4th australasian metabolomics symposium

Any publication included in this abstract book/ or opinions expressed therein

remain solely those of the author(s). Such publications have been included

only for ease of reference and academic purposes.

Edited by:

Prof. Dr. Teh Lay Kek

Prof. Dr. Mohd Zaki Salleh

Prof. Dr. Jean-Frédéric Weber

Published by:

Penerbit Universiti Teknologi MARA (UPENA),

Universiti Teknologi MARA,

40450 Shah Alam,

Selangor Darul Ehsan

Printed by:

Printing Unit,

Faculty of Art & Design,

Universiti Teknologi MARA (UiTM),

40450 Shah Alam, Selangor.

ISBN 978-967-363-331-9

© 2012 Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy,

Universiti Teknologi MARA, Malaysia.

i

4th Australasian Metabolomics

Symposium 2012

Welcome Note

Assalamualaikum w.b.t. and a very good day, Distinguished guests, speakers, lecturers, MyMS members and researchers. It is my great pleasure to welcome you all to the 4

th Australasian

Metabolomics Symposium and Workshops. I am pleased that the symposium and workshops could take place here at our beloved UiTM Puncak Alam campus and hope that you are pleased with our hospitality. I was informed that, this is the first of its kind of event to be held here in UiTM and in Malaysia. The 4

th Australasian Symposium was previously

held in New Zealand and Australia. I appreciate the effort that the MyMS committee took to realise this event and hope that this effort continues in

future. The 4

th Australasian Metabolomics Symposium and Workshops provides a golden platform for all to

exchange knowledge and to build networking. I would like to encourage all researchers to take advantage of this platform to meet colleagues from your own speciality and also to discuss with experienced Professors and researchers in Metabolomics on potential research collaboration. Seeing the prospect of the “omics” field, the researchers at UiTM have come to realise the advantage of metabolomics and are working on metabolomics based researches. To share the beauty of metabolomics, I was told that the committee has attempted to replicate the spirit of the original “Symposium” written by Plato, the great Greek philosopher for benefits of the research society as a whole. As a result, they have organised the symposium and workshops to discuss on the selected theme of “Moving Forward to Integrate Systemic Biology into Post-Genomics Era”. Your strong support has made the 4

th Australasian Metabolomics Symposium and Workshops a record

breaking event. The quality of technical programme is world class and the spectrum of topics is current and broad. Two workshops were added to the programme to make this symposium strong and wholesome. The invited speakers include Prof. Dr. David Wishart (University of Alberta, Canada), Prof. Dr. Rudolf Grimm (Agilent Technologies, Adjunct Professor to University California Davis), Dr. Matthias Pelzing (Bruker Daltonics, Australia), Dr. Ute Roessner (University of Melbourne), Prof. Dr. Thomas Hennessy (Adjunct Professor to PROMISE, UiTM), Dr. Fang Fang (Bruker Biospin, Germany), Prof. Dr. Robert Trengove (Director for the Murdoch Separation Science and Metabolomics Laboratory and the node leader for the Murdoch Node of Metabolomics Australia) and Assoc. Prof. Dr. Markus R. Wenk (National University of Singapore) are globally renowned for their expertise in the metabolomics field. I hope you enjoy your participation in the 4

th Australasian Metabolomics Symposium and Workshops and

hope that you‟re being here at our Puncak Alam campus would be a memorable one. With this, I officiate the 4

th Australasian Metabolomics Symposium and Workshops.

Thank you.

Dato’ Prof. Ir. Dr. Sahol Hamid Abu Bakar FASc, PEng, DSPN, DJN, DSM, BCN Vice Chancellor, Universiti Teknologi MARA Malaysia

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Welcome Note

Assalamualaikum w.b.t and good day. Distinguished speakers, researchers, lecturers and MyMS members. I would like to welcome you to the 4

th Australasian Metabolomics

Symposium and Workshops where this would be the first time it is held in Malaysia, at UiTM Puncak Alam Campus. My sincere congratulation and appreciation to the organising committee for all the hard work done in making this event a success. Hopefully, we will continue to excel with passion and enthusiasm in our research studies. To all participants of the symposium and workshops, I wish you a rewarding journey throughout the five days of learning and sharing, enriching your

minds on " The art of metabolomics: moving forward to integrate systemic biology in post-genomics era” - aptly used as the conference theme. To guests of UiTM, I wish you a pleasant stay in our green campus here in Puncak Alam. To guests of the country, I wish you lots of memorable experience in Malaysia, and I hope that you will come back to savour more of our Malaysian hospitality. The symposium and workshop mark the milestones of the Malaysian Metabolomics Society (MyMS) gathering renowned scientists from everywhere to meet and share their knowledge and experiences. MyMS was registered with the Registrar Office of Society and it symbolises the desire of the academics in Malaysia to enhance metabolomics related researches and to integrate this tool into the post genomic era, to help provide more understanding towards what is known through genomics. MyMS vowed to take the challenge of realising the mission of gathering the scientists to share knowledge, to collaborate and to accelerate metabolomics related researches. MyMS aims to foster and inculcate good research culture among the young generation and provide or facilitate them with the tools they need. I cannot think of a better symbol for the goals of this society but to seed and sustain the reformation that requires each of us who carries the flame and serves as a torchbearer for metabolomics research in Malaysia. I hope events such as this will provide us with the opportunity to share our experiences, learn from one another, and progress debates in a positive, cooperative manner in moving forward with excellence in research. Best of luck as we all work together in dispelling myths and increasing the credibility and reputation of MyMS as a whole. Thank you. Prof. Dr. Mohd Zaki Salleh Founding President of MyMS

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Welcome Note

A very good day to distinguished guests, speakers, MyMS members, and researchers. Ladies and gentlemen, welcome to the 4

th Australasian Metabolomics

Symposium and Workshops held at UiTM Puncak Alam. I would like to begin by thanking the people who had initiated the idea of this symposium, Dr. Ute Roessner, Prof. Dr. Thomas Hennessy and Prof. Dr. Mohd Zaki Salleh. Many must have wondered why the symposium is named as the 4

th Australasian Symposium, and what has it got to do with Malaysia.

For your information, the first three installments were conducted in New Zealand and Australia. It is our good friend, Dr. Ute Roessner, who suggested for the symposium to be held for the first time in Malaysia. This

happened over a coffee talk when a few of us were discussing on how to move forward metabolomics in Malaysia. Metabolomics is truly a useful tool for developing and testing theories and hypotheses and the number of such studies increases fast. The emergence of various “omics” studies share one goal, which is to discover the cellular processes and identifying potential biomarkers and providing answers to our research questions. This symposium marks an important milestone for the efforts of Malaysian Metabolomics Society and Pharmacogenomics Centre in bringing together local researchers interested in metabolomics to share and learn from the experts we invited from overseas. We sincerely hope that this kind of platform remains effective, robust and enduring in promoting progress in science. The keywords in the title for this gathering “The art of metabolomics: moving forward to integrate systemic biology in post-genomics era” were selected specifically to stress this very point. I am indeed very pleased to see so many colleagues and friends together at this event. I would especially like to welcome those attendees and speakers from abroad who have kindly come to share their wisdom and insight on metabolomics. I hope this event marks the beginning of our friendships and more collaboration work among us can be initiated and realised. My sincere gratitude goes to the eminent speakers in accepting our invitation. Your presence is the climax of this event. Thank you Prof. Dr. David Wishart (University of Alberta, Canada), Prof. Dr. Rudolf Grimm (Agilent Technologies, Adjunct Professor to University California Davis), Dr. Matthias Pelzing (Bruker Daltonics, Australia), Dr. Ute Roessner (University of Melbourne), Prof. Dr. Thomas Hennessy (Adjunct Professor to PROMISE, UiTM), Dr. Fang Fang (Bruker Biospin, Germany), Assoc. Prof. Dr. Robert Trengove (Director for the Murdoch Separation Science and Metabolomics Laboratory and the node leader for the Murdoch Node of Metabolomics Australia) and Assoc. Prof. Dr. Markus R. Wenk (National University of Singapore). It is indeed an honour and a privilege to have you here with us, sharing your precious time and experience with us. Ladies and gentlemen, aside from the symposium we have also arranged workshops including the Pre-Symposium Workshop. I do hope that all of us would take the opportunity to gain as much knowledge as we can from the line of programs we have arranged. The event would provide an opportunity for all of us to meet with professors and experts in metabolomics to discuss more and to integrate knowledge in order to move forward in mastering the metabolomics field. I would once again thank everyone for making this event a success. I would like to extend my gratitude to the Vice Chancellor of UiTM, Assist. Vice Chancellor of Puncak Perdana and Puncak Alam of UiTM and Dean of Faculty of Pharmacy for the endless support. I would like to extend my gratitude to our generous sponsors ranging from companies throughout Malaysia especially our Platinum Sponsors, Agilent Technologies and Bruker Corporation. This proves that our good effort is highly supported by the private sector as they appreciate the benefit of the symposium as an excellent venue to discuss scientific and technological collaboration across the Pacific. While I am saying thanks it would be remiss of me if I did not express my gratitude for all the hard work and commitment of the organising committee for making this event a success. In particular, I would like to thank Prof. Dr. Mohd Zaki Salleh and Prof. Dr. Jean-Frédéric Weber, who have been supportive of this event and

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given a lot of ideas to ensure smooth running of the event. My appreciation goes as well to the secretariat steered by Fazleen, Richard, Dr. John Shia, the committee members including Assoc. Prof. Dr. Kalavathy Ramasamy, Assoc. Prof. Dr. Tang Thean Hock, Assoc. Prof. Dr. Kamarulzaman, Dr. Siti Azma Jusoh, Dr. Wan Iryani, Ruzianisra, Zafirah Liyana, Dr. Adzrool, Mr. Mior, Mr. Azanizam, ICT team of Faculty Pharmacy, Puan Noridah. And last but not least, I owe my gratitude to all the students of Pharmacogenomics Centre who had been running around and for the hard work done these past couple of months to prepare an outstanding event for the benefit of all. Thank you Sharina, Salleh, Shafiq, Rose, Izwan Ismail, Lian Shien, Ruhil, Erda, Hanif, Husaini, Ikhwan, Rahimi, Hafiq, Lan, Dr Zakaria, Izwan Yusof, Syafirul, Aida, Lyn, Norleen, Leya, Lala, Irma and Azwin. Lastly, I hope that this two-day symposium and the Pre-Symposium Workshop and Workshop will be a fruitful one. In short, please enjoy this gathering and to the young scientists, do learn from the eminent researchers. Many of you must have heard of the eminent speakers through their publication and I believe for some of you it is really an ecstasy to be able to meet them in person. I sincerely apologise if there is any shortcoming of this event. Let‟s make this symposium a platform to share and exchange information for better research. Thank you.

Prof. Dr. Teh Lay Kek Chairperson Organising Committee

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Symposium 2012

Welcome Note

With the greatest pleasure I welcome you to the symposium in conjunction with the 4

th Australasian Metabolomics Symposium hosted by Prof. Dr.

Mohd. Zaki and Prof. Teh from the Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy at the Universiti Teknologi MARA. We are especially excited to bring the newly founded Malaysian Metabolomics Society (http://www.mymetabolomics.org/) alive by bringing metabolomics experts together with researchers interested in using metabolomics as a new tool for their research programs. The Australian and New Zealand Metabolomics Network (http://www.anzmn.com.au) is proud to be part of the exciting developments in the metabolomics field in Malaysia and are looking forward to the event allowing exchange of knowledge and building new bridges and friendships!

We hope you have a great time and enjoy the Symposium! Thank you.

Dr. Ute Roessner Co-Chairperson Organising Committee Australian and New Zealand Metabolomics Network

http://www.mymetabolomics.org/

http://www.anzmn.com.au/

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Acknowledgements

The MyMS Organising Committee expresses its sincere appreciation to everyone who has given invaluable support, time and cooperation:

Y. Bhg. Dato’ Prof. Ir. Dr. Sahol Hamid Abu Bakar Vice-Chancellor, UiTM Malaysia

Y. Bhg. Prof. Dr. Azni Zain Ahmed Deputy Vice-Chancellor (Academic and International), UiTM Malaysia

Y. Bhg. Prof. Ir. Dr. Hj. Abdul Rahman Omar Deputy Vice-Chancellor (Research and Innovation), UiTM Malaysia Y. Bhg. Prof. Dr. Azizul Halim Yahya Assistant Vice Chancellor (Puncak Alam and Puncak Perdana Campus), UiTM Malaysia

Y. Bhg. Prof. Dr. Abu Bakar Abdul Majeed Assistant Vice Chancellor (Research), UiTM Malaysia Y. Bhg. Prof. Dr. Aishah Adam Dean of Faculty of Pharmacy, UiTM Malaysia

Y. Bhg. Prof. Dr. Mohd Zaki Salleh Head of PROMISE, (Puncak Alam Campus), UiTM Malaysia

Plenary Speakers:-

Prof. Dr. David Wishart Dr. Fang Fang Assoc. Prof. Dr. Markus R. Wenk Dr. Matthias Pelzing Assoc. Prof. Dr. Robert Trengove Prof. Dr. Rudolf Grimm Prof. Dr. Teh Lay Kek Prof. Dr. Thomas Hennessy Dr. Ute Roessner

Pre- workshop speaker:-

Prof. Dr. Jean-Frédéric Weber

Workshop Speakers:-

Christopher Bowen Robin Philp Assoc. Prof. Dr. Robert Trengove Prof. Dr. Thomas Hennessy Dr. Ute Roessner

Platinum sponsors

Agilent Technologies (M) Sdn. Bhd. Bruker (M) Sdn. Bhd.

Sponsors

Next Gene Scientific Sdn. Bhd. Analisa Resources (M) Sdn. Bhd. Medigene Sdn. Bhd.

Leco Saintifik Maju Sdn. Bhd.

http://www.google.com.my/url?sa=t&rct=j&q=&esrc=s&source=web&cd=8&ved=0CIgBEBYwBw&url=http%3A%2F%2Fmystarjob.com%2Fjob%2Ffeaturedcompany.aspx%3Feid%3D4363&ei=MqwcUKqHO8_HrQfuyYGADg&usg=AFQjCNFeeqk7LA0JpqoU9A2H7DxN67u2kA&sig2=RgTG_eAxIlg1LnPmadLIoQ

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Organising Committee

Advisor : Prof. Dr. Mohd Zaki Salleh

: Prof. Dr. Aishah Adam

Chairperson : Prof. Dr. Teh Lay Kek

Co-chairperson : Dr. Ute Roessner

Secretary : Ms. Fazleen Haslinda Mohd Hatta

: Mr. Richard Muhammad Johari James

Treasurer : Assoc. Prof. Dr. Kalavathy Ramasamy

: Assoc. Prof. Dr. Tang Thean Hock

Scientific committee : Prof. Dr. Mohd Zaki Salleh

: Prof. Dr. Jean-Frédéric Weber

: Dr. Wan Iryani Wan Ismail

: Dr. John Shia Kwong Siew

: Dr. Zakaria Bannur

: Mrs. Ruzianisra Mohamed

Program and abstract book : Ms. Erda Syerena Rosli

: Ms. Nur Jalinna Abdul Jalil

: Ms. Asbiyatulaida Derahman

Registration : Mrs. Zafirah Liyana Abdullah

: Mrs. Siti Nooraishah

: Ms. Nur Azwin Ismail

: Ms. Nornazliya Mohamad

Protocol : Mr. Azanizam Ismail

: Ms. Irma Syakina Mohd Zaki

: Mr. Mohd Shafiq Aazmi

Hospitality : Mrs. Nurul Aqmar Mohd Nor Hazalin

: Ms. Sharina Hamzah

: Mr. Mohd Syafirul Shamsuddin

: Ms. Rose Iszati Ismet Nayan

Exhibition (sponsors) : Mrs. Lee Lian Shien

: Mrs. Norleen Mohamed Ali

: Mr. Muhammad Husaini Ismail

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ICT, Art & Design : Mr. Mohd Izwan Mohamad Yusof

: Mr. Mohd Ikmal Hanif Abdul Khalid

: Mr. Mohamad Izwan Ismail

: Dr. Adzrool Idzwan Ismail (Photography and video)

: Assoc. Prof. Dr. Kamarulzaman Md. Isa (Photography & video)

Publicity : Dr. Siti Azma Jusoh

: Mr. Mohd Salleh Rofiee


: Mrs. Noridah Md Lasam

Social and tour : Ms. Ruhil Nadirah Che Omar


Conference workshop : Mr. Mohd Ikhwan Ismail



: Mr. Mohd Salleh Rofiee

: Mr. Hasbullani Zakaria

: Mr. Muhamad Hafiq Zulkifli

Food and beverages : Mr. Mohd Rahimi Muda



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Malaysian Metabolomics Society (MyMS) About Metabolomics related research is dated back in 1970 by the first paper published by Pauling. In 2001, the term "metabolomics" was introduced by Oliver Fiehn and defined as "a comprehensive and quantitative analysis of all metabolites" in a system. Later in 2004, Metabolomics Society was founded by Rima Kaddurah-Daouk, Associate Professor, Duke University Medical Center. And in 2007, Professor Dr David Wishart, project leader for The Human Metabolome Project released the first draft of the human metabolome comprising of 500 metabolites, 1,200 drugs and 3,500 food components. Now the database has grown to cover more than 7900 metabolites (http://www.hmdb.ca/). Watching the impressive movement in metabolomics by scientists all over the world and with the potential of metabolomics in closing the gaps of information currently faced with other “omics”, a group of researchers in Malaysia came together to establish the Malaysian Metabolomics Society (MyMS). The founding President, Prof. Dr. Mohd Zaki Salleh together with Prof. Dr. Teh Lay Kek, advised by Dr. Ute Roessner and Prof. Dr. Thomas Hennessy took the challenge to steer the protemp committee comprised of 20 academics with 30 members from all background. The society is registered later with the Registry of Societies of Malaysia in 2011. The members are professionals working in various fields including Life Sciences, Chemistry, Pharmacy and Medicine. It is an independent, Non-Governmental Organisation and a Non-Profit Organisation governed by Registry of Societies of Malaysia. One of the important function of this society is to gather scientists in Malaysia interested in metabolomics to work together and establish or contribute to the metabolome database, to help the scientific committee move further in unravelling disease mechanisms, discover new and more sensitive and specific biomarker to help diagnose disease quicker and monitor drug responses and disease progresses in patients. Vision To become a platform for gathering scientists and enhance metabolomics-based researchers Missions

To enhance metabolomics-related researchers in Malaysia. To become the platform for knowledge sharing among the researchers in the field of metabolomics. To organise seminars, symposiums, workshops. To facilitate networking between scientists and researchers in different organisations locally and

internationally.

