throughput, quantitative
technologies for biochemical phenotyping of large sets of samples and for systems biology
through standardization and combination of state–of-art technologies, (ii) identification of
metabolites in human biofluids, plants, microorganisms and animal cell extracts, through the
implementation and maintenance of centralized and open spectral repositories for metabolome
annotations, (iii) large-scale flux profiling and sub-cellular fluxomics through integration of
analytical data from multiple analytical devices, (iv) access to high-impact services to the national
scientific community and industrial actors and, (v) attracting and training a new generation of
scientists and students.
24
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A3. Metabolomics the way forward
Royston Goodacre*
School of Chemistry, and Manchester Institute of Biotechnology, University of Manchester, 131
Princess Street, Manchester, M1 7DN, UK
*E-mail: [email protected] (corresponding author)
Abstract
Metabolomics is a growing discipline that allows the analysis of the thousands of
structural different small molecules found within a biological system. These metabolites can be
measured using a variety of different analytical approaches including NMR spectroscopy or
alternatively can be separated and quantified using gas or liquid chromatography coupled to
mass spectrometry, after which the hope is that specific biomarkers can be discovered and linked
to health, nutrition and disease [1].
This presentation will provide an overview of metabolomics and will give a flavor of how
data are generated and analyzed. The role of metabolomics in nutrigenomics will also be
discussed, as well as the concepts of the human being considered a superorganism [2] and all the
complexities this generates that need to be overcome to understand human health and disease.
During the presentation, I will also discuss the bioanalytical pathway [3] that we have developed
as part of our human serum metabolome project (http://www.husermet.org/) that is need in
order to negotiate the metabolite superhighway such that rigorously identified metabolites are
discovered that can be used for understanding normal population behaviours and for disease
stratification [4].
Finally I will give a very brief introduction to the Metabolomics Society.
References
1. Goodacre, R. Vaidyanathan, S., Dunn, W.B., Harrigan, G.G. & Kell, D.B. (2004)
Metabolomics by numbers - acquiring and understanding global metabolite data. Trends
in Biotechnology 22, 245-252.
2. Goodacre, R. (2007) Metabolomics of a superorganism. Journal of Nutrition 137, 259S266S.
3. Dunn, W.B., Broadhurst, D., Begley, P., Zelena, E., Francis-McIntyre, S., Anderson, N.,
Brown, M., Knowles, J.D., Halsall, A., Haselden, J.N., Nicholls, A.W., Wilson, I.D., The
Husermet consortium, Kell, D.B. & Goodacre, R. (2011) Procedures for large-scale
metabolic profiling of serum and plasma using gas chromatography and liquid
chromatography coupled to mass spectrometry. Nature Protocols 6, 1060-1083.
4. Dunn, W.B., Lin, W., Broadhurst, D., Begley, P., Brown, M., Zelena, E., Vaughan, A.A.,
Halsall, A., Harding, N., Knowles, J.D., Francis-McIntyre, S., Tseng, A., Ellis, D.I., O’Hagan,
S., Aarons, G., Benjamin, B., Chew-Graham, S., Moseley, C., Potter, P., Winder, C.L., Potts,
C., Thornton, P., McWhirter, C., Zubair, M., Pan, M., Burns, A., Cruickshank, J.K., Jayson,
G.C., Purandare, N., Wu, F.C.W., Finn, J.D., Haselden, J.N., Nicholls, A.W., Wilson, I. D.,
Goodacre, R. & Kell, D.B. (2014) Molecular phenotyping of a UK population: defining the
human serum metabolome. Metabolomics, in press
25
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A4. Metabolic network modelling and Food Toxicology
Florence Vinson1, Ludovic Cottret2, Benjamin Merlet1, Yoann Gloaguen3, Clément
Frainay1, Nathalie Poupin1, Daniel Zalko1, and Fabien Jourdan1*
1
INRA UMR1331 - Toxalim - MetaboHub, Toulouse, France
INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441, Castanet-Tolosan,
F-31326, France
3
Glasgow Polyomics, University of Glasgow, 120 University Place, Glasgow G12 8TA
* Speaker
Mailing address: Fabien Jourdan, INRA UMR1331 TOXALIM-MetaboHub, 180 Chemin de
Tournefeuille F31027 Toulouse Cedex 3, France
*Email: [email protected] (corresponding author)
2
Abstract
Metabolomics is a powerful tool to detect if there is a significant metabolic shift when an
organism is exposed to chemical pollutants. To do so, lists of significantly modified metabolites,
constituting metabolic fingerprints, are built using Nuclear Magnetic Resonance and/or Mass
Spectrometry. It then allows classifying individuals based on the dose, frequency, and period of
exposition to xenobiotics. Nevertheless these biomarkers cannot directly be used to decipher the
mechanistic changes occurring in the organism. This interpretation is of outmost importance
since endocrine disruptors like Bisphenol A may have long term impacts and lead to metabolic
diseases. Our aim is to develop modelling approaches to help in understanding which parts of the
metabolism are affected by chemical pollutants.
To interpret global and untargeted data generated by metabolomics we need to consider
metabolism at once and not to focus on individual metabolic pathways. To do so we use
metabolic network modelling which gathers all metabolic reactions an organism can use for
catabolic and anabolic processes. Human genome scale metabolic network recently published
contains 7440 reactions and 2626 metabolites. The challenge is then to mine this network in
order to find which cascades of reactions are responsible of biomarker concentration changes.
We propose algorithms to build interpretable sub-networks from the whole network by
extracting tenths of meaningful reactions. We will show that, based on sub-network
comparisons, it is then possible to point out specific metabolic shifts induced by different doses
of Bisphenol A. Finally, since reconstruction of metabolic networks is achieved using genomic
information, it establishes a link between reactions, proteins and genes. Network can then be
used as a relevant framework for polyomics studies.
All these bioinformatics methods and metabolic networks are accessible through the
MetExplore web server we are developing in the framework of MetaboHub.
26
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A5. Untargeted metabolomic approaches and data mining tools for
marker discovery in nutrition
B. Lyan1,2, F. Giacomoni1,2, C. Joly1,2, C. Migné1,2, J. F. Martin1,2, M. Pétéra1,2, J. Bourseau3,
A. Napoli3, J. L. Sébédio2, B. Comte2, E. Pujos-Guillot 1,2 *
1
MetaboHUB Clermont-Ferrand, INRA, University Blaise Pascal, F-63122 Saint-GenèsChampanelle, France
2
INRA, UMR1019, UNH, F-63000 Clermont-Ferrand, France
3
LORIA, B.P. 239, F-54506 Vandoeuvre-lès-Nancy, France
*E-mail: [email protected] (corresponding author)
Abstract
Metabolomics is a powerful tool in nutrition research for characterizing complex
phenotypes and identifying biomarkers of specific physiological responses to different diets in
the context of molecular pathways, physiology and health status. However, nutritional
metabolomics is particularly challenging because of the low amplitude of effects induced by
nutritional interventions or transitions, in comparison to the large variability in samples due to
intra/inter-individual variations but also collection, storage and pre-treatment. Untargeted
approaches using multiple analytical platforms are particularly of relevance to obtain a large
analytical coverage of metabolites, which is essential because of the extreme chemical diversity
of the endogenous and food metabolomes, ranging from sugars and lipids to polyphenols.
However, in this context, important criteria for selection of an untargeted method are not only
based on analytical coverage, but also on robustness of sample pre-treatment and on analytical
reproducibility in terms of retention times, masses and signal intensities.
We will illustrate the use of UPLC/HRMS and GC/MS based untargeted strategies for the
study of food exposure, as well as for the characterization of metabolic phenotypes associated
with development or prevention of chronic metabolic diseases. Indeed, the determination and
the annotation of plasma and urine metabolic profiles constitute a powerful data-driven
approach for the discovery of potential biomarkers of food intake and/or metabolic changes.
However, irrespective of the type of analytical techniques used, these metabolomic strategies
generate large amounts of data that need analyses and integration to extract the biologically
meaningful information and enable the discovery of new knowledge. We will show that
application of numerical and symbolic data mining methods based on clustering and pattern
mining/formal concept analysis are particularly relevant to discover classes and sub-classes of
individual phenotypes, as well as guide visualization and interpretation of data from nutritional
metabolomics.
27
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A6. Lipidomic analysis of palm oil variability
L. Fouillen1*, G-F. Ngando-Ebongue2, Y. Yuang1, V. Arondel1 and R. Lessire1
1
Laboratoire de Biogenèse Membranaire, UMR 5200, Université de Bordeaux/CNRS, CS20032,
33140 Villenave d’Ornon, France, 2 Centre de Recherches sur le Palmier à Huile de la Dibamba,
Institut de Recherche Agricole pour le Développement, BP243 Douala, Cameroon.
*E-mail: [email protected] (corresponding author)
Abstract
Palm oil is one of the most widely used vegetal oil in the human diet. Triacylglycerols
(TAGs), the molecules composing the oil, contain three fatty acids esterified to a glycerol
backbone. Palmitic and oleic acids (C16:0 and C18:1, respectively) represent about 85% of palm
oil total fatty acids. It has been questioned whether the high level of saturated palmitic acid (4555%) might lead to increased coronary heart disease risk, which has generated suspicion in
consumers for food that contains palm oil. A research program is currently being developed in
Cameroon to investigate natural variability of fatty acid composition, with the long term aim of
decreasing saturated fatty acid levels in palm oil through conventional breeding.
Thus, we have analyzed oils from different genotypes commonly used for palm oil
production for fatty acid content by GC-FID and for TAG composition by ESI-MSn. Differences
between the various genotypes in the level of fatty acid distribution and content as well as in the
TAGs composition have been observed. The variability for palmitic acid ranges from 15 to 45%
and for oleic acid from 40 to 60%. The TAG composition showed more than 20 molecular TAG
species, with high content of TAG 52:2, TAG 54:3 and TAG 54:4. Our results show that variability
of fatty acid and TAG compositions exists, which will be useful to select genotypes more
appropriate for human and industrial uses. Moreover, these data will allow us to study in more
details the biochemical mechanisms responsible for this variability.
28
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A7. Novel Metabolites Involved in Pathogen-Host Interaction and Disease
Control
Naweed Naqvi*, Rajesh Patkar, Ziwei Qu
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore
117604
*Email: [email protected] (corresponding author)
Abstract
The rice-blast fungus, Magnaporthe oryzae, is a hemibiotroph that utilizes specialized
infection-structures (the appressoria) to breach the host cuticle. Blast disease is a major
deterrent to crop production and food security. Using mutant screening and chemical genomics,
we identified Abc3 transporter function as being essential for pathogenesis in M. oryzae (1). We
developed a novel platform technology to identify the Abc3 efflux Transport Substrate. ATS is a
Digoxin-like steroidal glycoside, and essential for the host penetration step of Magnaporthe
pathogenesis (2). ATS functions in regulating ion-homeostasis across appressorial plasma
membrane and subsequently acts as an elicitor/effector of the host defense response during the
biotrophic phase of rice blast. Interestingly, ATS (and digoxin) possesses a potent antifungal
activity and is capable of inhibiting growth and pathogenesis in rice blast and Candida species (US
Patent No. 20120003261).
Recently, we identified an Antibiotic Biosynthesis Monooxygenase (ABM) as a
Cytochrome P450-related function essential for Magnaporthe pathogenesis. ABM
monooxygenase activity is directly involved in the biosynthesis of a novel metabolite that is
secreted into the host cells to suppress the defense/resistance response therein. ABM plays an
important role in the transition from biotrophy to necrotrophy in the blast disease cycle. Thus,
the blast fungus directly interferes with hormonal signaling involved in pathogen recognition and
defense system in rice. Our novel disease control strategies aim to disrupt such highly specific
and intricate chemical communication at the host-pathogen interface.
References
1. Sun, C.B., Suresh, A., Deng, Y.Z., Naqvi, N.I. (2006) A Multidrug Resistance Transporter in
Magnaporthe is required for Host Penetration and for Survival during Oxidative Stress.
The Plant Cell 18, 3686-3705.
2. Patkar, R., Xue, Y., Shui, G., Wenk, M., Naqvi, N.I. (2012) Abc3-mediated Efflux of an
Endogenous Digoxin-like Steroidal Glycoside by Magnaporthe oryzae is Necessary for
Host Invasion during Blast Disease. PLOS Pathogens 8(8), e1002888.
29
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A8. Mass spectrometry based metabolomics and lipidomics for the study
rare diseases
Benoit Colsch1, Samia Boudah1, 2 Etienne Thévenot3, Alexandre Seyer4, Simon Broudin4,
Lydie Oliveira5, Florence Castelli1, Jean-Claude Tabet1,6, Christophe Junot1*
1
LEMM-CEA-Saclay, MetaboHUB-Paris, France; 2GlaxoSmithKline - Centre de recherche
F.Hyafil,France; 3 Laboratory of Software Intensive Technologies (LIST), CEA-Saclay, MetaboHUBParis, France; 4 Profilomic SA, France ; 5CEA-iRCM-GReX, France ;6 Laboratoire de Chimie
Structurale Organique et Biologique, IPCM/CNRS, Université Pierre et Marie Curie, France.
*Email: [email protected] (corresponding author)
Abstract
The metabolome is the set of small molecular mass compounds found in biological
media, and metabolomics, which refers to as the analysis of metabolome in a given biological
condition, deals with the large scale detection and quantification of metabolites in biological
media. It is a data driven and multidisciplinary approach combining analytical chemistry for data
acquisition, and biostatistics, informatics and biochemistry for mining and interpretation of these
data. Since the middle of the 2000’s, high resolution mass spectrometry (HRMS) is widely used in
metabolomics, mainly because the detection and identification of metabolites are improved
compared to low resolution instruments. Furthermore, thanks to their versatility, HRMS
instruments are the most appropriate to achieve an optimal metabolome coverage, at the border
of other omics fields such as lipidomics.