Committee

President Prof. Dr. Mohd Zaki Salleh

Deputy President Assoc. Prof. Dr. Zainul Amiruddin Bin

Zakaria Vice President Prof. Dr. Teh Lay Kek

Secretary Dr. John Shia Kwong Siew

Deputy Secretary Richard Muhammad Johari James

Treasurer Assoc. Prof. Dr. Kalavathy Ramasamy

Deputy Treasurer Selene Tan Shiau Leng

International Advisors Prof. Dr. Thomas Hennessy Dr. Ute Roessner

Committee members Dr. Wan Iryani Wan Ismail Dr. Siti Azma Jusoh Dr. Mohd Shihabuddin Mohamed

Noorden Fazleen Haslinda Mohd Hatta Seetha Ramasamy Tee Ting Yee

http://www.hmdb.ca/


Symposium 2012

Table of Contents

Page

Welcome Note

Vice Chancellor Universiti Teknologi MARA i

President of MyMS ii

Chairperson Organising Committee iii

Co-chairperson Organising Committee v

Acknowledgements vi

Organising Committee vii

Malaysian Metabolomics Society (MyMS) ix

Symposium Programme

Tuesday (4th September 2012) 1

Wednesday (5th

September 2012) 2

Plenary Speakers 3

Abstract

Plenary Lectures 7

Oral Communications

Day 1 18

Day 2 26

Poster Communications 36

Appendices

List of Participants xi

Floor Plan xvii

Contacts Number (Secretariat of Symposium) xviii

Conference Evaluation Form -

MyMS Membership Form -

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Symposium 2012

Symposium Programme Tuesday (4

th September 2012)

Time Programme

0830 - 0930 Registration

0930 - 1015 Plenary Lecture 1: Prof. Dr. David Wishart

Comprehensive Characterisation of the Human Biofluid Metabolomes

1015 - 1100 Plenary Lecture 2: Prof. Dr. Rudolf Grimm

Multi-omics Studies of the Oldest Human Mummy the Tyrolean Iceman or Oetzi

1100 - 1130 Tea Break/Poster Session

1130 - 1215

Plenary Lecture 3: Dr. Matthias Pelzing

Ultrafast Statistical Profiling: FT-ICR Based Profiling of Myxobacteria Natural Products for Rapid Detection and Identification of Marker Compounds

1215 - 1300 Plenary Lecture 4: Dr. Ute Roessner

The Potential of Metabolomics in Systems Biology

1300 - 1430 Lunch Break/Poster Session

1430 - 1515

Plenary Lecture 5: Dr. Fang Fang

Innovative NMR Metabolomics for Food Safety and Quality Control and Newborn Urine Screening


1530 - 1600 Talk by Representative of Sponsor (platinum)

1615 - 1730 Oral Communication 1

1800 - 2230 Symposium and Workshops Officiated by Vice Chancellor UiTM/Cultural Dance/Dinner (BBQ)/Social Events

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Symposium 2012

Symposium Programme

Wednesday (5

th September 2012)

Time Programme

0830 - 0930 Registration

0930 - 1015

Plenary Lecture 6: Assoc. Prof. Dr. Robert Trengove

Metabolomics and the Nexus with Residue Analysis: The Study of Health Impacts of Residue Exposure

1015 - 1100 Plenary Lecture 7: Assoc. Prof. Dr. Markus R. Wenk

Lipidomics, New Tools and Applications


1130 - 1215

Plenary Lecture 8: Prof. Dr. Thomas Hennessy

The Application of Mass Profiler Professional in Biomarker Discovery: A Statistical Analysis & Visualisation Software Tool in PTSD Metabolomics

1215 - 1300 Plenary Lecture 9: Prof. Dr. Teh Lay Kek (PROMISE, UiTM)

Moving Forward with Metabolomics: Scenario in Malaysia

1300 - 1430 Lunch Break/Poster Session

1430 - 1500 Talk by Representative of Sponsor (Platinum)

1515 - 1630 Oral Communication 2

1630 - 1730 Round up Discussion

Tea Break/Adjourn

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Plenary Speakers

Prof. Dr. David Wishart

A professor in the Department of Computing Science and Biological Sciences at the University of Alberta, Edmonton.

Wishart's research interests are very broad, touching on areas relating to nanobiology, genomics, proteomics, metabolomics, bioinformatics and systems biology. His particular interests lie in developing novel methods or improved experimental techniques to facilitate the understanding of biological systems at the molecular (or nano) scale. His research includes the Human Metabolome Project and the Human Metabolite Database Project, among others.

Dr. Fang Fang

An application Chemist for Bruker BioSpin since 2008.

Her research interests include Metabonomics and Mixture Analysis. She is the expert in the areas related to High Resolution Magic Angle Spinning (HR-MAS-NMR). She is involved in studies of food safety and quality control (juice, honey, beer, wine, milk powder, cheese, gutter oil), as well as in clinical biofluid screening (inborn errors, epidemiology, infections and cancer).

Assoc. Prof. Dr. Markus R. Wenk

Associate Professor of Biochemistry at National University of Singapore, Director of SLING and Privatdozent at the University of Basel.

Markus Wenk obtained his PhD in Biophysics from the Biozentrum at the University of Basel, Switzerland in 1997. He then moved to Yale University where he initially was a postdoctoral fellow and subsequently a research faculty scientist in the Department of Cell Biology. In 2004, he took up a position as an Assistant Professor at the National University of Singapore (Yong Loo Lin School of Medicine, Department of Biochemistry and Faculty of Science, Department of Biological Sciences). He is currently an Associate Professor, Assistant Head of Department of Biochemistry and member of the Executive Committee of NGS, the NUS Graduate School for Integrative Sciences and Engineering. Since 2005, he is Privatdozent in the Faculty of Science at the University of Basel. In this capacity, he has initiated and is co-directing an international joint graduate program between Singapore and Switzerland. He is also founder and organiser of the biennial International Singapore Lipid Symposium and Executive Editor of Progress in Lipid Research. Dr. Markus had introduced and established novel techniques for analysis of phospholipids metabolism pertinent to membrane dynamics at the neurological nerve terminal. His work resulted in scientific publications which have major impact on conceptual advancements in the field of neurobiology and lipid metabolism. He is now spearheading novel approaches in global analysis of lipids (lipidomics) for applications in drug and biomarker development with relevance to various disease areas.

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Symposium 2012

Dr. Matthias Pelzing

An application Manager for Bruker Daltonics Asia Pacific.

Matthias Pelzing received his diploma degree in chemistry and his Ph.D. degree in mass spectrometry at the University of Leipzig, Germany in 1991 and 1994. In his Ph.D. thesis he studied waste products in semiconductor industry with mass spectrometric methods in the group of Prof. R. Herzschuh. From 1995 to 1998 he worked as a Postdoctoral Fellow at Institute of Tropospheric Research on characterisation of atmospheric aerosol particles with different mass spectrometric techniques. In 1999 he joint Bruker Daltonik where he worked as applications scientist with focus on coupling techniques of separation systems to API-MS systems. Since January 2006 he has been working as Application Manager for Bruker Daltonics Asia Pacific/ Japan based in Melbourne/Australia. Over the last 10 years, his main research interests are in method development of hyphenated separation techniques coupled to mass spectrometry.

Assoc. Prof. Dr. Robert Trengove

Director for the Murdoch Separation Science and Metabolomics Laboratory and the node leader for the Murdoch Node of Metabolomics Australia.

Robert Trengove has pioneered the development of MS-based „omics 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. Professor Trengove has published many high-impact journal articles and has ongoing commercial contract research arrangements with major industry players in the petrochemical, clinical and animal health pharmaceutical sector.

Prof. Dr. Rudolf Grimm

Worldwide Proteomics and Metabolomics Market Development Manager and Director of Science and Tech at Agilent Technologies. Adjunct Professor at the

University of California in Davis.

Rudolf Grimm received his Ph.D. in Biology from the University of Munich/Germany. After completing a post-doc at the University of Freiburg/Germany and Riken Institute in Tokyo/Japan he joined Hewlett-Packard as a senior life science application chemist in 1991. In 1998, he became the head of protein chemistry at the Munich based proteomics company Toplab. In 1999, he joined Hexal Pharma to establish the Biotech Laboratories for the successful development of generic recombinant protein drugs (Biosimilars). In 2002, he re-joined Agilent Technologies as the worldwide proteomics and metabolomics market development manager. In 2009 he became a director of science and technology at Agilent Technologies. In 2010, Rudi became an Adjunct Professor at the University of California in Davis and in 2011 an Adjunct Professor at Chungnam National University in Daejeon/South-Korea. Since 2010, he managed the strategic academic collaborations in Asia-Pacific.

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Prof. Dr. Teh Lay Kek

A Professor of the Faculty of Pharmacy at University Teknologi MARA (UiTM), Puncak Alam Campus, Selangor, Malaysia.

She graduated with a BPharm (Hons.) from the University of Nottingham, UK in 1995 and completed her Master of Clinical Pharmacy in 1997 from Universiti Sains Malaysia (USM). In 2002, she obtained her Doctor of Philosophy certificate in Pharmacology from USM. She started her career as a lecturer in University of Malaya in 1998. She moved to UiTM as a senior lecturer in 2003 and was promoted to position as an associate professor in 2008 and to professor in 2012. She is one of the active founding members of the Pharmacogenomics Centre (PROMISE) established in 2008. She is also one of the founding members of the Malaysian Society for the Advancement of Pharmacogenomics and Malaysian Metabolomics Society (MyMS). Her research interests are in experimental and clinical pharmacogenomics as well as metabolomics.

Prof. Dr. Thomas Hennessy

Application scientist for life sciences high-end mass spectrometry applications. Adjunct Professor at Universiti Teknologi MARA, Malaysia.

Thomas Patrick Hennessy graduated with a M.Sc in Chemistry (Diplom) in 1999 and received his PhD in Chemistry with the dissertation title “Peptide mapping employing analytical & micro-separation techniques” in 2002. Key aspects of his research comprise translational research and clinical applications using -omics technologies. His tutoring and teaching experience features advanced vocational training at international symposia and conferences as well as international workshops in separation science in the post-genome era. The requirements of increasingly complex separation systems and platforms frequently result in organising advanced vocational short courses and workshops co-lecturing alongside renowned experts from industrial co-operation partners and academic experts on multi-dimensional separation technologies hyphenated to mass spectrometry. Moreover, his lectures cover biochemistry, inorganic & analytical chemistry as well as basic quantum mechanics.

Dr. Ute Roessner

Head of the Metabolomics Facility at the Australian Centre for Plant Functional Genomics. One of the pioneers of metabolomics technology developments

especially in the plant research / agricultural fields.

Dr. Roessner obtained her PhD in Biochemistry at the MPI for Molecular Plant Physiology in Germany, where she developed novel GC-MS method to analyse metabolites in plants. The field of metabolomics emerged together with the application of sophisticated data mining, and it is today an important tool in biological sciences, systems biology and biomarker discovery. In 2003, Dr. Roessner moved to Australia where she established a GC-MS and LC-MS based metabolomics platform at the University of Melbourne, node of the Australian Centre for Plant Functional Genomics. Since 2007, Dr. Roessner has also been involved in the setup of Metabolomics Australia (MA), an Australian federal government investment, through Bioplatforms Australia Ltd. and currently she leads the MA node at the University of Melbourne.


Symposium 2012

ABSTRACTS Plenary Lectures

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Symposium 2012

Plenary Lectures

No. Name Title

1. David Wishart Comprehensive Characterisation of the Human Biofluid Metabolomes

2. Rudolf Grimm Multi-omics Studies of the Oldest Human Mummy the Tyrolean Iceman or Oetzi

3. Matthias Pelzing Ultrafast Statistical Profiling: FT-ICR Based Profiling of Myxobacteria Natural Products for Rapid Detection and Identification of Marker Compounds

4. Ute Roessner The Potential of Metabolomics in Systems Biology

5. Fang Fang Innovative NMR Metabolomics for Food Safety and Quality Control and Newborn Urine Screening

6. Robert Trengove Metabolomics and the Nexus with Residue Analysis: The Study of Health Impacts of Residue Exposure

7. Markus R. Wenk Lipidomics, New Tools and Applications

8. Thomas Hennessy The Application of Mass Profiler Professional in Biomarker Discovery: A Statistical Analysis & Visualisation Software Tool in PTSD Metabolomics

9. Teh Lay Kek Moving Forward with Metabolomics: Scenario in Malaysia

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Comprehensive Characterisation of the Human Biofluid Metabolomes

David Wishart

Depts. of Computing Science and Biological Sciences, University of Alberta, Edmonton, AB, Canada.

Corresponding author: [email protected]

Abstract The most accessible material for metabolomic studies in humans are biofluids. In particular, blood, urine, saliva and cerebrospinal fluid (CSF) are relatively easy to obtain and are routinely used in many clinical and metabolomic studies. However, despite their ubiquity and clinical utility, surprisingly little is (or has been) known about their full chemical composition. As part of an ongoing effort begun in 2005, we have been comprehensively characterising the metabolomes of human CSF, blood, urine and saliva using a broad array of experimental techniques. This experimental work has been closely coupled with an extensive review of the literature. In this presentation I will describe our progress towards this goal and our specific methodologies. In particular I will focus on our recent advances in characterising the human CSF and human urine metabolomes. Comprehensive knowledge of human biofluid metabolomes, along with their “normal” concentration ranges opens the door to the discovery of novel disease biomarkers and biomarker profiles. In this presentation I will also describe the potential impact of knowing what is detectable at certain concentration thresholds in terms of the development of automated metabolomic detection and quantification for different platforms. Keywords: biomarker; human biofluids; metabolomic

9


Symposium 2012

Multi-omics Studies of the Oldest Human Mummy the Tyrolean Iceman or Oetzi

Rudolf Grimm1,3,6*, Rick Reisdorph2, Roger Powell2, Spencer Mahaffey2, Hyun Joo An3,6, Grace Ro3, Sureyya Ozcan3, Carlito Lebrilla3, Christian Reiter4 and Thomas Bereuter4,5 ,

Rob Moritz7, Michael Hoopman7, Holly Ganz3, Bart Weimer3, David Bishop8, Philip Doble8, Amaury Gassiot9, Markus Wenk9, Theo Sana1, Frank Maixner10, Albert Zink10

1Agilent Technologies, Santa Clara, USA; 2National Jewish Health Institute, Denver, USA; 3UC Davis, Davis, USA; 4Medical University of Vienna, Austria; 5Technical University Graz, Austria;

6Chungnam National University, Daejeon, South-Korea; 7ISB, Seattle, USA; 8University of Technology Sydney, Australia; 9National University of Singapore, Singapore, 10Institute for

Mummies and the Iceman, EURAC, Bolzano, Italy.

*Corresponding author: [email protected]

Abstract Ötzi the Iceman is a well-preserved natural mummy of a man who lived about 5300 years ago. The mummy was found in September 1991 in the Ötztal Alps, hence Ötzi, near the Similaun mountain and Hauslabjoch on the border between Austria and Italy. He is the oldest known natural human mummy. We performed the first multi-omics study of this famous mummy including genomics, proteomics, glycomics, metabolomics, lipidomics and metallomics approaches. Complex multi-omics data will be presented from samples taking out of his stomach. Interesting insights will be given in his last meal(s) and health conditions before he got killed. Glycomics data will be also presented from a tissue sample taken out of his hip region and compared to data generated from two 2400 years old Siberian ice mummies and a contemporary mummy. Keywords: multi-omics; Ötzi the Iceman

ii

10


Symposium 2012

Ultrafast Statistical Profiling: FT-ICR Based Profiling of Myxobacteria Natural Products for Rapid Detection and Identification of Marker

Compounds

Matthias Pelzing1*, Daniel Krug2,3, Thomas Hoffmann2,3, Aiko Barsch4, Matthias Witt4, Rolf Müller2,3

1Bruker Biosciences Pty Ltd, Australia; 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany; 3 Saarland University, Saarbrücken, Germany; 4Bruker Daltonik

GmbH, Bremen, Germany.


Abstract Myxobacteria represent an important source of novel natural products exhibiting a wide range of biological activities. Some of these so-called secondary metabolites are investigated as potential leads for novel drugs. Traditional approaches to discovering natural products mainly employ bioassays and activity-guided isolation from different myxobacterial isolates, but genomics-based strategies become increasingly successful to reveal additional compounds. These "metabolome-mining" approaches hold great promise for uncovering novel secondary metabolites from myxobacterial strains, as the number of known compounds identified to date is often significantly lower than expected from genome sequence information. Currently, successfully established methods are based on LC-QTOF-MS analysis requiring about 20 min analysis times per sample. Facing several thousand myxobacterial strain isolates as well as numerous genetic knock out mutant strains available to analyse for the presence of novel secondary metabolites, analysis time becomes a bottleneck. In this proof of concept study we analysed several metabolite extracts from genetic knock-out mutants by ultra-high resolution FT-ICR direct infusion measurements, requiring only about 1 min / sample to measure. Principal Component Analysis (PCA) of the acquired MS spectra showed a clustering of the samples according to the bacterial genetic background, i.e. wild type and mutants could be differentiated. PCA loadings pointed to compounds responsible for this differentiation. By measuring these regions of interest in continuous accumulation of selected ions (CASI) mode, the sensitivity and resolution of these mass ranges could be enhanced significantly. The instrument resolving power of R>750.000 provided isotopic fine structure information. This enabled to literally “read out” the correct elemental composition for the target compounds. We have demonstrated for myxoprincomide, a recently discovered myxobacterial secondary metabolite, that this approach enables an unequivocal molecular formula generation for a compound with MW >1000. The ultra high resolution of an FT-ICR instrument enables for the first time an elemental composition determination for a molecular weight range where mass accuracy of existing mass spectrometers would be not enough for an unequivocal decision. The presented “Ultrafast Statistical Profiling” workflow enables a rapid profiling of complex metabolite extracts and identification of relevant marker compounds by making use of the ultrahigh resolution and the wide dynamic range provided by the FT-ICR technology – addressing two of the major bottleneck in metabolomics, sample throughput and compound identification.

Keywords: FT-ICR technology; myxobacteria; Ultrafast Statistical Profiling

11


Symposium 2012

The Potential of Metabolomics in Systems Biology

Ute Roessner

Australian Centre for Plant Functional Genomics and Metabolomics Australia, School of Botany, the University of Melbourne, 3010 Victoria, Australia.


Abstract In the last two decades, significant progress has been made in the sequencing of the genomes of a number of different organisms. Simultaneously, large investments have occurred in the development of high throughput analytical approaches to analyse different cell products, such as those from gene expression (transcriptomics) as well as proteins (proteomics) and metabolites (metabolomics). These „omics approaches, when used in combination with sophisticated bioinformatics tools for data mining and interpretation, are considered important tools to be applied and utilised to understand the biology of an organism and its response to environmental stimuli or genetic perturbation. The ability to measure all elements of a biological system, such as DNA, mRNA, proteins, metabolites and structural elements such as the cytoskeleton, cell walls and membranes, and also to determine the relationship of those elements to one another as part of the system‟s response to various stimuli is called Systems Biology. In this paper I want to discuss the importance of metabolomics as a major contributor in any systems biology study and explore an example from our own research how we extend the potential of metabolomics by combining it with genetics to understand adaptation and tolerance to drought in wheat, a major food crop. Keywords: adaptation; bioinformatics tools; metabolomics; tolerance

mailto:[email protected]

12


Symposium 2012

Innovative NMR Metabolomics for Food Safety and Quality Control and Newborn Urine Screening

F Fang*, B Schuetz, C Cannet, D Krings, H Schaefer, M Moertter, M Spraul

Bruker BioSpin GmbH, Germany.