Our team is involved in the development of tools based on liquid chromatography
coupled to HRMS for the profiling of metabolites and lipids in various human biological media,
such as plasma, cerebrospinal fluids and red blood cell extracts. We have also developed spectral
databases for annotation of data sets and metabolite identification.
The aim of this lecture will be to present HRMS based tools for metabolomics and lipidomics, and
their relevance to the field of biomarker discovery for the diagnosis and follow-up of pathologies.
This will be done through studies developed at the laboratory, mainly in the field of neurological
diseases.
30
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A9. Lipidomics : a key tool for human health.
Pauline LE FAOUDER 1, Aude DUPUY1, Fabien RIOLS1, Véronique ROQUES1, Nicolas
CENAC 2, Thierry LEVADE 3, Hervé GUILLOU 4 , Bernard PAYRASTRE 5, Justine BERTRANDMICHEL1*
1
MetaboHUB-MetaToul-LIPIDOMIQUE, Inserm U1048, 1 Av. J Poulhes, 31 432 Toulouse, France
CPTP, Inserm U1043,CHU Purpan, 31 024 Toulouse, France
3
CRCT, Inserm U1037, 1 Av. J Poulhes, 31 432 Toulouse, France
4
Toxalim UMR 1331, INRA , 180 Chemin de Tournefeuille, 31 027 Toulouse, France
5
I2MC, Inserm U1048, 1 Av. J Poulhes, 31 432 Toulouse, France
*E-mail: [email protected] (corresponding author)
2
Abstract
Lipidomics is an emerging field, where the structures, functions and dynamic changes of
lipids in cells, tissues or body fluids are investigated. To this end, methods should be able to
quantify numerous lipid molecular species. There are two main difficulties to face: the wide
variability of the lipid structures and the large range of quantities of these molecules in biological
samples. There are virtually no common features between sterols, fatty acids, sphingolipids and
glycerolipids: despite their solubility in organic solvents, their polar and chromatographic
properties are very different. A good example that illustrates the wide range of concentrations is
phosphrylated sphingoid bases which are 1,000-10,000-fold less abundant than the major
sphingolipid sphingomyelin. These 2 points cause real difficulties to quantify lipids. Global
approaches are complex, as minor lipid species which can be key biomarkers in health are often
erased from the data. So we chose targeted approaches to be able to quantify minor lipids.
Sample preparation is a key point of lipidomics, usually a liquid-liquid extraction is performed
which can be completed with a solid phase extraction to pre-concentrate the sample. Hydrolysis
and derivatisation steps can be added if necessary. Then classical chromatographic tools are used
to separate molecules with gaseous or liquid phase depending on the lipid family. While universal
detectors like corona or flame ionization are still used, mass detectors are now most popular. We
will present here the way to analyze sterols, fatty acids and their oxidized derivatives,
glycerolipids and sphingolipids in biological samples, with a focus on some applications in cancer,
inflammation 1 and metabolic diseases 2.
References
1. Gobbetti T, Le Faouder P, Bertrand J, Dubourdeau M, Barocelli E, Cenac N, Vergnolle N.
Polyunsaturated fatty acid metabolism signature in ischemia differs from reperfusion in
mouse intestine.PLoS One. 2013
2. Ducheix S, Montagner A, Polizzi A, Lasserre F, Marmugi A, Bertrand-Michel J, Podechard
N, Al Saati T, Chétiveaux M, Baron S, Boué J, Dietrich G, Mselli-Lakhal L, Costet P,
Lobaccaro JM, Pineau T, Theodorou V, Postic C, Martin PG, Guillou H, Essential fatty acids
deficiency promotes lipogenic gene expression and hepatic steatosis through the liver X
receptor., J Hepatol. 2013
31
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A10. Urinary metabolomics revealed arsenic exposure-related metabolic
alteration: a proof-of-concept study in a Chinese male cohort
Jie Zhang*, Weipan Xu, Liangpo Liu, Qingyu Huang, Meiping Tian, Heqing Shen
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of
Sciences, China
*E-mail: [email protected] (corresponding author)
Abstract
The general population may be exposed to arsenic through various sources. Although
targeted urinary biomonitoring of arsenic species provides the most accurate assessment of
arsenic exposure, the arsenic-related metabolic perturbations are required to be evaluated. The
objective of this proof-of-concept study is to investigate arsenic-induced phenotypic metabolome
changes. Urinary arsenic species such as inorganic arsenic, methylarsonic acid, dimethylarsinic
acid and arsenobetaine were quantified in a Chinese adult male cohort using high performance
liquid chromatography (HPLC)-inductively coupled plasma-mass spectrometry. Urinary
metabolomes were analyzed using HPLC-quadrupole time of flight mass spectrometry (qTOF-MS)
and the arsenic-related metabolic changes were investigated by using a partial least squares
discriminant analysis model. After adjustment for age, BMI, smoking and drinking, five potential
biomarkers of testosterone, hippurate, acetyl-N-formyl-5-methoxykynurenamine (AFMK), serine
and guanine were identified from 61 candidate metabolites, which indicated the exposure to
arsenic induced endocrine disruption and oxidative stress in humans. Some dose-dependent
trends have been also observed. The combined pattern of testosterone, hippurate, serine and
guanine gave an AUC of 0.91 with sensitivity and specificity = 88% and 80%, respectively. We
have demonstrated that metabolomics can be used to identify arsenic-related changes at the
biomolecular level. The nature of the arsenic-related metabolic changes and trends may be used
to further refine population based risk analysis of arsenic exposure.
32
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A11. A13. Noninvasive urinary metabonomic diagnosis of human bladder
cancer
Kishore Kumar Pasikanti1, Kesavan Esuvaranathan2, Yanjun Hong1, Paul C. Ho1, Ratha
Mahendran2, Lata Raman Nee Mani2, Edmund Chiong2, and Eric Chun Yong Chan1*
1
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive
4, Singapore 117543
2
Department of Surgery, National University Health System, 5 Lower Kent Ridge Road, Singapore
119074.
*E-mail: [email protected] (corresponding author)
Abstract
Cystoscopy is the gold standard clinical diagnosis of human bladder cancer (BC). As
cystoscopy is expensive and invasive, it compromises patients’ compliance towards surveillance
screening and challenges the detection of recurrent BC. Therefore, the development of a
noninvasive method for the diagnosis and surveillance of BC and the elucidation of BC
progression become pertinent. In this study, urine samples from 38 BC patients and 61 non-BC
controls were subjected to urinary metabotyping using two dimensional gas chromatography
time-of-flight mass spectrometry (GC×GC/TOFMS). Subsequent to data preprocessing and
chemometric analysis, the orthogonal partial least squares discriminant analysis (OPLS-DA, R2X =
0.278, R2Y = 0.904 and Q2Y (cumulative) = 0.398) model was validated using permutation tests
and receiver operating characteristic (ROC) analysis. Marker metabolites were further screened
from the OPLS-DA model using statistical tests. GC×GC/TOFMS urinary metabotyping
demonstrated 100% specificity and 71% sensitivity in detecting BC while 100% specificity and
46% sensitivity were observed via cytology. In addition, the model revealed 46 metabolites that
characterize human BC. Among the perturbed metabolic pathways, our clinical finding on the
alteration of the tryptophan-quinolinic metabolic axis in BC suggested the potential roles of
kynurenine in the malignancy and therapy of BC. In conclusion, global urinary metabotyping
holds potential for the noninvasive diagnosis and surveillance of BC in clinics. In addition,
perturbed metabolic pathways gleaned from urinary metabotyping shed new and established
insights on the biology of human BC.
33
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A12. Natural variation of lipidomes
Markus R Wenk1,*
1
Department of Biochemistry, Yong Loo Lin School of Medicine, and Department of Biological
Sciences, National University of Singapore
*E-mail: [email protected] (corresponding author)
Abstract
Once viewed simply as a reservoir for carbon storage, lipids are no longer cast as
bystanders in the drama of biological systems. The emerging field of lipidomics is driven by
technology, most notably mass spectrometry, but also by complementary approaches for the
detection and characterization of lipids and their biosynthetic enzymes in living cells (Wenk 2010
Cell 143(6):888-95).
Our recent results show extensive diversity in circadian regulation of plasma lipids and
evidence for different circadian metabolic phenotypes in humans (Chua et al 2013 Proc Natl Acad
Sci U.S.A. 110(35):14468-73). I will also introduce a strategy for capture of phospho-monoester
lipids. Using this enhanced workflow we identified novel forms of sphingosine-1-phosphates, in
tissue from human, mouse and fruit fly, respectively.
Understanding better the fundamentals of natural variation in lipidomes as well as
specific recognition of individual lipid species are the scientific aims of SLING, the Singapore
Lipidomics Incubator (http://lsi.nus.edu.sg/corp/research-programmes/sling/). This centre is a
global magnet for collaborating parties in lipidomics – from academia and industry – delivering
new technologies and intellectual capital. SLING organizes the international Singapore Lipid
Symposium (ISLS), a major symposium in lipidomics research in Asia Pacific
(http://www.lipidprofiles.com/index.php?id=82) and ‘i c lipid’, an intensive immersion course in
mass spectrometry based lipidomics (http://www.lipidprofiles.com/index.php?id=139).
34
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A13. Application of metabolic fingerprinting for food quality assessments
Eiichiro Fukusaki*
Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
*E-mail: [email protected] (corresponding author)
Abstract
Foods are generally multi-functional commodities which consist of multi components. It
is almost impossible to perfectly elucidate the relationship between multi components and multi
functions. Therefore, qualitative study of food function is very much difficult. A conventional
reductionism would not be effective for food function study. Food sensory panel is very
important for food quality evaluation. To organize good sensory test, skilled veterans must
participate. But to keep enough number of skilled veteran with good condition is difficult.
Alternative ways for food sensory test would be required. By the way, recently metabolomics is
thought to be a promising tactics for further understanding genomic information. Metabolomics
would be useful information in the integration of ome-information such as transcriptome and/or
proteome. However, metabome itself can be also applicable without any ome-information.
Particularly metabolic fingerprinting can be available in various application areas. Using metabolic
fingerprinting technology, we can observe relationship between patients’ metabolome and their
disease condition. It indicates usage of metabolome for early diagnosis system for some disease.
The metabolic fingerprinting is also applicable to food materials. We can quantitatively guess
several food functions including sensory value by means of metabolic fingerprinting. We
conducted GC/MS and LC/MS to analyze compounds in food both qualitatively and quantitatively.
The organized metabolome matrix was subjected to some multivariate analysis including PCA,
PLS and OPLS. We have already reported metabolomics oriented sensory prediction of green tea,
water melon, Japanese-sake, cheese, soy-sauce etc. I will introduce a principle of food metabolic
fingerprinting. And some experimental example of them will be delivered.
References
1. Putri SP, Yamamoto S, Tsugawa H, Fukusaki E., J Biosci Bioeng. 2013 Jun;115(6) 9-16.
“Current metabolimics:Technological Advance”.
2. Putri SP, Nakayama Y, Matsuda F, Uchikata T, Kobayashi S, Matsubara A, Fukusaki E., J
Biosci Bioeng. 2013 Jun;115(6):579-89. “Current metabolimics:Practical application”.
35
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A14. Metabolomics and systems biology in the context of food toxicology
D. Zalko1*, C. Canlet2, N.J. Cabaton1, M. Tremblay-Franco2, M. Audebert1, N. Poupin1, E.
Jamin2, R. Gautier2, L. Debrauwer2, F. Vinson1, F. Jourdan1.
1
MeX team, UMR 1331 Toxalim INRA-INPT, 31027 Toulouse cedex 3, France
MetaboHUB Toulouse, CNRS, INRA, INSA, Université Paul Sabatier, 31077 Toulouse cedex 04,
France
*E-mail: [email protected] (corresponding author)
2
Abstract
Novel questions in toxicology have emerged regarding chemicals that interfere with the
homeostasis of living systems. Many man-made molecules present in the environment and the
food-web are suspected EDC (Endocrine Disrupting Chemicals), and there's a growing awareness
of the public regarding the risks associated with these bioactive substances. The evaluation of
EDC sets new challenges for toxicologists as well as health risk assessment agencies, as illustrated
by ongoing controversies on the “low-dose” effects of EDC1. Even for the best known model
xeno-estrogens, mechanisms of low-dose metabolic modulation are only poorly understood, and
the observation of non-monotonic dose-response curves (in vitro and in vivo)2,3 suggests a
metabolic modulation based on multiple targets. Consequently, the debate has gradually evolved
towards a major question currently discussed by toxicologists: should such effects be considered
as adverse effects or are they simply an adaptive response with no health consequences? We
previously demonstrated that the modulation produced by very low doses of EDC could be
detected when exposure occurs during the perinatal period1. It is expected that the use of human
metabolically competent cell lines, combined with high resolution spectral analyses, will allow, in
a first step, to identify "metabolic fingerprints" of chemical pollutants. Then, based on a
reconstruction of the metabolic network, and on comparative studies of closely related
xenobitics (in terms of structure and/or effects), evidence for metabolic networks modulation
should highlight major disruption routes of cellular metabolism. The use of non targeted
approaches also opens the way to a better understanding of the mechanisms of toxicity of low
doses of exposure, and of the effect of mixtures. Ultimately, in vitro and in vivo approaches
should be combined for building novel strategies to characterize the danger associated with the
exposure to major groups of chemicals.
References
1. Vandenberg, L.N., Maffini, M.V., Sonnenschein, C., Rubin, B.S., Soto, A.M. (2009). Endocr
Rev 30, (1), 75-95.
2. Cabaton, N.J., Canlet, C., Wadia, P.R., Tremblay-Franco, M., Gautier, R., Molina, J.,
Sonnenschein, C., Cravedi, J.P., Rubin, B.S., Soto, A.M., Zalko, D. (2013). Environ Health
Perspect. 586-93.
3. Shioda, T., Rosenthal, N.F., Coser, K.R., Suto, M., Phatak, M., Medvedovic, M., Carey, V.J.,
Isselbacher, K.J. (2013).
36
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A15. Use of metabolomics for gene function discovery in a dwarf rice
mutant
Rengasamy Ramamoorthy1, Amit Rai1, Peter Benke2, S. Ramachandran3, Sanjay Swarup1,2,
PRAKASH P. KUMAR1,2,3*
1
Department of Biological Sciences, Faculty of Science, National University of Singapore,
Singapore 117543
2
NUS Environmental Research Institute, National University of Singapore
3
Temasek Life Sciences Laboratory, 1 Research Link, Republic of Singapore
*E-mail: [email protected] (corresponding author)
Abstract
A dwarf mutant rice oscyp96B4 was identified from a phenotype screen of Ds insertion
lines. The homozygous mutant plants also had reduced panicle size and seeding rates compared
to the Nipponbare wild-type rice plants (WT). Molecular data showed that this mutant had a
single copy Ds insertion in OSCYP96B4 gene. The gene function is unknown, but it is predicted to
encode for a cytochrome P450 protein. Cytochrome P450s are monooxygenases, a family of
membrane-bound heme-containing proteins that play various roles such as biosynthesis and
metabolism of lipids, plant hormones and secondary metabolites. Quantitative Real-Time-PCR
and Northern blot analysis showed that OsCYP96B4 was expressed in all the WT tissues tested,
with high expression level in the reproductive organs and low level in the roots. Since functions of
CYP96 gene family in rice are unknown, we employed a metabolomics approach to identify the
probable function(s) of OSCYP96B4 gene. Total metabolite profiling and comparison was done in
WT, mutant, dsRNAi, complementation line, and ectopic expression line of OSCYP96B4 gene. The
LC-MS-based metabolomics analysis yielded ~8,000 ‘features’. The metabolomics-based plant
clustering was consistent with observed phenotypes, and this has led to identification of several
affected metabolic pathways in the mutant. Our data suggest that metabolomics is a useful tool
to characterize mutants when the function of the affected gene is unknown.
References
1. Ramamoorthy R, Jiang S-Y, Ramachandran S (2011). Oryza sativa Cytochrome P450
Family Member OsCYP96B4 Reduces Plant Height in a Transcript Dosage Dependent
Manner. PLoS ONE 6(11):e28069 doi:10.1371/journal.pone.0028069
37
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A16. Nutritional metabolomics: an integrative understanding of metabolic
disease development
E. Pujos-Guillot1,2, M. Rambeau1, C. Joly1,2, B. Lyan1,2, J. F. Martin1,2, M. Pétéra1,2, A.
Napoli3, J. L. Sébédio1, S. Czernichow4, M. Zins4, M. Goldberg4, B. Comte1*
1
INRA, UMR1019, UNH, F-63000 Clermont-Ferrand, France
MetaboHUB Clermont-Ferrand, INRA, University Blaise Pascal, F-63122 Saint-GenèsChampanelle, France
3
LORIA, B.P. 239, F-54506 Vandoeuvre-lès-Nancy, France
4
UMS11 Cohortes en population, INSERM/Université Versailles Saint-Quentin, F-94807 Villejuif,
France
*E-mail: [email protected] (corresponding author)
2
Abstract
Life course is a continuum of consecutive transitions with multifactor lifestyle changes
potentially impacting well-being and health status. Among multiple factors, shifts in dietary
pattern, food composition and processing, are causing nutritional transitions. In the context of
deleterious behavior, these transitions led to energy imbalance with an increasing prevalence of
non-communicable chronic diseases. The complexity of diseases brings challenges for an
integrative understanding of the metabolic processes contributing to the development of these
disorders. Metabolomics offers now the possibility of characterizing global alterations associated
with disease conditions or nutritional exposition and transition, and of identifying early and/or
predictive biomarkers of disease development.
In this context, intervention studies are essential to analyse specific nutrition effects, to
discover potential new biomarkers and identify their role in the pathophysiological mechanisms.
Using this approach, we showed in normal and overweight volunteers submitted to two months
of overfeeding, that metabolomics allows identifying trajectories that reveal early subtle
metabolic dysregulations, not detected by classical biochemical approaches, linked to differences
in metabolic plasticity.
To obtain a more comprehensive picture of nutritional exposure as well as a better
predictive capacity of (bio)markers, complementary study designs in cohorts are mandatory.
Within the French Gazel cohort, we will show that multidimensional model building, consisting in
integrating heterogeneous data (i.e. metabolic, metabolomic, clinical and functional but also
socio-economic data and food patterns) will bring new tools to better predict metabolic
syndrome development. These models will allow revealing determinants and modulators of
changes, and evaluating the contribution of nutrition as a modifiable risk factor, in disease
development.
This work was performed through the technical and scientific support of MetaboHUB. It
illustrates the constructive collaborations between the infrastructure and research units in order
to tackle challenging scientific questions in nutrition.
38
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A17. A totally new way to measure the germination of grain using
metabolomics
O.A.H.Jones1*, M. Burke1
1
School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia
*E-mail: [email protected] (corresponding author)
Abstract
The pre-harvest sprouting of grains frequently causes significant losses to grain growers
worldwide. The problem can influence suitability of grain for processing and hence marketability
and returns. It can occur throughout the grain growing regions of Australia and the result damage
has adversely affected as much as 25 % of production in some seasons. Visual assessment of
sprouting is ineffective because grains showing no external signs may still have sprouted
sufficiently so that it is no longer possible for quality products to be made. As an example, it is
known that Asian noodles are particularly sensitive to the presence of sprouted grain. Most
procedures to determine whether a grain has germinated have been based around measuring the
effects of particular enzymes that breakdown starch (α-amylase). This has been done either
directly or indirectly, however, despite protracted efforts to develop enhanced ways to measure
germination over the last 50 years, there are currently no highly accurate or reliable approaches
available.
The present work uses Infrared and Raman Spectrometry based metabolomic methods to
measure germination of barley and wheat over 24 hours. Our results indicate that infrared
spectrometry is not only able to tell if grain has germinated or not but also gives an indication of
how far the germination process has progressed The technique therefore has the potential to
form a simple dockside test that would allow segregation and minimising the comingling of sound
and mildly sprouted grain. As a result grower returns can be maximised through ensuring
customer satisfaction in the many markets where sprouting causes major problems with
potentially large benefits to the grain industry.
References
1. Jacobsen, J.V., Pearce, D.W., Poole, A.T., Pharis, R.P. and Mander L.N. (2002) Abscisic
acid, phaseic acid and gibberellin contents associated with dormancy and germination in
barley. Physiologia Plantarum 115, 428–441.
39
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A18. Metabolomic aspects of symbiosis and transgenomic interactions in
Mammals
H.R. Tang1,2*, Y.L. Wang2
1
Metabolomics and Systems Biology Laboratory, School of Life Science, Fudan University,
Shanghai; 2 CAS Key Lab of Magnetic Resonance in Biological Systems, Wuhan Institute of Physics
and Mathematics, Chinese Academy of Sciences, Wuhan, China.
*E-mail: [email protected] (corresponding author)
Abstract
All mammals are regarded as “superorganisms” consisting of two distinct but integrated
parts including hosts themselves and symbiotic gut microbiota with the symbiosis established
interactively through co-evolution and mutual selections. Mammals harbor trillions of microbes
with thousands of microbial species in their gastrointestinal tract (GIT) which are also known as
the gut microbiota, microparasites and microbiomes. In normal human GIT, there are more than
one kilogram of microbes having ten times more cells than hosts. The gut microbiota co-develop
with their hosts’ growth playing essential roles in many aspects of mammalian physiology and
having profound effects on the hosts’ health. Therefore, microbiomes are regarded collectively as
an “essential organ” or extended genomes, transcriptomes, proteomes and metabonomes for
their mammalian hosts. The physiology and health of mammals in entirety have to be understood
by taking into consideration of hosts, symbiotic microbes and their interactions. However, it is
nontrivial to completely define the genomes of these microbiomes as for their hosts. Neither can
their composition, transcriptomes and proteomes be defined in details since many species
cannot be cultured ex vivo. Nevertheless, mammal and gut microbiota co-metabolism is essential
for symbiosis or transgenomic interactions. This presentation will briefly introduce the
importance of gut microbiota in mammalian physiology and pathophysiology. Then we will
discuss some recent results on the mammalian metabolic interactions with their gut microbiota
in terms of the environmental effects on human health with particular focus on the regulatory
functions of gut microbiota in rodents and humans. Technical aspects will also be dealt with in
the same context.
References
1.
2.
3.
4.
FPJ Martin, et al, Mol Systems Biol, 3, 112, 2007.
M Li, et al, PNAS, 105, 2117, 2008.
Y Tian, et al, J Proteome Res, 11,1397, 2012.
Y Zhao, et al, J Proteome Res, 12,2987, 2013.
40
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A19. Metabolomic applications in bioprocessing and clinical research
Y.S. Ho1,*, W.P.K. Chong1, C.C. Teo1, S.W. Chen1, S.Y. Mak1, E. Tan1, N. Basri1, E. Peh1, A.HM. Tan1, S. Xu1,2, D-Y. Lee1,3, T.H.K. Lim4, D.M.Y. Tan5, K-P. Lam1,2
1
Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01 Centros, Singapore
138668; 2Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599;
3
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117576; 4Department of Pathology, Singapore General Hospital,
20 College Road, Academia, Level 7, Singapore 169856; 5National Cancer Centre, 11 Hospital
Drive, Singapore 169610.
*E-mail: [email protected] (corresponding author)
Abstract
Research at the Bioprocessing Technology Institute (BTI) encompasses various aspects of
biopharmaceutical drugs production, including the engineering of novel cell lines and
biomolecules, the optimisation of therapeutics production and purification in microbial and
mammalian systems and the characterisation of bioprocesses and products using a suite of
analytical and “-omics” technologies. Of these “-omics” technologies, metabolomics is considered
the closest representation of the phenotype of the system, as metabolite levels are often seen as
the final response of biological systems to genetic or environmental alterations.
A liquid-chromatography mass spectrometry (LC-MS) based metabolomics platform was
initially established at BTI, with the primary objective of guiding bioprocess development for the
Chinese Hamster Ovary (CHO) cell system – the most widely used host for large-scale production
of biopharmaceutical drugs. The platform led to the successful identification of key metabolites
impacting cell growth, as well as novel cell engineering targets, subsequently resulting in a cell
line with improved growth characteristics and increases in recombinant therapeutic protein yield.
More recently, the metabolomics platform has also been extended to biomarker
discovery and the elucidation of disease mechanisms in various clinical applications. To
accommodate the profiling of clinical samples such as primary cells and biofluids that are highly
limited in nature, additional platform capabilities aimed at increasing metabolite coverage and
reducing the sample volume required for analysis were developed.
This presentation will chronicle the evolvement of our platform, from its initial
beginnings as a supporting “-omics” based technology for bioprocess optimisation, to its more
recent applications in the clinical field.
41
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A20. Metabolomics driven the discovery of functional small molecules
towards a diversity of biological innovations
Leyu Yan, Wenna Nie, Xiaojuan Guo, Tianbing Guan, Qiang Li, Jiaoyan Chen, Haitao Lu*
Laboratory for Functional Omics and Innovative Chinese Medicines, Chongqing University
Innovative Drug Research Centre, Chongqing, 401331, China
*E-mail: [email protected] (corresponding author)
Abstract
In vivo small molecules as necessary intermediates are involved in numerous critical
metabolic pathways and biological processes associated with many essential biological functions
and events1,2. In recent years, There is growing evidence to manifest that metabolomics has being
emerged as a powerful tool for facilitating the discovery of functional small molecules which
provides novel insights into a diversity of biological concerns involving disease diagnosis,
therapeutic discovery and toxicology3,4. Yet, metabolomics holds promise in promoting widely
biological innovations in a real world besides terming biomedical niche, which also yields a
wealth of information about food and environmental questions concerning safety and toxicology.
In this proposed presentation, brief introduction to metabolomics science shall be given to
highlight its concept, protocol, manipulation and biological application for the first instance, and
then a line of typical examples will be adopted to demonstrate how metabolomics combining
with bioinformatic/genetic strategy was employed by our group to facilitate the discovery of
functional small molecules whose pattern changes can efficiently dissect the complex
mechanisms implicated in varied biological events via targeting the most affected metabolic
pathways, such as disease progression, drug toxicity, pathogen virulence and drug discovery as
well..
References
1. Nie, W.N., Yan, L.Y., Lee, Y. H., Guha, C., Kurland, I. J., and Lu, H.T. * (2014). Advanced
mass spectrometry based multiple omics technologies to exploring the pathogenesis of
hepatocellular carcinoma. Mass Spectrometry Reviews, Jun 2, doi: 10.1002/mas.21439,
Epub ahead of print. (*Corresponding Author).
2. Yan, L. Y., Nie, W.N., Parker, T., Upton, Z., Lu, H. T. * (2013) Mass spectrometry based
metabolomics facilitates the discovery of in vivo functional small-molecules with a
diversity of biological contexts. Future Medicinal Chemistry, 5, 1953-1965.
(*corresponding author).
3. Lv (or u), H. T*., Hung, C. S., and Henderson, J. P.* (2014): Metabolomic Analysis of
Siderophore Cheater Mutants Reveals Metabolic Costs of Expression in Uropathogenic
Escherichia coli. Journal of Proteome Research, 13, 1397-1404.(*Corresponding Author).
4. Lv (or u), H. T.* (2013): Mass spectrometry based metabolomics towards understanding
of gene functions with a diversity of biological contexts, Mass Spectrometry Reviews, 32,
118-128. (*Corresponding Author).
42
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A21. Urine, metabolite profiling, its applications
Eng Shi Ong
Singapore University of Technology and Design, 20 Dover Drive, Singapore 138682.