Abstract NMR is established as an efficient analytical tool for metabolomics analysis based on its highest reproducibility and transferability. Smallest changes of concentrations of many compounds in biofluid can then be observed in untargeted and targeted screening with one experiment. Based on its unmatched reproducibility, NMR based metabolomics is possible to visualise smallest changes in multiple metabolite concentrations simultaneously, defining the strength of NMR as a multimarker analytical tool to detect all types of deviations, even those, previously unknown. To fulfil the stringent requirements of statistical data evaluation, Standard Operation Procedures (SOPs) from sample handling/preparation to post processing have been developed. The need of complete standardisation will be shown through a NMR based Metabolomics case study of urine based newborn screening for inborn errors of metabolism. Given the fact, with minimised sample preparation NMR can deliver high throughput under full automation. The cost of per sample analysis is low, however the information content obtained is unique. Such a quality control system for fruit juices is already available under full automation from measurement to final report. The same technical procedure has been applied to wine-, milk product-, and edible oil screening. Examples are explained for every food category mentioned. All applications mentioned are strictly following standard operation procedures, such allowing to transfer statistical models and quantification routines between different instruments of the same field strength and different labs. Keywords: inborn errors; NMR based metabolomics; urine

13


Symposium 2012

Metabolomics and the Nexus with Residue Analysis: the Study of Health Impacts of Residue Exposure

Bruce Peebles1,2, Catherine Rawlinson1,2, Joel Gummer1,2, Garth Maker1,2,3, Ian

Mullaney2,4, Robert Trengove1,2*

1Murdoch University Metabolomics Australia Node, Murdoch University, 90 South Street, Murdoch WA 6150, Australia; 2Separation Science and Metabolomics Laboratory, Murdoch University, 90 South Street, Murdoch WA 6150, Australia; 3School of Biological Sciences and Biotechnology, Murdoch University, 90 South Street, Murdoch WA 6150, Australia; 4School of Veterinary and

Biomedical Sciences, Murdoch University, 90 South Street, Murdoch WA 6150, Australia.


Abstract The "omics", particularly proteomics and more recently metabolomics have been drivers in recent instrumental development, leading to faster chromatography, accurate mass and high resolution mass analysers, and equally as important robust sample interfaces for minimal sample preparation. Alongside the hardware developments there have been considerable developments in software development for mass spectral deconvolution, data visualisation, and mapping of analytes onto biochemical pathways to identify which pathways are modified 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". 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. This presentation will address the approach and the challenges that need to be addressed to facilitate the use of “untargeted methods”. In particular the different approaches required for GC/MS based vs. LC/MS based metabolomics. Instrumentation optimisation will be discussed in the context of combination of targeted compound analysis and metabolite profiling. Collaborative pathways to advance this approach will be discussed. Keywords: GC/MS; LC/MS; metabolomics; untargeted methods


14


Symposium 2012

Lipidomics, New Tools and Applications

Markus R. Wenk

Department of Biochemistry, Yong Loo Lin School of Medicine, and Department of Biological Sciences, National University of Singapore.


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 characterisation of lipids and their biosynthetic enzymes in living cells. The development of these integrated tools promises to greatly advance our understanding of the diverse biological roles of lipids (Wenk 2010 Cell 143(6):888-95). Understanding better the fundamentals of natural variation in lipidomes as well as specific recognition of individual lipid species are main scientific aims of SLING, the Singapore Lipidomics Incubator (http://www.sling.nus.edu.sg/). Shaped by a five year competitive research program supported by the National Research Foundation and the National University of Singapore, this centre is a major global magnet for collaborating parties in lipidomics – from academia and industry – delivering new technologies and intellectual capital. Keywords: lipidomics; lipids

15


Symposium 2012

The Application of Mass Profiler Professional in Biomarker Discovery: A Statistical Analysis & Visualisation Software Tool in PTSD

Metabolomics

G Hamuni1, A Karabatsiakis2, S Kadereit3, R Durairaj4, TP Hennessy5*, G Currie6, IT Kolassa2

1Department of Psychology, Clinical & Neuropsychology, Universität Konstanz; 2Institut für Psychologie und Pädagogik, Klinische und Biologische Psychologie, Universität Ulm;

3Zukunftskolleg,Fachbereich Biologie,Universität Konstanz; 4Information Technology & Services, Strand Life Sciences, Bangalore; 5Life Sciences Group, Agilent Technologies, Singapore; 6Life

Sciences Group, Agilent Technologies, Melbourne.


Abstract Biomarker discovery can be performed at all levels of information flow in biological systems. Integrated Biology then is the discipline compiling and extracting meaningful data to comprehensibly describe any such system under scrutiny. However, in the quest for biomarkers it is desirable to obtain and retain only information which shows variance in expression/abundance levels at the expense of all other information. Metabolite Biomarker discovery is the pursuit to display differential abundance metabolite levels. MS based differential metabolite profiling is one way of monitoring up- and down-regulation between samples. Provided any perturbation causes a response in the system and here in particular at the metabolite level this variation may be monitored over time. Mass Profiler Professional (MPP) software is used to cluster biological and technical replicates and differentiate them in terms of the time domain of any variance occurring between samples where time course experiments are concerned or likewise expression/abundance levels where differential expression is the result of a treatment or a disease state. Only the molecular entities that show defined significant differences between samples are selected and retained by the MPP statistical analysis and visualisation platform and ultimately identified. The resulting identified metabolites are candidate biomarkers for the response of the system to the perturbation. The profiling strategy is, hence, ideally suitable to investigate experimental designs to study disease states in particular and any differential expression/abundance scenario in general. The analysis strategy on the statistical analysis level (MPP) will be exemplified via software data (profiling, molecular feature extraction, filtering, principle component analysis (PCA), condition- and mass-tree generation aka hierarchical clustering analysis (HCA), fold-change analysis, volcano plot representation) detailing the workflow to answer questions dedicated to Post Traumatic Stress Disease (PTSD) metabolomics. Keywords: Mass Profiler Professional (MPP); metabolite biomarker discovery; Post Traumatic Stress Disease (PTSD)


16


Symposium 2012

Moving Forward with Metabolomics: Scenario in Malaysia

LK Teh*, MZ Salleh*

Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, Universiti Teknologi MARA Malaysia.

Corresponding authors: [email protected]/[email protected]

Abstract Metabolomics is an important functional genomics tool which received great attention and emphasis in recent years. Many researchers in Malaysia were working on metabolomics of plant until 2009 when it was applied into clinical researches. Now, there is a wide range of its application in plant, microorganisms and human with large metabolic complexity. The benefit of integrating metabolomics with other “omics” is believed to provide comprehensive insights for a variety of cellular metabolic processes in a cell, organ or organism; filling the missing gaps of genomics, transcriptomics and proteomic analyses. In plant science, metabolomics is useful to provide clues to understand the metabolic regulatory complex and its physiological state in response to the environmental stress. Understanding the environmental stressors which control the production of beneficial metabolites or factors affecting the growth and yield of the plant would be of great importance in the field of agricultural science. Similarly, the pharmaceutical industry would cast interest in the mechanism of actions of these valuable metabolites with potential use as therapeutic agents. While in clinical researches, translating metabolomics to discover biomarkers for bedside testing is greatly desired as currently available diagnostic tools for many diseases lack accuracy or are invasive. In PROMISE, we are investigating the metabolic profiles of disease causing pathogens to understand their pathogenecity and virulence. We are also integrating genomics with metabolomics in search for biomarkers and understanding the mechanisms involved in protection and susceptibility to diseases in a study which involves the local aborigines. Several diseases or health issues would be of focus, which include cancers, cardiovascular diseases, obesity and intellectual capabilities. In collaboration with other researchers, we are looking into the use of metabolomics in improving yield and nutrient value of crops/marine products and cataloguing mechanisms of the biological activities of local herbs. Keywords: disease markers; metabolomics; pathogenecity; virulence



Symposium 2012

ABSTRACTS Oral Communications

18


Symposium 2012

Oral Communications Day 1

No. Presenter Title Reference No.

1. Azizan et al. Footprinting Profiling of L. lactis in Response to Derivatisation Techniques

MYMS-001po/m

2. Hamzah et al. Potential Biomarkers for the Monitoring of Kidney Function in Kidney Transplant Patients

MYMS-002po/m

3. Ibrahim et al.

Partial Least Square-Discriminant Analysis and Youden Index for Metabolomic Analysis of NMR Spectra of Exhaled Breath Condensate of Asthmatics and Healthy Controls

MYMS-003po/m

4. Ismail et al. Effect of Tocotrienol Rich Fraction (TRF) on Oxidant-Antioxidant Equilibrium in Reducing Carbamazepine (CBZ) Induced Lipid Peroxidation

MYMS-004po/m

5. Khalid et al.

Manipulation of In Vitro Cultures of Bosenbergia rotunda for Enhanced Production of Targeted Bioactive Compounds in the Phenylpropanoid Biosynthesis Pathway

MYMS-005po/m

6. Laith et al.

Understanding the Metabolic Response of Catfish (Clarias garipenus) to Elizabethkingia meningoseptica Infections via Liquid Chromatography–Mass Spectrometry (LC/MS-QTOF)

MYMS-006po/m

7. Rosli et al. Metabolites Profile of Tissue and Plasma in Breast Cancer Patients Using Metabolomic Approach: Role of Eicosanoid Metabolism

MYMS-007po/m

19


Symposium 2012

Footprinting Profiling of L. lactis in Response to Derivatisation Techniques

Kamalrul Azlan Azizan*, Syarul Nataqain Baharum, Normah Mohd Noor

Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600, Bangi,

Selangor, Malaysia.


Abstract In general, most of the known low molecular weight metabolites are non-volatile thus require chemical derivatisation to increase the volatility of the metabolites especially for GC based analysis. Here we described the usage of an optimised protocol of conventional technique of TMS derivatisation with shaking and recently introduced MCF derivatisation to determine the major groups of metabolite that were produced in the fermented broth cultivated by the bacterium. The footprinting metabolites of mesophilic L.lactis grown at 30°C with shaking and 37°C was profiled using chemical derivatisation prior to gas chromatography mass spectrometry analysis. It was observed that profiled groups of metabolites were according to the specific chemical derivatisation used. TMS agent detected ranges of metabolites including alcohols, aldehydes, ketones and sugars compared to MCF agent which were limited to amino and non-amino acids. In additional, the major metabolites that contributed towards separation and discrimination in Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) is further demonstrated and validated. Keywords: L.lactis; MCF derivatisation; TMS derivatisation


20


Symposium 2012

Potential Biomarkers for the Monitoring of Kidney Function in Kidney Transplant Patients

S Hamzah1*, JKS Shia1, G Ahmad2, HS Wong3, TP Hennessy1,4, LK Teh1,

MZ Salleh1*

1Pharmacogenomics Research Centre (PROMISE), Faculty of Pharmacy, Universiti Teknologi MARA, 42300 Puncak Alam, Selangor; 2Institute of Nephrology and Urology, Hospital Kuala Lumpur, 50586 Kuala Lumpur; 3Department of Nephrology, Hospital Selayang, 68100 Batu

Caves, Selangor; 4Life Sciences Group, Agilent Technologies, Singapore.

*Corresponding authors: [email protected]/[email protected]

Abstract Kidney transplant patients are exposed to various complications such as immunological rejection, ischemia or reperfusion injury and immunosuppressant induced nephrotoxicity. Currently, the diagnosis of allograft rejection and toxicity of chronic calcineurin inhibitor relies on invasive procedure of biopsy. Therefore in this study, a metabolomics approach was explored to search for compounds that have potential to be further developed into biomarkers to monitor transplanted patients. Serum was obtained from kidney-transplant patients treated with tacrolimus and healthy volunteers. Fifty-five biological replicates of patients and 20 biological replicates of controls as well as technical triplicates were analysed using LC/MS-QTOF (Agilent Tech). Samples were injected after protein precipitation with acetonitrile and centrifuged for 10 min at 8,500×rpm at 4°C. The chromatography was performed on an Agilent 1200 Series using an XDB C18 4.6 µM, 150×1.8 mm (Agilent Tech.). A total of 9 groups of compound were found to have potential to be developed into biomarkers. One of the most interesting compounds that were found to be differentially expressed between the patients and healthy volunteers is Diacylglycerol (DAG). DAG carries the potential to be used as a marker to monitor the suppression level of the immune system. Comparison of metabolite profiles between patients with episode of rejection and stable patients revealed high abundance of Sphingosine-1-phosphate among patients who experienced rejection. This compound is potentially a biomarker to predict allograft rejection since it has been shown to carry various immune modulatory functions. Glycerophosphocholines (GPC) is also another prospective group of compound that exhibited great potential due to its association with tacrolimus dose-adjusted level and CYP3A5 genotypes demonstrated in this study. The present study demonstrated the potential use of metabolomics approach for the search of candidate biomarkers to monitor kidney transplant patients. However, all of these potential compounds need to be further validated before translating into clinical practices. Keywords: DAG; glycerophosphocholines (GPC); kidney-transplant patients; potential biomarkers; rejection; sphingosine-1-phosphate


21


Symposium 2012

Partial Least Square-Discriminant Analysis and Youden Index for Metabolomic Analysis of NMR Spectra of Exhaled Breath Condensate

of Asthmatics and Healthy Controls

B Ibrahim1, 2*, M Nilsson 1, 3, SJ Fowler 1, 4

1University of Manchester, Manchester Academic Health Science Centre, and NIHR Respiratory and Allergy Clinical Research Facility, University Hospital of South Manchester, Manchester, UK;

2School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia; 3School of Chemistry, University of Manchester, UK; 4Lancashire Teaching Hospitals NHS Foundation Trust,

Preston, UK.


Abstract Multivariate data analysis and chemometrics are keystone statistical techniques used to identify markers of diseases in metabolomic studies. Previously, using principal component analysis (PCA), logistic regression and receiver operating characteristic (ROC), we have shown the ability of nuclear magnetic resonance (NMR) spectroscopy of exhaled breath condensate (EBC) in discriminating asthmatics from healthy controls. The aim of this analysis is to employ partial least square-discriminant analysis (PLS-DA) and Youden Index to re-evaluate this data. The NMR spectra of EBC were binned to 0.02 ppm and PCA was applied initially to identify outliers. Multivariate modelling was performed on a “training set” (70% of the total sample) in order to produce a discriminatory model classifying asthmatics from healthy controls, and the model tested in the remaining subjects. PLS-DA identifies a linear regression model between predictors and binary outcome while Youden Index (sensitivity+specificity-1) is a summary measure of ROC which represents the maximum potential effectiveness of a marker. In the training set, a model (R2Y=0.43 and Q2Y=0.27) discriminating asthmatics (n=57) and controls (n=22) was developed with an area under the receiver operating curve (AUROC) of 0.93. The highest Youden index (0.744) was observed at a predicted value of 0.72, which corresponded to a sensitivity of 77% and a specificity of 96%. In the test set (asthmatics, n=22; controls, n=12) the AUROC was 0.85, thus providing external validation for the model. The sensitivity and the specificity were 77% and 92% respectively. Response permutation test (100 permutations) proved internal validity of the model (R2Y-intercept <0.3 and Q2T-intercept <0.05).The results were superior to previous analysis of the same data set. PLS-DA and Youden Index are robust methods for metabolomic analysis of NMR spectral data to discriminate asthmatics from healthy controls. These techniques also provide for internal and external validation. Keywords: asthma; exhaled breath condensate; metabolomics; partial least square-discriminant analysis; Youden Index


22


Symposium 2012

Effect of Tocotrienol Rich Fraction (TRF) on Oxidant-Antioxidant Equilibrium in Reducing Carbamazepine (CBZ) Induced Lipid

Peroxidation

MI Ismail1*, ES Rosli1, A Derahman1, M Nornazliya1, Z Bannur1, MS Rofiee1, ZA Zakaria2, TP Hennesy1,3, MZ Salleh1, LK Teh1*

1 Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA

(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Biomedical Sciences, Faculty of Medical and Health Sciences, Universiti Putra Malaysia, Malaysia; 3Life

Sciences Group, Agilent Technologies, Singapore.


Abstract Carbamazepine (CBZ) is commonly used to treat epilepsy but it ranked as one of the top 10 drugs that caused fatal cutaneous drug reactions which include Steven Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN). CBZ induced toxicities has been hypothesised to be related to the oxidant-antioxidant imbalances. Therefore, we aim to evaluate the potential role of Tocotrienol Rich Fraction (TRF) in balancing the oxidant-antioxidant impairment induced by CBZ using 48 male Sprague Dawley (SD) rats. Seven groups of rats comprising of 6 rats per cages were studied. A control group and 6 different groups of rats treated with 3 different dosages of CBZ (50, 100 and 200 mg/kg) and CBZ + TRF were conducted. Organs which include liver, kidney, brain were collected from each rat on day 7 post treatment. All organs were weighed then freeze snapped using liquid nitrogen. The organs were immersed into 0.9% NaCl and stored at -80°C before subjected to 6 series of extraction solvent which include ethyl acetate, hexane, ethanol, methanol, dichloromethane and distilled water. Supernatant were pooled, dried, reconstituted with mobile phase and injected to the LC/MS-QTOF for analysis. A total of 389 metabolites were identified, 61 metabolites were related to CBZ treatment. The biochemical pathways including tryptophan metabolism, purine metabolism and fatty acid (FA) metabolism were shown to be affected in rats treated with CBZ. TRF was found to down-regulate FA metabolism which is anticipated to have protective role in reducing lipid peroxidation especially in the brain. In addition, the significant reduced expression of glutamylphenylalanine also suggest protective role of TRF in nephrotoxicity. However, further study to evaluate the potential use of TRF as health supplement in attempts to prevent or reduce toxicities is required. Keywords: antioxidant; carbamazepine; metabolomic; SJS-TEN; TRF



23


Symposium 2012

Manipulation of In Vitro Cultures of Bosenbergia rotunda for Enhanced Production of Targeted Bioactive Compounds in the Phenylpropanoid

Biosynthesis Pathway

N Khalid*, N Yusof, Md Mustafa N, SM Wong and EC Tan

Centre for Biotechnology in Agriculture Research, Institute of Biological Sciences, Faculty of Science, University Malaya, 40603 Kuala Lumpur, Malaysia.


Abstract Boesenbergia rotunda (BR), a medicinal ginger, with bioactive compounds such as alpinetin, cardamonin, pinostrobin, pinocembrin, panduratin A and 4-hydroxypanduratin A have shown to have antibacterial, antifungal, anti-inflammatory, anticancer, and antioxidant properties. In our work, panduratin A and 4-hydroxypanduratin A exhibited anti-dengue NS2B/ NS3 protease activity. However, naturally these compounds are not feasible for future drug applications. The aim of this work is to enhance the production of panduratin A and 4-hydroxypanduratin A through chemical elicitation and genetic engineering. Initially cell suspension cultures of Bosenbergia rotunda was established for chemical elicitation and genetic manipulation studies. Cultures were initiated in shake flasks and growth was optimised in 5 litre bioreactors. Phenylalanine was used as an elicitor to enhance the production of panduratin A and 4-hydroxypanduratin A. proteomic approaches to identify proteins that may be involved in the biosynthesis of these compounds were carried out. Following image analysis, protein spots whose expressions were found to be regulated were identified using Matrix Assisted Laser Desorption-Ionisation tandem mass spectrometry. Transcriptomic expression on cell cultures treated with phenylalanine was also studied. Embryogenic callus from cell suspension was regenerated for genetic transformation studies. In this work, the effects of initial inoculation volume, temperature and speed of agitation on cell growth, total and selected flavonoid in suspension cultures of B. rotunda were determined. The high performance liquid chromatography analysis showed that 2% inoculation volume induced significantly high accumulation of biomass and flavonoid in the cells. For proteomics studies, thirty four proteins were identified. Eleven of the proteins involved in the phenylpropanoid biosynthetic pathway are related to the biosynthesis of cyclohexenyl chalcone derivatives. Transcriptomics data analysis has provided more information on the phenylpropanoid pathway. As for plant regeneration, embryogenic callus derived from cell suspension has been successfully optimised at a high frequency. This study has provided information on the possible enzymes involved in the phenylpropanoid pathway leading to the production of panduratin A and 4-hydroxypanduratin A. Growth of suspension cultures were optimised both in small and large scale production. Regenerable cell suspension cultures have also been established which will allow the expression of targeted genes for enhanced accumulation of the bioactive compounds using genetic transformation. Keywords: 4-hydroxypanduratin; biosynthesis pathway; Bosenbergia rotunda; panduratin A; phenylpropanoid


24


Symposium 2012

Understanding the Metabolic Response of Catfish (Clarias garipenus) to Elizabethkingia meningoseptica Infections via Liquid Chromatography–Mass Spectrometry (LC–MS QTOF)

Laith AA1*, Najiah M1, Sifzizul TTM2, LK Teh3, MZ Salleh3

1Department of Fisheries and Aquaculture, Faculty of Fisheries and Aqua Industry, 2 Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu 21030 Terengganu Malaysia 3 Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi

MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.