*E-mail: [email protected] (corresponding author)
Abstract
To evaluate and monitor body homeostasis and other processes in metabolism, urine has
been an effective biofluids. Urine has a number of advantages over other biofluids for diagnostics
purposes as a result of to its non-invasive nature. The formation of urine in the human body,
various aspects of analysis of urinary metabolites including sampling and traditional approaches
before presenting recent developments using metabolite profiling will be emphasized. Currently,
the advancement of liquid chromatography tandem mass spectrometry (LC/MSMS), the
establishment of standardized chemical fragmentation libraries, computing power, novel dataanalysis approaches with pattern recognition tools have made mass spectrometry-based
metabolomics the latest sought-after technology. Metabolites present in the urine samples
collected can vary widely and normalization for these effects will be presented. In addition, it will
allow for the ability to dynamically catalogue and quantify small amount of cellular metabolites in
urine samples. Case studies for colon cancer and others will be presented. It is noted that all
these major advancement with mass spectrometry-based metabolomics will lead to new
biomarkers and novel biochemical insights.
References
1. Z Jiang, F Liu, ES Ong, SFY Li, Metabolic profile associated with glucose and cholesterol
lowering effects of berberine in Sprague–Dawley rats. Metabolomics 2012, 8 (6), 10521068
2. Liu F, Gan PP, Wu H, Woo WS, Ong ES, Li SF. A combination of metabolomics and
metallomics studies of urine and serum from hypercholesterolaemic rats after
berberine injection. Anal Bioanal Chem. 2012 May;403(3):847-56.
43
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A22. Biostatistics for biomarker discovery and phenotype prediction
Etienne A. Thévenot1,*, Natacha Lenuzza1, Aurélie Roux2, Samia Boudah2, and Christophe
Junot2
1
Laboratory of Software Intensive Technologies (LIST) and 2 Drug metabolism research laboratory
(LEMM), MetaboHUB Paris, CEA, 91191 Gif-sur-Yvette, France
*E-mail: [email protected] (corresponding author)
Abstract
Biomarker discovery through metabolomics profiling shows promising results for the
diagnostics of diseases such as cancer, Alzheimer's disease, or diabetes. Identification of an
effective biomarker from thousands of raw LC-MS signals critically depends upon advanced
statistical strategies for data processing, analysis, and the development of robust models. In
particular, one of the major threats is bias resulting from confounding factors.
To characterize the physiological variations of the human metabolome, we conducted a
study of individual urine samples from more than 200 volunteers (CEA employees). Acquisitions
in the positive and negative ionization modes were performed in a total of three batches.
Preprocessing and annotation with public and in-house databases resulted in the structural full or
partial identification of 400 metabolites.
The variations of these metabolites associated to age, body mass index (BMI) and gender
were analyzed with a three-step statistical methodology. First, analytical drifts (decrease of the
sensitivity of the instrument over time during each run) and batch effects (offset from one run to
the other) were corrected by using normalization approaches. Second, significance of the
variation of each metabolite concentration associated to each factor (and their interactions) was
determined by univariate hypothesis testing. Third, orthogonal partial least-squares (OPLS)
algorithms were implemented to build and validate robust predictive multivariate models.
Almost all gender-specific metabolites detected had higher concentrations in males
compared to females, and included steroid hormones (e.g. testosterone) and the energy
metabolism (lipids, dipeptides, and nucleosides). A decrease of metabolism with age was
observed (steroid hormones, amino acids, nucleoside derivatives), with the exception of
exogenous metabolites such as caffeine. Altogether these results provide a critical comparison of
statistical approaches for biomarker discovery applied to the characterization of the human
metabolome.
44
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A23. Metabolomics-A great tool for generating hypothesis
Y. L. Wang*, H. D. Li, H. R. Tang
Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic
Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan
Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, P. R. China
*E-mail: [email protected] (corresponding author)
Abstract
Metabonomics is the science that studies dynamic alterations of metabolites in a cell,
organ or entire organism1,2. The definition of the metabonomics was first given in 1999 as “the
quantitative measurement of the time-related multiparametric metabolic response of living
systems to pathophysiological stimuli or genetic modification”2. Technically, metabonomics
investigations usually consist of the collection of metabolic profiles using nuclear magnetic
resonance spectroscopy or mass spectrometry techniques and analysis of the collected data
using appropriate multivariate statistical techniques. Metabonomics is a great tool for generating
credible hypothesis. Recently, we have applied this technique to investigate interactions between
host and hepatitis B virus (HBV). We characterize the metabolic features of host cells expressing
the HBV virion using metabonomics, and then, identified important metabolic pathways related
to the infection. Then we performed proteomics investigation to validate relevant metabolic
pathways that were changed after HBV infection. Based on the metabolic and proteomics data,
we found that HBV replication induces the promotions of central carbon metabolism,
biosynthesis of nucleotides and total fatty acids; HBV up-regulates the biosynthesis of
hexosamine and phosphatidylcholine through activating glutamine-fructose-6-phosphate
amidotransferase 1 (GFAT1) and choline kinase α (CHKA), respectively. In order to validate the
importance of GFAT1 and CHKA in the replication of HBV, we conducted further molecular
biological assays on the system. The activations of GFAT1 and CHKA are known to contribute to
carcinogenesis. Our results also indicate that HBV-induced hepatitis and fatty liver could be
partially attributed to GFAT1 activated hexosamine biosynthesis. Furthermore, we demonstrate
that GFAT1 and CHKA are two potential targets for treating HBV infection. This study provides
new insights into the pathogenesis of HBV-induced diseases and the discovery of drug targets.
We show in our work that metabonomics is a great tool for generating credible hypothesis.
References
1. Nicholson, J. K., et al., 'Metabonomics': understanding the metabolic responses of living
systems to pathophysiological stimuli via multivariate statistical analysis of biological
NMR spectroscopic data. Xenobiotica 1999, 29 (11), 1181-1189.
2. Tang, H. R., et al., Metabonomics: a revolution in progress. Prog Biochem Biophys, 2006,
33 (5), 401-414.
45
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A24. Model systems for the metabolomics study of environmental
exposure
Sarah Hayton1,2, Nicola Parkinson1,2, Stephen Harrison1,2, Stephanie Meilke1,2, Thomas
Dignan1,2, Catherine Rawlinson1,2, Joel Gummer1,2,3, Ian Mullaney1,2, Garth Maker1,2,
Robert Trengove1,3*
1
Separation Science & Metabolomics Laboratory, Murdoch University
Veterinary and Life Sciences, Murdoch University
3
Metabolomics Australia, Western Australian Node
*Email: [email protected] (corresponding author)
2
Abstract
We are at the emergence of the capability to simultaneously measure residue and
environmental analytes and profiles of primary and secondary metabolites. We can start to
investigate from both directions or investigate "cause" and "effect" simultaneously.
The life sciences, particularly proteomics and more recently metabolomics have been
drivers in recent instrumental development, leading to faster chromatography, accurate mass
and high resolution mass analyzers, and equally as important robust sample interfaces for
minimal sample preparation. Alongside the hardware developments there have been
considerable developments in software for mass spectral deconvolution, data visualisation, and
mapping of analytes onto biochemical pathways to identify which pathways are modifed in a
challenged sample vs a control sample. For instance, this allows the mechanisms of disease
progression to be dissected and potential management strategies and potential therapeutic
targets to be identified.
These same instrumental developments have been utilised by the residue and
environmental communities with the development of multiresidue methods that screen for in
excess of 1000 analytes in a single analysis. Metabolomics seeks to identify changes in response
to some stimulus, whilst residue and environmental analysis seeks to identify all possible residue
and environmental "stimulus".
This presentation will focus on, and address, the approach and the challenges to facilitate
the use of both “targeted” and “untargeted methods”. In particular the different approaches
required for GC/MS based vs LC/MS based metabolomics. Instrumental optimisation will be
discussed in the context of combination of targeted compound analysis and metabolite profiling.
46
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A25. Environmental metabolomics to understand resource partitioning
strategies in algae
Swarup S1, 2, 3 *, Pan Di1, Amit Rai2, Umashankar S1, 2, 3, Vello V4, Kumar V1, Benke P1,
Phang S M4, Williams RBH3
1
NUS Environmental Research Institute, National University of Singapore, Singapore 117411; 2
Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore,
Singapore 117543; 3 Singapore Centre for Environmental Life Sciences Engineering (SCELSE),
Nanyang Technological University, Singapore 637551; 4 Institute of Ocean and Earth Sciences,
University of Malaya
*E-mail: [email protected] (corresponding author)
Abstract
Cellular resources in organisms are shared and reallocated dynamically to adapt to
environmental pressures. We show the metabolic resource partitioning strategies used by algae
in diverse environmental conditions, using two case studies (i) during harmful algal bloom for cell
multiplication and toxin production, (ii) influence of habitat and growth on microalgae to guiding
bioprocess development for biofuel production.
(i) Microcystis aeruginosa PCC7806, a toxic model cyanobacterium strain, has been used
in our laboratory to study the transcriptome and metabolome dynamics of cyanobacterium
under different light and nitrogen conditions that trigger algal bloom. It was noticed that light
influences the cellular processes mainly through transcriptional changes, while nitrogen’s effects
are mainly seen at metabolome level. Under increased light conditions, both the microarray and
metabolic profiling data suggested that combating oxidative stress, while maximizing growth is
the preferred strategy for cyanobacteria. The most significant change in metabolites observed
was for amino acids, and therefore, protein biosynthesis and amino acid metabolism were
identified as pathways significantly altered during the shift from low to high microcystin
conditions. High amino acid concentration at high nitrogen concentration is associated with low
mcy gene expression. These results suggest that when there is more nitrogen available, the
cellular strategy is to use more amino acids for protein synthesis, rather than microcystin
biosynthesis.
(ii) Algal growth and lipid production are interlinked, and play a critical role during biofuel
production processes. Here, we used untargeted high-resolution mass spectrometry to
understand the metabolic differences at exponential and stationary growth stages using 22
Chlorella strains collected from S.E. Asia. This analysis revealed strain-specific signatures of
metabolic reprogramming strategies, and associations between physiochemical measures and
metabolic pathways. These findings will aid the development of efficient bioprocess strategies to
improve algal bio-products.
47
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A26. Non targeted metabolomics for assessment of environmental
exposure to contaminants and their biological effects: towards exposomics
L. Debrauwer1*, E. Jamin1, C. Canlet1, M. Tremblay-Franco1, N. Bonvallot2, C. Chevrier2, S.
Cordier2, J.P. Cravedi3
1
MetaboHUB Toulouse, CNRS, INRA, INSA, Université Paul Sabatier, 31077 Toulouse cedex 04,
France
2
INSERM UMR 1085 IRSET – EHESP, 35042 Rennes, France
3
MeX team, UMR 1331 Toxalim INRA-INPT, 31027 Toulouse cedex 3, France
*E-mail: [email protected] (corresponding author)
Abstract
The use of pesticides and related environmental contaminations can lead to human
exposure to various molecules, which could be responsible for adverse effects. However, human
health risks associated with multi-exposure to complex mixtures are currently under-explored,
and the following questions remain challenging: Can we assess the exposure to pesticides from
biofluids in an untargeted way? What is the influence of exposures to multiple pesticides on the
metabolome? What mechanistic pathways could be involved in the metabolic changes observed?
To provide answers to these questions, both NMR and MS tools were developed, based on urine
samples from 338 early pregnant women belonging to the PELAGIE cohort, which were classified
in 3 groups according to the surface of land dedicated to cereal farming in their town of
residence.
For non-targeted exposure assessment, an LC-MS workflow was developed, integrating
untargeted data acquisition, identification of pesticides metabolites (which structures could be
unknown) using an upgradable list of metabolites to seek in urine samples, and statistical
treatment of data. The groups of individuals could be separated according to their exposure to
pesticides, on the basis of metabolites representing 2 fungicides (azoxystrobin, fenpropimorph)
usually used on cereal cultures. The relevance and limitations of this innovative approach will be
discussed.
To evidence possible impacts of the exposure on global metabolism as revealed in urine,
NMR-based analyses were performed. Global NMR fingerprints were treated using supervised
multivariate statistics (PLS-DA, polytomous regressions) to discriminate the 3 groups of women,
after restriction of our sample to subjects with 10-11 weeks amenorrhea and 20-30 body mass
index (N=123) for reducing inter-individual variability. The most statistically significant changes
(decreasing trend) were observed for glycine and citric, hippuric and lactic acids, indicating that a
complex exposure to pesticides could induce glycine depletion (aminoacid involved in xenobiotics
biotransformation) and disturbances of energy metabolism.
48
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A27. A study on Honeybee losses. First insight into environmental
interactions.
Céline DALLE1-3, Johan PANEK1, Angélique ABILA3, Hicham EL ALAOUI1, Marie LAGREE3,
Mounir TRAÏKIA2,3, Michaël ROUSSEL1, Frédéric DELBAC1 & Cyril JOUSSE 2,3*
1
Clermont University, Blaise Pascal University, « Microorganisms: Genome and Environment »
Laboratory, CNRS, UMR 6023, F-63000 CLERMONT-FERRAND, FRANCE
2
Clermont University, Blaise Pascal University, Institute of Chemistry of Clermont-Ferrand, CNRS,
UMR 6296, F-63000 CLERMONT-FERRAND, FRANCE
3
Platform of Exploration of Metabolism, Blaise Pascal University, F-63000 CLERMONT-FERRAND,
FRANCE
Email: [email protected] (corresponding author)
Abstract
The honeybee (Apis mellifera) colony losses recorded during the last decades represent a
major issue regarding agricultural yields, apiculture and biodiversity conservation. The origin of
these losses is thought to be multicausal, with a strong emphasis on pesticides and parasites.
Among the parasites, the microsporidian Nosema ceranae is the etiologic agent of the Nosemosis
disease in A. mellifera. This obligate intracellular parasite develops in the epithelial cells of adult
honeybee mid-gut. The infection can increase energetic demand in bees, suppress the immune
response and may affect pheromone production. Such disturbances may have major
consequences on bee metabolic pathways. Here we present an emerging project for that
purpose. We have decided to perform metabolomics using different tissues, hemolymph (bee
biofluid) and gut. Hemolymph is collected by centrifugation and gut by dissection. Samples can be
analysed trough Metabolic Profiler (LC/MS-NMR) and/or HR-MAS. After data treatment and
multivariate analyses (PCA, PLS), databases have to be screened on the highest relevant signals.
Validation on putative metabolites is usually conducted using 2D-NMR and orbitrap HRMS
experiments. Then, hyphenation techniques permit to increase metabolic coverage and a better
chance for stress biomarker discovery.
References
1. Aliferis KA, Copley T, Jabaji S (2012) Gas chromatography-mass spectrometry metabolite
profiling of worker honey bee (Apis mellifera L.) hemolymph for the study of Nosema ceranae
infection. J Insect Physiol 58, 1349–1359.
2. Dussaubat C, Maisonnasse A, Crauser D, Beslay D, Costagliola G, Soubeyrand S,
Kretzchmar A, Le Conte Y (2013) Flight behavior and pheromone changes associated to
Nosema ceranae infection of honey bee workers (Apis mellifera) in field conditions, J
Invertebr Pathol 113, 42-51.
3. Goblirsch M, Huang ZY & Spivak M (2013). Physiological and behavioral changes in honey
bees (Apis mellifera) induced by Nosema ceranae infection. PLoS ONE 8, e58165.
49
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A28. Comprehensive investigations of cellular metabolic networks using
13
C-fluxomics and applications to systems microbiology and biotechnology
JC. Portais1,2*, L. Peyriga1, F. Bellvert1, S. Sokol1, S. Heux2, F. Letisse2, L. Gales1, H. KulykBarbier1, E. Cahoreau1.
1
MetaboHUB Toulouse, CNRS, INRA, INSA, INSERM, Université Paul Sabatier, 31077 Toulouse
cedex 04, France
2
Team MetaSys, Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, CNRS, INRA,
INSA, F-31076 Toulouse, France
*E-mail: [email protected] (corresponding author)
Abstract
Metabolism is a basic cellular function that sustains survival, growth and adaptation of
living organisms. At the cellular level, metabolism is organized as a network, i.e. a complex set of
biochemical reactions that are tightly interconnected. Currently, the general – design – principles
of organization, functioning and adaptation of metabolic networks are not well understood. Such
knowledge is critical to get better understanding of the role of metabolism in the adaptation of
organisms to their environment or to develop efficient and cost-effective biotechnological
processes. In the recent years, we have developed a wide range of approaches (metabolomics,
fluxomics) to provide detailed measurement of the response of metabolic networks to changes in
the genome or environment. These methods can be combined with other omics tools (e.g.
transcriptomics, proteomics, etc.) and to metabolic modelling to provide systems biology
approaches for in-depth, comprehensive understanding of the organization, functioning and
adaptation of cellular metabolism. This understanding is used to get efficient control and
optimization of the metabolism of industrially-relevant microorganisms (e.g. E. coli, yeast). The
use of systems-level studies makes it possible to comprehensively understand not only the
impact of pathway engineering within the context of the entire host metabolism, but also to
diagnose stresses due to product synthesis or to extreme environmental conditions, thereby
providing the rationale for efficient and cost-effective optimization of production systems
(knowledge-based metabolic optimization). In this lecture, the benefit and limits of these
approaches will be discussed, with a particular emphasis on 13C-fluxomics.
50
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A29. Response of cloud microorganisms to atmospheric stresses: the case
study of cold shock
Anne-Marie Delort1,2,3*, Cyril Jousse1,3, Céline Dalle1, Isabelle Canet1, Marie Lagrée3,
Mounir Traïkia1,3 , Bernard Lyan4 , Martine Sancelme1, Pierre Amato1,2 .
1
Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448,
F-63000 Clermont-Ferrand , France
2
CNRS, UMR 6296, ICCF, BP 80026, F-63171 Aubière, France
3
MetaboHUB, PFEM, Université Blaise Pascal, BP 80026, F-63171 Aubière, France
4
MetaboHUB, PFEM, INRA-Clermont-Theix, Saint-Genes Champanelle, France
*E-mail: [email protected] (corresponding author)
Abstract
Microorganisms have been known for a long time as important actors in aquatic and
terrestrial ecosystems. Their discovery is rather recent in the atmosphere and more particularly
in clouds (1). Our team has isolated around 700 microbial strains from cloud waters at the puy de
Dôme summit during the last ten years, among them, Pseudomonas is one of the major
encountered genus (2). Atmosphere is a really specific and versatile compartment on earth
where microorganisms are facing various stresses including dryness, pH, UV, cold shocks…etc.
Then, a key question is to understand how microorganisms can resist to such stresses and survive
under such harsh conditions.
In this work, we have selected a Pseudomonas syringae strain, isolated from cloud waters
to study the influence of temperature and identify cold shock biomarkers through metabolomics
experiments.
This strain was incubated at 5°C and 17°C, these temperatures correspond respectively to
the annual and the summer temperature means. Bacterial extracts were analyzed using a LC/MSNMR facility (Metabolic Profiler®), and multivariate analyses (PCA, PLS-DA). We highlighted
different groups of relevant biomarkers. Some of them have been already described for cold
shock stress: they include trehalose, betaïne, carnitine (cryoprotection ), organic acids, ATP,
sugars (energetic metabolism ), insaturated lipids (membrane fluidity) and gluthatione (oxidative
stress metabolism). Surprisingly, we also found short peptides described for the first time as cold
shock biomarkers. One of these peptides was identified as a virulence factor (Tabtoxin).
References
1. Delort et al., Atmos. Res., 2010, 98, 249-260.
2. Vaitilingom et al., Atmos. Environ., 2012, 56, 88-100.
51
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A30. Analysis of leachates from gasification of solid wastes by
metabolomics techniques
S. N. Lee1,3, W. L. Zhang1,3, L. Rong2,3, T. Maneerung3, C. H. Wang2,3, K. G. Neoh2,3, and S.
F. Y. Li1,3*
1
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4
Engineering Drive 4, Singapore 117576
3
NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1,
Singapore 117411
*E-mail: [email protected] (corresponding author)
2
Abstract
Leachate samples from fly ash and bottom ash obtained by gasification of solid
wastes were analyzed by non-targeted screening using liquid chromatographyquadrupole-time of flight-mass spectrometry (LC-QTOF-MS). The results were used to
determine which organic compounds could contribute to the toxicity of the leachates of
solid waste gasification. Subsequently, the effects of the leachate on mortility and
immobility of Daphnia mangna were evaluated as a method for monitoring water quality,
and as a screening method for toxicity assessment of solid waste re-utilization.
52
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A31. Methods for dealing with batch effects in metabolome data
S. Umashankar1, S. Swarup1,2, R. B. H. Williams2,*
1
Department of Biological Sciences, National University of Singapore, Singapore 117411;
Singapore Centre for Environmental Life Sciences Engineering (SCELSE), National University of
Singapore, Singapore, 117456
*E-mail: [email protected] (corresponding author)
2
Abstract
High-throughput mass spectrometry based experiments generate large and complex
data. Combining data from such experiments performed over time (weeks/months) or in
different batches present numerous challenges. Due to the massively parallel nature of
metabolomics data, such differences in experimental condition can induce a structure into the
data that is of non-biological origin. These effects are often overlooked in current pipelines, but
can easily contribute to artefactual signals being attributed to biological . Using untargeted
metabolite profile data from 22 Chlorella strains, compared over two growth stages and run in
four batches, we show removal of run-day effect, using a filtering procedure based on the
singular value decomposition, that demonstrably preserves biological signal in the experiment, as
measured by inter-strain variation. Our approach will be broadly applicable in metabolomics
analysis, and highlights the importance of carefully dissecting the possible source into batch
variation at the both the design and analysis stages of a study.
53
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
A32. Metabolomics-driven strain improvement of higher alcohols in
Escherichia coli
Sastia Prama Putri1, Naoki Kawase1, Shingo Noguchi1, Claire Shen2, James Liao2,
Takeshi Bamba1, Eiichiro Fukusaki1*
1
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1Yamadaoka,
Suita, Osaka 565-0871, Japan; 2 Department of Chemical and Biomolecular Engineering,
University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA
90095, USA
*E-mail: [email protected] (corresponding author)
Abstract
The concerted effort for bioproduction of higher alcohols to replace fossil fuels has
translated into successful production of higher alcohols based on biochemical knowledge and
stochiometric arguments. To date, the selection of targets for strain improvement has been
based at its best on expert knowledge, but to a large extent also on ‘educated guesses’ and ‘trials
and errors’. Metabolomics, the global quantitative assessment of metabolites in a biological
system in combination with multivariate data analysis (MVDA) tools, allow the replacement of
current empirical approaches by a scientific approach towards the selection and ranking of
targets. In addition, metabolomics provides experimental data for identifying unknown and
under-appreciated reactions in the metabolic network. Previously, Escherichia coli strains capable
of producing 1-propanol from glucose had been constructed by incorporating synergy as a design
principle of strain engineering. 1-propanol production was achieved via 2-ketobutyrate (2KB)
from two pathways, native threonine pathway and non-native citramalate pathway from
Methanococcus jannaschii. The strain possessing two pathways resulted in a higher titer of 1propanol (2.5 g/L/day), more than 3-folds compared with the production from a single pathway
(0.8 g/L/day). Interestingly, SYN12 strain bearing overexpression of both pathways and SYN3
strain bearing overexpression of only citramalate pathway with a native threonine pathway had
the same production performance. Here, we demonstrate the utility of metabolomics analysis to
validate the importance of synergy for 1-propanol production, to effectively identify 2KB
accumulation as the bottleneck step of 1-propanol production in strain SYN12, and to uncover
the underlying reason of this bottleneck.
54
MERLION METABOLOMICS WORKSHOP SINGAPORE
Abstracts for
Young Researchers Session
55
2014
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y1. A comparative urine metabolic profiling of tuberculosis patients and
healthy individuals
Mrinal Kumar Das1*, Subasa Chandra Bishwal1, Aleena Das1, George M. Varghese2,
Bhaswati Pandit3, Ranjan Nanda1
1
Immunology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB),
Aruna Asaf Ali Marg, New Delhi, India
2
Department of Medicine, Christian Medical College, Vellore, Tamilnadu, India
3
National Institute of Biomedical Genomics, Kalyani, West Bengal, India
*E-mail: [email protected] (Presenter)
Abstract:
Tuberculosis (TB) is a curable disease and timely diagnosis could save many precious
lives. However, most common TB diagnostic methods e.g. Sputum microscopy have limitations
like low sensitivity and other logistic issues. Moreover, metabolite markers from non-invasively
collected biofluid like urine hold promise of an easy to use, cost-effective and indextrous method
for TB diagnosis. Research from our lab has demonstrated presence of distinct urine volatile
signature in TB patients (Bandey et al., 2011). Here we are reporting deregulated non-volatile
polar molecules in urine of TB patients.
Urine samples (n=30; TB/Healthy: 17/13) were profiled using gas chromatography and
mass spectrometry (GC-MS) preceded by trimethylsilyl derivatization. Raw data were
preprocessed using ChromaTOF. The final metadata with the aligned molecules present in >50%
of at least one class were exported as a .csv file for further analysis. Important molecules were
identified using multi-variate analysis reported earlier by Das et al 2014. Pathway enrichment
assay was carried out using a sub group of these selected molecules.
Preprocessing of the raw data file resulted a 30x233 matrix and out of which 182
variables tentatively identified based on library search. A set of 21 molecules with VIP score more
than 1.5 were selected as important molecules. Calculated area under curve (AUC) of receiver
operating characteristic curve (ROC) was 0.96. Pathway enrichment showed altered lipid and
amino acid metabolism in TB patients and corroborates findingof Zhou et al (2013).
Our findings provide evidence that deregulated urine metabolites of TB patients may be
useful as an alternate solution to develop new and easy to use TB diagnostic assay.
References:
1. Banday, K.M., (2011) Anal. Chemistry 83, 5526–5534.
2. Das, M.K., (2014) Anal. Chemistry 86, 1229−1237.
3. Zhou, A., (2013) J. Prot. Research 12, 4642−4649
56
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y2. Metabolomics study of a mouse model with influenza virus induced
lung injury
Liang Cui1, Dahai Zheng1, Yadunanda Kumar1, Yie Hou Lee1, Wanxing Eugene Ho2,
Jian Zhu Chen1,3, Choon Nam Ong2,4*, Steven R. Tannenbaum1,5*
1
Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research &
Technology (SMART), Singapore
2
Saw Swee Hock School of Public Health, National University of Singapore, Singapore
3
Departments of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,
United States of America
4
NUS Environment Research Institute, Singapore
5
Departments of Biological Engineering and Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts, United States of America
*E-mail: [email protected] (Presenter)
Abstract
Influenza virus infection (IVI) can cause lung injury, which may progress to
respiratory failure and fatal outcome. At present, the molecular interactions between host and
influenza virus and corresponding metabolic processes involved in the viral pathogenesis are
poorly-elucidated. We conducted a mass spectrometry-based metabolomics analysis on serum,
bronchoalveolar lavage fluid (BALF) and lung tissue from a murine IVI model of lung damage and
repair. To capture the dynamic metabolic responses to IVI and discover reliable differential
metabolites related to influenza pathophysiology, biological samples were collected at 0, 6, 10,
14, 21 and 28 days post IVI, representing infection stages which correspond to ‘maximal viremia’,
‘peak body weight loss’, ‘maximal inflammatory lung damage’, and ‘recovery phase’ of the
infection. Significant metabolome changes with over 100 identified differential metabolites were
found upon IVI, covering a variety of metabolite classes. Meanwhile, distinctive metabolite
profiles were observed in serum, BALF and lung, and certain metabolic changes were lungspecific, indicating the necessity of parallel metabolic profiling of both biofluids and lung tissues.