Abstract Safety and quality fish product is an important subject in aquaculture as the global demand for fish products has doubled and is still rising. Catfish (clarias carepinus) is one of the world‟s most important staple meats, providing food to more than a billion peoplen but the pathogenicity of Elizabethkingia meningoseptica, a significant bacterial group for human clinical infection, in fish has not been established. In order to better understand the mechanism of infection and immunity of fish to bacteria, a study was undertaken to identify metabolites that are related to infection and resistance. A two-step approach using LC/MS Q-TOF system was employed. Differential expression analysis of metabolite profiles for samples was conducted using Mass Profiler Professional (Agilent Tech.) software. The differentially expressed metabolites were further analysed using quadrupole time-of-flight (Q-TOF) MS/MS. Two metabolic molecules, Alpah- Amyrin and Guanosine, were examined. The fish metabolic profiles were studied by top-down chemometric analysis. The LD50 was estimated at 4x105 cfu mL-1. Experimental infection of E.meningoseptica on juvenile catfish was carried out via intraperitoneal (i.p.) route and immersion (i.m.). E.meningoseptica injected via i.p. route was found to be more potent than i.m. in causing mortality to juvenile catfish. There was a significant difference (p < 0.05) in the rate of mortality between i.p. and i.m. group. A significant difference (p < 0.05) was also observed within immersion groups. Fish in i.p. group showed subcutaneous haemorrhages and lesion in kidney, while the i.m. group showed hypertrophy, necrosis and removal of dermal layer. In the present study, clear differences in the metabolite profiles of the different isolates (skin and kidney) were detected. Guanosine was down-regulated in the serum of catfish infected by E.meningoseptica isolated from kidney, but it was up-regulated in catfish isolated from skin. On the other hand, alpha-amyrin was up-regulated in serum of catfish infected by E.meningoseptica isolated from the skin, but it was down-regulated in catfish isolated from kidney. Alpha-amyrin has strong anti-inflammatory activity, is a PKA inhibitor as well as a selective protease inhibitor. It has been reported to prevent molecule oxidation due to UV-B irradiation and prevent skin damage through prevention of lipid peroxidation and inhibition of PGE2 release. It also has broad-spectrum analgesic properties. Guanosine, a purine nucleoside, has important functions in the cellular metabolism and is used to synthesise enzyme inhibitors, antiviral agents, and anticancer agents. We conclude that damage in the skin of catfish infected with E.meningoseptica increased its alpha-amyrin molecules as a response to repair skin damage. Increase in guanosine levels indicates that there was severe tissue damage in the kidney. Keywords: catfish; LC-MS QTOF; LD50; metabolomics; serum


25


Symposium 2012

Metabolites Profile of Tissue and Plasma in Breast Cancer Patients Using Metabolomic Approach: Role of Eicosanoid Metabolism

ES Rosli1*, NI Mohamed1, MZ Salleh1, JKS Shia1

, M Rohaizak2, NS Shahrun2, JJ Saladina2, H Roslan3, TP Hennessy1,4, LK Teh1*

1Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA

(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Surgery, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur,

Malaysia; 3UKM Medical Biology Institute (UMBI) and Department of Medicine, Universiti Kebangsaan Malaysia Medical Centre, (UKMMC), Kuala Lumpur, Malaysia; 4Life Sciences Group,

Agilent Technologies, Singapore.


Abstract Breast cancer is one of the leading causes of death among women worldwide. Many studies had utilised genomics, transcriptomics and proteomics approaches to understand the regulation and pathogenesis of the disease. The information is hoped to provide insights to better therapy outcomes and to help strategise preventive measures. Biomarkers useful for clinical monitoring of the efficacy of drug treatment among breast cancer patients are heavily searched by researchers. In this study, we used metabolomics platform to help identify the metabolite profiles which could be relevant biomarkers in understanding the variation of disease. Ethics approvals from Research Ethics Committee of UiTM and HUKM were obtained. For this study, tumour and normal tissues of breast cancer patients were collected from the same patient. Blood was collected from patients prior to treatment and healthy volunteers based on the inclusion and exclusion criteria. Differential expressions of metabolites between the two tissues and serum for the patients and healthy volunteers were investigated. Samples were injected into LC/MS QTOF and the data was analysed using Mass Profiler Professional (MPP) software for differential analysis. These metabolic datasets were mined using a range of pattern recognition techniques, including hierarchical cluster analysis, principal component analysis, partial least squares and neural networks. Increment of several metabolites in tumour samples has highlighted lipid and eicosanoid metabolic pathways as potential perturbation in the metabolism of tumour cells in comparison to normal. Further validation on the metabolites is required before they can be used as breast cancer biomarkers. Keywords: breast cancer; biomarker; LC/MS QTOF; lipid and eicosanoid metabolic pathway; metabolomics

26


Symposium 2012

Oral Communications Day 2


8. Mohamad Yusof

et al. Activity-Guided Isolation of the Chemicals Constituents of Muntingia calabura using Formalin Test

MYMS-008po

9. Aqilah et al. Isolation and Identification of Aeromonas hydrophila Isolated from Tilapia, Oreochromis niloticus

MYMS-009po

10. Izwan et al. Soapdenovo Provides Lower Number of Scaffolds and Contigs: An Example of Optimum Assembly for Low Yield Sequencing Data of Acinetobacter baumannii

MYMS-010po

11. Lee et al. Utilising a Genomics Approach to Investigate the Mechanism of Antibiotic Resistance in Staphylococcus aureus

MYMS-011po

12. Hanif et al. Comparison between r2cat and OSLay Software for Contigs Arrangement

MYMS-012po

13. Rose Ismet et al.

Whole Genome Sequencing Report of a Malaysian Aborigine Individual and Annotation of Medico-Genomic Association Using In-House Genomic Bioinformatics Workflow

MYMS-013po

14. Hatta et al. The Patterns of Four Natural Variations in Human CYP2C9 Genes across Three Populations

MYMS-014po

27


Symposium 2012

Activity-Guided Isolation of the Chemicals Constituents of Muntingia calabura Using Formalin Test

MI Mohamad Yusof1*, N Ahmad2, ZA Zakaria3*, MZ Salleh1, LK Teh1

1Pharmacogenomics Centre, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam,

42300, Selangor, Malaysia; 2Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, 40450, Selangor, Malaysia; 3Department of Biomedical Sciences, Faculty of Medicine and Health

Science, Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia.


Abstract Muntingia calabura is known locally as Kerukup Siam. It has been claimed by the Peruvian folklore to possess medicinal values which include soothing gastric ulcers, relieving headache and cold, reducing swelling of the prostate gland and antiseptic. This study focuses on understanding the antinociceptive effects of the extract using Sprague Dawley (S.D.) rats. In the present study, activity-guided of methanol extract of Muntingia calabura collected in Shah Alam, Malaysia were evaluated for their antinociceptive property using the formalin test. Seven fractions of petroleum ether extract labelled with A, B, C, D, E, F and G were separately treated (orally) with distilled water or 10 % DMSO as negative controls and morphine and aspirin as the positive control. Fraction D showed most significant antinociceptive activity when compared to another fraction. At the dose of 300 mg/kg, fraction D and morphine showed no significant difference in first phase, while in second phase, fraction D and aspirin showed no significant different. One new compound together with three known compounds namely 8-hydroxy-6-methoxyflavone, 5-hydroxy-3, 7, 8-trimethoxyflavone (methylgnaphaliin), 3, 7-dimethoxy-5-hydroflavone and 2‟, 4‟-dihydroxy-3‟-methoxychalcone were isolated from fraction D. Keywords: antinociceptive; formalin test; methanol extract; Muntingia calabura


28


Symposium 2012

Isolation and Identification of Aeromonas hydrophila Isolated from Tilapia, Oreochromis niloticus

NI Aqilah1*, YS Yong1, AI Zamani1, HH Ruhil3, M Nadirah2, M Najiah1

1Department of Aquaculture Science; 2Department of Fisheries Science, Faculty of Fisheries and

Aqua-Industry, Universiti Malaysia Terengganu, 21030, Mengabang Telipot, Terengganu, Malaysia; 3Faculty of Veterinary medicine, Universiti Malaysia Kelantan, Locked Bag 36,

Pengkalan Chepa 16100 Kota Bharu,Kelantan, Malaysia.


Abstract This study was conducted to isolate and identify Aeromonas hydrophila from 30 tilapia fry in Terengganu, Malaysia. Isolated bacteria were preliminarily identified using GSP agar and thirteen isolates that showed yellow colony were selected and sub-cultured for further identification using morphological, biochemical and physiological tests. The phenotypic relationships among isolates were studied using numerical taxonomy and performed by Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Data coefficient (SD) was used to determine the similarity between the strain and the data was then compared with previous studies according to Holt el al. (1994) and Buller (2004). Results show the yellow colony isolates on GSP agar were identified as Aeromonas hydrophila. Based on numerical taxonomy, isolates show 93.75% and 92.86% similarities from identification done by Holt et al. and Buller, respectively. From the result, UPGMA and Dice coefficient were used to estimate the similarities between the samples based on phenotypic characteristics. Keywords: Aeromonas hydrophila; data coefficient; Oreochromis niloticus; UPGMA


29


Symposium 2012

Soapdenovo Provides Lower Number of Scaffolds and Contigs: An Example of Optimum Assembly for Low Yield Sequencing Data of

Acinetobacter baumannii

Izwan I*, LK Teh, LS Lee, Hanif AK, Irma SZ, MZ Salleh*

Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA

(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.


Abstract Low sequencing yield has been a bane for bioinformaticians, as it limits the output of the data analysis that follows. This paper aims to provide a comparison between two well-received de novo assemblers in order to aid in maximising the use of such low yield data. Sequencing data of an Acinetobacter baumannii strain was selected with data size approximately half of the expected output, and an overall coverage of at least 50X. The 36 bp short reads were sequenced using the Illumina GA IIx. The quality scores of the reads were analysed via FastQC, and the sequences were assembled using both SOAPdenovo (V1.05) and VelvetOptimiser (V2.2.2). The number of contigs/scaffolds produced using the two software were compared. The optimal syntenic layout and number of ORFs were compared using OSLay and PRODIGAL, respectively. The assembly using SOAPdenovo produced 320 scaffolds, whereas VelvetOptimiser yielded 3091 contigs. Analysis using OSLay revealed better syntenic layout between SOAPdenovo scaffolds and references A.baumannii ACICU, AYE, ATCC 17978, and SDF. Using the gene prediction script PRODIGAL, 4497 ORFs for SOAPdenovo-assembled sequences and 4208 ORFs for VelvetOptimiser-assembled sequences were generated. It is concluded that SOAPdenovo is more suited for handling low yield data for tertiary analysis. Keywords: Acinetobacter baumannii; Soapdenovo; VelvetOptimiser

mailto:[email protected]/

30


Symposium 2012

Utilising a Genomics Approach to Investigate the Mechanism of Antibiotic Resistance in Staphylococcus aureus

LS Lee1,2*, LK Teh1, ZF Zainuddin2, MZ Salleh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia.


Abstract With the rapidly decreasing cost of whole genome sequencing, genomics has become a viable approach in examining various facets of the mechanism of antibiotic resistance of pathogenic bacteria. We seek to examine the utility of this approach by sequencing a methicillin-resistant Staphylococcus aureus (MRSA) strain isolated from a patient with septicaemia in Kuala Lumpur. The strain was typed using multi-locus sequence typing (MLST) and disk susceptibility tests were performed to identify its resistance pattern. The genome sequence was obtained using 104 Mb of paired-end (300 bp spacing) data from the Illumina GA IIx platform with 36-bp reads. Sequence data were assembled using the CLCBio Genomics Workbench. The resultant 195 contigs were overlaid with the Mu50 reference sequence using OSLay to generate fourteen scaffolds. Gaps were closed using Sanger sequencing. The genome was annotated using BASys and RAST. The clinical isolate, termed MRSA PR1, has been identified as sequence type 239 (ST239), a health care-associated MRSA lineage prevalent in Asia, South America, and Eastern Europe that is typically defined by its extended antibiotic and antiseptic resistance pattern. This is consistent with the findings of the disc susceptibility tests that show that MRSA PR1 is resistant to oxacillin, cefuroxime, gentamicin, erythromycin, ciprofloxacin and co-trimoxazole. The MRSA PR1 genome consists of a 2,725,110-bp circular chromosome with a GC content of 32.6%. It contains 2638 CDS and 19 rRNA features. MRSA PR1 harbours SCC mec type III, which consists of a composite element comprising SCCmec and SCCmercury. This region harbours ccrC, pI258 and Tn554 as well as several genes involved in cadmium resistance. Several resistance features were identified such as the qacA gene which confers resistance to certain antiseptics via an export-mediated mechanism, and norA which confers resistance to hydrophilic quinolones. This study provided the whole genome sequence of a locally isolated MRSA strain and identified several key features that are involved in conferring antibiotic resistance. Keywords: antibiotic resistance; genomics; methicillin resistance; MRSA; resequencing; Staphylococcus aureus; whole genome sequencing



31


Symposium 2012

Comparison between r2cat and OSLAY Software for Contigs Arrangement

Hanif AK*, LS Lee, Izwan I, MH Zulkifli, Azwin Ismail, Irma SZ, LK Teh, MZ Salleh*




Abstract With the high throughput sequencing technology, it has become easier and faster in obtaining accurate sequences of a genome. This results in tremendous short reads of the sequenced genome. The data will be assembled using de novo assembler in acquiring novel genes or variants of the organisms. The contigs that are assembled would be mapped to related species of high homology before annotation is performed. In this study, the characteristics of the contigs obtained using r2cat and OSLay will be compared. Proteus mirabilis, Klebsiella pneumonia, and Streptococcus agalactiae were isolated from samples obtained from septicaemic patients at Hospital Putrajaya and Hospital Universiti Sains Malaysia. DNA was extracted and libraries were prepared for whole genome sequencing using Genome Analyser IIx. Output of sequencing data was assembled using CLCBio version 4.9 and Velvet Optimiser. The contigs of P. mirabilis, K. pneumonia, and S. agalactiae was assorted using r2cat and OSLay then validated using CLCBio in map to reference method. The characteristics compared using r2cat and OSLay include contigs arrangement, number of scaffold produced, total contigs used. For P. mirabilis strain PM001, OSLay produced nine scaffolds using 96 contigs; the remaining 30 contigs were not able to be mapped to reference. Meanwhile, r2cat produced one scaffold using 125 contig and leaving one contig unmapped. For K. pneumonia, OSLay produced twelve scaffolds using 78 contigs out of 122 contigs while r2cat produced one scaffold without any contig unused. Lastly, for S. agalactiae, OSLay produced six scaffolds and 10 out of 76 contigs were left unused. However, using r2cat software, scaffolds produced was invalid as more than 80% of the reads were not mapped due to wrong contigs arrangement. Therefore, OSLay is the preferred choice and provide users the ability to change parameters for the assembly to achieve optimum result. Keywords: K. pneumonia; OSLAY; P. mirabilis; r2cat; S. agalactiae


32


Symposium 2012

Whole Genome Sequencing Report of a Malaysian Aborigine Individual and Annotation of Medico-Genomic Association Using In

House Genomic Bioinformatics Workflow Rose Ismet1*, Husaini I1, M Nornazliya1, Shafiq A1, Z Bannur1, LS Lee1, LK Teh1, Zafarina Z2, Norazmi MN2, BP Hoh3, Aminuddin A3, Fadzilah MN3, T Rahman3, R Razali3, Adzrool

Idzwan Ismail4, Kamarudzaman Md. Isa4, Mustaffa Halabi Azahari4, MZ Salleh*

1 Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2School of Health

Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; 3Faculty of Medicine, UiTM Sungai Buloh, Selangor, Malaysia; 4Faculty of Art & Design, UiTM Shah Alam, Selangor, Malaysia.

*Corresponding authors: [email protected]/zakisalleh@ puncakalam.uitm.edu.my

Abstract We believed that the Malaysian Aborigine is a unique group of population, especially the studied sub-tribes who are decreasing in population number. It is known that the Aborigine‟s genetic prehistory is affected by genetic drift and natural selection and their gene pool is richly textured by biocultural and evolutionary processes as well as gene flow or diffusion. However, there is no whole genome sequencing data of the Malaysian Aborigine available to date. Having the genetic blueprint of the aborigine could facilitate the understanding of disease and xenobiotic association as every disease carries its own genetic element be it as an inherited trait or by a response to environmental stressors. This in turn could lead to the understanding of why these aboriginal populations decrease in numbers. What is observed at present is that the Malaysian Aborigine is presented with conditions including G6PD deficiency, hepatosplenomegaly as well as skin infections. Resistance to malaria has also been observed among the Temuan presented with G6PD deficiency and elliptocytosis. By performing whole genome sequencing, these findings could be further confirmed. Thus, we aim to understand the disease risk associated with the Malaysian Aborigine which could contribute to their fragility and endangerment. We have sequenced a Che Wong subject using Next-Generation Sequencing (NGS) platform where the data was then subjected to primary analysis utilising the CASAVA v1.8.2 software. Data generated for the sample is at a satisfactory quality with 39.3x coverage. A total of 3,786,707 SNPs was successfully called. Short INDELs (< 300 bp) called include 337,269 insertions and 349,602 deletions. Larger structural variants (> 300 bp) detected include 3 insertions, 2,306 deletions and 1,282 CNVs. Further downstream analysis is warren in order to identify the structural variants associated with diseases among the Che Wong sub-tribe. The ability to identify the genetic variants that contribute to disease and variable pharmacological responses would aid the understanding of disease risk and susceptibility. Keywords: disease risk and susceptibility; Next-Generation Sequencing; pharmacological responses; structural variants


mailto:[email protected]*

33


Symposium 2012

The Patterns of Four Natural Variations in Human CYP2C9 Genes across Three Populations

Fazleen HM Hatta1, 2*, LK Teh1, MZ Salleh1, M Goktas4, U Yasar4, HK Roh3, E Aklillu2, L

Bertilsson2

1Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Division of Clinical

Pharmacology, Department of Laboratory Medicine, Karolinska Institutet–Karolinska University Hospital, Huddinge 141 86 Stockholm, Sweden; 3Division of Clinical Pharmacology, Department of

Internal Medicine, Gachon University Hospital, Incheon, Korea; 4Department of Pharmacology, Hacettepe University Faculty of Medicine, Sihhiye, 06100 Ankara, Turkey.