Amongst various altered metabolic pathways, amino-sugar and nucleotide-sugar metabolism,
purine and pyrimidine metabolism, sphingolipid metabolism, pantothenate and CoA biosynthesis,
and tryptophan-kynurenine metabolism were identified to be involved in lung injury and
recovery. These results advance current molecular understanding on host and influenza virus
interactions, and provide mechanistic insights into influenza virus-induced lung injury and repair
process.
References
1. Kumar, Y., Cui, L., Limmon, G.V., Liang, L., Engelward, B.P., et al. (2014) Molecular analysis
of serum and bronchoalveolar lavage in a mouse model of influenza reveals markers of
disease severity that can be clinically useful in humans. PLoS ONE 9: e86912.
2. Cui L, Lee YH, Kumar Y, Xu F, Lu K, et al. (2013) Serum metabolome and lipidome changes
in adult patients with primary dengue infection. PLoS Negl Trop Dis 7: e2373.
57
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y3. Metabolomics Studies of Urine and Plasma of Patients with Chronic
Kidney Disease by LC-MS
F. Liu1,2, X. Yin1, J. T. M. Ong1, J. B. W. Teo3, S. F. Y. Li1,2*
1
NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, National
University of Singapore, Singapore 117411; 2 organization and address , Calibri 11 pt
2
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
3
Department of University Medicine Cluster, National University Hospital, 5 Lower Kent Ridge
Road, Singapore 119074
*E-mail: [email protected] (corresponding author)
Abstract
Chronic kidney disease (CKD) is a condition characterized by the progressive loss in renal
function over a period of months or years which eventually leads to End Stage Renal Disease.
Currently diagnostic biomarkers is not sensitive, therefore the aim of this study was to (i)
characterize the wide-ranging metabolic changes occurring in CKD through non-targeted
metabolomics; (ii) determine if there is any difference in the pathological manifestation of CKD in
the non-diabetic group as compared to the diabetic group; (iii) identify metabolites for use as
biomarkers of kidney function. To understand the system’s metabolic changes associated with
CKD, we analysed the metabolomics changes of plasma and urine samples of patients with nondiabetic and diabetic CKD using LC-QTOF-MS. Statistical analyses such as OPLS-DA models were
used to compare the metabolomics profiles and select the metabolites that showed significant
change between diseased and control groups. The results presented here reveal alterations in
uremic toxins, amino acids, glucose and ceramides in the plasma of CKD patients and decreased
urinary excretion of bile acids, carnitines and vitamin E metabolites. These metabolomics
alterations collectively indicate a state of oxidative stress, inflammation and acidosis in the
pathophysiology of CKD.
References
1. Weiss, H. R.; Kim, K. (2012) Metabolomics in the study of kidney diseases. Nat. Rev.
Nephrol 8, 22-33.
58
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y4. Metabolomic Studies of Acute Coronary Syndrome Patients using LCMS
X.J. Yin1, 2*, F.Y.S. Li1,2,3
1
NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, National
University of Singapore, Singapore 117411;
2
Department of Chemistry, Block S5-02-03, 3 Science Drive 3, National University of Singapore,
Singapore 117543
3
Shenzhen Engineering Laboratory for Eco-efficient Polysilicate Materials, School of Environment
and
Energy, Peking University Shenzhen Graduate School, Shenzhen, P.R.C. 518055
*E-mail: [email protected] (corresponding author)
Abstract
Discovery of new biomarkers is critical for early diagnosis of acute coronary syndrome
(ACS). Recent advances in metabolomic technologies have drastically enhanced the possibility of
improving the knowledge of its physiopathology through the identification of the altered
metabolic pathways. In this study, peripheral plasma samples from 25 ST segment elevation ACS
patients and non-ACS patients were collected before and 24 hours after the angiography.
Analysis by liquid chromatography–mass spectrometry (LC–MS) and orthogonal partial least
squares (OPLS) permitted the identification of many metabolites with statistical differences
(p<0.05) between experimental groups. Additionally, validation by LC–MS permitted us to
identify a potential panel of biomarkers formed by various amino acids and glucose metabolism.
This panel of biomarkers reflects the oxidative stress and the hypoxic state that suffers the
myocardial cells and consequently constitutes a metabolomic signature of the atherogenesis
process that could be used for early diagnosis of ACS.
References
1. Roger, V. L., Go, A.S., Lloyd-Jones. D.M., Adams. R.J., Berry. J.D., Brown, T.M., Carnethon,
M.R., Dai, S., de Simone, G., Ford, E.S., Fox, C.S., Fullerton, H.J., Gillespie, C., Greenlund,
K.J., Hailpern, S.M., Heit, J.A., Ho, P.M., Howard, V.J., Kissela, B.M., Kittner, S.J., Lackland,
D.T., Lichtman, J.H., Lisabeth, L.D., Makuc, D.M., Marcus, G.M., Marelli, A., Matchar, D.B.,
McDermott, M.M., Meigs, J.B., Moy, C.S., Mozaffarian, D., Mussolino, M.E., Nichol, G.,
Paynter, N.P., Rosamond, W.D., Sorlie, P.D., Stafford, R.S., Turan, T.N., Turner, M.B.,
Wong, N.D., Wylie-Rosett, J. and American Heart Association Statistics Committee and
Stroke Statistics Subcommittee. (2011). Heart disease and stroke statistics-2011 update:
A report from the American Heart Association. Circulation, 123(4), e18–e209
2. Bassand, J. P., Hamm, C. W., Ardissino, D., Boersma, E., Budaj, A., Fernández-Avilés, F.,
Fox, K.A., Hasdai, D., Ohman, E.M., Wallentin, L. and Wijns, W. (2007). Guidelines for the
diagnosis and treatment of non-ST-segment elevation acute coronary syndromes.
European Heart Journal, 28, 1598–1660
59
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y5. LC-MS based metabolomics approach to study biomarkers of aging and
chronic kidney disease
E. Simsek1, L. F. Y. Sam2,*
1,2
NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, National
University of Singapore, Singapore 117411
*E-mail: [email protected] (corresponding author)
Abstract
Aging is a complex process involving the accumulation of diverse detrimental changes in
biological systems gradually so significant changes in cellular metabolism are produced
throughout aging. Normal aging process also often accompanies various diseases such as
Alzheimer’s and Parkinson’s disease, diabetes and kidney disease. To investigate metabolic
changes involved in aging process and chronic kidney disease (CVD), human plasma samples of
healthy people and CKD patients of various ages, namely 40s, 50s, 60s and 70s, obtained from a
local hospital in Singapore. The samples were analyzed using liquid chromatography coupled with
quadrupole time-of-flight mass spectrometry. Multivariate statistical analysis of the data using
principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) revealed separation between healthy versus CKD subjects and different age groups
among healthy subjects. Metabolic features responsible for the separation between groups were
chosen using variable importance for the projection (VIP) plot and searched against accurate
mass metabolite libraries of METLIN metabolite database and Human Matabolome Database
(HMDB) for identification. The study showed that it is possible to study biomarkers and related
biological mechanisms related to aging and CKD with a mass spectroscopy based metabolomics
approach.
References
1. Dunn, W. B. et al. Procedures for large-scale metabolic profiling of serum and plasma
using gas chromatography and liquid chromatography coupled to mass spectrometry.
Nat. Protoc. 6, 1060–83 (2011).
2. Mishur, R. J. & Rea, S. L. Applications of mass spectrometry to metabolomics and
metabonomics: detection of biomarkers of aging and of age-related diseases. Mass
Spectrom. Rev. 31, 70–95 (2012).
3. Zhu, Z.-J. et al. Liquid chromatography quadrupole time-of-flight mass spectrometry
characterization of metabolites guided by the METLIN database. Nat. Protoc. 8, 451–
60 (2013).
60
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y6. Comprehensive metaoblomics study of the acute inflammatory
response in a porcine model of combat trauma injury
A.K.C. Laserna1, G. Fang1, J. Wu2, Y. Lai2, R. Ganapathy 2, S.M. Moochhala2, S.F.Y. Li1*
1
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore
117543; 2 Defence Medical & Environmental Research Institute, DSO National Laboratories, 27
Medical Drive, Singapore 117510
*E-mail: [email protected] (corresponding author)
Abstract
Severe injuries, such as trauma injury, are among the leading causes of morbidity and
mortality worldwide.[1, 2] Injuries sustained in combat scenarios are even more dangerous given
that the wounding agents are intentionally more lethal and the resources available for treatment
are limited. Accompanying such severe injuries is the activation of the body’s immunologic and
inflammatory response. Normally, they are meant to facilitate healing and protect the body from
further damage. However, once they proceed uncontrolled, systemic inflammatory response
syndrome (SIRS) may ensue subsequently leading to damage of multiple organs, and possibly
death. The identification of biomarkers that can serve as indicators of the extent and progression
of the inflammatory response and the onset of organ failure can help in determining the
appropriate mode of intervention in the combat field, thereby improving patient outcome. In this
study, we have taken an untargeted metabolomic approach using the complementary techniques
of NMR and LC-MS to see the metabolic perturbations occurring in a simulation of combat
trauma injury using a pig (porcine) model. Based on an established complex combat trauma
injury model [3], pigs were subjected to femur fracture and soft-tissue injury with 60% blood loss,
followed by a 30-minute shock period, and induced hypothermia. Plasma samples before and
after injury, as well as sham samples, were analyzed to evaluate the changes in the metabolic
profiles of the pigs. Significant changes in several metabolites were observed in response to the
trauma injury. Correlation analysis with cytokines and some established protein markers revealed
potential biomarkers of systemic inflammatory response syndrome (SIRS) and organ-specific
injury.
References
1. Global burden of disease: 2004 update. [Accessed 16 June 2011]; Available from:
http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/index.h
tml
2. The global burden of injuries. American Journal of Public Health, 2000. 90(4): p. 523-526.
3. Cho, S.D., et al., Reproducibility of An Animal Model Simulating Complex Combat-Related
Injury in A Multiple-Institution Format. Shock, 2009. 31(1): p. 87-96.
61
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y7. Development and Applications of CE-QqQMS-based Metabolomics for
Cancer Research
K. Kami1,*, K. Sasaki1, T. Fujimori1, H. Yamamoto1, Y. Ohashi1
1
Human Metabolome Technologies, Inc., Mizukami 246-2, Kakuganji, Tsuruoka, Yamagata 9970052, Japan
*E-mail: [email protected] (corresponding author)
Abstract
Most metabolites comprising primary energy metabolism such as glycolysis, pentose
phosphate pathway, and the TCA cycle are anionic compounds; however, concentrations of most
these metabolites in typical mammalian cells and tissues are often low and difficult to be
measured by conventional metabolomics platforms. Here, we developed a capillary
electrophoresis-triple quadrupole mass spectrometry (CE-QqQMS)-based metabolomics
technology, which improved detection sensitivity of 63 anions up to 291-folds compared to a
conventional time-of-flight mass spectrometry-based analysis. The method was then applied to
profile metabolomic changes of cell lines treated by a Mek inhibitor, which significantly downregulated glycolysis and nucleotide synthesis in addition to oxidative stress and amino acid
imbalance. The CE-QqQMS was also used to elucidate drug toxicity against cancer metabolism in
EGFR sensitive/insensitive cell ilnes1. Paired non-tumor and tumor tissues of lung, prostate, and
gastric cancer patients were also characterized. The results not only distinguished tumor from
non-tumor profiles but also delineated within-tumor differences associated with their tissue
types or differentiation status2; considerably high glycolytic intermediate, nucleotide, and
glutathione levels in differentiated (DT) tumor tissues and rather low energy and redox statuses
in undifferentiated (UT) ones, of which trends were also observed in DT and UT cell lines. Taken
together, CE-QqQMS-based metabolomics is promising for elucidating energy metabolic changes
caused by the molecular target drug and for profiling tissue samples for searching correlations
between certain metabolite sets and clinical parameters.
References
1. Makinoshima, H., Takita, M., Matsumoto, S., Yagishita, A., Owada, S., Esumi, H. and
Tsuchihara, A. (2014) Epidermal Growth Factor Receptor (EGFR) Signaling Regulates
Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma. J. Biol. Chem. 9,
2370-2378.
2. Kami, K., Fujimori, T., Sato, H., Sato, M., Yamamoto, H., Ohashi, Y., Sugiyama, N.,
Ishihama, Y., Onozuka, H., Ochiai, A., Esumi, H., Soga, T., Tomita, M. (2013) Metabolomic
Profiling of Lung and Prostate Tumor Tissues by Capillary Electrophoresis Time-of-flight
Mass Spectrometry. Metabolomics. 9(2): 444-453.
62
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y8. Merging and sharing metabolomics analysis tools with Galaxy:
transparent, reproducible, open ’omics.
R. L. Davidson1,2, R. J. M. Weber3, P. Li2, M. R. Viant3,*
1
NERC Biomolecular Analysis Facility - Metabolomics Node (NBAF-B), School of Biosciences,
University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; 2GigaScience,BGI-Hong Kong Co.
Ltd, 16 Dai Fu Street, Tai Po Industrial Estate, NT, Hong Kong; 3School of Biosciences, University of
Birmingham, Birmingham B15 2TT, UK
*E-mail: [email protected] (corresponding author)
Abstract
’Omics technologies rely heavily on data and computational analyses. Both of these
research objects should be citable and openly available if science is to be reproducible. There has
been recent policy in e.g. the US and UK that publicly funded research should deposit data in
open repositories. The MetaboLights database (1) is now available for metabolomics data.
However, there has been no such promotion of software and workflows.
Here we bring a full workflow of metabolomics analysis tools into the Galaxy workflow
system (2) for the first time. We have implemented the pipeline for 2 widely used non-targeted
modalities, direct infusion mass spectrometry (DIMS) using Fourier transform ion cyclotron
resonance and liquid chromatography mass spectrometry (LC-MS). These pipelines have been
refined within our laboratory over the last several years, and include preparatory processing of
the raw data files (SimStitch (3), XCMS (4)), preparation of the X matrix, e.g. normalisation and
scaling, automated multivariate statistical analysis and also metabolite identification (MI-Pack
(5)). These pipelines represent the core requirements, from start to end, of a “metabolomics
analysis” for either data type.
With this we hope to initialise the move towards standardised, sharable, transparent
workflows in metabolomics while providing a much more intuitive interface for researchers
without programming experience and ultimately providing a platform that can integrate this
‘omics with the many others already existing in the Galaxy environment.
References
1. Haug, K et al. Nucl. Acids Res. (2013) doi: 10.1093/nar/gks1004.
2. Goecks, J et al. Genome Biol. (2010) 11(8):R86
3. Southam, A.D. et al. Anal. Chem. (2007) 79, pp. 4595–4602
4. Smith, C.A. et al. Anal. Chem. (2006), 78, pp. 779–787
63
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y9. Computational methods to study the effect of perturbations on
metabolic phenotypes
S Umashankar1, 2, RBH Williams3, S Swarup1, 2, 3,*
1
Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore, 14
Science Drive 4, Singapore 117543; 2 NUS Environmental Research Institute (NERI), National
University of Singapore, #02-01, T-Lab Building, 5A Engineering Drive 1, Singapore 117411; 3
Singapore Centre for Environmental Life Sciences Engineering (SCELSE),Nanyang Technological
University, SBS-01N-27, 60 Nanyang Drive, Singapore 637551;
*E-mail: [email protected] (corresponding author)
Presenter: Umashankar Shivshankar ([email protected])
Abstract
Organisms respond to genetic or environmental perturbations by modulating their cellular
metabolism. Changes to these metabolic processes are elicited through the regulation of gene,
protein and metabolite levels. Metabolites which form intermediates or end products of these
regulatory processes, can be regarded as the ultimate biochemical phenotype of a system.
Analysis of metabolomics data in combination with other omics or metadata provide vital insights
on how genetic information and environmental factors can influence these processes. Thus, we
developed and applied specific data analysis strategies for:
• Identifying non-biological sources of variation in untargeted metabolome surveys using
multivariate statistical techniques. A filtering procedure based on the singular value
decomposition was developed to remove these batch effects while permitting recovery
of signal of biological origin.
• An untargeted metabolite survey of algae- Chlorella species, to investigate the strain
specific signatures of metabolic reprogramming strategies, and associations between
physiochemical measures and metabolic pathways. These findings were used to identify
metabolic correlates of biotechnology related traits.
• To understand the effect of genetic perturbation on metabolic network of Arabidopsis, a
data-dependent multi-omics strategy that combines the genomic potential and gene
expression outcomes with comprehensive metabolite profiling was developed. This
analysis helped discover coordinated regulation of metabolic pathways associated with
innate immunity and secondary metabolism. Furthermore, analysis of metabolic network
perturbation caused by overexpression of a metabolic enzyme GLDC, revealed novel
metabolic reprogramming strategies identifying the gene responsible for non-small cell
lung cancer.
References
1.
2.
Zhang W, Shyh-Chang N, Yang H, Rai A, Umashankar S, Swarup S, Lim E, Lim B.
“Glycine Decarboxylase activity drives non-small cell lung cancer tumor-initiating cells
and tumorigenesis”. , Cell (2012)
Rai A, Umashankar S, Sanjay Swarup. “Plant Metabolomics: From Experimental
Design to Knowledge Extraction”. Legume Genomics (2013)
64
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y10. Metabolomics Investigation of Green Algae upon Exposure to
Pesticide
Yan GAO1,*, Qingsong LIN3, Sam Fong Yau LI 1,2*
1
Department of Chemistry, National University of Singapore, 3 Science Drive 2, Singapore,
117543
2
NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, National
University of Singapore, Singapore 117411
3
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4,
Singapore, 117543
*E-mail: [email protected] (corresponding author)
Abstract
Algae are an important component of biological monitoring programs for evaluating
water quality because of their short lifecycle, rapid reproduction rate and tendency to be most
directly affected by physical and chemical environmental factors1. In this study, metabolomics
technique is applied to green algae to investigate metabolites changes upon exposure to
pesticide. Different concentrations of diazinon (0.01 ppb, 0.1 ppb and 10 ppm) were used to treat
green algae Chlorella Vulgaris at different time points(24 h, 48 h, 72 h and 96 h), respectively. LCMSTOF was used to detect metabolite changes in green algae. Clearly separation between
control group and treatment groups (0.01 ppb, 0.1 ppb and 10 ppm) can be observed using PCA
plot, but the separation among different treatment groups is not good. This may indicate that
even lower diazinon concentration (0.01ppb) can cause metabolite changes. Data analysis of
different time points and potential biomarkers is still in progress.
References
1. Genevieve M. Carr, James P. Neary etc, Water Quality for Ecosystem and Human Health,
2008, United Nations Environment Programme Global Environment Monitoring
System/Water Programme.
65
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y11. A Combination of NMR and LC-MS Reveals Metabolic Differences in
Toxin and Non-Toxin Producing Microcystis sp.
W.L. Zhang 1,2, B.H. Fu 1, S.F.Y. Li 1,2,3 *
1
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore
117543.
2
NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1,
Singapore 117411.
3
School of Environment and Energy, Peking University Shenzhen Graduate School, University
Town, Xili, Nanshan District, Shenzhen P.R.C. 518055.
*E-mail: [email protected] (corresponding author)
Abstract
Harmful algal blooms predominately formed by Microcystis sp. which produces potent
microcystins (MCs) has become an emerging global health issue in freshwater habitats.[1]
Extensive research efforts have been carried out to understand the biological role of MCs and
environmental conditions that stimulate toxin productions.[2] In this presentation, a combination
of nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography mass
spectrometry (LC-MS) was employed to reveal the metabolic differences of toxic and non-toxic
Microcystis strains. Under similar growth conditions, the cell density of toxic strains is 2-3 folds
lower than non-toxic strains, suggesting that the presence of MCs suppress cell growth. The
substantial absence (p<0.001) of carbohydrates (sucrose, maltotriose, glucose and glycogen), and
amino acids (lysine, aspartate and glutamate) suggest photoinhibition in toxic strains. As MCs are
known to produce reactive oxygen species (ROS), and that ROS are one of the main agents
responsible for photoinhibition, we reveal toxin-induced photosynthesis impairment.
Photosynthesis perturbation would have influence the toxic strains to seek for alternative energy
source. Decrease in fatty acids content in toxic strains indicate that Microcystis sp. undergo βoxidation to generate acetyl-coA, which is later used for the biosynthesis of poly(3hydroxybutyrate), a carbon and energy reserve material found in cyanobacteria. The absence of
fatty acids with several phospholipid precursors such as sn-glycerol-3-phosphate, phosphocholine
and choline also suggest that the decrease in phospholipids would compromise membrane
integrity in Microcystis sp., thus result in lower cell density in toxic strains.
References
1.
Cong LM, Huang BF, Chen Q, Lu BY, Zhang J, Ren YP (2006) Determination of trace
amount of microcystins in water samples using liquid chromatography coupled with triple
quadrupole mass spectrometry. Anal Chim Acta 569 (1-2):157-168.
2.
Neilan BA, Pearson LA, Muenchhoff J, Moffitt MC, Dittmann E (2013) Environmental
conditions that influence toxin biosynthesis in cyanobacteria. Environ Microbiol 15 (5):12391253.
66
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y12. QuoA expression in Arabidopsis leads to phenolamide channeling to
lignins and coordinated effects at key branch points
Amit Rai1, Sheela Reuben1, Sanjay Swarup1, 2, 3*
1
Department of Biological Sciences, National University of Singapore, Singapore
([email protected]; [email protected]; [email protected]); 2Singapore Center for
Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
([email protected]); 3NUS Environmental Research Institute, National University of Singapore,
Singapore ([email protected]),
*E-mail: [email protected] (Presenter); [email protected] (corresponding author)
Abstract
Metabolic perturbations by a gain-of-function approach serves as a tool to alter steady
states levels of metabolites and query network properties without effecting native enzyme
complexes. In this study, targeted genetics and metabolomics approach was used to understand
the network properties and perturbation response of phenylpropanoid metabolism pathways. A
novel quercetin oxidoreductase, QuoA, from Pseudomonas putida, utilizes quercetin, a
metabolite from phenylpropanoid network released in rhizosphere, and converts it to
naringenin1. Since phenylpropanoid biosynthesis in plants involve formation of quercetin from
naringenin, we envisaged that QuoA expression in plants will provide us with a genetic tool to
“reverse” this biosynthetic step. Therefore, we created transgenic lines expressing bacterial QuoA
in Arabidopsis, thus effectively reversing the biosynthesis of quercetin. We selected
QuoA transgenic lines for low, medium, and high expression levels of QuoA RNA, which also had
corresponding levels of QuoA activity and hypocotyl coloration resulting from increased
anthocyanin accumulation. Stems of all three QuoA lines showed increased lignifications and
tensile strength in the same order as of QuoA RNA and activity levels2. Comparing gain-offunction transgenic lines, expressing QuoA with loss-of-function line at systems level revealed, (i)
metabolite flux from phenolamide into lignin pathways resulted in lignification and increased
stem stiffness and (ii) co-ordinated metabolite-to-gene-expression regulation at four key branch
points in the phenylpropanoid network. This approach provides dual benefit of imparting stem
strength through lignification and improved stress tolerance by up-regulation of flavonoids at the
same time.
References
1. Pillai BV, Swarup S. 2002. Elucidation of the flavonoid catabolism pathway in
Pseudomonas putida PML2 by comparative metabolic profiling. Applied and
Environmental Microbiology.
2. Reuben S, Rai A, Pillai BV, Rodrigues A, Swarup S. 2013. A bacterial quercetin
oxidoreductase QuoA-mediated perturbation in the phenylpropanoid metabolic network
increases lignification with a concomitant decrease in phenolamides in Arabidopsis.
Journal of Experimental Botany.
67
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y13. Optimization of metabolite extraction in Pseudomonas aeruginosa
PAO1 transition from planktonic to biofilms
Saravanan Periasamy1, Victor J. Nesati1, 4, Harikrishnan A.S. Nair1, 3, Staffan Kjelleberg1, 2,
5
, Sanjay Swarup1, 4, and Scott A. Rice1, 2, 5*,
1
Singapore Centre on Environmental Life Sciences Engineering and 2School of Biological Sciences,
Interdisciplinary Graduate School, Nanyang Technological University, Singapore, 4 NUS
Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, National University of
Singapore, Singapore 117411,5Centre for Marine Bio-Innovation and School of Biotechnology and
Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia.
*E-mail: [email protected] (corresponding author)
3
Abstract
Bacteria predominantly grow as surface attached biofilms and, just like other surface
attached organisms, such as corals, bacteria undergo dispersal events to allow them to seek out
and colonize new sites. In this way, bacteria display a biphasic lifecycle, free swimming
planktonic cells (dispersal propagules) and surface attached communities. Not surprisingly,
bacteria have been shown to express different genes and proteins in the two life phases. Biofilms
are important because they impart increased stress tolerance to those microbial populations and
communities, including the widely reported increase in antibiotic resistance. As a consequence
of their enhanced survival, biofilms pose significant risks where they are responsible for more
than 80% of infections and are common problems in a range of industrial settings. Thus, there is
a strong motivation to determine how and why bacteria form biofilms and to understand how
biofilms are functionally different from planktonic cells. Recent advances in metabolomics
methods now allows for detailed studies of the physiology of the populations in both the
planktonic and biofilm stages of growth which may provide invaluable information on differences
between these two states. Here, we compare the metabolomes of planktonic and biofilm
populations of Pseudomonas aeruginosa PAO1. To facilitate these studies, we have further
included the wild-type PAO1 and an isogenic wspF mutant that overproduces c-di-GMP and is a
hyperbiofilm forming strain. For metabolomics comparison of biofilms and planktonic cells, we
first evaluated several extraction methods, including I) Heat Ethanol and Water (HEW), II)
Methanol, Chloroform and Water (MCW) (50:50), III) Acetonitrile Methanol and Water (AMW)
(40:40:20). Liquid chromatography coupled to mass spectrometry (LC-MS) was used for an
untargeted survey of the global metabolites as well as a targeted approach, focused on
nucleotides for the comparison of planktonic culture and biofilms. A principal component,
multivariate analysis was used to compare samples and to identify features of interest for further
interrogation. As expected, the metabolite profiles of the planktonic cells were significantly
different from the biofilm populations. For example, both wild type PAO1 and wspF mutant
strains showed distinct metabolic profiles between planktonic and biofilm states. Further, results
showed that metabolite profiles for biofilms of the wild-type and the wspF mutant indicated the
two strains show distinct metabolite patterns. For example, 25 metabolite features were
significantly different between the two strains. Metabolites that were significantly higher in the
biofilm included 20 metabolite features, suggesting that the purine biosynthetic pathway was
highly induced during biofilm growth. Our data demonstrated that distinct metabolites features
were play an important role in cellular metabolism during biofilm formation.
68
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
References
1. Gjersing, E., Herberg, J.L., Horn, J., Schaldach, C.M., and Maxwell, R.S. (2007) NMR
Metabolomics of planktonic and Biofilm modes of growth in Pseudomonas aeruginosa.
Analytical Chemistry 79, 8037-8045.
2. Koex, M.M., Muilwijk, B, Vander werf, M.J., and Hankemeier, T. (2006) Microbial
metabolomics with gas chromatography/Mass spectrometry. Analytical Chemistry 78,
1272-1281.
3. Cordell, R.L., Hill, S.J., Ortor, C.A., and Barretr, D.A. (2008) Quatitative profiling of
nucleotides and related phosphate-contating metabolites in cultured mammalian cells by
liquid chromatography tandem electrospray mass spectrometry. Journal of
Chromatography 871, 115-124.