Abstract With more than 30 variant alleles, there are at least eight known single nucleotide polymorphisms (SNPs) that can cause poor drug metabolism by CYP2C9 enzymes. But interestingly, previous studies revealed unique interindvidual and interethnic variations observed within a population and among populations of selected individuals that are not carrying a defective allele. In this study, we aim to search for a relationship between inter-population metabolism of CYP2C9 and its genotypes. DNA from 85 Swedish, 128 Korean and 51 Turkish healthy volunteers were assessed for four natural variations [SNP 1 (IVS1+83T>C), SNP 2 (IVS2+73T>C) and SNP 3 (IVS6+95A>G) and SNP 4 (IVS8–109A>T)] of the CYP2C9 gene. PCR/RFLP and allele specific PCR methods were developed to screen the healthy volunteers excluding individuals carrying CYP2C9*2 or *3 alleles. Metabolic Ratio of losartan/metabolite E-3174 was used as a phenotyping measure. We

found a significant relationship between SNP 4 (IVS8–109A>T) and CYP2C9 activity (2 test, p=0.011) in the Swedes. No significant relationship between any of the SNPs and MR was

established for Korean and Turkish individuals (2 test, p=0.154 and 0.355 respectively). Although a majority of 14% of Swedes and 16% of the Turks are wild type for all four variants, a higher number of 40% of the Korean are wild type for these variations. We also found that SNP 1, 2 and 3 are very rare in the Korean population (1.6%, complete linkage). We found that SNP 4 IVS8 -109 T allele is associated with a higher MR of CYP2C9 in healthy Swedish subject, but further investigations need to be carried out to established a molecular explanation for inter-population differences of CYP2C9 catalysed metabolism. Haplotypes based on SNPs 1-4 did not seem to contribute to variation in the MR of the Koreans nor play a role in determining the MR of the Korean or Turkish individuals. Keywords: CYP2C9; Koreans; metabolic ratio; natural variations; Swedes; Turkish

Designed by: Mohd Izwan Ismail, PROMISE


Symposium 2012

ABSTRACTS Poster Communications

36


Symposium 2012

Poster Communications


1. Ashrafzadeh et al. Non-labelled Relative Comparison of Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) Sperm Proteins for Fertility Molecular Marker Discovery

MYMS-001pt

2. Hashim et al. Metabolite Profiles of Agarwood Leaves Aqueous Extracts

MYMS-002pt

3. Mei et al. Lipid Profiling At Three Main Stages of Oil Palm Fruit Mesocarp Development

MYMS-003pt

4. Bannur et al. Identifying Potential Biomarkers for Colorectal Cancer Using Metabolomics Approach

MYMS-004pt

5. Derahman et al. Search of Serum Markers for Aspirin Resistance in Cardiovascular Disease (CVD) Patients Using Metabolomics Approach

MYMS-005pt

6. Ebrahimi et al. Reproducibility and Stability of NMR Spectroscopy and the Method Development for Urine Metabolomic Analysis

MYMS-006pt

7. Nornazliya et al.

Comparison of the Metabotypes of the Malaysian Aborigines with the Urban Healthy Individuals and Cardiovascular Patients: Signature of Longer Life Span?

MYMS-007pt

8. Rofiee et al. Alteration of Metabolite Profile in Acetaminophen Induced Hepatotoxic Rats

MYMS-008pt

9. Sundram et al. (E)-labda-8(17),12-diene-15,16-dial Production from Curcuma mangga (Mango ginger) In Vitro Cultures

MYMS-009pt

10. Nurul Shahfiza et

al.

Discrimination between Infected Dengue Patient and Healthy Individual Based On Chemometry of

1H NMR

Spectroscopy MYMS-010pt

11. Ali et al. The Use of LC-MS QTOF-Based Metabolomics to Define the Potential of Probiotic against Colorectal Cancer Xenograft in the Mouse Model

MYMS-011pt

12. Mohamed et al. Patients with Combined Variant of CYP2D6 and Wild-type ABCB1 C3435T Developed Early Recurrence and Metastasis: Role of Sphinganine

MYMS-012pt

13. Enche Ady et al.

Metabolic Profiling for Early Detection of Alzheimer‟s Disease

MYMS-013pt

14. Noorzaid Muhamad

et al. Some Aspect of Urea Metabolism in Parasites Helminth Teladorsagia circumcincta

MYMS-014pt

15. Shafiq et al. Molecular Characterisation of Previously Untypeable Rotavirus

MYMS-015pt

37


Symposium 2012

16. Irma et al. Insight into Genome and Pathogenicity of Opportunistic Human Pathogen, Streptococcus agalactiae

MYMS-016pt

17. Husaini et al. ANNOVAR: A Useful Tool for Functional Annotation of Genetic Variants of a Human Genome

MYMS-017pt

18. Zulkifli et al. Whole Genome Sequencing and Comparative Analysis of Local Klebsiella pneumoniae Strain against Klebsiella pneumoniae Ntuh-K2044

MYMS-018pt

19. Azwin Ismail et al. Identification of Interesting Genomic Properties of Clinical Isolates of Mycobacterium tuberculosis

MYMS-019pt

20. Norfatimah et al. Comparative Study Using Bioinformatics Pipeline for Malaysian Mahseer (Tor tambroides) Mitogenome Assembly

MYMS-020pt

21. Hasbullani et al.

HLA-B*1502 is a Significant Marker for Carbamazepine-induced Stevens Johnson Syndrome (SJS): Pre- Implementation Trial of HLA-B*1502 Pharmacogenotyping in a Local Hospital in Malaysia

MYMS-021pt

22. Muda et al. Genetic Polymorphism of CYP3A4*18 and CYP3A5*3 in Malaysian Kidney Transplant Patients

MYMS-022pt

23. Abdul Jalil et al. The Implication of the Polymorphism of UGT1A6 among Cardiovascular Disease (CVD) Patients treated with Aspirin

MYMS-023pt

24. Che Omar et al. Genetic polymorphism of CYP2C19 among the Cardiovascular Patients of Three Ethnic Groups in Malaysia

MYMS-024pt

25. Mohamed Ali et al. Development of a Pharmacogenotyping Method for Detection of HLA Polymorphism in Personalising Allopurinol Therapy

MYMS-025pt

26. SS Mamat et al. Hepatoprotective Activity of Methanol Extract of Melastoma malabathricum Leaves on Paracetamol-Induced Liver Injury in Rat

MYMS-026pt

27. Samuel Oii et al. Chemopreventive Potential of Melastoma malabathricum Leaves Extract in DMBA/Croton Oil-Induced Mouse Skin Carcinogenesis

MYMS-027pt

28. Bendt et al. Mycolic Acids as Diagnostic Markers for Tuberculosis Case Detection and Drug Efficacy

MYMS-028pt

29. Narayananaswamy

et al.

A Sensitive and Efficient Method for the Quantification of Phosphorylated Sphingoid Bases in Biological Samples

MYMS-029pt

38


Symposium 2012

Non-Labelled Relative Comparison of Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) Sperm Proteins for Fertility

Molecular Marker Discovery

Ali Ashrafzadeh1*, Sheila Nathan1, Iekhsan Othman2, Ting Yee Tee2, Saiful Anuar Karsani3

1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti

Kebangsaan Malaysia, Selangor, Malaysia; 2School of Medicine and Health Sciences, Monash University, Sunway Campus, Kuala Lumpur, Malaysia; 3Institute of Biological Sciences, Faculty of

Science, & University of Malaya Centre for Proteomics Research, University of Malaya, Kuala Lumpur, Malaysia.


Abstract Malaysia is still reliant on importation of up to 80% of the nations‟ dairy and beef requirements. In an attempt to improve local beef and dairy production, cross breeding with high producing breeds is generally favoured. However, fertility of the crossbred progenies is low as a result of the high humidity and temperature. This study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous breed (Kedah Kelantan) and the low fertile crossbred cattle (Mafriwal) to identify proteins that may be potentially fertile and heat tolerance molecular marker(s). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Protein separation was conducted and gel bands were processed for in-gel digestion. For each breed, mass spectrometry data was acquired over 11 replicates. Analysed data identified compounds of different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins, fifteen motility and fertility related proteins were up-regulated in Kedah Kelantan. Sperm motility evaluation by CASA (Computer Assisted Semen Analysis) confirmed significantly higher motility in Kedah Kelantan sperm compared to Mafriwal. Further analysis of these proteins will allow us to evaluate their potential as selective markers indicating highly fertile and more environmentally compatible bulls for breeding purposes in tropical areas. Keywords: Bos indicus; Bos Taurus; mass spectrometry; molecular marker(s); sperm proteome

39


Symposium 2012

Metabolite Profiles of Agarwood Leaves Aqueous Extracts

YZH-Y Hashim1,2*, Ismail I1, Salleh F1, 2

1Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology

Engineering, Kulliyyah of Engineering, International Islamic University Malaysia, P.O. Box 50728, Kuala Lumpur, Malaysia; 2International Institute for Halal Research and Training

(INHART), Ground Floor, Block E0, Kulliyyah of Engineering Building, International Islamic University Malaysia, P.O. Box 50728, Kuala Lumpur, Malaysia.


Abstract

Agarwood has been known for its invaluable aromatic resin while other parts of the plant are not fully utilised. Although there are various ethnopharmacological evidences describing the health beneficial effects of other parts of the plant, very few scientific studies were undertaken to confirm these claims. This study aims to investigate the metabolites present in water extract of agarwood leaves. Fresh agarwood leaves from uninoculated trees were collected, washed and dried at room temperature for two weeks prior to grinding. The powdered leaves (1 g) were then extracted in a volume of distilled water (30 ml, 40 ml and 50 ml) at 120 rpm, 30 °C for 16 hours. Filtrate was centrifuged at 22000 g for 15 min and supernatant collected. For gas chromatography mass spectrometry analysis, 150 µl of supernatant was transferred to 1.5 ml reaction tube, dried using vacuum concentrator (30°C) for 2 hours and derivatised. The derivatised sample was transferred to the GC vial prior to analysis in Agilent 7890A GC system coupled to 5975C Inert XL MSD using HP-5MS column. Data was then subjected to Partial Least Square Discriminant Analysis (PLSDA) using SIMCA-P+ v12.0 (Umea, Sweden). Although all extracts shared compounds such as propanoic acid, aspartic acid, proline and fructose oxime, PLSDA clearly separated the compounds extracted based on ratio of solid to solvent with R2 of 0.698 and Q2 of 0.743. Samples with 1:40 ratio gave the highest metabolite number ranging from 39 to 64 and were dominated by gluconic acid, hexadecanoic acid, mannonic acid and dodecyl acrylate. The metabolites revealed in the leaves water extract may further be isolated for use as raw ingredients. This would add value to the agarwood leaves which otherwise would be considered as waste. Keywords: agarwood; aqueous extract; gas chromatography mass spectrometry; metabolites

40


Symposium 2012

Lipid Profiling At Three Main Stages of Oil Palm Fruit Mesocarp Development

Theresa Ng Lee Mei*, Tiong Soon Huat, Neoh Bee Keat, Teh Huey Fang, Thang Yin

Mee, Mohd Amiron Ersad, David R. Appleton

1Sime Darby Technology Centre Sdn. Bhd., 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh Silikon, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia


ABSTRACT Oil palm is well known for its effectiveness as oil storage whereby more than 90% of its oil is stored in fruit mesocarp. The oil accumulates over time at different developmental stages starting from the lag stage [11 to 14 week after anthesis (WAA)] to lipid biosynthesis stage [15 to 19 WAA] and finally to the ripening stage [20 to 24 WAA]. In this study, an extensive lipid research is carried out using targeted metabolomics approach on all the three main stages of oil palm fruit mesocarp development. Lipids analysis comprised of the total oil content, fatty acid composition, triacylglycerols (TAG) and phospholipids. The metabolites were extracted and profiled by multiple platforms such as the Gas Chromatography – Mass Spectrometry (GCMS) and Liquid Chromatography – Mass Spectrometry (LCMS). Results showed that the total oil content increases from first to the third phase with palmitic acid recorded the highest composition followed by oleic and stearic acids respectively. At the same time, the amount of phospholipids decreases while TAG accumulation increases, as oil is deposited in the form of TAG across the three main stages. In all, the different lipid class of oil palm fruit mesocarp development has been successfully classified and would definitely be useful for palm genotypic studies. Keywords: metabolites; oil palm; triacylglycerols

41


Symposium 2012

Identifying Potential Biomarkers for Colorectal Cancer Using Metabolomics Approach

Z Bannur1*, H Hashim1, MZ Salleh1, TP Hennessy1, 2, JKS Shia1, Azmi MN3, Zailani MH3,

Ramasamy P3, ZA Zakaria1,4, LK Teh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Life Sciences Group, Agilent Technologies, Singapore; 3International Islamic University Malaysia, Kuantan 25710

Malaysia; 4Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.


Abstract Colorectal cancer (CRC) is one of the most common malignant diseases among men and women in Malaysia. Management of CRC among different individuals is complicated as it depends on many factors such as stage of cancer, environmental factors and type of regimen used to treat colorectal cancer. Systematic approaches using specific biomarkers to evaluate the risk for individual patients are therefore desired. The emerging field of LC/MS based global metabolomics approach provides a platform to help unravel the mechanisms involved in the diseases and is useful to understand various biological processes in the living system as they are the intermediates and the end products of metabolism. This study aims to identify potential biomarkers for CRC by comparing the metabolites profiles between colorectal cancer patients treated with 5-fluorouracil (CRC-5-FU) and healthy control using metabolomics approach. Several classes of metabolites that were down regulated in the CRC-5-FU group include dicarboxylic acid (DA), polyunsatureated fatty acids (PUFA) and amino acids and conjugates. Conversely, metabolites that were up-regulated in CRC-5-FU include uremic toxins, bilirubin, bile acid and its conjugates and fatty acids. Our results confirmed the alteration of metabolic profile in patients with colon carcinoma which involve the alteration of pathways in the metabolism of bile acid and fatty acid as well as glycolysis pathways. The distinctive metabolite profiles of CRC-5-FU patients and healthy control provides clues of several biomarkers or pattern for detection of cancer.

Keywords: 5-FU; biomarkers; CRC; glycolysis pathway; metabolomics

mailto:[email protected]%20/


42


Symposium 2012

Search of Serum Markers for Aspirin Resistance in Cardiovascular Disease (CVD) Patients Using Metabolomics Approach

A Derahman1*, MZ Salleh1, O Maskon2, NJ Abdul Jalil1, RN Che Omar1, MI Ismail1, ES

Rosli1, M Nornazliya1, MS Rofiee1, Z Bannur1, TP Hennessy3, JKS Shia1, LK Teh1*

1Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Cardiology, Hospital Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia; 3Life Sciences

Group, Agilent Technologies, Singapore.


Abstract Cardiovascular disease (CVD) is the number one cause of death which covers a wide array of disorders, including diseases of the cardiac muscle and vascular system. Aspirin has been used as an analgesic-antipyretic for a long time and with lower doses, it is useful to prevent blood clots. However, in some patients, aspirin fails to protect them from thrombotic complications; this phenomenon is known as „aspirin resistance‟. Current study aims to identify biomarkers involved in aspirin resistance in which they can be used to identify who would response better to aspirin and those who may not response, so alternative drugs can be given at initiation of drug treatment for prevention of thrombotic complications. Resistance was defined in this study as occurrence of secondary cardiovascular events despite initiation of aspirin therapy in patients. Ethics approval were obtained from Research Ethics Committee of Universiti Teknologi MARA (UiTM) and Hospital Universiti Kebangsaan Malaysia (HUKM) for the conduct of this project. Blood serum was clean up using cold acetonitrile. Samples were then reconstituted with mobile phase and were injected into LC/MS-QTOF. Data was acquired using Agilent MassHunter Data Acquisition software. Data was analysed using software which includes Agilent MassHunter Qualitative Analysis, Agilent Mass Profiler Professional and Agilent METLIN Professional Metabolite Database. A total of 106 metabolites were detected. Dendrogram of Hierarchical Clustering Analysis (HCA) was used to visualise the up- and down-regulation of the expression of metabolites. The browser of the Human Metabolome Database (HMDB) pathway identified ten pathways and eight metabolites of interest. Of these metabolites, bilirubin and glycocholic acid were down-regulated while deoxycholic acid glycine conjugate and prostaglandin G2 were up-regulated. Prostaglandin G2 which is involved in arachidonic acid pathway may possibly have relationship with aspirin resistance. However, this requires further validation before it can be used as biomarker in monitoring aspirin resistance. Keywords: aspirin resistance; biomarkers; cardiovascular disease (CVD); metabolomics



43


Symposium 2012

Reproducibility and Stability of NMR Spectroscopy and the Method Development for Urine Metabolomic Analysis

F Ebrahimi1*, KL Chan1, F Fang2, P Sprenger2, V Murugaiyah 1, CH Teh2, B Ibrahim1*

1School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia; 2Bruker

Malaysia.


Abstract Accuracy and stability of NMR spectroscopy are vital in urine metabolomics study. Meticulous method development further ensures the achievement of reliable results. The clustering of sample groups on the NMR spectra in Orthogonal Partial Least Squares (OPLS) graph could provide some insight on NMR stability and accuracy. The present study determined the stability and reproducibility of the applied methodology including the use of a 500 MHz NMR instrument for urine metabolomics analysis. Twenty urine samples of healthy volunteers were collected and stored in a -80°C freezer. Prior to NMR analysis, the samples were thawed and eight samples were pooled together and vortex-mixed. Eight samples of 900 µl were taken from the pooled sample and mixed with 100 µl of urine buffer (containing phosphate buffer, sodium azide and trimethylsilyl propanoic acid) in eppendorf tubes. After 10 min of centrifugation, a 600 µl of the supernatant was transferred to a 5 mm NMR tube. Another 12 individual samples (unpooled) were also prepared in the same manner. The temperature of the NMR spectrometer was calibrated at 300K, and phase correction, baseline correction, tuning and matching, shim optimisation, water suppression and acquisition parameter optimisation were done. The NMR spectra were binned to 0.04 ppm and the data were analysed using OPLS-Discriminant Analysis (OPLS-DA) to determine the clustering and separation between samples. The NMR spectra exhibited peaks of high reproducibility. OPLS-DA analysis yielded a two-component model with R2Y=0.99 and Q2Y=0.75. A tight clustering was observed in the pooled samples whilst the unpooled samples were loosely clustered. A clear separation between pooled and unpooled groups was also observed. The results showed that the applied urine metabolomics technique and the NMR spectrometer were stable and reproducible to differentiate between groups. Future works on medicinal plants and their effects on rat urine metabolomics are on-going. Keywords: method development; Orthogonal Partial Least Squares; reproducibility; urine metabolomics



44


Symposium 2012

Comparison of the Metabotypes of the Malaysian Aborigines with the Urban Healthy Individuals and Cardiovascular Patients: Signature of

Longer Life Span?

M Nornazliya1*, MZ Salleh1, MI Ismail 1, MS Rofiee1, ES Rosli1, A Derahman1, Z Bannur1, Rose Ismet1, Husaini I1, Shafiq A 1, Aminuddin A2, Fadzilah MN2, T Rahman2, R Razali2,

Adzrool Idzwan Ismail3, Kamarudzaman Md. Isa3, Mustaffa Halabi Azahari3, TP Hennessy1,4, LK Teh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Faculty of Medicine, UiTM Sungai Buloh, Selangor, Malaysia; 3Faculty of Art & Design, UiTM Shah Alam, Selangor,

Malaysia; 4Life Sciences Group, Agilent Technologies, Singapore.


Abstract Orang Asli are believed to have different metabotypes compared to the urban people based on their lifestyles, habitat, culture, and nutrition behaviour. The urban people are faced with many diseases such as cardiovascular diseases and cancer, while the Orang Asli was commonly reported to have skin infection, worm infestations and tuberculosis. These diseases could be due to the differences in lifestyles. Currently, data about cardiovascular diseases in Orang Asli are lacking and this lead to the hypothesis that the Orang Asli may be protected from these diseases due to their lifestyles and diet behaviour. This study is preliminary and aims to investigate the differences in the metabotypes of the Orange Asli and a group of cardiovascular patients using global metabolomics approaches. Protein precipitation of serum sample was done using acetonitrile and the samples were reconstituted with mobile phase prior analysis using LC/MS-QTOF. The metabolic profiles of the Orang Asli were compared with the profiles of the urban healthy individuals and cardiovascular patients. Principal component analysis (PCA) showed an obvious clustering of metabolites in the PCA score plot. ANOVA results showed a significant difference in the expression of 92 out of 338 metabolites detected among the Orang Asli, healthy urban and urban patients with cardiovascular diseases (p<0.001). One particular metabolite of interest is indoleacrylic acid which was down-regulated in both healthy urban and Orang Asli and up-regulated in CVS patients. Indoleacrylic acid has been reported to inhibit tryptophan synthetase and thus resulting in lower level of tryptophan and its metabolites such as serotonin and melatonin. Hence this may cause depression and sleep disorders in those with lower serotonin and melatonin respectively. Interestingly, lower tryptophan signifies longer life span, and potential protection from insulin resistance. This result is preliminary and requires further validation of its significance in protecting Orange Asli from life-styles related diseases, but it provides insight for the researchers to navigate deeper for more answers. Keywords: cardiovascular; metabolomics; lifespan; Orang Asli; tryptophan


45


Symposium 2012

Alteration of Metabolite Profile in Acetaminophen Induced Hepatotoxic Rats

MS Rofiee1*, MI Ismail1, ES Rosli1, JKS Shia1, MZ Salleh1, ZA Zakaria2, TP Hennesy1,3,

LK Teh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2 Department of Biomedical Sciences, Faculty of Medical and Health Sciences, Universiti Putra Malaysia, Malaysia; 3Life

Sciences Group, Agilent Technologies, Singapore.


Abstract Acetaminophen (N-acetyl-p-aminophenol) or paracetamol is usually used as an analgesic and antipyretic. Acetaminophen is safe at standard recommended dose, but it may produce acute centrilobular hepatic necrosis, when given at high doses in humans and experimental animals. This study aims to further understand the mechanism and metabolic changes occur due to hepatic necrosis induced by acetaminophen. LC/MS-QTOF was used to profile the characteristic changes in the endogenous metabolites in response to acetaminophen overdose in rats. Hepatotoxicity was induced in a group of rats (6 rats) by oral administration of acetaminophen (300 mg/kg). Body weight, liver weight of the rats and gross picture of the liver were documented at 48 hours after administration of acetaminophen to confirm hepatotoxicity. Serum was collected pre and post toxicity event. Protein precipitation of serum sample was performed using cold acetonitrile. Sample were dried and reconstituted with mobile phase prior analysis using LC/MS-QTOF. A total of 133 compounds satisfying a p-value with cut off 0.05 and fold change cut off 2 were identified. Principal component analysis (PCA) showed an obvious clustering of metabolites for pre and post toxicity event. Compounds up-regulated in post toxicity event include p-acetamidophenyl glucoronide, p-acetamidophenol, 5‟-hydroxyhydrodolasetron, 2-hydroxy-4 methlvaleric acid and Stearoylcarnitine; while two compound (cytosine and 5,8-heptadecadiynoic acid) was down regulated in the treated group. However, further study is required to validate the function of each of these compounds. Keywords: APAP; liver toxicity; metabolomics


46


Symposium 2012

(E)-labda-8(17),12-diene-15,16-dial Production from Curcuma mangga (Mango ginger) In Vitro Cultures

Tamil CM Sundram1*, Lee Guan Serm 2, Sri Nurestri Abdul Malek 2, M Suffian M Annuar1,

Norzulaani Khalid1*

1CEBAR, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; 2Biochemistry Department, Faculty of Science, University of Malaya, 50603,

Kuala Lumpur, Malaysia.

*Corresponding author: [email protected]/[email protected]

Abstract Curcuma mangga Val., (Mango ginger) has been used traditionally as a seasoning for food and treatment for stomach aches, fever, and cancer related diseases. (E)-labda-8(17),12-diene-15,16 dial is one of the major compounds isolated from rhizomes of Curcuma mangga. Malek et al. (2011) has demonstrated that this bioactive compound showed cytotoxic effects against six human cancer cell lines. In this research, callus and cell suspension cultures of Curcuma mangga were established to be tested as an alternative source of (E)-labda-8(17), 12-diene-15,16 dial. In vitro cultures will be advantageous compared to field grown rhizomes since in vitro sources is possible to ensure continuous supply of consistently high quality compounds. Friable calli were initiated from small and active shoot buds of the rhizomes, when cultured on MS (Murashige and Skooq) basal medium supplemented with 1 mg/L 2.4-D and 3% sucrose under dark condition. The growth of callus culture was assessed and harvested fortnightly at different growth stages. After 4 to 8 weeks, rapidly growing friable callus were also transferred and maintained in MS liquid medium with 1 mg/l 2.4-D and 30 g/L sucrose to obtain liquid suspension culture. Rapidly proliferating and dense cells were harvested every three days. (E)-Labda-8(17), 12-diene-15,16 dial was extracted from cells, callus, rhizomes and shoots using solvent extraction method. Gas Chromatography (GC) and Gas Chromatography Flame Ionisation Detector (GCFID) was performed to examine and compare the quantity of (E)-labda-8(17), 12-diene-15,16 dial production in cells and callus induced at different growth period with field grown rhizomes and shoot buds. The results revealed the presence of (E)-labda-8(17), 12-diene-15,16 dial in all materials tested and were in comparable amounts. With this findings, further work on the enhancement of the compound could be carried out through elicitation and metabolic engineering. Keywords: (E)-labda-8(17); 12-diene-15,16 dial; callus; cancer; cell suspension; gas chromatography; in vitro; mango ginger


47


Symposium 2012

Discrimination between Infected Dengue Patient and Healthy Individual Based On Chemometry of 1H NMR Spectroscopy

Nurul Shahfiza1, Maulidiani2, Hasnah Osman3, Khozirah Shaari2, TS Chow4, TH Tang1,

Abdel-Hamid AZ1*

1Infectious Disease Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia,

Penang, Malaysia; 2Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia; 3School of Chemical Sciences, Universiti

Sains Malaysia, Penang, Malaysia; 4 Infectious Disease Unit, Hospital Besar Pulau Pinang, Penang, Malaysia.


Abstract Dengue is a major health and pressing threat to Malaysia. The increasing incidence makes the early diagnosis important as supportive treatment can substantially lower the risk of developing severe disease. Therefore, the metabolomics approach was applied to investigate the metabolite candidates that are potentially useful as indicators for dengue disease. Forty-seven patients diagnosed with dengue fever at Hospital Pulau Pinang and forty-one healthy individuals were included in this study. Medical record was used to characterise the disease and provisional diagnosis. Urine samples were collected from confirmed dengue patients. A combination of 1H NMR spectrometry and multivariate statistical analysis (SIMCA) was used to differentiate the metabolic profiles of dengue infected and non-dengue infected subjects. The metabolic compounds responsible for the differentiation were identified based on 1H NMR chemical shifts and compared to Human Metabolome database (HMDB). Orthogonal partial least square discriminant analysis (OPLS-DA) of the 1H NMR spectra showed a clear discrimination between the dengue infected and non-dengue infected subjects with the total R2Y (cum) value of 0.935, and the total Q2Y (cum) value is 0.832. The S-plot visualises that carnitine, O-phosphocholine, O-acetylcholine were the metabolites contribute most to the separation. This study demonstrated that the metabolites derived from the urinary metabolite profiling approach holds a potential of using a combination of 1H NMR spectroscopy and multivariate data analyses in differentiating dengue infected and non-dengue infected subjects. However, further validation of the metabolites is required for confirmation. Keywords: 1H NMR spectroscopy; dengue; metabolomics; multivariate statistical analysis; orthogonal partial least square discriminant analysis

48


Symposium 2012

The Use of LC-MS QTOF-Based Metabolomics to Define the Potential of Probiotic against Colorectal Cancer Xenograft in the Mouse Model

Azidah Ali1, Kalavathy Ramasamy1*, LK Teh2, Vasudevan Mani3

MZ Salleh2, Abu Bakar Abdul Majeed3

1Collabrative Drug Discovery Research (CDDR) Laboratory, Faculty of Pharmacy, Uitm Puncak

Alam, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak

Alam, Selangor Darul Ehsan, Malaysia; 3Brain Research Laboratory, Faculty of Pharmacy, Uitm Puncak Alam, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.


Abstract Numerous studies have shown that there exists a potential for foods that contain probiotics to affect the host by changing the colonic microbiota in a way that might prevent colorectal cancer (CRC). Probiotic is defined as live microbial food ingredients which upon ingestion may be beneficial to the health of the host. Most of the earlier studies of probiotic effects on CRC were with regards to the reduction of cancer cell viability or tumour size and monitored only specific end products such as amino acids or bile acids. The interrelationship between the cellular effects of probiotics and the metabolites produced have not been well documented and is clearly a challenge. Metabolomics utilises fundamental analytical techniques to probe the chemical fingerprint of the sample and has been shown to be effective tool for disease diagnosis, biomarker screening and characterisation of biological pathway. In this study, metabolic profiling of serum male mice was carried out to assess the effects of probiotics on human colorectal cancer xenograft model using liquid chromatography-mass spectrometry quadrupole time of flight (LC/MS-QTOF). Mice were divided into 6 groups and administered with normal saline (control group); fermented and unfermented soymilk with probiotics, lactic acid bacteria (LAB 12 and LAB PC) for 4 weeks before tumour induction and the treatment was continuously given until the experiment was terminated at the end of 7 weeks. Data were analysed using principle component analysis (PCA) and clustering analysis. PCA showed the separation between the control group and treatment group. In addition, four metabolites (docosatriynoic acid, nanodecanoic acid, ladderane and octadecadienal) were found to be higher in treatment group with p<0.005. This study highlights the potential of metabolomics for assessing the probiotic effect in an animal model of xenograft models and exploring other potential metabolic biomarkers. Keywords: lactic acid bacteria; metabolomics; probiotics; serum; xenograft model


49


Symposium 2012

Patients with Combined Variant of CYP2D6 and Wild-type ABCB1 C3435T Developed Early Recurrence and Metastasis: Role of

Sphinganine

NI Mohamed1*, MZ Salleh1, Rohaizak M2, Shahrun NS2, Saladina JJ2, Roslan H3, Sood S4, Rajoo TS5, Muniandy SP5, Henry G5, Ngow HA6, KT Hla U 7 Din J7, TP Hennessy1,8,

LK Teh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Surgery, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Kuala Lumpur,

Malaysia; 3UKM Medical Biology Institute (UMBI) and Department of Medicine, Universiti Kebangsaan Malaysia Medical Centre, (UKMMC), Kuala Lumpur, Malaysia; 4Faculty of Medicine,

Universiti Teknologi MARA (UiTM), Selangor Darul Ehsan, Malaysia; 5 Department of Surgery, Selayang Hospital, Kuala Lumpur, Malaysia; 6Department of Internal Medicine, Kulliyah of Medicine, International Islamic University Malaysia (IIUM)/Tengku Ampuan Afzan Hospital (HTAA), Pahang Darul Makmur, Malaysia; 7Department of Surgery, International Islamic

University Malaysia (IIUM), Pahang Darul Makmur, Malaysia; 8Life Sciences Group, Agilent Technologies, Singapore


Abstract Tamoxifen has been widely used as the standard adjuvant therapy for breast cancer patients who are estrogen receptor positive. However, resistance towards tamoxifen therapy was found to underlies the cause of treatment failure. Earlier findings by our group reveal that patients with combination of variant alleles of CYP2D6 and wild-type of ABCB1 C3435T developed early recurrence and metastasis. Therefore, analysis on the differential expression of the metabolites was performed to understand the role of CYP2D6 and P-glycoprotein in regulation of metabolism among the patients which affect the risks of recurrence and metastasis. Plasma samples from 31 breast cancer patients treated with tamoxifen were analysed using LC/MS-QTOF (Agilent Tech). Samples were injected after protein precipitation with acetonitrile and centrifugation for 10 min at 10,000×rpm at 4°C. The chromatography was performed on Agilent 1200 Series using an XDB C18 4.6 µM, 150×1.8 mm. The MS data was analysed using Mass Profiler Professional (MPP) and Metlin Personal Metabolites Database. Combined partial least square discriminant analysis (PLS-DA) and hierarchical clustering with Bonferroni correction was done. Patients were grouped according to the variation of genotypes. Analysis on metabolite clustering revealed that patients with variant alleles of CYP2D6 and wild-type of ABCB1 C3435T have low level of vitamin D, bile acid glycine conjugate as well as several sphingoid bases. Sphinganine has been reported to inhibit growth and induce apoptosis of tumourigenic human breast epithelial cells (HBEC) compared to normal HBEC cells. Sphinganine is also a substrate of P-glycoprotein which was pumps out from the cells. The correlation of the lower level of sphinganine and higher risk of recurrence and metastasis in breast cancer patients however require further analysis using larger sample size. Proven the theory, supplementation with sphinganine for patients who carry variant alleles of CYP2D6 and wild-type of ABCB1 C3435T may improve the therapy outcome. Keywords: ABCB1 C3435T; breast cancer; CYP2D6; LC/MS-QTOF; tamoxifen



50


Symposium 2012

Metabolic Profiling for Early Detection of Alzheimer’s Disease

Che Nor Adlia Enche Ady1, Kalavathy Ramasamy1, Chin Ai-Vyrn2, Mohd Nazif Darawi1,

Vasudevan Mani1, Shahrul Bahyah Kamaruzzaman2, Tan Maw Pin2, LK Teh3, MZ Salleh3, Abu Bakar Abdul Majeed1

1Brain Research Laboratory, Faculty of Pharmacy, UiTM Puncak Alam, 42300 Bandar Puncak

Alam, Selangor Darul Ehsan, Malaysia; 2Department of Medicine, Faculty of Medicine, University Malaya, 50603 Kuala Lumpur; 3Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy,

University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.


Abstract Currently there is no cure for AD. Early diagnosis and intervention are the best options but the current neurophysiological evaluation to identify patients with AD is usually for those at their advanced stage and is still defined by signs and symptoms. Convenient non-invasive blood based test would clearly be valuable in the early detection of patients with memory deficits. A need for better biomarkers is also critical to provide insight into disease progression and for the development and testing of drugs. In the present preliminary study, metabolomics is employed to determine the metabolite profile of healthy and probable Alzheimer‟s disease (AD) patients. Serum metabolite profiles from patients with probable Alzheimer‟s disease (n=6) and age-matched healthy control (n=6) were obtained using liquid chromatography-mass spectrometry quadrupole time of flight (LC/MS-QTOF) platform. Data were analysed by using principle component analysis (PCA) and unpaired t-test. PCA showed the separation between AD patients and healthy control. Several metabolites (1-linoleoylphosphatidylcholine and docosatriynoic acid) were found to be significantly (P<0.05) lower in AD patients. However, the study is just at the initial stage and we hope to identify potential new blood-based biomarkers indicative of AD using metabolomic approach. Keywords: Alzheimer‟s disease; biomarkers; diagnosis; metabolomics; serum


51


Symposium 2012

Some Aspect of Urea Metabolism in Parasites Helminth Teladorsagia circumcincta

Noorzaid Muhamad1*, S Brown2, HV Simpson3

1Royal College of Medicine Perak , Universiti Kuala Lumpur, Malaysia; 2School of Human Life

Sciences, University of Tasmania, Launceston, Australia; 3Institute of Food, Nutrition & Human Health, Massey University, New Zealand.


Abstract Infective L3 T. circumcincta secreted or excreted urea at one quarter of the rate of NH3/NH4

+. The rate of urea accumulation in the medium was about 84 pmol h-1 (103 larvae)-1 over 4 hours, corresponding to about 11 nmol h-1 mg-1 protein. We were unable to detect urease activity, but urea production by both creatine amidinohydrolase and arginine amidinohydrolase could be detected. The apparent Km and Vmax of creatine amidinohydrolase were 1.1 mM and 48 nmol h-1 mg-1 proteins, respectively, and the activity was greatest at pH 8. The apparent Km and Vmax of arginine amidinohydrolase were 0.7 mM and 62 nmol h-1 mg-1 proteins, respectively, and the activity was greatest at pH 7.9. The activity of creatine amidinohydrolase and arginine amidinohydrolase was sufficient to account for the rate of urea secretion or excretion. Keywords: arginine amidinohydrolase; creatine amidinohydrolase; Teladorsagia circumcincta; urea


52


Symposium 2012

Molecular Characterisation of Previously Untypeable Rotavirus

Shafiq A1,*, Zuridah Hassan1, Carl Kirkwood2

1Faculty of Health Sciences, UiTM Puncak Alam, 42300

Puncak Alam, Selangor Malaysia; 2Enteric Virus Research Group, Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville,

Victoria, Melbourne.


Abstract Group A rotavirus (RV) is a causative agent for acute gastroenteritis among children under the age of five. Due to genetic drift and shift on VP7 and VP4 gene of rotavirus, some strains were untypeable using currently available primers. Thus molecular strategy for genotyping needs to be revised. Newly designed VP7 and VP4 primer pairs to identify uncommon strains will minimise percentage of untypeable cases. Surveillance study conducted in 1996 using rotavirus positive stool samples from Kuala Lumpur General Hospital identified eight samples which were G untypeable and five samples were P untypeable. In this study, these untypeable samples were re-examined using RNA-PAGE and re-typed using the same VP7 and VP4 primer pairs with different PCR optimisation. RNA-PAGE profiles using mini gel electrophoresis revealed all the eight (100%) G untypeable samples were Long e-type. For P untypeable, three samples (60%) were Long e-type and another two samples (40%) were degraded. Even though all eight G untypeable RNAs were intact in RNA-PAGE, only six (75%) were G1 and two (25%) samples were still untypeable. For P typing, three (60%) samples were P [8] and another two (40%) samples were negative when tested with RT-PCR. Using new VP7 primer pair, two G untypeable samples could be retyped as G1. An alternative new VP4 primer pair that was designed to retype all the three (60%) reconfirmed all as P [8] for the second time. All these untypeable samples were G1P [8] strains. Different brands of reagents, new primer sets, optimised two steps RT-PCR and direct genogroup assignation were able to circumvent untypeable problem. The new VP7 and VP4 primer pairs designed in this study could be used as alternative primers for characterisation of VP7 and VP4 gene as part of continuous surveillance activity to differentiate co-circulating strains. Keywords: G1; Rotavirus; RNA-PAGE; RT-PCR; P [8]


53


Symposium 2012

Insight into Genome and Pathogenicity of Opportunistic Human Pathogen, Streptococcus agalactiae

Irma SZ*, LS Lee, Azwin Ismail, Hanif AK, Izwan I, MH Zulkifli, MZ Salleh and LK Teh




Abstract Streptococcus agalactiae is well adapted, asymptomatic coloniser of the human intestinal tract population. However, S. agalactiae has subsequently emerged as a significant cause of human diseases. Now it is one of the most common causes of life-threatening invasive bacterial infections such as septicaemia, pneumonia and meningitis in neonates. It is also an important cause of morbidity and mortality in pregnant women and adults with underlying chronic diseases. However, the mechanism and genetic elements that are responsible for the virulency of these bacteria may vary in different geographical areas. Therefore, this study aims to uncover the genetic elements that contribute to acquisition of virulency and panthogenicity of Streptococcus agalactiae by using whole genome sequencing approach. Whole genome sequences of a clinical isolate of Streptococcus agalactiae was obtained using Illumina sequencing platform. The open reading frames were defined using Prodigal while their positions were visualised via Artemis. Genome annotation was done by CLCbio Genomics Workbench 5 and BASys. The assembly result obtained a total of 52,832,486 reads which cover a total of 1.9 Mbp. The genome has a GC content of 37% and functional genome analysis annotated more than 2699 of total genes which may be important in the pathogenecity of Streptococcus agalactiae. The genome of Streptococcus agalactiae was successfully sequenced, assembled and annotated using next generation sequencing strategy established in Pharmacogenomics Centre (PROMISE). We believed that whole genome sequencing is a way forward in development of countermeasures against these pathogenic bacteria. Keywords: pathogenicity; Streptococcus agalactiae; virulence; whole genome sequencing


54


Symposium 2012

ANNOVAR: A Useful Tool for Functional Annotation of Genetic Variants of a Human Genome

Husaini I*, Rose Ismet, Shafiq A, LK Teh, MZ Salleh*




Abstract High-throughput sequencing platforms produce huge amounts of data from diverse genomes including human, animals and plants. The data include genetic diversity of the genomes such as single nucleotide variations (SNVs) and other structural variations (SVs). Above all that, characterising the genetic variations and pinpointing functionally important variants are necessary to further assist in understanding the evolution and disease susceptibility. Here we described the usage of ANNOVAR to functionally annotate the genetic variants of the Malay genome data. Variant calls file (.vcf) of the Malay genome was generated using Burrows-Wheeler Alignment and SAMtools programs prior to annotation using ANNOVAR. A total of 3,847,934 genetic variants including mainly single-nucleotide variations (SNVs) and insertions-deletions (indels) were identified. The variants were functionally annotated using three main options in ANNOVAR which are; gene-based, region-based and filter-based annotation. Our analysis identified 20,070 variants in exonic regions using gene-based annotation. Region-based annotation identifies variants in specific genomic regions through mapping against the Databases of Genomic Variants and GWAS catalogue. A total of 1,357,949 and 2867 variants were found to match against each database respectively. Filter-based annotation was used to annotate all variants against dbSNP (SNP database) and SIFT (Sorting Tolerant From Intolerant). A total of 3,471,680 SNPs matched against the dbSNP and 375,239 were found to be novel. SIFT annotation on the other hand was performed to assign functionally important scores to non-synonymous SNPs (ns-SNPs). SIFT score more than the threshold value of 0.05 is regarded as benign. The annotation resulted in SIFT scores for 10,609 ns-SNPs. In conclusion, ANNOVAR is a useful tool to characterise genetic variations and provide insights into further analyses of human genome. The novel variants discovered will be further analysed to identify amino acid changes, to elucidate their effects on protein functions and association with diseases. Keywords: annotation; ANNOVAR; functional; non-synonymous SNPs; SNP


55


Symposium 2012

Whole Genome Sequencing and Comparative Analysis of Local Klebsiella pneumoniae Strain against Klebsiella pneumoniae Ntuh-

K2044

MH Zulkifli*, LS Lee, Hanif AK, Izwan I, Azwin Ismail, Irma SZ, Shafiq A , LK Teh, MZ Salleh*




Abstract Klebsiella pneumoniae is a Gram-negative bacterium which can cause several of diseases such as pneumoniae, urinary tract infection, bacteraemia, liver abscess and meningitis. Some studies reported that the clinical pattern of community-acquired K. pneumoniae infection varies with geographical areas. The virulence and pathogenic properties and mechanisms of different bacterial strains might vary in different strains. The aim of this study was to perform whole genome sequencing of a locally isolated K. pneumoniae and its comparative analysis against K. pneumoniae NTUH-K2044. K. pneumoniae which had caused septicaemia in a patient was identified and cultured. DNA was extracted and sequenced using Genome Analyser IIx (Illumina). The quality of the sequencing output was determined using FastQC. Ambiguous sequences were trimmed before assembly and mapped to K. pneumoniae NTUH-K2044. Then, the assemblies of trimmed-sequences were performed using CLCBio: CLC Genomic Workbench 5.1. The annotation was performed using Rapid Annotation Subsystem Technology (RAST) and then the genome sequence was visualised using Arthemis. The sequencing produced an output of 6,271,798 reads. FastQC revealed 56% of GC content for this sample while the reference genome has 57%. After trimming, 6,228,844 reads were produced with 99.32% of trimming efficiency. The assembly result showed that 122 contigs have been produced with size of 5,305,345 bp. Annotation results showed 4,876 protein-encoding genes, 4819 coding sequences and 56 RNAs for local sequence meanwhile 4,983 protein-encoding genes, 4871 of coding sequences and 111 RNAs in reference sequence. We conclude that there are significant differences between this particular strain and the reference sequence in terms of genetic contents. Hopefully, this could provide better knowledge regarding the virulence and pathogenicty of Klebsiella pneumoniae. Keywords: CLCBio; Klebsiella pneumonia; next-generation sequencing; Velvet; whole genome sequence


56


Symposium 2012

Identification of Interesting Genomic Properties of Clinical Isolates of

Mycobacterium tuberculosis

Azwin Ismail1*, LS Lee1,2, Irma SZ1, MH Zulkifli1, Izwan I1, Hanif AK1, Shafiq A1, LK Teh1, Norazmi MN2, Zainuddin F. Zainul2, Tang TH3, Najimuddin N4, Ngeow YF5, MZ Salleh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; 3 Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang, Malaysia; 4School of Biological Sciences, Universiti

Sains Malaysia, Penang, Malaysia; 5Faculty of Medicine Building, University of Malaya, Malaysia.


Abstract Tuberculosis meningitis (TBM) is the most severe extrapulmonary complication of Tuberculosis caused by the obligate human pathogen, Mycobacterium tuberculosis (MTB). It has distinctive rod shaped with rich lipid cell wall. It is widely studied due to its virulence and high prevalence which had contributed to expanding global health crisis and re-emergence of TB cases. A whole genome sequencing study using clinical isolates of Mycobacterium tuberculosis was conducted with the aim to investigate and provide new insight to the pathogenicity and virulence of this pathogen. Mycobacterium tuberculosis LRGS_TB008 was isolated from human cerebrospinal fluid in a patient diagnosed with tuberculosis. The DNA was sequenced using a Genome Analyser IIx (Illumina) sequencer. The reads were assembled using CLCBio (CLC Genomics Workbench version 5.1) and annotated using BASys and RAST. The whole genome data generated approximately 41X depth of coverage assuming a genome size of 4 megabases. The genome sequence assembled comprised of a circular chromosome of 4,411,532 bp, with high G+C content of 67%, 4,231 potential protein-coding sequences and 48 RNAs genes. Annotation of LRGS_TB008 genome analysis was performed using the reference genome sequence of M. tuberculosis H37Rv. The TB008 genome was most similar to strain H37Rv with 99% nucleotide sequence identity over the alignable sequences. A total of 672 new SNPs were identified when LRGS_TB008 were compared with M. tuberculosis H37Rv genomes. However, further validation on the functionality of the SNPs is required. Keywords: Mycobacterium tuberculosis, pathogenicity; virulence; whole genome sequencing

mailto:[email protected]%20/%[email protected]

57


Symposium 2012

Comparative Study Using Bioinformatics Pipeline for Malaysian Mahseer (Tor tambroides) Mitogenome Assembly

Norfatimah MY1,2*, LK Teh3, MZ Salleh3, Mat Isa MN4 and Siti Azizah MN2

1Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, 40450, Malaysia; 2School of

Biological Sciences Universiti Sains Malaysia Penang 11800, Malaysia; 3Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 4Malaysia Genome Institute, Kajang, 43000,

Malaysia.


Abstract Animal mitochondrial DNA has been studied extensively to address problems in population genetics and systematic of closely related species and individuals within a species as well as to detect heteroplasmy levels (mitochondrial diseases) in human. The goal of this study is to develop computational methods to analysed NGS data generated for Tor tambroides (Malaysian Mahseer) mitochondrial genomes (mitogenome). Two assembly approaches which is Velvet 1.2.07 (Linux based platform) and CLC Genomics Workbench 4.9 (automated commercial software) for the mitogenomic references mapping assemblies were developed, applied and compared. Total Genomic DNA of a single individual sample was isolated from the caudal fin using a tissue DNA extraction kit (Biotools, Madrid, Spain) and produced at least 5µg in 50µl for the concentration and must be in the ratio of 1.8-2.0 at A260/280. The DNA sample was then prepared using Illumina Truseq kit and sequenced by Illumina Genome Analyser IIX with 2x100 bp read length (Paired-End). The sample was sequenced in one lane to obtain a high amount of data. Before the sequence reads were assembled, the reads were subjected to quality control procedures to avoid the presence of low quality bases. The total number of reads after trimmed by using both approach is 75 million from the total reads of 80 million with the average length of 94.85 and the total bases are 7.1 Gb. The most related cyprinidae family, Cyprinus carpio with ID NC_001606 was choose and mapped to Tor tambroides data to perform assembly. Results shows only 2.2 Mb were mapped to the reference mitogenome by CLC and 1.6 Mb by Velvet. Total reference coverage for both approach are slightly difference with 134 X compared 95 X for CLC and Velvet respectively. A detailed study on the bioinformatics pipeline for mitogenomic study would allow comparison to be made with various bioinformatics tools which could generate high-quality mitogenome. Keywords: CLC Bio; mitogenome; NGS; Tor tambroides; Velvet


58


Symposium 2012

HLA-B*1502 is a Significant Marker for Carbamazepine-Induced Stevens Johnson Syndrome (SJS): Pre-Implementation Trial of HLA-B*1502

Pharmacogenotyping in a Local Hospital in Malaysia

Hasbullani Z1, SNA Syeith1, RN Che Omar1, LK Teh1, Fazleen HMH1, Sharina H1, Syafirul MS1, Shanthi Datuk Puvana Raja2, Md Hanip Rafia2, MI Ismail1, MZ Salleh1


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Institute of Neurology, Hospital Kuala Lumpur, Wilayah Persekutuan, Malaysia.


Abstract Stevens Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) are two fatal adverse drug reactions (ADR) encountered in some patients prescribed carbamazepine (CBZ) or phenytoin. FDA USA had published a medical alert which recommended HLAB*1502 screening before these 2 drugs were prescribed to patients of Asian ancestry as the likelihood of SJS/TEN were highly associated with HLAB*1502. The aim of this study is to investigate the relevance of carbamazepine-induced severe cutaneous adverse drug reaction with HLA-B*1502 in a cohort of patients. Seventy five (75) patients attending clinics at Department of Neurology, Hospital Kuala Lumpur (HKL) were recruited. DNA was extracted from blood samples obtained from each patient. Patients‟ genotypes were determined by Alleles Specific Polymerase Chain Reaction (AS-PCR) developed in-house. Among the 75 patients, 41 were newly registered patients and genotype screening was conducted before patients were prescribed with CBZ. Among the patients in this group, 21.95% were positive for HLAB*1502. In another cohort in which CBZ had been withdrawn due to SJS/ TEN; all of them (6) were positive for HLAB*1502. One patient with positive HLAB*1502 however did not develop SJS/TEN and is therefore tolerant. The calculated ratio of patients at risk of developing SJS/TEN based on this small samples size is 238.33 (95% CI : 8.6783 – 6545.42; p =0.0012). We conclude that the implementation of HLA-B*1502 screening is necessary to avoid patients at risk of SJS/TEN from being prescribed CBZ due to the high odd ratio of observed in this study. Keywords: ADR; CBZ; HLA-A*1502; SJS-TEN



59


Symposium 2012

Genetic Polymorphism of CYP3A4*18 and CYP3A5*3 in Malaysian Kidney Transplant Patients

MR Muda, S Hamzah, JKS Shia, LK Teh, MZ Salleh



*Corresponding author: [email protected]/[email protected]

Abstract Tacrolimus has been widely used in combination with other immunosuppressant to prevent allograft rejection after transplantation. It is characterised by narrow therapeutic window that makes the needs for therapeutic drug monitoring essential to prevent rejection or toxicity event. Extensive researches had shown the involvement of cytochrome P450 (CYP) enzymes in contributing to interindividual differences of pharmacokinetic and bioavailability of tacrolimus in kidney transplant patients. CYP3A4 and CYP3A5 are the major enzymes of CYP3A subfamily that are abundantly expressed in the liver and have the capacity to metabolise approximately up to 50 % of marketed drugs including tacrolimus. Based on previous studies, it has been suggested that polymorphism of CYP3A5*3 affected the expression of the enzyme by producing cryptic splicing site while polymorphism of CYP3A4*18 causes amino acid alteration that produce non-functional enzyme. The objective of this study is to investigate the influence of genetic polymorphism of CYP3A5*3 and CYP3A4*18 toward tacrolimus level among kidney transplant patients in Malaysia population. Eighty-kidney transplant patients treated with tacrolimus were recruited. DNA was extracted from peripheral venous blood using salting out method. They were genotyped for CYP3A5*3 and CYP3A4*18. The genotypes were correlated with tacrolimus dose adjusted trough levels (dose/body weight) per at 3-, 6-, 9-, and 12-month post-transplantation. At 3-month post transplantation, tacrolimus dose adjusted trough levels were higher for patients with CYP3A5*3/*3 genotype compared to those with CYP3A5*1/*1 genotype (2.31; 95% CI 1.6-3.6 vs. 0.75; 95% CI 0.56-1.7) ng/ml/mg/kg, p< 0.0001). Similar pattern were observed for the association of tarcolimus level with genotypes at 6-, 9-, and 12-months post-transplantation. CYP3A4 polymorphism has no significant correlation with the tacrolimus dose adjusted trough level. In conclusion, patients who carry CYP3A5*3/*3 require lower doses of tacrolimus to reach target trough level as compared to those with CYP3A5*1/*1 genotype. Keywords: tacrolimus; CYP3A polymorphism; kidney transplantation

60


Symposium 2012

The Implication of the Polymorphism of UGT1A6 among Cardiovascular Disease (CVD) Patients Treated with Aspirin

NJ Abdul Jalil1*, MZ Salleh1, O Maskon2, A Derahman1, RN Che Omar1, H Zakaria1, JKS

Shia1, LK Teh1*

1Pharmacogenomics Centre (PROMISE), Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Cardiology,

Hospital Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia.


Abstract UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major enzyme involved in the glucuronidation of salicylic acid. UGT1A6 gene which encodes UGT1A6 is polymorphic. The single nucleotide polymorphisms (SNPs) of UGT1A6 gene that play important role in the metabolism of salicylic acid include UGT1A6*2 (A541G, rs2070959) and UGT1A6*3 (A522C, rs1105879). The aim of this study is to determine the allelic frequency of UGT1A6 gene in Malaysian population. The project was approved by relevant Research Ethics Committee. A total of 151 patients with cardiovascular disease who were treated with 75 -150 mg daily dose of aspirin were recruited. DNA was extracted from the blood samples and genotyped for the single nucleotide polymorphisms (SNPs) using polymerase chain reaction (PCR). Analysis of UGT1A6 genotypes in these patients revealed that 71 were homozygote UGT1A6*1/*1. Four percent (4%) were heterozygote UGT1A6*1/*2, 14% were heterozygote UGT1A6*1/*3, and 28% were heterozygote UGT1A6*2/*3. Of the 151 patients, only 10 patients were homozygous for UGT1A6*3/*3. No homozygous UGT1A6*2/*2 was detected. The allele frequencies of UGT1A6*1, UGT1A6*2 and UGT1A6*3 were 0.6 (95% CI 0.08-0.96), 0.2 (95% CI 0.01 – 0.87) and 0.2 (95% CI 0.01 – 0.87) respectively. Allele frequencies of UGT1A6*1 were 0.56, 0.62 and 0.40 in the Malays, Chinese and Indians respectively. The frequency of 0.2 for all the 3 ethnic groups. The Indians have the highest frequency of UGT1A6*3 [0.40; 95% CI 0.22 – 0.40) compared to the Malays (0.30) and Chinese (0.20) UGT1A6 is highly polymorphic in Malaysia and therefore may have important implication in personalised medicine to ensure safe and cost effective therapy. Keywords: aspirin; cardiovascular disease; Malaysian population; UGT1A6


61


Symposium 2012

Genetic polymorphism of CYP2C19 among the Cardiovascular Patients of Three Ethnic Groups in Malaysia

RN Che Omar1*, NJ Abdul Jalil1, A Derahman1, H Zakaria1, O Maskon2, JKS Shia1, MZ

Salleh1, LK Teh1*


(UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; 2Department of Cardiology, Hospital Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia


Abstract Cytochrome P450 is 2C19 is a highly polymorphic enzyme that cause marked interindividual and interethnic variation in the metabolism and disposition of many therapeutic agents. The goal of this study was to determine the frequencies of important allelic variant of CYP2C19 in a cohort of cardiovascular patients attending follow up clinic at Hospital UKM. This project is approved by local Research Ethics Committee. Patients were explained the study protocol and written informed consents were obtained before the study. A total of 150 cardiovascular patients were recruited. They were unrelated Malaysians aged 18–65 years. Blood sample were obtained from antecubital vein for DNA extraction. All samples were genotyped for single nucleotide polymorphisms (SNPs) of CYP2C19*2 (rs4986893) and CYP2C19*3 (rs4244285) using polymerase chain reaction (PCR). The allele frequencies for CYP2C19*1 are 81%, 75% and 71% in the Malays, Chinese and Indian respectively. CYP2C19*2 was present at 16%, 17% and 26% in the Malays, Chinese and Indian respectively; and carriers of CYP2C19*3 comprised of 3%, 8% and 3% in the Malays, Chinese and Indian respectively. There were significant differences in the poor metaboliser genotypes (*2/*2, *2/*3, *3/*3) between the Malays and Indians (Chi square test, P ≤ 0.05). We conclude that frequencies of CYP2C19 genotypes were different among the three major ethnic groups in Malaysia. This result is useful for clinical practitioners in understanding the differences in drug responses observed among their patients and the potential of molecular diagnostics in personalised drug therapy. Keywords: cardiovascular disease; CYP2C19; poor metaboliser



62


Symposium 2012

Development of a Pharmacogenotyping Method for Detection of HLA Polymorphism in Personalising Allopurinol Therapy

N Mohamed Ali*, LK Teh*, Fazleen HM Hatta, S Hamzah, MZ Salleh




Abstract Previous studies performed in Europe and Israel had shown that allopurinol is commonly associated with drug induced Stevens - Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN). Human leukocyte antigen (HLA) A33 and B58 was the first genetic variants associated with allopurinol induced skin eruption. On the other hand, researchers in Taiwan (Han Chinese population), Thailand and Korea showed that there is a strong association of HLA-B*5801 allele and severe cutaneous adverse drug reaction (SCAR). The pathogenesis of allopurinol induced cutaneous reaction is consistent with delayed type immune mediated reaction. Observed familial predisposition in conjunction with the pathogenesis of allopurinol induced cutaneous reaction, alludes to the potential of genetic based immunological markers. The aim of this study is to develop an accurate PCR based method to determine the genetic variants of HLA-B*5801 which can be used for association study of HLA polymorphism with allopurinol induced cutaneous adverse drug reaction in Malaysian population. Genomic DNA was extracted from the whole blood sample using salting out method. Allele specific polymerase chain reaction (PCR) method was developed based on specific 3‟ end primer mismatch for tagging SNPs of HLA-B*5801. To avoid any misinterpretation of the PCR result, positive control containing DNA fragment of tagging SNPs for HLA-B*5801 were cloned into plasmid. Both the positive and negative controls were used for quality assurance and were included in each PCR run. Method was validated by direct sequencing. The method developed is useful to study the association of HLA polymorphism with the susceptibility of allopurinol induced cutaneous adverse drug reactions. The assay developed can be used in clinical practice to help identify patients at risk of SCAR and thus reduce cost of patient care. This will help realise personalised medicine and improve patient‟s quality of life. Keywords: human leukocytes antigen (HLA); polymerase chain reaction (PCR); severe cutaneous adverse reaction (SCAR); Steven – Johnson Syndrome (SJS); Toxic Epidermal Necrolysis



63


Symposium 2012

Hepatoprotective Activity of Methanol Extract of Melastoma malabathricum Leaves on Paracetamol-Induced Liver Injury in Rat

SS Mamat1*, N Mohtarrudin2, ZA Zakaria1*

1Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2Department of Pathology, Faculty of Medicine& Health Sciences,Universiti Putra Malaysia, 43400 UPM , Serdang Selangor , Malaysia.


Abstract The aim of the present study was to determine the hepatoprotective effect of methanol extract of Melastoma malabathricum L. leaves (MEMM; family Melastomaceae) against paracetamol (PCM)-induced liver toxicity. The dried, grinded leaves of M. malabathricum (40 g) were soaked with 800 ml methanol (1:20 (w/v)) for 72 hours at room temperature. The supernatant was collected, filtered using cotton wool followed by Whatman No. 1 filter paper, and finally subjected to the rotary evaporation process. This process was repeated for another two times. The vehicle (10% DMSO), 200 mg/kg silymarin or MEMM (50, 250 and 500 mg/kg) were administered orally once daily for 7 successive days in rats (n=6). Hepatotoxicity was induced by the administration of PCM (3 g/kg) 3 hours after the last MEMM administration. On the 9th day, blood was collected followed by the immediate sacrifice of animals to collect the liver for histopathological examination. Hepatic enzymes, AST (aspartate aminotransferase), ALP (alkaline phosphatase) and ALT (alanine aminotransferase), were assessed as indicators of liver damage. The results obtained showed that pre-treatment with MEMM significantly (p<0.05) reduced the PCM-induced serum levels of hepatic enzyme markers compared to negative control group (P<0.05). Liver histopathology also showed that MEMM reduced the incidence of liver lesions including hepatic steatosis, lymphocyte infiltration and marked necrosis as compared to negative control group. In conclusion, the methanol extract of leaves of M. malabathricum exerts potential hepatoprotective property that warrants further investigation. Keywords: hepatoprotective activity; liver enzyme markers; Melastoma malabathriucm; methanol extract; paracetamol

64


Symposium 2012

Chemopreventive Potential of Melastoma malabathricum Leaves Extract in DMBA/Croton Oil-Induced Mouse Skin Carcinogenesis

Samuel Oii KK*, R Rodzi, F Othman, ZA Zakaria

Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra

Malaysia, 43400 UPM Serdang, Selangor, Malaysia.


Abstract The ideas that cancer is preventable had contributed to research on plant-based products for chemoprevention. Melastoma malabathricum known as “Pokok Sendudok” in Malays community is a plant used traditionally to treat diarrhoea, dysentery, cuts, wounds, and other ailments. Previous studies had shown M.malabathricum exhibited in-vitro anti-proliferative activity. The aim of this study was to investigate the anti-carcinogenic activity of methanol leaves extract of M.malabathricum (MMLE) in two stage mouse skin carcinogenesis model. The skin tumours were initiated by a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) topically on shaved dorsal skin, followed by twice weekly administration of 1% croton oil (100 μg/100 μl) as tumour promoter. A varying doses (30, 100 and 300 mg/kg of body weight) of MMLE were tested on DMBA/croton oil-induced mouse skin carcinogenesis. Tumours were examined and measured weekly for 15 weeks. At the end of the experiment, animals in carcinogen control developed tumour burden of 5.33 ± 1.36, in contrast mice treated with 30, 100 and 300 mg/kg MMLE developed tumour burden of 4.40 ± 1.68, 1.16 ± 0.41 and 1.00 ± 0.13 with percentage of tumour incidence of 62.5%, 37.5% and 12.5% respectively. Mice treated with 100 mg/kg and 300 mg/kg showed higher tumour volume per individual tumour in contrast to carcinogen control, although they are significantly different (P<0.05). Histopathological findings of skin showed different degree of suppression of MMLE on DMBA/croton oil-induced hyperplasia and papillomatosis in contrast to carcinogen group. Results from the study indicated pre-treatment of MMLE 30 minutes prior administration of croton oil resulted in protection against skin carcinogenesis in a dose-dependent manner. Keywords: 7, 12-dimethylbenz[a]anthracene; chemopreventive; croton oil; Melastomaceae; Melastoma malabathricum, methanol extract

65


Symposium 2012

Mycolic Acids as Diagnostic Markers for Tuberculosis Case Detection and Drug Efficacy

Guanghou Shui1*, Anne K Bendt1*, Ignasius A Jappar1, Hui Ming Lim1, Marie Laneelle3, Maxime Hervé5, Laura E Via4, Gek Huey Chua1, Martin W Bratschi1,10, Siti Zarina Zainul Rahim6, Ang Lay Teng Michelle6, Soo-Hee Hwang7, Jong-Soek Lee8, Seok-Yong Eum8,

Hyun-Kyung Kwak8, Mamadou Daffé3, Véronique Dartois2,5, Gerd Michel9, Clifton E. Barry III4, Markus R Wenk1,2,10

1Yong Loo Lin School of Medicine, National University of Singapore, Department of Biochemistry

and 2Department of Biological Sciences, National University of Singapore, Singapore; 3Département Mécanismes Moléculaires des Infections Mycobactériennes, IPBS-UMR, Toulouse Cedex, France; 4Tuberculosis Research Section, Laboratory of Clinical Infectious Disease, NIAID,

NIH, USA; 5Novartis Institute for Tropical Diseases, Singapore; 6Department of Microbiology, National University of Singapore, Singapore; 7National Masan Tuberculosis Hospital, Masan, Republic of Korea; 8International Tuberculosis Research Center, Masan, Republic of Korea;

9Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland; 10Swiss Tropical and Public Health Institute, University of Basel, Socinstr. 57, P.O. Box, 4002 Basel.


Abstract Tuberculosis, which is caused by infection with Mycobacterium tuberculosis, remains a major global health threat, with over nine million new cases and close to two million deaths annually. Detection of active tuberculosis cases in endemic countries still relies on sputum smear microscopy, which only has an average sensitivity of 65% and which is highly dependent on the individual examiner. The more sensitive culture methodology on the other hand takes weeks, thus further delaying initiation of treatment and potential spread of pathogens. Better diagnostics are therefore urgently needed. Since a high percentage of patients are co-infected with HIV, diagnostics should be suitable for immunocompromised individuals. Furthermore, a specific molecular marker which reflects clearance of bacteria from lungs would be helpful to monitor treatment response and drug efficacy in pre-clinical and clinical trials. Keywords: diagnostic marker; lipidomics; mass spectrometry; Mycobacterium tuberculosis; mycolic acids


66


Symposium 2012

A Sensitive and Efficient Method for the Quantification of Phosphorylated Sphingoid Bases in Biological Samples

*Pradeep Narayanaswamy1*, Husna Begum2, Bowen Li3, Federico Torta4, Rudolf Grimm5, Markus

R. Wenk1-3

1Department of Biological Sciences, National University of Singapore, 2NUS Graduate School, National University of Singapore, 3Department of Biochemistry, National University of Singapore,

4Mechanobiology Institute, National University of Singapore, Singapore, and 5Agilent Technologies, Santa Clara, USA.


Abstract Sphingosine-1-Phosphate (S1P) is an important bioactive lipid mediator and a normal constituent of human plasma. S1P is synthesized from sphingosine by the enzyme Sphingosine Kinase and its biological activity is modulated through binding to specific receptors. S1P plays important physiological roles in the regulation of cell growth, differentiation, motility and survival; in addition it participates in pathological conditions like autoimmunity, cancer, angiogenesis and myocardial infarction. Thus S1P gained clinical importance as biomarker for diseases. For the detection of S1P in biological matrices we developed a simple, quick, sensitive and reliable assay by using an Agilent HPLC-Chip MS system after derivatisation of the sample. New method is a valuable tool for the detection of S1P levels from small amounts of human and mouse plasma samples. Keywords: angiogenesis; myocardial infarction; sphingosine-1-phosphate

Designed by: Mohd Izwan Ismail, PROMISE


Symposium 2012

APPENDICES

xi


Symposium 2012

List of Participants

No. Name Email Organisation

1. Aishah Adam (Prof. Dr.)

[email protected]

Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

2. Anne Kathrin Bendt (Dr.)

[email protected] Department of Biochemistry, Yong Loo Lin School of Medicine, and Department of Biological Sciences, National University of Singapore.

3. Ali Ashrafzadeh [email protected] School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Selangor, Malaysia

4. Asbiyatulaida Derahman

[email protected] Pharmacogenomics Centre, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

5. Azidah Ali [email protected] Collaborative Drug Discovery Research, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

6. a Azizah Othman [email protected] Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

7. Baharudin Ibrahim [email protected] School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia

8. Che Nor Adlia Enche Ady

[email protected] Collaborative Drug Discovery Research, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

9. Chin Siok Fong (Dr.)

[email protected]

Sime Darby Technology Centre Sdn. Bhd., 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh Silikon, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

10. Christopher Bowen

[email protected] Agilent Tech. Australia

11. David Ross Appleton

[email protected]


12. David Wishart (Prof. Dr.)

[email protected] Department of Computing Science and Biological Sciences at the University of Alberta, Edmonton

13. Dayana Sazereen Md Hasni

[email protected] Collaborative Drug Discovery Research, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

14. Delphia Shem [email protected]

Agro-Biotechnology Institute, Malaysia (ABI), Serdang, Selangor, Malaysia

15. Erda Syerena Rosli


16. Fang Fang (Dr.) [email protected] Application Chemist for Bruker BioSpin, GmbH Silberstreifen 4 76287, Rheinstetten, Germany

17. Fauziah Abdullah [email protected] Phytochemistry Programme, Natural Products Division, FRIM, Kepong, Selangor, Malaysia








xii


Symposium 2012

18. Fazleen Haslinda Mohd Hatta


19. Forough Ebrahimi [email protected] Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia

20. Gunaletchumy

Selva Perumal [email protected]

Department of Medical Microbiology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia

21. Hanum Ya’akub [email protected] Collaborative Drug Discovery Research, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

22. Hasbullani Zakaria [email protected] Pharmacogenomics Centre, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

23. Hemalatha Visuvalingam

[email protected] Universiti Sains Malaysia, Penang, Malaysia

24. Irma Syakina Mohd Zaki


25. Izmil Haikal Zainol [email protected] Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

26.

Jean-Frédéric Weber

(Prof. Dr.)

[email protected]

Atta-ur-Rahman Institute for Natural Product Discovery, Level 9, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

27. John Shia Kwong Siew (Dr.)


28.

Kalavathy Ramasamy (Assoc. Prof. Dr.)


29. Kamalrul Azlan Azizan

[email protected] Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia

30. Laith Razzak (Dr.) [email protected]

Department of Fisheries and Aquaculture, Faculty of Fisheries and Aqua Industry, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia

31. Lee Lian Shien [email protected] Pharmacogenomics Centre, Level 7,Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

32. Lim Vuanghao [email protected]

Advanced Medical & Dental Institute, Universiti Sains Malaysia, No 1-8 (Lot 8), Persiaran Seksyen 4/1, Bandar Putra Bertam, Kepala Batas. Pulau Pinang, Malaysia

33. Loke Mun Fai (Dr.) [email protected] Department of Microbiology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia

34. Lydiatul Shima

Ashari [email protected]

School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia

35. Mailina Jamil [email protected] Herbal Products Development Programme, Natural Product Division, FRIM, Kepong, Selangor, Malaysia














xiii


Symposium 2012

36. Markus R. Wenk [email protected]

Department of Biochemistry, Yong Loo Lin School of Medicine, and Department of Biological Sciences, National University of Singapore

37. Matthias Pelzing (Dr.)

[email protected] Application Manager for Bruker Daltonics Asia Pacific/ Japan, Bruker Biosciences Pty Ltd, Australia

38. Mohamad Izwan Ismail


39. Mohd Hafis Yuswan

[email protected] Agro-biotechnology Institute, Serdang, Selangor, Malaysia

40. Mohd Ikhwan Ismail


41. Mohd Ikmal Hanif Abdul Khalid


42. Mohd Izwan Mohamad Yusof


43. Mohd Nazri Hj. Abu


44. Mohd Rahimi Muda


45. Mohd Salleh Rofiee


46. Mohd Shafiq Aazmi


47. Mohd Zaki Salleh (Prof. Dr.)


48. Muhamad Hafiq Zulkifli


49. Muhammad Husaini Ismail


50. Nazrien Kaman [email protected] Agro-biotechnology Institute, Serdang, Selangor, Malaysia

51.

Noorzaid Muhamad (Assoc. Prof. Dr.)

[email protected] Royal College of Medicine Perak , Universiti Kuala Lumpur, Malaysia

52. Nor Amalina Ahmad Alwi


53. Norfatimah Mohamed Yunus

[email protected] Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia

54. Nor Izwani Mohamed



















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Symposium 2012

55. Nor Nadia Ban [email protected]

Collaborative Drug Discovery Research, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

56. Norleen Mohamed Ali


57. Norlelawati A.Talib

[email protected] Department of Basic Medical Sciences, Kulliyah of Medicine, International Islamic University Malaysia, Kuantan Campus, Kuantan, Pahang

58. Nornazliya Mohamad


59. Norzulaani Khalid (Prof. Dr.)

[email protected]

Centre for Biotechnology in Agriculture Research, Institute of Biological Sciences, Faculty of Science, University Malaya, Kuala Lumpur, Malaysia

60. Nur Azwin Ismail [email protected] Pharmacogenomics Centre, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

61. Nur Jalinna Abdul Jalil


62. Nur Syafiqah Rahim


63. Nur Syahidah [email protected]

Agro-Biotechnology Institute Malaysia (ABI)

64. Nurul Aqilah Iberahim

[email protected] Department of Aquaculture Science, Faculty of Fisheries and Aqua-Industry, University Malaysia Terengganu, Mengabang Telipot, Terengganu

65.

Nurul Aqmar Mohamad Nor Hazalin

[email protected]


66. Nurul Ashikin Md. Hazmi

[email protected] Agro-biotechnology Institute, Serdang, Selangor, Malaysia

67. Nurul Hamiyah Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

68. Nurul Shahfiza Noor

[email protected]

Advanced Medical & Dental Institute, Universiti Sains Malaysia, No. 1-8, Persiaran Seksyen 4/1, Bandar Putra Bertam, Kepala Batas, Penang

69. Pradeep Narayanaswamy

[email protected] Department of Biochemistry, Yong Loo Lin School of Medicine, and Department of Biological Sciences, National University of Singapore

70. Qamarul Hafiz Zainul Abidin


71. Rehvathy Vellayan [email protected] Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur

72. Richard Muhammad

[email protected] Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

73. Robert Trengove [email protected] Murdoch University Metabolomics Australia Node, 90 South Street, Murdoch WA 6150, Australia















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Symposium 2012

74. Robin Philp [email protected] Life Sciences Group, Agilent Technologies, Singapore

75. Roihanah Rodzi [email protected] Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

76. Rose Iszati Ismet Nayan


77. Rosmadi Yusoff (Dr.)


78. Rudolf Grimm [email protected] Agilent Technologies, Santa Clara, USA

79. Ruhil Hayati Hamdan

[email protected] Aquatic Animal Health Unit (AAHU), Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia

80. Ruhil Nadirah Che Omar


81. Ruzianisra Mohamed

[email protected] Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor DE, Malaysia

82. Salfarina Ramli [email protected]. my

Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor DE, Malaysia

83. Selene Tan [email protected] Agilent Tech. Malaysia

84. Sharina Hamzah [email protected] Pharmacogenomics Centre, Level 7, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

85. Siti Azma Jusoh (Dr.)


86. Siti Nooraishah Hussin

[email protected]


87. Siti Syariah Mamat

[email protected] Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

88. Suhana Samat Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia

89.

Tamil Chelvan Meenakshi Sundram

[email protected] CEBAR, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

90. Tan Hooi Poay [email protected] Phytochemistry Programme, Natural Products Division, FRIM, Kepong, Selangor, Malaysia

91. Tang Thean Hock (Assoc. Prof. Dr.)

[email protected]

Advanced Medical & Dental Institute, Universiti Sains Malaysia, No. 1-8, Persiaran Seksyen 4/1, Bandar Putra Bertam, Kepala Batas, Penang, Malaysia

92. Teh Lay Kek (Prof. Dr.)


93. Theresa Ng Lee Mei

[email protected]


94. Theresa Yap Wan Cheng

[email protected] Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur















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Symposium 2012

95.

Thomas P Hennessy (Prof. Dr.)

[email protected] Life Sciences Group, Agilent Technologies, Singapore

96. Ute Roessner (Dr.) [email protected] Australian Centre for Plant Functional Genomics and Metabolomics Australia, School of Botany, the University of Melbourne, 3010 Victoria, Australia

97. Wan Iryani Wan Ismail (Dr.)


98. Wong Wai Kuan [email protected] Universiti Malaya, Kuala Lumpur, Malaysia

99.

Yumi Zuhanis Has-Yun Bt. Hashim (Dr.)

[email protected]

Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia

100. Zafirah Liyana [email protected]


101. Zakaria M Bannur (Dr.)


102. Zainul Amiruddin Zakaria

[email protected] Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

103. Zam Zureena Mohd Rani

[email protected]

UKM Medical Biology Institute (UMBI) and Department of Medicine, Universiti Kebangsaan Malaysia Medical Centre, (UKMMC), Kuala Lumpur, Malaysia

104. Zu Zunoliza Abdullah [email protected]

Phytochemistry Programme, Natural Products Division, FRIM, 52109 Kepong, Selangor, Malaysia








xvii


Symposium 2012

Floor Plan

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Symposium 2012

Contact Number

Secretariat of Symposium

Secretariat Team members Contact number

Hospitality

(transport/ hotel)

: Mrs. Nurul Aqmar Mohd Nor Hazalin




019-3439154

017-6540441

013-9501647

017-3767660

Exhibition

(sponsors)

: Mrs. Lee Lian Shien

: Mrs. Norleen Mohamed Ali

: Mr. Muhammad Husaini Ismail

016-3053405

012-3457994

019-5279610

ICT

(for oral communication)

: Mr. Mohd Izwan Mohamad Yusof

: Mr. Mohd Ikmal Hanif Abdul Khalid


017-9383035

019-6784205

019-3663859


Symposium 2012

Conference Evaluation Form

1. Please rate each of the following:

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(comment/recommendation)?

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MyMS Application Form

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Membership Subscription fee is RM 50.00 per annum. In addition, there is a joining fee of RM 10.00

payable in the first year of membership. The fees are payable to Malaysian Society Metabolomics (UOB

362-3-000178-2).

Please post your application form and payment (crossed cheque) to:

Malaysian Metabolomics Society

Pharmacogenomics Centre (PROMISE),

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Universiti Teknologi MARA,

42300 Puncak Alam, Selangor.

Tel: +603-3258 4652

Fax: +603-3258 4602

4th australasian metabolomics symposium

Documents

australasian symposium

advantage of metabolomics

metabolomics laboratory

beauty of metabolomics

symposium strong

original symposium

universiti teknologi

shah alam