69
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y14. Time-course targeted metabolite profiling in Vitis vinifera cell
suspension cultures
Nay Min Min Thaw Saw1,*, Claudio Moser2, Pietro Franceschi1
1
Biostatistics and Data Management Group, Computational Biology, Fondazione Edmund Mach,
Via Mach1, 38010 San Michele all’ Adige (TN), Italy
2
Gene Functions, Genomics and Biology of Fruit Crop Department, Fondazione Edmund Mach,
Via Mach1, 38010 San Michele all’ Adige (TN), Italy
*E-mail: [email protected] (corresponding author)
Abstract
Grapes (Vitis vinifera) are rich sources of phenolic compounds including flavonoids and
non-flavonoids. Cell suspension cultures of grapes can be used as a model system for
investigating different factors that are able to induce and/or modify secondary metabolite
biosynthesis. The characterization of the full time-course metabolic profile of the grape
suspension is expected to give by far more information than measurements at one or two timepoints. In this communication we report on the optimization of a complete analytical protocol for
the time-course targeted metabolite profiling of Vitis vinifera cv. Pinot Noir suspension cultures.
Cell cultures of V. vinifera were grown for 15 days and the sampling was performed daily. The
experiment was performed in triplicates. The freeze-dried samples were extracted twice using
methanol with the help of sonication and subsequent centrifugation. The identification and
quantification of targeted metabolomics in the extract was performed by LC-MS/MS (Vrhovsek et
al, 2012) and NMR. Preliminary results indicate that at day seven the most abundant metabolites
were benzoic acid derivatives such as 4-aminobenzoic acid, vanillin and vanillic acid, and stilbenes
as well as flavan-3-ols and flavonols. Quercetin-type flavonols were found dominantly in all the
days during the growth curve. In addition, hesperidin, sinensetin and luteolin were detected as
major flavanones in the cell cultures.
References
1.
Urska Vrhovsek, Domenico Masuero, Mattia Gasperotti, Pietro Franceschi, Lorenzo
Caputi, Roberto Viola, Fulvio Mattivi (2012). A Versatile Targeted Metabolomics Method for the
Rapid Quantification of Multiple Classes of Phenolics in Fruits and Beverages. Journal of
Agricultural and Food Chemistry, 8831-8840.
70
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y15. Metabolomics-based multi-omics analysis of Wolfberry for optimal
IBD management
Wanping Aw1, 2, Huijuan Jia1, Shinji Fukuda2, Manaka Hanate1, Masaru Tomita2 and
Hisanori Kato1
1. Corporate Sponsored Research Program “Food for Life”, Organization for Interdisciplinary
Research Projects, The University of Tokyo
2. Institute for Advanced Biosciences, Keio University, Yamagata, Japan
Presenting Author
Wanping Aw, Ph.D.
Email: [email protected]
Mailing Address: Keio SFC Systems Biology Laboratory, Kugenumaishigami 1-13-6, Fujisawa,
Kanagawa, 251-0022, Japan
Abstract
Background and Objective
Wolfberry (WOL, Lycium barbarum), a traditional Chinese medicinal food, has been reported to
have antiaging, anticancer, health-promoting, and immune-boosting properties (1). We aimed to
elucidate the anti-inflammatory molecular mechanisms of WOL in a dextran sodium sulphate
(DSS) induced colitis model.
Methods
Seven-week-old male C57BL/6J mice were fed either 2% WOL or control diet for 1 week after
which colitis was induced by administering 1.5% (w/v) DSS for 9 days and Disease Activity Index
(DAI) was observed. Colonic and hepatic microarray (Mouse Genome 230 2.0, Affymetrix);
quantitative iTRAQ hepatic proteome; and capillary electrophoresis-mass spectrometry (CE-MS,
Agilent)-based metabolome analysis of plasma and liver metabolites were performed.
Results and Discussion
WOL significantly suppressed colon length decrease and DAI. Transcriptome and proteome
analysis have revealed their common patterns in the suppression of anti-inflammatory cytokines
by WOL supplementation. Unique to proteome and metabolome analysis were the amelioration
of oxidative markers. These clues from each analysis complement each other, suggesting WOL as
an effective nutraceutical in IBD management.
Conclusion
These findings will serve as a clarion call to promote integrated omics nutrition research to
propose dietary-intervention strategies to recover normal homeostasis in disease states and to
maintain healthy well-being via daily nutritional consumption.
References
1. Tang. WM., et al. A review of the anticancer and immunomodulatory effects of Lycium
barbarum fruit. Inflammopharmacology. 20 (6), 307-314 (2012)
2. Amagase. H and Farnsworth NR. A review of botanical characteristics, phytochemistry,
clinical relevance in efficacy and safety of Lycium barbarum fruit (Goji). Food Res Int. 44
(7), 1702-1717 (2011)
71
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Y16. Unraveling the metabolic strategies underlying pathogenesis in
cereal killer.
1*
1
2
2
Rajesh. N. Patkar , Z. Qu , P. Benke , S. Swarup , N. Naqvi1
1 Temasek Life Sciences Laboratory (TLL), 1 Research Link, Singapore
2 NUS Environmental Research Institute (NERI), Singapore
*E-‐mail: [email protected] (presenting author)
Abstract
Blast is a fungal disease of a wide variety of food crops including rice, wheat and
barley. Significant yield losses due to Blast disease make the causal fungal pathogen
Magnaporthe oryzae a serious threat to global food security. M. oryzae has a remarkable
ability to modulate the host immunity for its own advantage. Here, we found that loss of an
Antibiotic Biosynthesis Monooxygenase (Abm), from a secondary metabolism gene cluster,
led to fungal inability to suppress host innate immunity, and resulted in non-‐pathogenicity in
the mutant. Using a plant-‐based assay combined with chemical analysis tools, we identified
that Abm directly inactivates jasmonic acid (JA) into 12-‐hydroxyjasmonic acid (12-‐OH-‐JA) to
down-‐regulate the defense response in the host. Further systematic characterization showed
that M. oryzae expresses Abm intracellularly to synthesize endogenous 12-‐OH-‐JA, which is
secreted out at the time of invasion, most likely to suppress early wounding response in the
host plant. Interestingly, M. oryzae secreted Abm upon invasion, likely to suppress the plant
innate immunity by converting host JA to 12-‐OH-‐JA therein. This study not only suggests
fungal Abm as a potential fungicidal target towards disease control and food security, but
also provides a synthetic strategy for transformation of a versatile small-‐ molecule plant
hormone.
72
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Agilent Sponsored Talk
Two-dimensional liquid chromatography: a new technique for
metabolomics?
O.A.H.Jones1*,
1
School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia
* E-mail: [email protected]
Abstract
Multi-dimensional chromatography is a system in which more than one mechanism of separation
is applied to a sample, with each mechanism being considered an independent separation
dimension (1). Multi-dimensional gas chromatography is an established technique with a
distinguished history in metabolomics. However, as with all forms of GC, samples are limited to
those compounds that are, or can be made, volatile. In contrast, two-dimensional liquid
chromatography (LC×LC) is a relatively new technique and does not require derivatisation. We
have applied this method to the analysis of complex samples and demonstrate that the enhanced
resolution and peak capacity allow peaks to be detected that could not be separated using
standard, one-dimensional LC.
An offline reversed phase x reversed phase LCxLC method was developed and applied to
Agaricus bisporus mushrooms in order to demonstrate the potential of the technique and assess
the effect of UV irradiation on the mushroom's metabolic profile. The method allowed the
detection of 158 peaks in a single analytical run and aa total of 51 compounds including sugars,
amino acids, organic and fatty acids and phenolic compounds were identified. After irradiation of
the mushrooms with UV for 30 s the number of peaks detected decreased from 158 to 150; 47
compounds increased in concentration while 72 substances decreased. This is the first time that
two-dimensional liquid chromatography has been carried out for the metabolomic analysis of
mushrooms. The data provide an overview of the gain/loss of nutritional value of mushrooms
following UV exposure and demonstrate that the increased peak capacity and separation space of
LCxLC has great potential in metabolomics.
References
1. Stevenson, P.G., M. Mnatsakanyan, G. Guiochon, and R.A. Shalliker, (2010) Peak Picking
And The Assessment Of Separation Performance In Two-Dimensional High Performance
Liquid Chromatography. Analyst 135, p. 1541-1550.
73
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Technical Site Visit Information
Agilent LC/MS facility
Agilent Technologies Inc. (NYSE: A) is the world's premier measurement company and a
technology leader in chemical analysis, life sciences, electronics and communications.
The company's 18,500 employees serve customers in more than 100 countries. The
company is a trusted partner to thousands of the scientists and lab analysts worldwide
who are focused on basic research, drug discovery & development, food &
environmental testing, forensics and clinical research.
The Agilent LC/MS facility is located within the Agilent Life Sciences Group Order
Fulfillment Center, located at its Yishun, Singapore, site. The center will be responsible
for efficient management of the supply-chain network -- including planning,
procurement, value-added engineering and logistics. The center ensures that the LC/MS
facility is able to efficiently and effectively deliver configurable solutions to customers
worldwide while maintaining Agilent's hallmark of quality.
Information about Agilent is available at www.agilent.com.
74
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
NERI Laboratory at T-Lab and CREATE.
Laboratory and Technical Staff Support
The NERI Laboratory serves as a strong backbone analytical support for NUS researchers
in environmental research. It collaborates closely with different initiatives and
programmes as well as leading analytical equipment industries to bring the latest
advanced analytical and spectroscopic instruments. The instruments will allow
researchers to unravel and discover mysteries in their respective research and identify
traces of organic and inorganic compounds that may be previously undetected. Also, the
NERI Laboratory has a comprehensive sample preparation facility that expedites the
clean-up and extraction from complex sample matrices.
The NERI Laboratory is managed by a team of dedicated and experienced technical and
research staff. They provide expertise in assisting the NUS community and industry
engaged with NERI doing environmental research. They are also committed in ensuring a
high standard of occupational safety and health for staff and researchers.
Exchange of Technical Knowledge
As a focal point for NUS researchers on environmental research, NERI's laboratory is also
a facility where researchers from multi-disciplines (biologists, bioinformatics engineers,
analytical chemists, toxicologists, chemical and biomolecular engineers and
environmental engineers) gather and exchange technical knowledge to enhance research
in their respective fields. The more experienced research staff will often interact with
postgraduates to provide guidance and advice in their respective research studies.
Information about NERI is available at: http://www.nus.edu.sg/neri/index.html
75
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Sponsors’ Profile
The Merlion Metabolomics Workshop Organising
Committee gratefully acknowledges the generous support
from the following sponsors:
76
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Agilent Technologies Inc. (NYSE: A) is a leader in chemical analysis, life sciences, diagnostics, electronics and
communications. The company’s 20,600 employees serve customers in more than 100 countries. Agilent had revenues
of $6.8 billion in fiscal 2013. Information about Agilent is available at www.agilent.com.
Singapore is the hub for Agilent’s infrastructure, strategic supply chain, global logistics, treasury, business support as
well as sales and marketing services functions. The site also houses the life sciences manufacturing facility. The facility
was officially opened in 2010 by Minister, Prime Minister’s Office, Mr. S. Iswaran, and Agilent CEO, Mr. Bill Sullivan. The
manufacturing facility is ISO 9001 certified, ISO 13485 certified in FDA Class 1, in addition to certifications from Korean
FDA and Singapore HSA, it is also QSR compliant. The manufacturing facility produces Liquid Chromatography Mass
Spectrometers (LC/MS), Automation, Supercritical Fluid Chromatography (SFC) and Microarray Scanners, which are
shipped to customers globally.
77
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Waters Corporation is a publicly traded laboratory analytical instrument and
software company headquartered in Milford, Massachusetts. The company
employs more than 5,000 people, with manufacturing facilities located in Milford,
Taunton, Massachusetts; Wexford, Ireland; Manchester, England; and contract
manufacturing in Singapore.
Waters markets to the laboratory-dependent organization in these market areas:
liquid chromatography, mass spectrometry, supercritical fluid chromatography,
laboratory informatics, rheometry and microcalorimetry.
Website: http://www.waters.com/waters/home.htm?locale=en_US
Under the Thermo Scientific brand of Thermo Fisher Scientific, Inc. (NYSE:TMO),
we help scientists meet the challenges they face in the lab or in the field every
day. From routine analysis to new discoveries, our innovations help professionals
do the science they need to do, the way they want to do it. Our high-end
analytical instruments, laboratory equipment, software, services, consumables
and reagents help our customers solve complex analytical challenges, improve
patient diagnostics and increase laboratory productivity.
Website: http://www.thermoscientific.com/en/home.html
78
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Location Map of NUS-University
Town
Visitors can get to University Town (UTown) by:
A. MRT & Bus
From Buona Vista MRT Station - Bus 196
Walk to the bus stop opposite Buona Vista MRT Station located
near Exit D, Bus Stop No. 11369. Board Bus 196 (towards Clementi
Interchange). Alight 6 stops later at Bus Stop No. 19059. Walk
approximately 5 to 8 minutes to UTown.
From Clementi MRT Station - Bus 183
Walk to Clementi Bus Stop, Bus Stop No. 17179, from Clementi
MRT Station (Exit B). Board Bus 183 (towards Jurong East Temp
Interchange). Alight 3 stops later at Bus Stop No. 17099. Walk
approximately 8 to 10 minutes to UTown.
From Clementi Bus Interchange - Bus 96
Board Bus 96 (towards Clementi Interchange). Alight 3 stops later
at Bus Stop No. 17099. Walk approximately 8 to 10 minutes to
UTown.
From Kent Ridge MRT Station - NUS Internal Bus Shuttle
Walk to Kent Ridge Bus Stop, Bus Stop No. 18331, from Kent Ridge
MRT Station (Exit A). Board NUS Internal Bus Shuttle D2. Alight 5
stops later at UTown.
B. NUS Internal Bus Shuttle
Services D1 and D2
79
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
Internal Shuttle
Bus D1/D2
80
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
NOTES
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
81
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
82
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
83
MERLION METABOLOMICS WORKSHOP SINGAPORE
2014
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
_________________________________
84
Supported by:
Silver Sponsor:
Sponsors: