Research Highlights in 4Bs: Biosensors, Biodiagnostics, Biochips and Biotechnology by Dr. Subhash Bhore

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Proceedings of the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) held on the campus of AIMST University, Kedah, Malaysia, in April 2016

Editor Subhash Bhore

2016

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology Subhash Bhore (Editor) Published by AIMST University 2016 ISBN: 978-983-43522-8-8 (Print version) eISBN: 978-983-43522-7-1 (e-Book version)

Financial support for the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) and for this Book was provided by:

Conference was jointly organized by:

Conference was supported by:

Published by AIMST University Printed by AIMST University Copyright Copyright © 2016: This book is an ‘Open Access’ type of publication for the free and permanent unrestricted online access to scholarly research articles and or abstracts. Authors retain copyright to their work, and a license is applied which allows users to download, copy, reuse and distribute data provided the original article is fully cited. This open access aims to maximize the visibility of research articles and abstracts, much of which is from publicly funded projects. Disclaimer: The information provided in this book is designed to highlight the research findings, views and or perspectives of respective researchers. While the best efforts have been used in preparing this book, Editor and or Publisher make no representations or warranties of any kind and assume no liabilities of any kind with respect to the accuracy or completeness of the contents and specifically disclaim any implied warranties. Neither the Editor nor Publisher of this book shall be held liable or responsible to any person or entity with respect to any loss or incidental or consequential damages caused, or alleged to have been caused, directly or indirectly, by the information highlighted herein. Readers should be aware that the information provided in this book may change. All full articles and abstracts published in this book are deemed to reflect the individual views of the authors and not the official points of view, either of the Editor or of the Publisher.

Edited by Dr. Subhash J. Bhore Senior Associate Professor Chairman, the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia; Telephone No.: +604 429 8176; e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com

Front Cover Design Mr. Mahes D.S. AIMST University, Malaysia Edition First; July 26, 2016

Dedication This book is dedicated to all speakers, participants and organizing committee members of the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) in recognition of their contributions for making event.

this

conference

successful

Conference Organizing Committee

Chairman Dr. Subhash J. Bhore Co-chairman Dr. Matiullah Khan Secretary Ms. Kalaiselvee Rethinam Scientific Committee Snr. Prof. Dr. M. Ravichandran Prof. Dato' Dr. Mohd Zaki Salleh Prof. Dr. Uda Hashim Prof. Dr. Teh Lay Kek Dr. Subhash J Bhore Dr. K. Marimuthu Dr. Lee Su Yin Dr. V. Ravichandran Dr. Werasak S. Dr. P. Balakumar Dr. Benchaporn L. Dr. S. Kathiresan Dr. Kazi Selim Anwar Dr. Sivakumar Pendyala Dr. Ramesh Kumaresan Secretariat Dr. Annie Jeyachristy Dr. Heera Rajandas Treasurer Mr. Arvinth Murugiah Mr. Vijayan Krishnan Logistics Committee Ms. Musalinah Buzri Mr. V. Krishnan Ms. Yoganandhini Mr. Christapher V. Mr. Mahes D.S. Mr. G. Prabhakaran Dr. D. Jawahar

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Publicity & Exhibitions Committee Dr. Sivachandran P. Mr. Siventhiran B. Mr. M. K. Faiz Mr. Dhanaraj R. Mr. P. K. Karuna Reception Committee Ms. Sridevi Visvanathan Ms. Elil Suthamathi Dr. Tahmina Monowar Ms. Azdlina First Aid and Safety Committee Dr. Sawri Rajan Dr. Leela A. J. Mr. Maheswaran S.

Foreword

It is my great pleasure to write this foreword for this book; because, I have attended this conference and witnessed the success of the whole event. First of all, I would like to thank all speakers, delegates, young researchers and participants of the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) for sharing their research findings, views and perspectives in the domains of Biosensors, Biodiagnostics, Biochips and Biotechnology. The 3rdRC4Bs-2016 brought together the leading scientists, academicians, researchers, students, entrepreneurs and industry players in the field of Biosensors, Biodiagnostics, Biochips and Biotechnology to discuss the latest developments in the respective fields. This conference provided a wonderful opportunity for all the participants not only to present their research contribution and interact with eminent colleagues but also to widen their professional network. I want to convey my special thanks to YBhg. Dato' Prof. Dr. Asma Binti Ismail, Honourable Director General, Department of Higher Education, Ministry of Higher Education Malaysia and Prof. Eiichi Tamiya, Osaka University, Japan for delivering a key note address and visiting the AIMST University. I wish to thank all the scientists and researchers who have travelled from 13 countries to participate in 3rdRC4Bs2016. I also wish to thank all co-organizers and supporters for supporting the 3rdRC4Bs-2016. A proper documentation of scientific information and or events is highly important in this modern world and I am extremely happy to know that full-length articles and abstracts received from the participants of the 3rdRC4Bs-2016 are being published in this book. I want to record my special thanks to Senior Associate Professor Dr. Subhash Bhore, a highly committed organizing Chairman and Editor of this book for his efforts in bringing out this book to document the event. I also thank the purpose driven scientific committee, organising committee members and volunteers for their contribution. I am very sure that this book will serve as a reference to students, researchers, scientists and all other stakeholders of the Biosensors, Biodiagnostics, Biochips and Biotechnology sectors. Thank you,

Senior Professor Dr. M. Ravichandran Chief Executive & Vice-Chancellor, AIMST University, Malaysia

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Preface

The 21st century is the age of biological sciences and a special emphasis on biotechnology is clearly noticeable woldwide. Biotechnology do have a tremendous potential not only to address the challenges in agriculture and health care sectors but also to generate the new jobs and wealth (bio-economy). In fact, biotechnology has become an integral part of the knowledge-based economy of developing and developed countries. In biotechnology industry and health care sector, biosensors, biodiagnostics and biochips based technologies are playing an important role in providing the most innovative products and services. In April 2016, the 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 (3rdRC4Bs-2016) was held at the AIMST University which provided a platform for scientists, researchers, academicians, students and stakeholders to share their knowledge, challenges, recent advances and future perspectives in the multidisciplinary areas of biosensors, biodiagnostics, biochips and biotechnology (4Bs). The scientific programme of the conference was rich and wide-ranging with 2 keynote talks, 14 invited plenary talks, 38 technical papers and about 50 posters’ presentation. Prof. Asma (Ministry of Higher Education, Malaysia) delivered a keynote address and highlighted various aspects of ‘Moving the Regional Biotechnology and Bioeconomy Forward’. By giving examples of policies and long term vision plan implemented by Chinese Government for moving their national bio-economy forward, she highlighted that most of the Asian countries including Malaysia have a great potential and resources to boost regional bio-economy; however, the lack of alignment of the numerous policies, strategies and initiatives makes it a challenge to coordinate implementation. In a keynote address, Prof. Eiichi Tamiya (Osaka University, Japan) highlighted the achievements, challenges and latest trends in ‘nanotechnology oriented biosensors and biomedical applications’. In a plenary talk, Prof. Prakash Kumar (NUS, Singapore) highlighted that we need to use modern biotechnological approaches to enhance the agricultural productivity for the global food security and sustainability. All 14 plenary talks were very comprehensive and highlighted the latest trends, achievements and challenges in various domain of biosensors, biodiagnostics, biochips and biotechnology. This book contains full-length articles, talk abstracts of all invited speakers, and abstracts of all technical papers presented in oral and poster sessions. I wish to thank and acknowledge the support of all co-organizers, supporters, and AIMST University. Nevertheless, the success of the conference was possible because of the hard efforts of dedicated and committed members of the organizing committee team as well as volunteers and they deserve the appreciation for it. Subhash Bhore July 26, 2016 ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Contents Foreword ....................................................................................................................... v Preface .......................................................................................................................... vi Contents ......................................................................................................................vii Full Length Articles ..................................................................................................... 1 Relevance of Biotechnological Applications for Global Food Security and Sustainability.............................................................................................................. 1 Nano- and Bio-technological Advancement to assist in the Determination of Halal Products.................................................................................................................... 10 Bacteriophages for Biocontrol of Foodborne Pathogens: An Overview ................. 20 Cloning and Expression of the Urease Operon from Helicobacter pylori J99 ........ 33 Production of Butter Flavour Concentrate from Butter fat with Lactic Acid Bacteria by Solid Substrate Fermentation .............................................................................. 42 Reconfigurable Filter Bank for Accurate Spectral Decomposition of EEG Signals57 Coenzyme Q10 Dietary Supplementation during Antitubercular Therapy Prevents Renal Damage in Rats .............................................................................................. 67 Safe Water as the Key to Food Safety and Global Health ....................................... 77 The Kratom Plant [Mitragyna speciosa (Korth.)] Paradox: Beneficial or Detrimental? ............................................................................................................. 84 Abstracts (Keynote and Plenary Talks) ................................................................... 90 Moving the Regional Biotechnology and Bioeconomy Forward ............................ 90 Nanotechnology Oriented Biosensors and Biomedical Application ....................... 91 Current Progress in Cholera Diagnostics ................................................................. 92 Supercomputing in Biotechnology: Making Sense of Big Data .............................. 93 Molecular Approaches to Fundamental Studies on Biomarkers and Development of Sustainable Rapid Nano-biodiagnostics to Enteric Diseases for Low Resources Settings ..................................................................................................................... 94 Bio-Applications of Innovative Nano-materials ...................................................... 95 Aptasensors: Bench to Bedside and Beyond ........................................................... 96 Recent Progress in the Production of Biodegradable Plastics from Palm Oil in Malaysia ................................................................................................................... 97 Recent Advances in Biosensors Based on Enzyme Inhibition ................................ 98 Genomics of the Endangered Orang Asli: Disease Susceptibility and Sustainability .................................................................................................................................. 99 ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Highly Sensitive Detection of DNA Hybridization and Immunoassay Based on Nanomaterials ........................................................................................................ 100 Fungal Secondary Metabolites - A Pharmaceutical Chemist Perspective ............. 101 Foot-and-Mouth Disease: Current Scenario in Asia and Bangladesh ................... 102 Abstracts (Oral Presentations) ............................................................................... 103 Paper-based visual detection of Salmonella bacteria using Isothermal DNA amplification and magnetic beads aggregation ...................................................... 103 Development of a Reverse Hybridization Assay (RHA) for Simultaneous Identification of Salmonella Serotypes Causing Enteric Fever ............................. 104 Decrypting the Evolutionary Path of Antimicrobial Resistance of Acinetobacter baumannii via Next-Gen Sequencing .................................................................... 105 Isoluminol-functionalized gold nanoparticles and graphene oxide nanoribbons composite for development of enzyme-based electrochemiluminescence biosensors ................................................................................................................................ 106 Disposable Screen-Printed Electrodes Modified With Nanoparticles for Sucrose Sensor..................................................................................................................... 107 Analysis of Chalcone-Flavanone Isomerase (CHI) Gene cDNA Isolated from American oil-palm (Elaeis oleifera) Mesocarp Tissue cDNA Library ................. 108 Non-Protein coding RNA genes as novel diagnostic markers to detect pathogenic bacteria ................................................................................................................... 109 Herbal Based Stabilizers of Native and Misfolded State of Nuclear Co-repressor (N-CoR) ................................................................................................................. 110 Hepatoprotective effect of methanol extract of Polygonum minus leaves in carbon tetrachloride-induced liver damage in rats ............................................................. 111 Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail, Pomacea maculata ................................................................................................................. 112 Design and Characterization in Time of an On-off DNA Biosensor ..................... 113 Optimization of PCR for Rapid Detection of CTX-M Gene in ESBL Producing Klebsiella pneumoniae Clinical Isolates ................................................................ 114 Umami Tasting Detection Based Electrochemical Sensor .................................... 115 Detection of Salmonella enterica serovar Typhi Form Water Samples and Its Association with Geographical Clustering of Enteric Fever ................................. 116 The Fabrication of Membrane-Based Pneumatic Microvalves in Microfluidic System .................................................................................................................... 117 Role of Outermember Proteins (OMP) and Lipopolysaccharides (LPS) in Antibody Response Against Pasteurella multocida type B-2 in Bovines ............................. 118

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Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Prooxidant Properties .................................................................................................. 119 Sensitivity Analysis of Graphene Based Surface Plasmon Resonance Biosensor 120 Ameliorative Effect of Curcumin on Olanzapine Induced Obesity in Sprague Dawley Rats ........................................................................................................... 121 Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of Sudan I contamination in chili ............................................................................................ 122 Bioengineering of Tacca integrifolia (Bat flower): Effects of Hormones on in vitro Rooting and Production of Taccalonolides ............................................................ 123 Isolation, characterization and potential application of bacteriophages for phage therapy.................................................................................................................... 124 Eco-friendly Biosynthesis of Atrocarpus altilis Mediated Silver Nanoparticles Characterization and Evaluation of its Antimicrobial and Antioxidant Potential . 125 Mutiplex Isothermal Amplification for Detection of Melioidosis ......................... 126 Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves Nebulization and Phononic Crystal Structures ...................................................... 127 Development of a Novel Duplex PCR Assay for Specific Detection of Salmonella enterica subspecies enterica serovar Typhi Based on Single-Gene Target ........... 128 Assessment of Biodiesel Properties From the FAME Composition of a Malaysian Rhodophyte (Kappaphycus sp.) ............................................................................. 129 Generation of RNA Aptamers Against Mycobacterium tuberculosis Secretory Protein ESAT-6 - a Preliminary Study .................................................................. 130 An Expression Analysis of Salmonella Pathogenicity Island (SPI)-Derived NonProtein Coding RNAs in S. Typhi Biofilm formation ........................................... 131 Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood for Personalized Medicine ........................................................................................... 132 Development of Rapid Diagnostic Detection for Salmonella enterica Subspecies enterica Serovar Paratyphi A using Cross Priming Amplification ........................ 133 Conversion of Rice Husks to Polyhydroxyalkanoate (PHA) ................................. 134 Salmonella typhimurium Detection Based on Electrochemical Immunoassay using Methylene blue/MWNTs/Magnetic Particle .......................................................... 135 Electrochemical Characterisation and Determination of Mycobacterium tuberculosis by Voltammetry at Polymer Nanocomposite modified Platform ...... 136 Abstracts (Poster Presentations) ............................................................................ 137 Cloning, Over-expression, and Purification of Hfq Protein from Klebsiella pneumoniae ............................................................................................................ 137

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In vitro Anti-oxidant Assay, HPLC Profiling of Polyphenolic Compounds, AAS and FTIR Spectrum of Malaysian Solanum torvum Swartz fruit .......................... 138 Phytochemical Analysis and Antioxidant Activity of Malaysian Medicinal Plant Abroma augustum Leaf Extract ............................................................................. 139 Reduced Reproductive Function up to Three Generations of Rats Due to Paternal Heroin Addiction ................................................................................................... 140 Optimization of Cryopreservation Using Different Cryoprotective Agents and Differential Temperatures on Freeze Dried Probiotics .......................................... 141 Development of a Reusable Electrochemical Immunosensor for Direct Detection of Small Organic Molecules ....................................................................................... 142 Over-expression and Purification of an RNA Chaperone, Hfq Protein of Proteus mirabilis ................................................................................................................. 143 Peritoneal Mast Cell Stabilization and Toxicological Properties of the Ethanolic Extract of Solanum trilobatum Linn. ................................................................... 144 Understanding the Host-Pathogen Interaction in Klebsiella pneumoniae Infected Rat Model via Metabolomics Approaches ............................................................. 145 New PDE4 Inhibitors: Design, ADMET and Docking Studies on Chalcones and Flavones for Anti-Inflammatory Activities ........................................................... 146 Effect of Telfaira occidentalis in Mice Fed Aflatoxin Contaminated Feed .......... 147 Accuracy of Rapid Point-of-Care Diagnostic Tests for Acquired Immune Deficiency Syndrome - A Systematic Review and Meta-analysis ........................ 148 Biocontrol of Macergen Infestation on Plants using Bacteriophage Cocktail ....... 149 Oral Bacterial Diversity Study in Malay Ethnic Group in Malaysia ..................... 150 Isolation and Characterization of Seven Lytic Bacteriophages As Candidates for Phage Therapy ....................................................................................................... 151 Transcriptome Analysis for the Identification of Novel ncRNAs in Acinetobacter baumannii .............................................................................................................. 152 Computational Modelling of the Newly Synthesized Chalcone Derivatives in Inhibiting 5-lipoxygenase ...................................................................................... 153 Computational Design of Flavone and Chalcone Derivatives as Cyclooxygenase-2 (COX-2) Inhibitor .................................................................................................. 154 Identification of Novel npcRNA Candidates in Klebsiella pneumoniae ............... 155 Arduino Microcontroller Based Heart Rate Monitor using Fingertip Sensors ...... 156 Transcriptome Analysis of Proteus mirabilis during Oxidative Stress Adaptation ................................................................................................................................ 157 Safety and antiobesity effect of Garcinia atroviridis ............................................ 158

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Development of Ochratoxin A Detection Based on Electrochemical Sensor by Using Au-ball Labels ............................................................................................. 159 Effect of Different Diets on the Growth and Survival of Silver Arowana (Osteoglossum bichirrosum) .................................................................................. 160 Effect of Aqueous and Methanol Extracts of Polygonum minus Leaves on DrugInduced Hepatotoxicity in Rats .............................................................................. 161 Identification of Novel Non-protein Coding RNAs (ncRNAs) in Staphylococcus haemolyticus Biofilm ............................................................................................. 162 Application of a Novel Lytic Bacteriophage Strain as Biocontrol Agent for Water Sanitization ............................................................................................................ 163 Phytochemical Analysis and Pharmacological Screening (Dopamine level) of the Ethanolic Extract of Solanum trilobatum Linn. ..................................................... 164 Computational Analysis of Common Bean (Phaseolus vulgaris L.) S-adenosyl-Lmethionine dependent methyltransferase gene cDNA Isolated from Bean-pod-tissue cDNA Library ........................................................................................................ 165 Biochemical Changes in African catfish, Clarias gariepinus Exposed to Buprofezin.............................................................................................................. 166 Haematological Changes in African catfish, Clarias gariepinus exposed to Buprofezin.............................................................................................................. 167 Identification of Novel Non-coding RNA (ncRNA) from Tannerella forsythia ... 168 Microbial Load, Antimicrobial Sensitivity and Plasmid Profiles of Vibrio cholerae in Fruit Juice .......................................................................................................... 169 Isolation of Arsenite Resistant Bacteria from Ground Water and Soil of Dhaka, Bangladesh ............................................................................................................. 170 Compared to Serum Media, Serum-Free Medium Enhanced the Generation of Mesenchymal Stem Cells Derived from Full-Term Human Amniotic Fluid ........ 171 Hybrid Assembly of Salmonella enterica subsp. enterica ser. Typhi Isolates PM016/13 and B/SF/13/03/195 from a Typhoid Outbreak in Pasir Mas, Kelantan in 2013........................................................................................................................ 172 Identification and Characterization of Novel Non-protein Coding RNAs in Salmonella serovar Typhi Biofilm ........................................................................ 173 Identification of Novel Non-coding RNA (ncRNA) from Bacillus thuringiensis . 174 Morphological and Differential Protein Expression Analysis of Placentas from Term and Spontaneous Preterm Labor with Intact Membrane .............................. 175 Expression of Salmonella Typhi Ty21 TolC Protein in Different Host Cells ....... 176 Stemness of Spontaneously Transformed Murine Bone Marrow Mesenchymal Stem Cells is Maintained Upon Prolonged In Vitro Expansion ...................................... 177

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16S Ribosomal DNA Sequence Based Identification of Bacterial Endophytes Isolated from Seeds of Starfruit (Averrhoa carambola L.).................................... 178 An Alternative Method of Screening the M2/ANXA5 Haplotype for Repeated Pregnancy Loss ...................................................................................................... 179 Computational Analysis of Class IV Chitinase Gene cDNA Isolated from American Oil Palm (Elaeis oleifera) Fruit Mesocarp Tissue cDNA Library......................... 180 In Silico Analysis of Omega-6 Fatty Acid Desaturase Gene cDNA Isolated from Common Bean (Phaseolus vulgaris L.) Pod Tissue cDNA Library ...................... 181 Computational Analysis of Common Bean (Phaseolus vulgaris L.) Palmitoyltransferase Gene cDNA Isolated from Bean Pod Tissue cDNA Library ................................................................................................................................ 182 Annotation of Elaeis oleifera CAP cDNA and its Deduced Amino Acid Sequence using Computational Tools .................................................................................... 183 Comparison of Methods for Pure Quality RNA Isolation from Polysaccharide Rich Averrhoa carambola L. Fruits (Starfruits) ............................................................. 184 Appendices ................................................................................................................ 185 Appendix I: Brief biography of keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016. .......................................................................................... 185 Appendix II: A copy of scientific programme of the 3rdRC4Bs-2016................... 198

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Men love to wonder, and that is the seed of science. --- Ralph Waldo Emerson

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Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016), P1-9

Relevance of Biotechnological Applications for Global Food Security and Sustainability Prakash P. Kumar* Department of Biological Sciences, National University of Singapore, 10 Science Drive 4, Singapore 117543;*corresponding author, e-mail: dbskumar@nus.edu.sg

ABSTRACT In this review, I will briefly discuss the use of biotechnology in the recent past for crop plant improvement with some specific examples of successes. These will include brief discussions on herbicide/insect resistance, drought tolerance, and golden rice. Some of the main objections raised against GM crops will be examined briefly to see if these objections have reliable scientific basis. Breeding in a number of animal and plant species has been revolutionized by the emergence of DNA markers such as single nucleotide polymorphism (SNP) arrays. The method is based on the prediction of genetic merit by incorporating relationships among individuals based on SNP array data. This process is known as genomic selection. Some of the current developments in the field, including genome-wide association studies (GWAS) tools applied for crop plant improvement will be discussed with an example of a recent study on oil palm. Another area that is making rapid progress in biotechnology is genome editing. Genome editing tools that are currently being developed include transcription activatorâ&#x20AC;&#x201C;like effector nucleases (TALENs), zinc-finger nucleases (ZFN) and Clustered regularly-interspaced short palindromic repeats (CRISPR) paired with CRISPR-associated (Cas) nuclease system. Of these, the CRISPR/Cas9 tool, which is based on bacterial immune system, holds great promise for crop biotechnology as well as biomedical fields. The clustered repeats in the bacterial DNA correspond to captured DNA fragments of pathogens that invaded the cells. The Cas9 endonuclease detects these and using a guide RNA molecule with complementarity to the DNA, it serves to cut the new invaders with similar DNA material. This is now adapted to create specific breaks and repairs in the genomic DNA of eukaryotic cells, thereby achieving DNA editing. The tools are being refined such that when genome editing is finished, the resulting plants will be treated just as natural mutants rather than transgenic plants. Highlights of these biotechnological tools will be examined with a discussion on how these can contribute to future global food security. Keywords: Agriculture; Biotechnology; DNA; Food; GM technology; Sustainability.

PREAMBLE The use of genetically modified (GM) crops has been made into a controversial topic that elicits quite polarized views. This article is

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based on a plenary talk delivered at a conference. Based on scientific evidence and first-hand experience with GM plant production, the intended take-home message of this talk was that we should not hesitate to

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Global Food Security and Sustainability use modern technologies for crop improvement. It is acknowledged that having adequate tests to verify the general safety of the GM crops is important, but once proven safe (or not significantly different from the conventional crop variety), the GM crop should be used as a valuable alternative. With judicious use of technology and crop management practices, we can achieve global food security. GM FOOD What is Genetic Modification (GM) and is it vastly different from conventional plant breeding in its principle? Crop modification has been ongoing for thousands of years. The use of everchanging technologies has been a constant theme in this as with all other modern human endeavors. We recognize that use of any novel technology involves risks and benefits, it is up to us to evaluate them and decide to adopt them if the benefits vastly outweigh risks. Also, human ingenuity is such that technology can be constantly refined to minimize the initial risks to negligible levels as was done with the minimization of radiation emission in the present day cell phone or mobile phone technology. The yield enhancement as a result of the Green Revolution has reached its limit now. Although it is known that crop plants have not reached their biological upper limits for yield increase, any further increases in crop yield will have to rely on novel tools. One of the relatively recent novel technologies is genetic modification or genetic engineering (also known as biotechnology, recombinant DNA technology, etc.). Two main approaches in GM plant production are: a) Modifying existing genes in plants - e.g., fruit ripening by modifying ethylene biosynthesis or ethylene action, and ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar b) Expression of introduced (foreign) gene to confer a new/modified trait to the crop plant, e.g., carotenoid production in the endosperm tissue of the Golden Rice grains. The main examples of GM plants The main examples of GM plants that have been commercially cultivated for the last 20 or so years include herbicide resistant and insect resistant plants. The major crops with such GM varieties include corn, soybean, canola and cotton. In 2013, GM crops were grown in about 175 million hectares of land by 18 million farmers in 27 countries (James, 2013). The global market value of GM crops for 2013 was estimated to be about US$15.6 billion. Some of the other traits being introduced to crop plants include drought tolerance, disease tolerance (both fungal and viral), resistance towards root nematodes, modified nutritional parameters such as altered lipid profiles in oil crops and fortified grains. Although the Golden Rice has been fully developed and safety tested â&#x20AC;&#x201C; it has gone through the so called deregulation process mandatory for GM crops â&#x20AC;&#x201C; it has not yet received the necessary Government approvals. The Golden Rice has been touted as a staple grain that will lead to vast improvements in the nutritional status (with respect to vitamin A and iron) of millions of poor people in Asia and Africa. Nevertheless, it has not been released to the growers. Scientists at the International Rice Research Institute, Philippines (IRRI) have developed Golden Rice varieties suitable for several rice growing regions with the help of generous funding from philanthropic organizations such as the Bill and Melinda Gates Foundation. Therefore, when approved, the Golden Rice seeds can be distributed to growers without profiteering. The IRRI has established a highly positive and remarkable reputation for successfully 2

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Global Food Security and Sustainability releasing the flood tolerant SUB1 rice that was developed by non-GM methods (marker assisted breeding). However, it must be highlighted that this could not have been achieved without data from GM tools. Prior studies using molecular biological tools helped to identify the flood tolerant variant of the Sub1a gene in rice. Experimental GM rice plants demonstrated that when this resistant form of the gene was introduced to flood sensitive rice varieties they could be turned into flood tolerant forms. Therefore, without GM tools scientists could not have so easily developed the marker assisted SUB1 rice varieties in such a short time span. These varieties dubbed ‘Scuba Rice’ are being grown for the past several years by thousands of happy farmers in millions of hectares of land, for example, in the flood prone areas of India and Bangladesh. OTHER GM FOOD Besides the examples cited above, there are several other minor successes in GM plants including the virus resistant GM papaya in Hawaii, the delayed ripening tomatoes, as well as some ornamentals such as long-vaselife carnation flowers and the Blue Rose marketed by Suntory Biotechnology company. The use of GM products such as recombinant proteins, mainly enzymes in food industry is noteworthy development. GM or ‘Recombinant proteins’ from bacteria and fungi are used in cheese making. Cheese production requires the use of a protein called chymosin (also known as rennin or rennet). Chymosin was previously obtained only from calf stomachs, which had several implications including health concerns, conflict with nutritional preferences due to religious restrictions or personal choices of various groups of people. The use of chymosin from GM microbes has become ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar common in cheese making now, with estimated 70% to 90% of cheeses in the USA manufactured using such microbial GM rennet now. Also, the US Food and Drug Administration (FDA) has approved over 30 other recombinant enzymes for use in food production (including α-amylase, used to make glucose or fructose syrups). Another noteworthy development in the recent years is the FDA approval of the first transgenic animal for food use, namely, GM salmon. After an exhaustive and rigorous review that lasted nearly twenty years, the FDA has approved the use of fast growing GM salmon (AquAdvantage) in November 2015. FDA determined that the AquAdvantage salmon is as safe to be consumed by humans as the commercial non-GM Atlantic salmon. This was a longawaited positive decision welcomed by biotech scientists around the world. AGRICULTURAL ECONOMY How important are GM crops to the agricultural economy? An economic assessment of the impact of commercial agricultural biotechnology on global agriculture was done (Brookes and Barfoot, 2009). The study highlighted the significant impacts on the production of soybeans, corn, cotton and canola crops. According to this study, GM technology led to net economic benefits at the farm level of about US$10.1 billion in 2007. The other benefits associated with the use of GM had a positive impact to the tune of 25% of the total direct farm income benefit in the USA. Biotech crops contributed to increasing global production levels of the four main crops examined. For example, the use of GM soybeans corresponded to adding 68 million tons and GM corn added 62 million tons to global production levels in 2007. Their use was equivalent to 172 million kg 3

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Global Food Security and Sustainability reduction in pesticide use (which corresponds to 14% reduction in the environmental footprint associated with pesticide use). In addition, other factors from growing these biotech crops significantly reduced greenhouse gas emissions from agriculture equivalent to removing 5 million cars from the roads (Brookes and Barfoot, 2009). THE NEED TO INCREASE FOOD PRODUCTION Why not just stick to old ways of producing food? Why resort to new tools? According to a UN study, the world population is expected to grow to ~9.7 billion by 2050. According to that study, Africa and Asia would be adding 1.14 billion more people to their population. By 2050, Africa (2478 million) and Asia (5267 million) collectively will have a population size larger than the current world population.

Kumar Further complications are already evident from the global climate change. The predicted impacts of climate change include, reduced wheat production in South Asia by 2030, and rice production in Southeast Asia, particularly in the Greater Mekong Subregion (ADB, 2009). These will almost certainly lead to increased food prices. The ADB estimates that rice, wheat, and soybean prices could increase by 10%–50%, while corn price may double by 2050. This should be recalled in connection with the global food and energy price surge in 2007–2008, which exposed the vulnerabilities of households and of the international food and nutrition insecurity. Increase in extreme weather events (floods, droughts, typhoons) will have serious consequences for agriculture, food, and forestry production in the coming decades. Therefore, it is imperative that crop yield has to be raised to ensure food security. FOOD PRODUCTION AND WATER

Average Asian rice consumption is about 100 kg per person per year. The number of undernourished people in the world is close to 1 billion and nearly two-thirds of the world’s hungry people reside in Asia and the Pacific (FAO, 2009). POPULATION AND CLIMATE CHANGE V/S FOOD SECURITY It is well recognized now that crop yield has to be raised to ensure food security in the coming decades. Global food production will need to increase by 40% by 2030 to keep pace with global demand (FAO, 2009), because the world population is increasing. The maxim ‘Less food, more mouths to feed’ is made relevant when we realize the fact that the land area available for crop cultivation is continually decreasing as the population keeps increasing.

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The growing worldwide shortage of water is extremely worrisome. The global demand for water is estimated to double by 2050. However, about 35% of the world population is already facing water shortages. Asia is facing acute water shortage, with available freshwater being unevenly distributed. Under these circumstances, it is obvious that to grow more food with less water, crop productivity needs to be improved. However, the traditional breeding tools are often inadequate to achieve such spectacular increases in yield. Water and rice - how are the two related? To grow the current world production of approximately 500 million tons of rice it is estimated that about 1,750 km3 fresh water is needed per year [1 km3 = 1000 billion liters (=1012 liters)]. If we take overall agriculture, it accounts for about 80% of 4

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Global Food Security and Sustainability world water use (www.adb.org/publications/asian-waterdevelopment-outlook-2013). Currently, ~40% of this water is lost due to inefficient agricultural practices (crops and farm animals). The water required for production of some of the key agricultural produce illustrate the vastness of water needed in agriculture. Liters of water necessary per kg of produce (indicated in parentheses): for rice (2,500), other grains in a favorable climate (2,000), grains in arid climate (5,000) and beef (15,000). Therefore, the total world water use is 7,450 to 8,160 km3/year, and the water used for food production is estimated to be 6,390 to 7,200 km3/year, with domestic needs accounting for 180 to 344 km3/year (IRRI, and various other sources). Some of the major challenges for rice and grain production include abiotic and biotic stresses. The major abiotic stresses are (1) cold (a climatic factor that affects ~66% of land worldwide), (2) drought, (3) flood (~10% of yield loss, ~25% of the global rice crop lands sees floods each year – with ~20 million hectares of Asian rice land prone to flooding), (4) salinity (~10% of world’s land affected and ~12 million hectares in Asia affected by increased soil salinity). SUSTAINABLE FOOD PRODUCTION STRATEGIES There are several ways to increase crop yield. Four of the prominent options are mentioned here. (1) Expanding the cultivated area: land expansion can account for ~ 20% increase in yield. (2) Increasing the cropping intensity can contribute about 10% increase. (3) Reducing pre- and postharvest loss and wastage: 20 to 30% increase if all wastage is prevented, (4) Improving crop yields using latest knowledge and technologies. Therefore, about 40% increase ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar in crop yield has to be through plant breeding and biotechnology. In view of the above, the answer to the question of whether we should use novel technologies (e.g., GM technology) for crop improvement should be obviously affirmative! I cite the example of breast feeding v/s infant formula in support of this view. We all know breast feeding is the best nutritional option for infants. But, global market for baby food is huge (over US$10 billion/year) with 50% to 70% of it as the value of infant formula! North America and Europe constitute 33% of this market, while Asia forms 53% of the infant food market. Over 120 companies manufacture and sell infant formula! What has happened here is the need for convenience (in most cases, necessity in some) has led to the adoption of an alternative to breast feeding as an acceptable alternative for our infants. In a similar rational manner, we need to develop modern technologies for crop improvement and ensure food security. We should not hesitate to use new technologies to develop alternative options that should coexist with the established techniques. What are the other alternatives? Certainly, GM crops are not the only option to increase crop yield – they are an efficient and complementary tool to the established tools at our disposal. It is recognized that efficient crop management will improve yield to some extent. These include the use of right amount of chemical fertilizers and pesticides, because excess use of such agricultural chemicals leads to yield loss as well as air and water pollution. Ecological engineering is another concept being tested with positive effect. For example, the use of beneficial insects for pest control by growing flowering plants around the fields is one such ecological modification tool. This method helped to reduce pesticide use by 5

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Global Food Security and Sustainability ~21% in Vietnam according to a study coordinated by IRRI. Alternate Wetting and Drying (AWD) method is another crop management tool. AWD can lead to ~35% less water requirement for rice growing compared to the traditional farming method, with ~10% yield increase and attendant reduction in greenhouse gas (methane) emission from the fields. The reduction in methane emission is due to the reduction in methanogenic bacteria growing in the root zones of rice plants when cultivated under permanent submergence (leading to anaerobic soil favoring the methanogenic bacteria). The AWD management practice leads to reduced growth of such bacteria along with reduced water needs. Marker-assisted breeding, genome-wideassociation study (GWAS) leading to Genomic Selection (GS) are established as non-GM alternatives for crop improvement in the past decade or so (Meuwissen et al., 2001, Desta and Ortiz, 2014). Genomic Selection is based on the powerful tools of genome-wide prediction in plant improvement. GWAS helps to generate statistical models to predict how a plant will perform before conducting a genetic cross and field-testing of actual crops. This involves the combined use of genomic information, statistics, bioinformatics tools, and prediction-based breeding for crop improvement. Of course, this requires that the scientists establish robust association between DNA seqence (SNPs) and specific traits for a large number of breeding lines (1000s of individual plants). This will then be used to estimate Genomic Breeding Values (GEBV). New plants to be used for breeding will then be sequenced; for each new breeding line a DNA-profile will be determined and using such profiles from each line scientists will estimate GEBV for every trait of interest. Based on the genetic potential for the different traits the breeder ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar can design a new variety on the computer which combines plant lines with highest scores (using the predictive models built). One of the more recent GWAS studies with impressive results is on oil palm (Teh et al., 2016). Also, several improved crops generated using these techniques are being released and more will be coming in the near future. The gene that facilitates deep rooted phenotype beneficial to develop drought tolerant crops, e.g., DEEP ROOTED1 gene or DRO1 of rice was identified by GWAS approaches while as mentioned above, the SUB1 rice was developed by marker assisted breeding. Genetically modified (GM) crops: to use or not? Based on ample evidence from the use of GM crops during the past couple of decades it is safe to conclude that GM food can save millions of kids from dying due to malnutrition. The use of GM crops can also help to reduce pesticide usage and environmental pollution. In this connection it is worth remembering that technological advances are continually occurring. Accordingly, new genome editing methods may be seen as good alternative tools to generate the new generation, advanced crop plants. Besides GM, several non-GM strategies are being developed and some of the novel strategies for crop breeding by genome editing include: (a) TALENs (b) Zinc Finger Nucleases (ZFN) (Petolino and Kumar, 2016) and (c) CRISPR-Cas9 system (Chen and Bailey, 2016; Kleinstiver et al., 2016). TALEN stands for Transcription Activatorlike Effector Nucleases; while CRISPR/Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated(Cas) system. CRISPR/Cas9 was first named as Short Regularly Spaced Repeats (SRSR) in 2000. 6

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Global Food Security and Sustainability It was renamed as CRISPR in 2002. CRISPR/Cas9 induces double-strand break (DSB), generates deletion and/or insertion of short sequences at the break leading to altered gene of interest. In fact, the common feature of all these genome editing tools is that they induce DSBs in the genomic DNA at specific locations (gene of interest) – with or without short insertions. The DSB will then be repaired by one of two repair processes, namely, Non-Homologous End Joining (NHEJ), or Homology Dependent Repair (HDR). This results in gene editing. FUTURE PERSPECTIVES Gene-edited CRISPR mushroom has been generated recently and it was exempted from having to go through US Regulatory process; because, such organisms do not harbor foreign genes and are similar to induced mutants. The white button mushrooms generated have their polyphenol oxidase gene edited, which helps to prevent browning (Waltz, 2016). Scientific American (14 April 2016) also indicated that this is one of almost 30 genome edited organisms to avoid regulatory requirement currently. These represent the first few improved crops and more such crops will be released by various companies in the years to come (Wolt et al., 2016).

Kumar direct beneficiaries until now. But, when we carefully examine it there are already plenty of indirect benefits for all. GM papayas (ring-spot virus resistant) saved the papaya industry in Hawaii, GM crops will play an important role in protecting soil and water resources, minimizing crop diseases and attendant loss of produce, and responding to the pressures of climate change. Cultivation of Bt-cotton has reduced the amount of neurotoxic insecticide use by millions of kilograms every year in many countries where it is grown. Hence, one way of looking at it is that the concept of organic crops and GM crops coexist! GM crops do not pose any conflict with traditional or organic farming. A book published by Oxford University Press in April 2008 by authors Pamela Ronald and Raoul Adamchak (UC, Davis) advocates a food system that is organic and genetically engineered! They argue that a judicious blend of GM and organic farming representing two important strands of agriculture is key to helping feed the world's growing population in an ecologically balanced manner.

If they are safe why is there hesitation to use GM crops? One of the possible contributing factors is that the general public and most of the opponents of GM crops do not fully understand the underlying process. Perhaps equally importantly, consumers are not direct beneficiaries of the GM technology so far. GM crops of the past have been designed to enhance the productivity of industrial farming – so only the farmers who adopted the GM crops and the seed companies that sell them have been the

Even newer GM crops that will directly benefit the consumers are coming, for example, the purple tomatoes produced by Prof Cathie Martin’s lab, John Innes Centre, UK. These GM tomatoes have high amounts of plant nutrients (e.g., anthocyanins found in some berries), which are super foods that will confer tremendous health benefits to the consumers. The purple tomatoes were grown in controlled greenhouses in Canada and juice from these GM tomatoes is undergoing tests and evaluations. Clearly, this represents a ‘super food’ that could not be otherwise made available to us in plentiful quantities. They have already shown that inclusion of these high-anthocyanin GM tomatoes in the diet of cancer-prone mice can extend their healthy life-span by 30% - a most welcome

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Global Food Security and Sustainability prospect indeed! It is a firm belief of the author that people will line up for this type of GM superfoods and demand that more be supplied! From the foregoing discussion it is clear that the future global food supply can benefit greatly by the use of new initiatives in research. We should be open to employ a multidisciplinary approach involving molecular genetics, cell and developmental biology, systems biology, as well as ecology. GM crops have been grown and consumed for over 20 years now and the underlying technology is continually being improved. Hence, the emerging novel tools such as genome editing will help us alleviate food scarcity in the future decades. We should be mindful to develop ‘safe technology’ and develop suitable alternative strategies such as GM, molecular marker assisted breeding and genome editing. Collectively, these will serve us well in ensuring future food security. REFERENCES ADB

(2009). Operational plan for sustainable food security in Asia and the Pacific. Document downloadable from ADB web site. http://www.adb.org/publications

Brookes G, Barfoot P (2009). Global impact of biotech crops: Income and production effects, 1996-2007. AgBioForum 12(2), 184-208. Chen H, Bailey S (2016). Cas9, poised for DNA cleavage. Structural rearrangements explain how Cas9 cuts DNA. Science 351, 811-812. Desta ZA, Ortiz R (2014). Genomic selection: genome-wide prediction in plant improvement. Trends in Plant Science 19, 592-601. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Kumar FAO paper (2009). How to Feed the World in 2050? Document available at the FAO web site. http://www.fao.org/fileadmin/template s/wsfs/docs/expert_paper/How_to_Fee d_the_World_in_2050.pdf James, C. (2013). Global status of commercialised biotech/GM crops: 2013, ISAAA Brief No. 46. International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY. ISBN 9781-892456-55-9. www. isaaa. org/resources/publications/briefs/46, 2014. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016). High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495. Meuwissen THE, Hayes BJ, Goddard ME (2001). Prediction of total genetic value using genome-wide dense marker maps. Genetics 157, 1819– 1829. Pamela C. R. and Raoul, W. A. (2008). Tomorrow's Table: Organic Farming, Genetics, and the Future of Food, Oxford University press (OUP). Petolino JF, Kumar S (2016). Transgenic trait deployment using designed nucleases. Plant Biotechnology Journal 14, 503–509. Teh CK, Ong AL, Kwong QB, Apparow S, Chew FT, Mayes S, MohamedM, Appleton D, Kulaveerasingam H (2016). Genome-wide association study identifies three key loci for high mesocarp oil content in perennial crop

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Global Food Security and Sustainability oil palm. Scientific Reports 6, 19075 | DOI: 10.1038/srep19075 Waltz E (2016). Gene-edited CRISPR mushroom escapes US regulation. A fungus engineered using CRISPR– Cas9 can be cultivated and sold without oversight. Nature 532, 293.

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Kumar Wolt JD, Wang K, Yang B (2016). The regulatory status of genome-edited crops. Plant Biotechnology Journal 14, 510–518.

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016), P10-19

Nano- and Bio-technological Advancement to assist in the Determination of Halal Products Quamrul Hasan1, 2, * 1

College of Business, Universiti Utara Malaysia (UUM), 06010 Sintok, Kedah, Malaysia; 2Japan Halal Research Institute for Products and Services (JAHARI), Kobe, Japan; *corresponding author, e-mail: quamrul@uum.edu.my / quamrulhasan@gmail.com

ABSTRACT Aims: Halal industry science can be defined as the experimental investigation of the consumable product by using scientific (analytical) method to reveal its contents, thus assisting to determine the product is halal or not. This scope can be further extended to address any relevant issues on the halal products involving scientific and technological advancements. This definition is contrived for the first time in this article by the author. The present study was conducted in collaboration with university, industry, professional laboratories and governmental organization, which was aimed in finding out how effective the currently available analytical methods especially when the results were targeted to be used in the determination of the Halal products. Furthermore, in this study, the successful development of a protein-based (immunochromatography) test kit for the purpose was explained. Methodology and results: DNA extraction was performed using commercially available DNA extraction kit from QIAGEN or NEOGEN. The DNA extraction was performed in duplicate for each sample. PCR-conventional was performed using Thermal cycler (GeneAmpÂŽ PCR System 9700, Applied Biosystems). Porcine and Bovine specific primers for mitochondrial DNA sourced from Food and Agricultural Materials Center, Japan (FAMIC) were used. In addition, for comparison NEOGEN primer specific for porcine genomic DNA was used for one sample. A total of 4 commercial products (3 food/snack, 1 functional cosmetic) were tested in this study. Among these, except 1 marshmallow product which might be fish DNA positive, 3 were found as porcine DNA positive. One of the porcine DNA positive product failed to show the same result when it was tested using the commercial kit-NEOGEN- containing porcine genomic DNA. None of these products were found as bovine DNA positive. Conclusion, significance and impact of study: The determination of the Halal is a very sensitive issue. Therefore, in this study, we have concluded that in determination of the Halal in a processed and commercialized product by employing a single approach or method especially when targeting DNA is not enough to confirm the authenticity of the test result due to possible limitation of the method used. We have proven that the primers for mitochondrial DNAs sourced from FAMIC, Japan could be more reliable for the purpose. The effective collaboration between industry, academia and related professional organizations for developing innovative Halal test kit successfully is critical. Keywords: Biotechnology; Determination of halal products; Halal industry science; Nanotechnology.

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Determination of Halal Products INTRODUCTION As processed and prepared ready-to-eat food products with animal origins have become increasingly available to consumers, due to technological advances, the possibility of fraudulent adulteration and substitution of the expected species (source) with other sources has also increased. The same goes to confectionery and functional cosmetic products especially containing gelatin or collagen peptides. Such practices pose a substantial concern to consumers in terms of economic loss, allergies, religious observance, loss of traceability, and food safety. In many cases, porcine derivatives are used due to cheaper price and readily available. For Muslim consumers, the major authenticity concerns are in meat and meat products include porcine gelatin, collagen, fat, and so on. The analytical methods used for Halal authentication of meat and meat products include: Polymerase Chain Reaction (PCR); Enzyme Linked Immunosorbent Assays (ELISA); Mass spectrometry; Chromatography, Electronic nose and Spectroscopy. An overview of the currently available analytical methods or techniques is given in Table 1. The analytical methods currently in use to detect the presence of porcine materials are mainly protein and DNA-based. These are described below. DNA-based detection PCR is capable of amplifying very few copies of DNA and its detection limit is much lower than what is observed with protein based assays. PCR amplification is based on hybridization of specific oligonucleotides to a target DNA and synthesis of million copies flanked by these primers. The simplest PCR strategy applied to evaluate presence of any species in a meat product is the amplification of DNA fragments, followed by agarose gel electrophoresis for fragment size verification. To successfully ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Hasan detect a species with PCR, adequate genetic markers are chosen to develop the assay. Either nuclear or mitochondrial genes can be targeted (Fajardo et al., 2008). However, the use of mitochondrial DNA (Mt DNA) offers a series of advantages over cell nucleus DNA. Mitochondrial DNA facilitates PCR amplification, even in cases where the availability of DNA template after its extraction is insufficient for detection (Murugaiah et al., 2009). This is attributed to the fact that Mt DNA is several fold more abundant than that of nuclear genome; each mitochondrion is estimated to contain 2 to 10 Mt DNA (Murugaiah et al., 2009). Furthermore, Mt DNA evolves much faster than nuclear DNA and henceforth contains more sequence diversity facilitating the identification of phylogenetically related species (Fajardo et al., 2010; Girish et al., 2005; Murugaiah et al., 2009). Among the mitochondrial genes, cytochrome b (cyt b) (Aida et al., 2005; Murugaiah et al., 2009) and 12S rRNA (Chen et al., 2010; Girish et al., 2005) are the most commonly used markers in the development of DNA methods for meat species authentication. Protein-based detection Porcine protein, due to it is being cheap and readily available, might fraudulently be used to substitute other animal proteins. ELISA is the most commonly used method to detect animal proteins and a number of commercial immunoassays are available. Chen and Hsieh (2000) were the first ones to develop the immunoassay (ELISA) using a monoclonal antibody to a porcine thermostable muscle protein for detection of pork in cooked meat products. They observed no cross-reactivity with common food proteins. By employing this technology, the first pork detection kit was developed in Japan by a Japanese company (Okamoto, 2016). This kit is immuno-chromatographic employing nano-sized colloidal gold 11

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Determination of Halal Products particles to detect presence of pork in food samples. It can detect pork in both raw and cooked food. It allows rapid detection of pork in food samples at low cost without

Hasan using any special equipment or requiring skill. The basic principle of immunechromatographic kit is shown in Figure 1.

Table 1: Summary of analytical techniques applicable in the halal authentication of meat products♦. Authenticity issue Pork adulteration Species identification

Analytical Techniques PCR-RFLP Real time PCR

Species-specific PCR

Pork protein

Pork fat (lard)

RAPD PCR sequencing ELISA Chromatography Peptide examination Isoelectric focusing FTIR spectroscopy

DSC Electronic nose

Blood plasma

Isoelectronic focusing ELISA Immunodiffusion LC – MS/MS

References Murugaiah et al. (2009), Aida, Che Man, Raha, and Son (2007), and Aida et al. (2005) Martín et al. (2009), Kesmen, Gulluce, Sahin, and Yetim (2009), Tanabe et al. (2007), Fumière, Dubois, Baeten, von Holst, and Berben (2006), and López-Andreo, GarridoPertierra, and Puyet (2006) Soares, Amaral, Mafra, and Oliveira (2010), Alaraidh (2008), Che Man et al. (2007) and Montiel-Sosa et al. (2000) Martinez and Malmheden Yman (1998) Karlsson and Holmlund (2007) Chen and Hsieh (2000); Chen and Hsieh (2000) Chou et al. (2007) Aristoy and Toldra (2004) Hofmann (1985) Rohman, Sismindari, Erwanto, and Che Man (2011a, 2011b), Che Man, Abidin, & Rohman, 2010, Rohman and Che Man (2011a, 2011b), Rohman and Che Man (2009), Che Man, Gan, NorAini, Nazimah, and Tan (2005), Che Man, Syahariza, Mirghani, Jinap, and Bakar (2005) and Che Man and Mirghani (2001) Marikkar, Ghazali, Man, and Lai (2003) and Marikkar, Lai, Ghazali, and Che Man (2001) Nurjuliana, Che Man, and Mat Hashim (2011a), Nurjuliana, Che Man, Mat Hashim, and Mohamed (2011b), Che Man, Gan, et al. (2005), and Che Man, Syahariza, et al. (2005) Bauer and Stachelberger (1984) Church and Hart (1995) Price, Hart, and Church (1992) Grundy et al. (2007) and Grundy et al. (2008)

♦

Source: Nakyinsige et al. (2012).

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Hasan

Figure 1 The basic principle of the immuno-chromatographic kit.

MATERIALS AND METHODS Detection of Porcine and Bovine DNAs from different products containing gelatin/collagen from different sources DNA extraction was performed using DNA extraction kit from QIAGEN. About 0.25g DNA per sample was extracted. The DNA extraction was performed in duplicate for each sample. The conventional-PCR was performed using Thermal cycler (GeneAmpÂ® PCR System 9700, Applied Biosystems). Porcine and Bovine specific primers for mitochondrial DNA were used. Each primer sequence is shown in Table 2. In addition, in order to check that DNA was extracted successfully, the PCR using the primer for vertebrate detection was also performed. The PCR condition is summarized in Table 3.

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Table 2 Sequence of Porcine and Bovine specific primers. Target Primer Sequence Porcine Forward GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA Reverse GCT GAT AGT AGA TTT GTG ATG ACC GTA Bovine Forward GAC CTC CCA GCT CCA TCA AAC ATC TCA TCT TGA TGA AA Reverse CTA GAA AAG TGT AAG ACC CGT AAT ATA AG 13

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Determination of Halal Products Table 3 Condition of PCR Temperature Step (°C) Initial 94 denaturation Denaturation 94 Annealing 60 Extension 72 Final 72 Extension Hold 4

Hasan B. FAMIC method: Time

Cycle

1 min

30 sec 30 sec 30 sec

7 min

Primer:FAMIC Porcine mitochondrial DNA DNA: 20 ng PCR steps: Step



Comparative study on the PCR-based detection of the Porcine and Bovine genes using different primers

Initial denaturation Denaturation Annealing Extension Final Extension Hold

primer-

Temperature (°C)

Time

Cycle

9 min

92 60 72

30 sec 1 min 1 min

5 min



A. Neogen kit method: Primer: Neogen genomic DNA DNA: 20 ng PCR steps:

Porcine

primer-

Step

Temperature Time Cycle (°C) Initial 94 10 1 denaturation min Denaturation 94 15 sec Annealing 64 15 30 sec Extension 72 15 sec Final 72 3 1 Extension min Hold 4 

Primer:FAMIC Bovine mitochondrial DNA DNA: 20 ng PCR steps: Step Initial denaturation Denaturation Annealing Extension Final Extension Hold

Temperature (°C)

primer-

Time

Cycle

9 min

92 55 72

30 sec 30 sec 30 sec

5 min



RESULTS Detection of Porcine and Bovine DNAs from different products containing gelatin/collagen from different sources

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Determination of Halal Products Table 4: Summarized results of the detection of porcine, bovine and vertebrate DNA Porcine Bovine Vertebrate Sample 1 Not Not Detected* detected detected Sample 2 Detected* Not Not detected detected Sample 3 Detected* Not Not detected detected Sample 4 Detected* Not Detected* detected *When one of the test samples from duplicate was found positive, it was taken as the positive (as shown in the following three figures).

Hasan 3. Two products (one marshmallow, one functional cosmetic) showed positive result for vertebrate material. Only one of the two marshmallow products might contain fish material (gelatin) since it was found porcine negative.

Figure 3 Result of PCR using Bovine specific primer; Lane 1: Marker (25bp ladder), 2: Negative control, 3: sample 1 (n=1), 4: sample 1 (n=2), 5: sample 2 (n=1), 6: sample 2 (n=2), 7: sample 3 (n=1), 8: sample 3 (n=2), 9: sample 4 (n=1), 10: sample 4 (n=2), and 11: Positive control [Microchip electrophoresis system (MultiNA), Shimadzu Corporation]. Figure 2 Result of PCR using Porcine specific primer; Lane 1: Marker (25bp ladder), 2: Negative control, 3: sample 1 (n=1), 4: sample 1 (n=2), 5: sample 2 (n=1), 6: sample 2 (n=2), 7: sample 3 (n=1), 8: sample 3 (n=2), 9: sample 4 (n=1), 10: sample 4 (n=2), and 11: Positive control [Microchip electrophoresis system (MultiNA), Shimadzu Corporation]. Assumptions on the detection of porcine, bovine and vertebrate DNA from four commercialized products are listed below: 1. Not a single product showed positive result for bovine material. 2. Three products (one marshmallow, one Jelly, one functional cosmetic) showed positive result for porcine material. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Figure 4 Result of PCR using the primer that common to a vertebrate; Lane 1: Marker (25bp ladder), 2: Negative control, 3: sample 1 (n=1), 4: sample 1 (n=2), 5: sample 2 (n=1), 6: sample 2 (n=2), 7: sample 3 (n=1), 8: sample 3 (n=2), 9: sample 4 (n=1), 10: sample 4 (n=2), and 11: Positive control [Microchip electrophoresis system (MultiNA), Shimadzu Corporation]. 15

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Comparative study on the PCR-based detection of the Porcine and Bovine genes using different primers Result-1 (DNA concentration) The values are shown in the following table DNA extraction kit: Neogen (Speciation) Samples: No Sample ID DNA 260/280 (ng/Âľl) 1 M-A 12.66 1.50 2 M-B 22.44 1.38 3 T-A 10.49 1.63 4 T-B 12.19 1.52 Result-2 (PCR-Electrophoresis) A. Neogen kit method: As shown in the Figure 5, no porcine DNA was detected in any of the two samples tested in duplicates. B. FAMIC method: As shown in the Figure 6, Porcine DNA was detected for the two samples though in single from duplicate (MB and T-A). No bovine DNA was detected from the two samples tested.

Figure 5 Porcine DNA; PCR product size: 380-bp (housekeeping fragment), 314-bp (porcine fragment); from left: marker, M-A, M-B, T-A, T-B, positive control (380-bp, 314-bp).

Assumptions on the comparative study using different primers are listed below: 1. The porcine DNA was detected in the both samples (marshmallow M-B and T-A) when using only the FAMIC method. These results indicate that both the marshmallows might have a small amount of porcine DNA. 2. The bovine DNA was not detected in any of the two samples tested under the same condition. DISCUSSION In this study, we have reported on comparing the specific and qualitative detection methods for porcine and bovine materials in the processed food products (marshmallows ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Figure 6 Porcine DNA; PCR product size: 126-bp; from left: M-A, M-B, T-A, T-B, positive control. 16

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Hasan tested. PCR-based detection was performed by using PK mastermix/control POD from Neogen Europe Ltd., UK, and porcine and bovine specific primers from Food and Agricultural Materials Inspection Center (FAMIC), Japan.

Figure 7 Bovine DNA; PCR product size: 126-bp; from left: M-A, M-B, T-A, T-B, positive control and jelly containing gelatin) and functional cosmetic product containing collagen peptides by employing conventional PCR and two different primers (nuclear and mitochondrial genes). Earlier, two other groups also reported about the successful application of the conventional PCR in meat species detections including from the processed foods (Tanabe et. al., 2007; Matsunaga et. al., 1999). These conventional PCR methods are simple and useful. That is why we have employed these. The choice of the target gene and the design of the primers have a great impact on the sensitivity and specificity of a detection system. It is wellknown that very sensitive PCR assays can be established when the primer target is a multicopy gene, such as a mitochondrial gene (Holzhauser et. al., 2006). In this study, we chose both mitochondrial and nuclear (genomic) genes as the target to detect porcine and bovine materials for comparing the efficiency of these two primers while using the processed products. DNA extraction was performed by using the commercial kit from Neogen Europe Ltd., UK and following the manufacturerâ&#x20AC;&#x2122;s instructions. The DNA extraction was carried out in duplicate for each sample ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Among the four products tested, in this study, we found out at least three were porcine DNA positive (one marshmallow, one jelly, one functional cosmetic). These result surprised us since all these products were sourced from a strictly Halal-regulated Muslim dominated country though these were imported from other countries. Moreover, one product (functional cosmetic) claimed that it contained fish collagen from Japan, which we found porcine positive. Our results from this study proved that selection of the right primer is critical to obtain the authentic result: the porcine DNA was detected positive only when the FAMIC primer (mitochondrial DNA) was used and, in contrast, the result was negative using the NEOGEN kit primer (genomic DNA). This might be due to the higher sensitivity of mitochondrial gene target primer, which is a multicopy gene. In another study, we attempted to develop a rapid method for meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor (Ahmed et al., 2010). In a separate effort, a protein-basedimmuno-chromatographic porcine detection kit was successfully co-developed under a collaborative effort between two universities (in USA and Japan) and two companies in Japan (the methods and results were not available for this paper). This development was owned and sponsored by a Japanese company having a leading position in the precious metal business including the expertise in nano-gold technology. This company obtained the immune17

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Determination of Halal Products chromatographic know-how from a university in Japan. However, to develop a porcine detection kit, the missing part was the biotechnology: a monoclonal antibody to a porcine thermostable muscle protein for detection of pork in cooked meat products. To address this issue, the author of this paper was approached by the Japanese company who successfully made the connection with a university in USA already reported on the availability of this antibody and he coordinated the collaboration between the Japanese company and the US University. Finally, the effort was paid-off. About 2 years later, the Japanese company confirmed the successful development of porcine detection kit and announced it. Immediately, they were approached by a large US company (renowned analytical equipment manufacturer) for this technology, which resulted in to the successful commercialization of this protein-based (immuno-chromatographic) porcine detection kit in the global market. ACKNOWLEDGEMENTS The author gratefully acknowledges the cooperations from Professor Eiichi Tamiya at Osaka University, Japan; Professor Emerita Peggy Hsieh at Florida State University, USA; Mr. Masatoshi Watai at Japan Food Research Laboratories, Japan; Dr. Hiro Haraguchi at FASMAC Co., Japan; Dr. Koji Okamoto at Tanaka Precious Metal Co., Japan; and the Ministry of industry and primary resources, Brunei Darussalam without which this study would not have been possible. The author is thankful to Mr. Haikal Ismail at STML, Universiti Utara Malaysia for kindly assisting him in the preparation of this manuscript. REFERENCES

Hasan Meat species the loop amplification DNA sensor. 605.

identification based on mediated isothermal and electrochemical Food Control 21, 599-

Aida, A. A., Che Man, Y. B., Wong, C. M. V. L., Raha, A. R. and Son, R. (2005). Analysis of raw meats and fats of pigs using polymerase chain reaction for halal authentication. Meat Science 69(1), 47–52. Chen, F. C., and Hsieh, Y. H. P. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International 83(1), 79–85. Chen, S. Y., Liu, Y. P. and Yao, Y. G. (2010). Species authentication of commercial beef jerky based on PCR–RFLP analysis of the mitochondrial 12S rRNA gene. Journal of Genetics and Genomics 37(11), 763–769. Fajardo, V., González, I., Martín, I., Rojas, M., Hernández, P. E., García, T. et al. (2008). Differentiation of European wild boar (Susscrofa scrofa) and domestic swine (Susscrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes. Meat Science 78(3), 314–322. Fajardo, V., González, I., Rojas, M., García, T. and Martín, R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends in Food Science & Technology 21(8), 408–421.

Ahmed, M. U., Hasan, Q., Hossain, M. M., Saito, M. and Tamiya, E. (2010). ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Determination of Halal Products Girish, P. S., Anjaneyulu, A. S. R., Viswas, K. N., Shivakumar, B. M., Anand, M., Patel, M. et al. (2005). Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) of mitochondrial 12S rRNA gene. Meat Science 70(1), 107–112. Holzhauser, T., Stephen O., and Vieths, S. (2006). Polymerase chain reaction (PCR) methods for the detection of allergenic foods. In “Detecting Allergens in Food,” S.J. Koppelman and S.L. Hefle, edc. CRC Press, Boca Raton, pp. 125-143. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., & Shinmura, Y. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science 1, 143-148.

Hasan Radu, S. (2009). Meat species identification and halal authentication analysis using mitochondrial DNA. Meat Science 83(1), 57–61. Nakyinsige, K., Che Man, Y.B., & Sazili, A.Q. (2012). Halal authenticity issues in mean and meat products. Meat Science 91, 207-214. Okamoto, K. (2016). Development of Porcine Immunochromato. Bioindustry, April 2016, Vol. 33(4), 26-32, CMC Books, Tokyo. (in Japanese). Tanabe, S., Miyauchi, E., Muneshige, A., Mio, K., Sato, C., and Sato, M. (2007). PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods. Bioscience, Biotechnology, and Biochemistry 71(7), 1663–1667.

Murugaiah, C., Noor, Z. M., Mastakim, M., Bilung, L. M., Selamat, J., &

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Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

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Bacteriophages for Biocontrol of Foodborne Pathogens: An Overview Bhandare Sudhakar Ganapati* Faculty of Veterinary Medicine, University Malaysia Kelantan, Locked Bag 36, Pengkalan Chepa, 16100 Kota Bharu, Kelantan, Malaysia; *corresponding author, e-mail: sudhakar@umk.edu.my

ABSTRACT This article is an overview to depict the potential of bacteriophages as biocontrol agents in controlling the foodborne pathogens for enhancing the food safety. Pathogenic strains of Salmonella spp., Campylobacter spp., E. coli, Listeria spp., Vibrio spp. and many other foodborne bacteria are a significant cause of foodborne illnesses in humans worldwide and especially in the developing world. Antibiotic resistance is increasing in many foodborne pathogens and the development pathway for new antibiotics is time consuming. Thus very few new antibiotics are discovered in recent years. Hence there is an urgent need for alternatives to antibiotics and bacteriophage biocontrol is one of the viable alternatives to antibiotics especially for the multiple drug resistant pathogens. The concept of using bacteriophages as food safety tool is emerging rapidly and they are becoming the logical agents for targeted control of pathogenic foodborne bacteria to overcome the food safety concerns regarding the entry of such pathogens in food chain and thereby affecting the public health. Thus, the bacteriophage biology, their structure, morphology, classification, mode of action, their usage as biocontrol agents to control foodborne bacteria and their advantages are discussed in this paper. Keywords: Antibiotic resistance; Bacteriophages; Biocontrol; Food safety; Foodborne bacterial pathogens.

INTRODUCTION Antimicrobial or antibiotic resistance (AMR/ABR) is an increasing concern world over. The World Health Organization in its Global Surveillance Report on ABR states that “The problem is so serious that it threatens the achievements of modern medicine. A post-antibiotic era - in which common infections and minor injuries can kill – is a very real possibility for the 21st century.” The US President (Whitehouse, 2014) and UK Prime Minister (O’Neill, 2014) have already asked their Governments to prepare a roadmap to tackle this issue. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

The European parliament has asked the member states to specifically prioritize the development of phage therapy (Parliamentary Assembly, 2014). Pathogenic strains of Salmonella spp., Campylobacter spp., E. coli, Listeria spp., Vibrio spp. and many other foodborne bacteria are a significant cause of foodborne illnesses in humans worldwide and especially in the developing world. Such illnesses can be treated with antibiotic chemotherapy but over the years, there has been considerable misuse of antibiotics. Many strains of foodborne pathogens are becoming resistant to antibiotics. The progressive resistance of 20

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Phage Biocontrol of Foodborne Pathogens Salmonella to the antibiotics has been reported (Fluit, 2005). The multiple drug resistance in zoonotic non typhoidal Salmonella from food animals and food sources is of great concern all over the world due to their entry into human food chain (Oâ&#x20AC;&#x2122;Mahony et al., 2005). Similar antibiotic resistance is noticed in Campylobacter species (Bester and Essack, 2008), E. coli (Diarrassouba et al., 2007 and Saenz et al., 2001) and Listeria species (Li et al., 2007). Drug resistance emerges through excessive usage of antimicrobials in humans and in food animals, which imposes a selection pressure for bacteria that are resistant to antibiotics (Huges and Heritage, 2006). Particularly, in fish farming antibiotics are given to the whole population with inaccurate doses and thus healthy fishes along with sick ones are exposed to antibiotics and thus development of antibiotic resistance is inevitable (Sorum, 2008). In USA about 40% of total antibiotics produced are used on livestock and about 80% of that is used as growth promoters (Hileman, 1999). Thus, considering the threat to public health the European Commission has imposed an EU wide ban on the use of antibiotics as growth promoters in animal feed from January 1, 2006 (European Commission, 2006). The development pathway for new antibiotics is time consuming and very few new antibiotics are discovered in recent years. Hence, there is an urgent need for alternatives to antibiotics and bacteriophage biocontrol is one of the viable alternatives to antibiotics especially for the multiple drug resistant pathogens (Barrow, 2001; Matsuzaki et al., 2014). Lytic phages are responsible for limiting bacterial numbers in an aquatic environment with up to 80 % of mortality evidenced in the bacterial population (Weinbauer, 2004). Phages are strain specific and thus attempting a biocontrol of single strain of bacteria at a ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Bhandare time is advisable and if successful another strain can be tackled. Eventually, a cocktail of phages will give a broad-spectrum activity. Recently, the scientific community has witnessed the sudden surge of interest in bacteriophage research. Bacteriophage therapy is a promising and natural way of reducing Salmonella (Atterbury et al., 2007), Campylobacter (Atterbury et al., 2005) and E. coli (Raya et al., 2006) as a pre harvest treatment. While, post harvest application of phages to reduce Listeria monocytogenes (Leverentz et al., 2003) contamination in later stages of food production is logical. Phages can help to reduce specific bacterial load in food animals through proactive ante mortem interventions rather than reactive end product testing or treatment of patients. They can be applied directly to foods to extend the shelf life and also as hygiene indicators in assessing food quality (Hudson et al., 2005). The isolation and characterization of bacteriophages is uncomplicated and is possible on large scale (Toro et al., 2005). Hankin in 1896 first noticed the bactericidal activity of phages from the Ganges and Jumna river waters in India, which when filtered had antibacterial properties against Vibrio Cholerae. Phage therapy was pioneered by Felix Dâ&#x20AC;&#x2122;Herelle, in early 19th century and his major contributions came from his work in India and he could discover the phages in Ganges, the holy river in India (Summers, 2001). The former Soviet Union has been using phage therapy since 1920s (Chanishvili et al., 2001) but after the discovery of antibiotics in early nineteenth century scientists stopped working with phages until recently. The reappraisal of phage therapy in the Western world was done by H. Williams Smith and his colleagues for oral E. coli infection control in neonatal animals and for systemic 21

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Phage Biocontrol of Foodborne Pathogens infection control of E. coli in mice (Smith and Huggins, 1982; Smith and Huggins, 1983). The concept of using bacteriophages as food safety tool is emerging rapidly (Hagens and Offerhaus, 2008) and they are becoming the logical agents for targeted control of pathogenic foodborne bacteria to overcome the food safety concerns regarding the entry of such pathogens in food chain and thereby affecting the public health. There are regulatory concerns over the human phage therapy but their application for food safety has relatively less regulatory concern owing to the presence of phages already in the food environment.

Bhandare Phage structure and morphology They consist of a nucleic acid genome surrounded by a protein coat called as capsid. Many phages contain additional structures such as collar, tails, basal plate and spikes or fibers (Figure 1). Structure of phages may be icosahedrons, spherical shapes consisting of triangular faces, or filamentous or complex structures consisting of icosahedral heads with helical tails. Their genomes can consist of either DNA or RNA, single (ss) or double (ds) stranded, circular or linear (Nicklin et al., 1999).

BACTERIOPHAGE BIOLOGY Bacteriophages (in Greek language “phagein” means ‘to eat’ or ‘to devour’, thus they are bacteria eaters), normally abbreviated to phage are a family of naturally occurring viruses that can be isolated from all those habitats where bacteria can thrive. They are the most abundant in the environment and almost ten bacteriophages are supposed to be there for each bacterial cell (Skrunik and Strauch, 2006). Phages can persist outside the host cells under a great variety of conditions, and usually persist much better than their bacterial hosts under adverse conditions. Most phages are far more resistant to heat, freezing, radiation, chemical disinfection and natural inactivation than their host bacteria (Jofre and Muniesa, 2000). They can infect and kill specific bacteria and do not affect other bacteria or cells, meaning the dysbiosis (imbalance of commensal gut flora) often resulting from the use of broad spectrum antibiotics can be avoided. They are obligate intracellular parasites that are capable of existing as phage particles outside the bacterial cell but can only reproduce inside the cell. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Figure 1: Schematic representation of bacteriophage.

Phage classification The morphology of the phages helps in their classification. The International Committee on Taxonomy of Viruses (ICTV) has presently classified bacteriophages in to one order, ten families and forty genera (ICTV, 2011) based upon their symmetry, nucleic acid and other morphological features (Ackermann, 2006) (Table 1 and Figure 2)

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Phage Biocontrol of Foodborne Pathogens Table 1: Classification of bacteriophages as per ICTV (Source: Bhandare, 2015). Order

Caudovirales Myoviridae

dsDNA linear

Genome size (Kb) 31-317

Caudovirales Podoviridae

dsDNA linear

Caudovirales Siphoviridae

dsDNA linear

Unassigned Unassigned Unassigned Unassigned

Family

Genome

Corticoviridae dsDNA circular supercoiled Plasmaviridae dsDNA circular supercoiled Tectiviridae dsDNA linear Inoviridae ssDNA (+) circular

Unassigned

Microviridae

Unassigned

Cystoviridae

Unassigned

Leviviridae

ssDNA (+) circular dsRNA three linear segments ssRNA (+)

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Bhandare

Envelope Morphology

Virion size

Icosahedral head with tail

Icosahedral heads: 60-45 nm; Elongated heads: 80-110 nm; Tail: 16-20 × 80-455 nm Icosahedral heads: 60-70 nm; Tail: 10-20 nm Icosahedral heads: 40-80 nm; Tail: 5-10 × 100-210 nm 60 nm

16-78

Icosahedral head with short tail

21-134

Icosahedral head with long tail

Icosahedral

Yes

15 Inoviruses: 5.812.4 Plectroviruses: 4.58.2 4.4-6.1

No No

Quasi-spherical, pleomorphic Icosahedral Inoviruses: filamentous Plectroviruses: rod shaped

Icosahedral

66 nm Inoviruses: 7 × 700-3500 nm; Plectroviruses: 15 × 200400 nm 25-27 nm

6.4-7.1; 3.6-4.7; 2.6-3.2 3.5-4.3

Yes

Spherical

85 nm

Icosahedral 23

26 nm

50-125 nm

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Phage Biocontrol of Foodborne Pathogens

Bhandare

Figure 2: Schematic representation of major phage groups. (Source: Ackermann, 2006) Phages have binary (consisting of two parts), cubic, helical and pleomorphic symmetries. Most phages have double stranded (ds) DNA, but there are few phages with single stranded (ss) DNA, ssRNA or ds RNA. Few phages contain lipid containing envelope surrounding the capsid (the protein coat covering viruses). The majority (96%) of the phages are tailed phages (binary symmetry), which are classified into the order Caudovirales and three very large phylogenetically related families, while remaining 4% are other types. Taxonomic names of orders, families, and genera are typically constructed from Latin or Greek roots and end in -virales, -viridae and â&#x20AC;&#x201C; virus, respectively (Ackermann, 2006). Phage mode of action

Phages possess the ability to infect a bacterium and redirect the cell to synthesize phage components for replication. A typical life cycle of a phage (Figure 3) starts by adsorption of the phages on to a specific receptor structures on the surface of the bacterium. After attachment they inject their genetic material inside, which reprogrammes the bacterium for synthesis of phage nucleic acid and other molecules required for reproduction of complete viral particles and thereby stopping synthesis of host cell components. Then new phage particles are assembled and when the new viruses are mature, an enzyme is produced that digests the cell wall, allowing the phage to burst out of the cell. If bacteriophage greatly outnumber their hosts, with many phages attached to each cell, the bacteriumâ&#x20AC;&#x2122;s

Figure 3: Replication cycle of Bacteriophage.

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Phage Biocontrol of Foodborne Pathogens cell wall can become unstable and rupture, without replication of the viruses inside, this phenomenon is called â&#x20AC;&#x2DC;passive inundationâ&#x20AC;&#x2122; (Atterbury, 2006). The stages in the life cycle of the phages are recognized when virus particles infect cells in culture and are illustrated in Figure 4, which exhibits a one-step growth curve. This graph displays the results of a single round of viral multiplication in a population of cells. Following adsorption, the virus particles undergo uncoating and viral nucleic acid is injected into bacterial cell, thus there is no growth observed and the phenomenon is called eclipse. During the latent period, replication of viral nucleic acid and proteins occurs. The maturation period follows, when virus nucleic acid and protein are assembled in to mature virus particles. At this time, if the cells are prematurely lysed (by the use of chloroform for example), mature virus particles can be detected. Finally, release occurs, either with cell lysis (e.g. lytic phage) or without cell lysis (e.g. temperate phage). The timing of the one-step growth cycle varies with the Eclipse

Bhandare viruses as different viruses have different latent periods and burst sizes. With many bacterial viruses, the whole cycle may be complete in 30-60 min (Madigan et al., 2003). The burst size is the average yield of phage particles per cell, which is very important for determination of inoculum size in phage therapy. Many times small doses are not sufficient and even at large doses some times there is no active replication and therapeutic benefits are obtained by using very large or repeated doses of phage (Payne and Jansen, 2001). Large dosage may cause lysis from without so that bacterial cells are destroyed without any phage replication if the phages are applied at high (> 100) MOI i.e. Multiplicity of Infection, which means number of phage per bacterium. The latent period is the minimum length of time from the adsorption of phage to its host, until the release of newly formed phage particles, which is crucial for phage therapy because the inoculations given in right time taking latent period in to account would be more effective. Rise

Plaque forming units

Latent period

Extracellular phage Burst size

Intracellular phage

Time

Figure 4: One-step growth curve of bacteriophage replication (Source: Bhandare, 2015).

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Phage Biocontrol of Foodborne Pathogens BACTERIOPHAGES APPLICATION AS BIOCONTROL AGENTS TO CONTROL FOODBORNE BACTERIA There are several reports on successful usage of bacteriophages in controlling the foodborne pathogens and some of the important reports are discussed in this section. Phages can help to reduce Salmonella in poultry through proactive antemortem interventions rather than reactive end product testing or treatment of sick birds. Berchieri, et al. (1991) successfully used bacteriphages to control Salmonella colonization in the poultry gastrointestinal tract. The mortality was reduced from 60% to 3% although large numbers of phages were needed. Their study revealed that the production of large numbers of phages is practicable on farms. It was also noticed that phages spread readily between bacterially infected birds. Similarly, E. coli infection in the poultry can be controlled by using phage therapy. Toro, et al. (2005) stated that there is a beneficial effect of the phage treatment on weight gain performance in chickens along with reduction of Salmonella colonization. Huff, et al. (2006) used aerosol spray of phages to bring down the mortality of birds due to E. coli infection. The mortality was brought down to 7% in phage treated birds while it was 48% in the untreated ones. Campylobacter spp., the celebrity bug in poultry as referred by Butzler (2004) is also being targeted by the phage therapy. Wagenaar et al. (2005); Loc et al. (2005) and Atterbury et al. (2005) have carried out successful phage therapy trials against Campylobacter spp. in poultry. Amongst the phage therapy reports in large food animals Smith and Huggins (1983) showed an effectiveness of phages in treating experimental E. coli diarrhea in calves, piglets and lambs. Infections were

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Bhandare caused by three different E. coli strains in calves, piglets and lambs and a mixture of two phages were given orally to protect them. E. coli infection was reduced to cure the diarrhea in all three species. The phage resistant mutants were emerged in the calves and piglets but they were less virulent than their parent strains. A cocktail of phages significantly reduced the numbers of E. coli O157:H7 in the intestinal tract of sheep (Callway and colleagues, 2008). Even in the fisheries the successful phage therapy is carried out by Karunasagar et al. (2007). They could achieve biocontrol of pathogens in shrimp hatcheries by using bacteriophages (Douglas, 1974). Also, the Japanese researchers (Park et al., 2000; Park and Nakai, 2003) studied the potential use of phages to control the fish infections caused by Lactococcus and Pseudomonas by giving phages orally as phage impregnated feed. Largely it is a win-win situation for the mankind if phage therapy is thoroughly scrutinized for applications in the food animals and then used because it will help to reduce the antibiotic usage to avoid multiple drug resistance along with the reduction in foodborne pathogen load. Rather than applying the phages in live animals before their slaughter the application of phages after slaughter to the carcasses and to the food products during their processing prior to the consumption is one more option to get rid of foodborne pathogens (Thorns, 2000). The significant reductions in the pathogenic bacteria can be achieved by their bacteriophage biocontrol in food processing. In number of studies the poultry products are used for their surface decontamination by phages. Atterbury et al. (2006) achieved a reduction in S. enteritidis and S. typhimurium below the detectable levels by applying phages to the chicken skin. Similarly, Goode and colleagues (2003) could reduce the Campylobacter

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Phage Biocontrol of Foodborne Pathogens jejuni by 1-1.3 log10 CFU within 24 hours by application of phages to the surface of chicken skin. The phage biocontrol of Salmonella upon whole carcasses of broiler chickens and turkeys was achieved by Higgins et al. (2005). Phages have also been used to reduce the numbers of Salmonella from chicken sausages (Whichard et al., 2003). In case of beef the bacteriophage biocontrol has been attempted to extend shelf life by reducing spoilage organisms (Greer and Dilts, 2002). The numbers of E. coli O157:H7 from the beef surfaces were brought down to below the detectable levels by O’Flynn et al. (2004). A 3 log10 CFU reduction in the Listeria counts on salmon was achieved by Hagens and Loessner (2007) after applying high titres of phage. L. monocytogenes, is more likely to occur during food processing, and thus at this point of time phage biocontrol of this pathogen can be done successfully. AntiListeria phages as food additives have been approved by the US FDA by according it a Generally Recognized As Safe (GRAS) status and such products are available in the market for use in food processing. For control of biofilms in the food processing environment the phage application is being envisaged. Roy et al. (1993) and Hibma and colleagues (1997) have showed that the formation of Listeria biofilms in the food processing environments can be reduced by application of phages. A great deal of research is being carried out for non-thermal ways of controlling foodborne pathogens during processing for the want of maintaining nutritive values of food and phage-based non-thermal intervention is an ideal way in such situations.

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Bhandare ADVANTAGES OF USING PHAGES FOR FOOD SAFETY ♦ Phages are ubiquitous in the environment. Every known bacterium in nature is supposed to have its complementary phage and thus it is possible to use phage biocontrol against any type of bacteria. ♦ Bacteriophages are strain specific to an individual bacteria and they don’t harm the other beneficial bacterial flora of the gut as is the case with antibiotics which harm the commensal gut flora. They can also be given in cocktails for broad spectrum effect. ♦ Bacteriophages are self replicating and thus the given dosage can self amplify in due course of the treatment so that they effectively tackle the target bacteria. Also, they will only replicate till the target bacterium is present and thus they are naturally self limiting. ♦ As there is resistance development in bacteria for antibiotics, the phage resistance may also develop but it has been reported (Loc Carrillo et al., 2005; Atterbury et al., 2007 and Smith and Huggins, 1983) that such bacteria would have low virulence or less survival rate owing to the ‘fitness penalty’. Also, the resistance to phage does not affect their sensitivity to antibiotics and the relevant phages naturally evolve alongside as bacteria evolve resistance. ♦ There are no reports of allergic reactions to phage as in antibiotics and no serious side effects have been described so far either in animals or humans. This may be due to the abundance of phages in our environment and regular exposure of animals and humans to them.

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Phage Biocontrol of Foodborne Pathogens ♦ It is relatively cheaper and easier to produce phage preparations especially as topical applications for surface decontamination of foods. ♦ As the human phage therapy is fraught with the limitations of lack of public confidence and more regulatory concerns, the application of phages as biocontrol agents in food animals and in food processing is an ideal way of harnessing the potential of this nature’s weapon for food safety. Though not totally eliminated, the foodborne pathogens can be reduced to the acceptable levels by application of bacteriophages as pre-harvest and post-harvest interventions. Phages can be effectively used along with other food safety tools to protect public health. REFERENCES Ackermann, H.W. (2006). ‘Classification of bacteriophages’, In: Calendar, R. (Ed). The Bacteriophages. Oxford University Press, New York. pp. 816. Atterbury, R. J., Van Bergen, M.A., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer, A., Wagenaar, J.A., Allen, V.M., and Barrow, P.A. (2007). ‘Bacteriophage therapy to reduce Salmonella colonization of broiler chickens’, Applied and Environmental Microbiology 73, 4543. Atterbury, R.J. (2006). The age of phage? Poultry International 6, 18-22. Atterbury, R.J., Dillon, E., Swift, C., Connerton, P.L., Frost, J.A., Dodd, C.E., Rees, C.E. and Connerton,

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Bhandare I.F. (2005). ‘Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca’, Applied and Environmental Microbiology 71, 4885-7. Atterbury, R.J., Van Bergen, M.A., Ortiz, F., Lovell, M., Harris, J.A. and De Boer, A. (2006). ‘Control of Salmonella in poultry using bacteriophage’, In Proceedings of the 13th International Symposium Salmonella Salmonellosis. Colin, P., and Clement, G. (eds). Saint Malo, France: 10–12 May, pp. 579–580. Barrow, P. A. (2001). "The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection." Journal of Chemical Technology and Biotechnology 76(7), 677-682. Berchieri, A. Jr, Lovell, M.A. and Barrow, P.A. (1991). ‘The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium’, Research in Microbiology 142, 541-549. Bester, L.A. and Essack, S.Y. (2008). ‘Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry suppliers in KwaZulu-Natal, South Africa’, Journal of Antimicrobial Chemotherapy 62(6), 1298-1300. Bhandare, S. G. (2015). Biocontrol of V. cholerae using bacteriophage. PhD, Thesis. University of Nottingham, UK.

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Phage Biocontrol of Foodborne Pathogens Butzler, J.P. (2004). ‘Campylobacter, from obscurity to celebrity’, Clinical Microbiology & Infection 10, 868. Callaway, T.R., Edrington, T.S., Brabban, A.D., Anderson, R.C., Rossman, M.L. and Engler, M.J. (2008). ‘Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts’, Foodborne Pathogens and Disease 5, 183–191. Chanishvili, N., Chanishvili, T., Tediashvili, M. and Barrow, P.A. (2001). ‘Phages and their application against drug-resistant bacteria’, Journal of Chemical Technology and Biotechnology 76, 689–699. Diarrassouba, F., Diarra, M.S., Bach, S., Delaquis, P., Pritchard, J., Topp, E. and Skura, B.J. (2007). ‘Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolates from commercial broiler chicken farms’, Journal of Food Protection 70(6), 1316-27. Douglas, J. (1974). Bacteriophages. Chapman and Hall, London. pp. 2148. European Commission (2006). Ban on antibiotics as growth promoters in animal feed enters into effect. Press release of 22/12/2005. http://europa.eu/rapid/pressrelease_IP-05-1687_en.htm (accessed on 10/5/2016). Fluit, A.C. (2005). Towards more virulent and antibiotic-resistant Salmonella?

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Bhandare FEMS Immunology & Microbiology 43, 1-11.

Medical

Goode, D., Allen, V.M. and Barrow, P.A. (2003). ‘Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages’, Applied Environmental Microbiology 69, 5032-5036. Greer, G.G. and Dilts, B.D. (2002). ‘Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages, Journal of Food Protection 65, 861– 863. Hagens, S. and Loessner, M.J. (2007). ‘Application of bacteriophages for detection and control of foodborne pathogens’, Applied Microbiology and Biotechnology 76, 513–519. Hagens, S. and Offerhaus, M.L. (2008). ‘Bacteriophages - new weapons for food safety’, Food Technology 4, 4654. Hankin, M.E. (1896). The bactericidal action of the waters of the Jamuna and Ganga rivers on Cholera microbes. (Translated from the original article published in French, Ref. Ann. De I’Inst. Pasteur 10, 511. Hibma, A.M., Jassim, S.A.A. and Griffiths, M. W. (1997). ‘Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage’, International Journal of Food Microbiology 34, 197–207. Higgins, J.P., Higgins, S.E., Guenther, K.L., Huff, W., Donoghue, A.M.,

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Phage Biocontrol of Foodborne Pathogens Donoghue, D.J. and Hargis, B.M. (2005). ‘Use of a specific bacteriophage treatment to reduce Salmonella in poultry products’, Poultry Science 84, 1141–1145. Hileman B. (1999). ‘Livestock antibiotic debate heats up’, Chemical and Engineering News 25, 32–35. Hudson, J.A., Billington, C., CareySmith, G. and Greening, G. (2005). ‘Bacteriophages as Biocontrol Agents in Food’, Journal of Food Protection 68, 426-437. Huff, W.E., Huff, G.R., Rath, N.C., and Donoghue, A.M. (2006). ‘Evaluation of the influence of bacteriophage titer on the treatment of colibacillosis in broiler chickens’, Poultry Science. Vol. 85, pp. 1373– 1377. Huges

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Bhandare Karunasagar, I., Shivu M.M., Girisha, S.K. , Krohne, G. and Karunasagar, I. (2007). ‘Biocontrol of pathogens in shrimp hatcheries using bacteriophages’, Aquaculture 268, 288–292. Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladze, T., Yang, M., Saftner, R. and Sulakvelidze, A. (2003). ‘Biocontrol of Listeria monocytogenes on freshcut produce by treatment with lytic bacteriophages and a bacteriocin’, Applied and Environmental Microbiology 69, 4519. Li, Q., Sherwood, J.S. and Logue, C.M. (2007). ‘Antimicrobial resistance of Listeria spp. recovered from processed bison. Letters in Applied Microbiology 44(1), 86-91. Loc Carrillo, C., Atterbury, R.J., elShibiny, A., Connerton, P.L., Dillon, E., Scott, A. and Connerton, I..F. (2005). ‘Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens’, Applied Environmental Microbiology 71, 6554–6563. Madigan, M.T., Martinko, J.M. and Parker, J. (2003). Brock biology of microorganisms (Tenth edition). Pearson Education International, NJ, USA pp 959-960. Matsuzaki, S., J. Uchiyama, I. TakemuraUchiyama and Daibata, M. (2014). Perspective: The age of the phage. Nature 509(7498), S9.

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Phage Biocontrol of Foodborne Pathogens Nicklin, J., Graeme-Cook, K., Paget, T. and Killington, R. (1999). Instant notes in Microbiology. Bios Scientific Publishers, Oxford, UK. pp. 329-335. O'Flynn, G., Ross, R.P., Fitzgerald, G.F. and Coffey, A. (2004). ‘Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157: H7’, Applied Environmental Microbiology 70, 3417-3424. O'Mahony, R., Saugy, M., Leonard, N., Drudy, D., Bradshaw, B., Egan, J., Whyte, P., O'Mahony, M., Wall, P. and Fanning, S. (2005). ‘Antimicrobial Resistance in Isolates of Salmonella spp. from Pigs and the Characterization of an S. Infantis Gene Cassette’, Foodborne Pathogens and Disease 2(3), 274281. O’Neill, J. (2014). Antimicrobial resistance: Tackling a crisis for the health and wealth of nations. http://amrreview.org/sites/default/files/AMR Review Paper - Tackling a crisis for the health and wealth of nations_1.pdf. (Accessed on 10/5/2016). Park,

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Park, S.C., Shimamura, I., Fukunaga, M., Mori, K.I. and Nakai, T. (2000). ‘Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control’, Applied

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Parliamentary Assembly (2014). Phage Therapy, a Public Health Issue. http://assembly.coe.int/nw/xml/XRef /X2H-XrefViewPDF.asp?FileID=20704&lang= en, European Union. (Accessed on 10/5/2016). Payne, R.J.H. and Jansen, V.A.A. (2001). ‘Understanding bacteriophage therapy as a density-dependent kinetic process’, Journal of Theoretical Biology 208, 37-48. Raya, R. R., Varey, P., Oot, R.A., Dyen, M.R., Callaway, T.R., Edrington, T.S., Kutter, E.M. and Brabban, A.D. (2006). ‘Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep’, Applied and Environmental Microbiology 72, 6405. Roy, B., Ackermann, H.W., Pandian, S., Picard, G. and Goulet, J. (1993). ‘Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound’, Applied Environmental Microbiology 59, 2914–2917. Sáenz, Y., Zarazaga, M., Briñas, L., Lantero, M., Ruiz-Larrea, F. and Torres, C. (2001). ‘Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain’, International Journal of Antimicrobial Agents 18(4), 353-8.

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Phage Biocontrol of Foodborne Pathogens Skrunik, M. and Strauch, E. (2006). ‘Phage therapy: Facts and fiction’ International Journal of Medical Microbiology 296, 5-14. Smith, H.W. and Huggins, M.B. (1982). ‘Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics’, Journal of General Microbiology 128, 307-318. Smith, H.W. and Huggins, M.B. (1983). ‘Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets, and lambs’, Journal of General Microbiology 129, 2659-2675. Sorum, H. (2008). Antibiotic resistance associated with veterinary drug use in fish farms. Improving farmed fish quality and safety. Lie. Cambridge, England, Woodhead Publishing, pp. 157-182. Summers, W.C. (2001). Bacteriophage therapy. Annual Reviews in Microbiology 55, 437-451. Thorns, C.J. (2000). ‘Bacterial foodborne zoonoses’, Revue Scientifique et Technique (International Office of Epizootics) 19, 226-39.

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Bhandare Toro, H., Price, S.B., Mckee, S., Hoerr, F.J., Krehling, J., Perdue, M. and Bauermeister, L (2005). ‘Use of Bacteriophages in combination with Competitive Exclusion to reduce Salmonella from infected chickens’, Avian Diseases 49, 118-124. Wagenaar, J., Van Bergen, M.A., Mueller, M.A., Wassenaar, T. and Carlton, R. (2005). ‘Phage therapy reduces Campylobacter jejuni colonization in broilers’, Veterinary Microbiology 109, 275–283. Weinbauer, M. G. (2004). Ecology of prokaryotic viruses. FEMS Microbiol Rev. 28(2), 127-181. Whichard, J.M., Sriranganathan, N. and Pierson, F.W. (2003). ‘Suppression of Salmonella growth by wild-type and large plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters. Journal of Food Protection 66, 220– 225. Whitehouse (2014). Report to the President on Combating Antibiotic Resistance. White house report.https://www.whitehouse.gov/s ites/default/files/microsites/ostp/PCA ST/pcast_carb_report_sept2014.pdf. (Accessed on 10/05/2016).

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016), P33-41

Cloning and Expression of the Urease Operon from Helicobacter pylori J99 Mohamad CWSR.1, 2, *, Abdul-Manaf U.2 and Mat-Arip Y.2 1

Biomedical Electronic Engineering, School of Mechatronic Engineering, Pauh Putra Main Campus, Universiti Malaysia Perlis, Arau, 02600 Perlis, Malaysia; 2 School of Biology Science, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; *corresponding author, e-mail: robiah@unimap.edu.my

ABSTRACT Aims: Helicobacter pylori urease is one of the antigens found in H. pylori with strong immunogenic property. This present study was to clone the whole of urease operon (pET32UOA6) with biologically active recombinant urease enzyme complex (UreA/UreB). Methodology and results: A recombinant molecule of the full-length urease operon was constructed in vitro from the H. pylori J99 and expressed in Escherichia coli cells, BL21 (DE3). The potential colonies were screened for inserts by performing colony PCR using specific primer, restriction enzyme digestion and nested PCR. A positive urease operon transformed using pET32Ek/LIC vector carrying the recombinant gene of the full-length urease operon, 5.9 Kb. This positive urease operon was growth in LB-medium and at exponential-phase culture of recombinant urease operon was induced with 0.4mM isopropyl-beta-D-thiogalactoside (IPTG). Positive urease clone pET32UOA6 expressed both ureases, UreA and UreB. This meant that the cloned urease operon was functioning in E. coli cell. Therefore, cloning of the whole of urease operon (pET32UOA6) produced biologically active recombinant urease enzyme complex (UreA/UreB) and verified by immune functioning assay using commercial antibody H. pylori urease-Îą and commercial antibody H. pylori urease-Î˛. Other than that, the confirmation of recombinant protein of urease operon was demonstrated by protein sequencing. The results of amino acid alignment based on BLASTp between recombinant urease against H. pylori J99 urease show high percent identities, 99-100%. Conclusion, significance and impact of study: The production of recombinant UreA/UreB complex indicates that a fully functional urease operon or urease operon was successfully constructed. In addition, the constructed H. pylori urease replicon opened an opportunity for developing a genetically modified animal model to study H. pylori pathogenesis. Keywords: Escherichia coli; Helicobacter pylori, pET32 Ek/LIC; Recombinant protein; Urease.

INTRODUCTION Helicobacter pylori is one of the common bacterial infections in human and recognized as the etiologic agent for majority of upper gastro duodenal diseases. H. pylori has been

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established as the causative agent for acute or chronic gastritis (Mitchell et al., 1999) and could be further developed into peptic ulcer disease, gastric carcinoma and others upper gastro duodenal diseases (Kiesslich et al., 2005; Ardekani et al., 2013). According

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Cloning and Expression of the Urease to the World Health Organization (WHO) statistic, H. pylori infection is on the rise and proportional to the progress of a country. Almost 50% of the world's population is infected by H. pylori (Sasidharan et al., 2008). In Malaysia, H. pylori infection is on the raise as well. From the year 2000 until 2007, patients infected by H. pylori were 30.4% of the gastro duodenal cases reported (Sasidharan et al., 2008). Urease is one of the pathogenic factors that help H. pylori colonizes the epithelium in the acidic environment of the stomach (Ardekani et al., 2013). H. pylori urease displays enzyme-independent effects in mammalian models, mostly through lipoxygenases-mediated pathway (Uberti et al., 2013). The urease would induce edema, neutrophil chemotaxis and shows apoptosis inhibition reverted in the presence of the lipoxygenase inhibitors esculetin (Uberti et al., 2013). In addition to its involvement in the pathogenesis process of H. pylori infection, urease is also a target for vaccination development (Voland et al., 2006) besides being a suitable marker to use as a target protein for detecting presence of H. pylori infection among suspected gastrointestinal patient. In this study, urease operon were isolate and examined by colony PCR, restriction enzyme digestion and nested PCR with specific primer to confirm orientation of urease operon was done before continue to protein expression. The sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gel was performed to examine the immune functioning assay of recombinant urease operon of Helicobacter pylori J99 and protein sequencing to make sure this protein was functioning.

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Mohamad et al. MATERIALS AND METHODS Escherichia coli growth and maintenance The E. coli strains (Merck Inc.) used in this study and their genotypes are shown in Table 1. Escherichia coli strains were grown on LB broth at 37°C for 16 hours in a shaker incubator (Shel Lab, UK). For storage purposes, E. coli strains were stored in LB broth containing 50% glycerol (50%), then, kept at -80oC for long term storage. Table 1: Genotypes of E. coli strains. Strain Genotype Nova Blue endA1 hsdR17(rK12– mK12+) supE44 thi-1 recA1gyrA96 relA1 lac [F’ proA+B+ lacIqZΔ M15 ::Tn10] BL21 (DE3) F– ompT hsdSB (rB– mB–) gal dcm (DE3) Helicobacter pylori J99 growth and maintenance Helicobacter pylori J99 (ATCC 700824) was grown on Eugon agar with 10% human expired blood at 37°C, 10% CO2 and 100% humidity in an incubator (Shel Lab, UK). Subcultured was performed every four to seven days to maintain fresh bacterium. For storage purposes, H. pylori J99 strain was stored in TSB containing 20% glycerol (20%), then, kept at -20oC for short term storage and -80oC for long term storage. Plasmid cloning vector The plasmid cloning vector used in this study was pET-32-Ek/LIC (Merck, Germany). This vector contains 109aa Trx•TagTM which encodes for the 109 amino acid of thioredoxin protein. The pET-32Ek/LIC vector was specially designed for cloning and high-level expression of target protein. Thioredoxin protein and histidine tag would be fused to the target protein to enhance purification of the expressed proteins. 34

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Cloning and Expression of the Urease Genomic extraction of H. pylori J99 Genomic of H. pylori was extracted by employing GENEAll ExGene Cell SV kit (Intron, Korea). The extracted genomic DNA was then stored at -20oC for further analysis. This genomic extraction of H. pylori J99 was used as a template for amplification of urease gene. Primer pair (pET32UOHP-F & pET32UOHP-R) would amplify the urease operon. The specific PCR primers for urease operon with pET-32 Ek/LIC complimentary overhangs are shown in Table 2. Table 2: Specific PCR primers sequence with vector-compatible overhang. Primer Sequences pET32UOHP-F 5’ GAC GAC GAC AAG ATG AAA CTC ACC CCA AAA GAG 3’ pET32UOHP-R 5’ GA GGA GAA GCC CGG TTC AAA CCT TTT GCG TGG TGG 3’ Amplification of urease gene The total volume for PCR reaction was 25 µl [1x PCR buffer, 0.1 mM dNTPs (NHK Inc), 0.4 pmol of each pET32UOHP-F, pET32UOHP-R primers, 1 unit of i-Taq DNA polymerase (NHK Inc) and 50 to 100 ng of H. pylori J99 DNA template]. Gradient temperatures for amplification of urease operon were from 64oC to 74oC by using gradient PCR thermal cycler (Biometra, USA). PCR cycler was programmed for 30 cycles with 95oC for 1 minute, 57oC for 2 minutes for full length urease operon and 72oC for 1 minute. The final elongation was set at 72oC for 10 minutes. Negative control was included consisting of all mixture except the DNA template. After PCR ended, 1 µl of ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. the PCR product was analyzed on 1% agarose gel then stained with ethidium bromide (EtBr) and 1Kb DNA ladder (Promega, USA) was used as a marker. Preparations of pET32 Ek/LIC insert The purification PCR product of H. pylori urease operon by employing PCR DNA Extraction System (Intron, Korea). After that, the annealing procedure between purified PCR products (Ek/LIC insert) with pET32 Ek/LIC vector was follow according to manufacturing protocol (Merck Inc.). Transform recombinant into cloning host Three microliter (3 µl) of amplification product was transferred into a new 1.5 ml Eppendorf tube which had been cooled on ice and the remaining annealing product was stored at -20°C. An uncut plasmid (0.1 ng) was used as a control for transformation efficiency of the competent cells. Escherichia coli NovaBlue competent cells were thaw on ice before transformation procedure. Transformation involved mixing 50 µl of the competent cell with 2 µl of annealing product and mixed gently. After that, the mixture was incubated on ice for 5 minutes, then, heat-shocked for 30 seconds at 42°C. Immediately after heat-shock, the tube was placed on ice for 2 minutes. Next, 80 µl of LB was added and the mixture was incubated at 37°C, shaker at 250 rpm for 60 minutes. The mixture was spread onto LB agar plate containing ampicillin (100µg/mL). The plates were incubated overnight (16 - 20 hours) at 37°C. Screening of recombinant urease operon Colonies that formed had the possibilities of carrying the insert. Thus, the colonies were screened for inserts by performing colony PCR using specific primers as in Table 3. The potential clones were also subjected to restriction enzyme digestion to confirm the 35

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Cloning and Expression of the Urease presence of the inserts. The used of restriction enzymes either as single (Sal1) or double digestions (EcoR1/ Pst1 and EcoRV/Bgl II) followed the manufacturer protocol (Promega, Inc.). The orientation of the urease gene was determined so that it was in the correct reading frame by nested PCR. The specific primers cws1, cws2 and cws3 were used to confirm the target insert as shown in Table 3. Finally, the clones were verified using DNA sequencing which was carried out by a service provider, NHK Bioscience Solution Sdn Bhd. The selected recombinant plasmid was named as pET32UOA6 for urease operon recombinant for expression urease UreA/UreB. Table 3: Specific nested PCR primers used for insert confirmation. Gene Sequences cws-1-F 5-CGT TAT GTC CTT AAG GAA AAA AC-3’ cws-1-R 5-CCC ATG AGC GAT CGC TGG GTT AAT GG-3’ cws-2-F 5’- CCA TTA ACC CAG CGA TCG CTC ATG GG -3’ cws-2-R 5’- CTA TGG GGC ATG CTT ACG GTT AAG -3’ cws-3-F 5-CAA AGC TGA ATT CCA ACG ATC GCT TAA CCG TAA GCA TGC C-3’ cws-3-R 5-GGT TAA AAA GAC TCG AGG GTT TTT TAA TC-3’ Transformation of pET32UOA6 into expression host The recombinant plasmids were sub-cloned into an expression host, E. coli BL21 (DE3) following the manufacturer protocol (Promega Inc.) Expression of urease operon A single colony was inoculated into a universal bottle containing 5 ml of LB broth ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. with ampicillin (100µg/mL) and incubated at 250 rpm, 37oC for 3 to 4 hours until OD600 reached 0.4 to 1.0. Next, 2 ml of this culture was aliquot into Mc Courtney bottles and kept at 4oC overnight. The next day, 2 ml pre-culture from 4oC was centrifuged at 13 000xg for 15 seconds, the supernatant was discarded and the cell pellet was suspended with 2 ml LB broth with ampicillin (100µg/mL). The suspended cell with fresh media then inoculated into a 250 ml flask containing 50 ml LB broth with ampicillin (100µg/mL). The cells were grown at 37oC, 250 rpm until OD600 reached approximately 0.6 (~3 to 4 hours). One ml of this culture was removed as un-induced sample for SDS-PAGE analysis. Cells were induced with 0.4mM IPTG at 37oC, 250 rpm for 3 hours. Subsequently, the culture was incubated on ice for 10 minutes and harvested by centrifugation at 5000 x g for 20 minutes at 4oC. The cell pellet was washed with 0.25 culture volume of cold 20 mM Tris-HCl pH 8.0, then centrifuged at 5000 x g for 20 minutes at 4oC. The supernatant were discarded and the cells pellet was kept at 80oC for further analysis. The expression of urease protein was detected by Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDSPAGE). The preparation and the assembly of SDS-PAGE electrophoresis was carried out according to the manufacturer protocol (Bio-Rad, USA). Briefly, the resolving gel was at 12.5% with standard 4% stacking gel. Cell pellets were suspended with PBS before 5X SDS-PAGE sample buffers at the ratio 3:1 was added. Next, the sample was heated at 95oC for 5 minutes. After heated, this sample became viscous and 29cc gouge needle was used to reduce the viscosities of the sample. Twenty microliter of sample 36

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Cloning and Expression of the Urease was loaded per lane on the SDS-PAGE gels. The SDS-PAGE running buffer was filled into the bottom and upper chambers and all the bubble were removed from the wells of the gels using a syringe. The gel was run at 80V constant current for 35 minutes and 100V for 2 hours until the stacking dye had reached the end of the gel. After that, the gel was carefully removed from the glass plates and stained with Coomassie Blue stain for 1 hour and destained with destining solution overnight (~16 hours). The expression of constructed recombinant urease operon was important to measure the urease expression level and to know the biological active of the recombinant urease. Immune functioning assay The immune functioning assay was used to verify the recombinant protein of pET32UOA6. The electrophoresis transfer of proteins to 0.45 Âľm Polyvinylidene fluoride (PVDF) membrane (Millipore Inc.) was accomplished by a modification of the method described by Towbin et al. (1979). Protein was electro blotted from the gel onto PVDF membrane by electrophoresis transfer using a Mini Trans-boltÂŽ Electrophoretic Transfer Cell (Bio-Rad, USA). Lastly, the membrane was put in a plastic wrap and the image was captured within 1 minute using Gene Genius Bio Imaging System. Protein sequencing Recombinant urease clones were grown, induced with 0.4 mM IPTG, followed by SDS-PAGE as described in Section 3.6.9. After SDS-PAGE, the band which represented the recombinant UreA and UreB was purified and sent to 1st BASE, Inc. for protein sequencing. Finally, the amino acid sequence resulted from protein sequencing was aligned using Protein Basic Local Alignment Search Tool (NCBI BLASTp) of National Center for Biotechnology Information (Stephen et al., 1997) and this ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. protein sequencing was carried out to ensure the expressed recombinant urease was identical to H. pylori J99 urease protein. RESULTS AND DISCUSSION Determine of recombinant urease operon The urease operon was determined by their size based on urease gene sequence information, accessed from NCBI database (Accession No. NC_000921.1). Complete urease operon that encode for urease enzymes and accessory proteins. As shown in Figure 1, the presence of the urease operon PCR amplified was detected with the expected size of 5974 bp.

Figure 1: Amplification of urease genes from ATCC 00824 H. pylori J99. Lane 1: 1Kbp DNA ladder; Lane 2 to 7: amplification of urease genes; Lane 8: Negative control (lack of i-Taq DNA polymerase). Recombinant clone screening using PCR The positive colonies were selected and subjected to PCR reaction using specific primers (Table 3) to confirm the presence of the inserts. All selected colonies was had inserts with the expected size that represent the size of urease operon fragment. Restriction enzyme digestion analysis Two clones from each of the recombinants were further selected for verification. The plasmids from potential recombinant clone pET32UO were digested with restriction enzymes to further confirm the presence of urease gene fragments. A single digestion (Sal I) and double digestion (EcoRI & Pst1 37

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Cloning and Expression of the Urease or EcoRV & Bgl II) produced different restriction enzyme patterns due to the unique sites of these restriction enzymes on the plasmids. Table 4 shows the expected fragment sizes resulted from the chosen restriction enzymes. Furthermore, agarose gel electrophoresis verified the expected fragment sizes from restriction enzyme digestions, as shown in Figure 2. All of the selected clones indicated the presence of recombinant plasmid harbouring urease genes. Table 4: Restriction enzyme digestions of a selected potential recombinant clone. Expected Recombinant Restriction size clones enzyme fragment (kbp) pET32UO EcoRI & 1332 & Pst I 10559 EcoRV & Bgl II

5865 & 6026

Sal I

11891

Figure 2: Restriction enzyme analysis of potential recombinant clones. The restriction enzyme digestion of urease operon fragment. Lane 1: 1 Kb DNA ladder; Lane 2 and 6: undigested plasmids; Lane 3 and 7: EcoRI & Pst I digestion; Lane 4 and 8: EcoRV & Bgl II digestion; Lane 5 and 9: Sal I digestion and Lane 10: Îť Hind III DNA ladder. DNA Sequencing ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. Final verification of the recombinant plasmids was made by DNA sequencing on clone pET32UOA6. The DNA sequencing was performed using specific primers as shown in Table 3. The results of the DNA sequencing showed 99% nucleotide similarity for urease operon to H. pylori J99 genome when analyzed using NCBI BLAST program (Zheng et al., 2000). Expression of urease genes Plasmid pET32 Ek/LIC carries IPTG inducible T7lac promoter for protein expression (Merck, Inc.). The expressed protein from this plasmid would be a fusion protein of 109 amino acids thioredoxin to the protein of interest. Thus, the recombinant urease produced would be slightly bigger than the native urease Induction study of ureases production In this study, the expression of recombinant urease was used 0.4 mM and/or 1.0 mM IPTG and 2 and/or 3 hours induction time. As shown in Figure 4, bands representing both ureases, UreA and UreB, were detected on SDS-PAGE with approximate sizes of 45 kDa and 74 kDa, respectively. Clone pET32UOA6 expressed both ureases, UreA and UreB (Figure 4). This meant that the cloned urease operon was functioning in E. coli cell. The selected recombinant clones carrying correct urease gene fragments, as well as, complete urease operon were subjected to expression study. As shown in Figure 3, bands representing recombinant UreA and UreB were detected indicating the clones were carrying functional urease genes. Determination of immune functioning of the expressed ureases The sizes of H. pylori recombinant UreA and UreB (Figure 4) were bigger, more than 29 kDa and 62 kDa respectively due to the fused amino acids thioredoxin. Sometime, 38

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Cloning and Expression of the Urease the presence of additional amino acids fused to protein of interest could change the protein conformation. Therefore, it was essential to determine whether the expressed recombinant UreA and UreB still maintained their immune properties.

Figure 3: SDS-PAGE analysis for expression of recombinant urease protein. The expression profiles for (A) pET32UOA6 (B) pET32ureA3 and (C) pET32ureB2. Lane 1: DGelTM Marker; Lane 2: Pre-culture urease gene; Lane 3 and 6: uninduced culture; Lane 4: Induced culture with 0.4 mM IPTG at 2 hours; Lane7: Induced culture with 0.4 mM IPTG at 3 hours; Lane 5: Induced culture with 1.0 mM IPTG at 2 hours and Lane 8: Induced culture with 1.0 mM IPTG at 3 hours. Figure 4 shows immune functioning of UreA and UreB from cell crude of recombinant urease clones compared with other bacterial crude cells, as negative controls and H. pylori J99 crude cell as the positive control. Helicobacter pylori ureaseα and urease-β antibodies (Santa Cruz, Inc.) were very specific and sensitive to UreA and UreB of H. pylori J99 crude cell (lanes 8 and 18). Similar specificity and sensitivity were observed for recombinant UreA and UreB, expressed in full operon unit (lanes 2-3 and 12-13). As suggested by the manufacturer, crude cells from Salmonella (lanes 4 & 14), Pseudomonas (lanes 7 & 17) and E. coli (lanes 9 & 19) gave negative results that indicate H. pylori urease-α and urease-β antibodies were very specific and sensitive to the native, as well as, the recombinant ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Mohamad et al. ureases. These results confirmed the recombinant UreA and UreB maintained their immune properties.

Figure 4: Western blot analysis for determination of recombinant ureases immune functioning in of UreA (A) and UreB crude cell (B). Lane 1, 10, 11 and 20: Kaleidoscope Prestained protein ladder; Lane 2-3 and 12-13: pET32UOA6 cell crude; Lane 5-6: pET32ureA6 cell crude; Lane 15-16: pET32ureB2 cell crude; Lane 4 and 14: Salmonella cell crude; Lane 7 and 17: Pseudomonas cell crude; Lane 8 and 18: H. pylori J99 cell crude; Lane 9 and 19: E. coli cell crude. Immune functioning assay verified the recombinant UreA and recombinant UreB still maintained their antigenicity, equivalent to native enzyme regardless of the presence of additional amino acids fused to them. These were supported by immune functioning assay through Western blotting and previous work by Hu et al. (1992). Purification of both recombinant ureases did not affect the antigenicity, as evidence by Western blotting (Figure 4). Regardless of this condition, both recombinant ureases still maintained their antigenicity in immune functioning assay. Purification process failed to separate UreA from UreB since H. pylori urease-α and urease-β antibodies (Santa Cruz, Inc, USA) still detecting both 39

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of them in the immune functioning assay. Thus, clone pET32UO expressed and assembled the urease apoenzyme and perhaps together with the accessory proteins to form the holoenzyme.

This study was supported by the Universiti Malaysia Perlis for Academic Training Scheme Funding (SLAI).

The confirmation of recombinant urease protein was demonstrated by protein sequencing. The results of amino acid alignment based on BLASTp between recombinant urease against H. pylori J99 urease show high percent identities, 99100%. Thus, these results further confirmed the authenticity of the recombinant ureases similar to H. pylori J99 urease.

Ardekani, L. S., Gargari, S. L. M., Rasooli, I., Bazl, M. R., Mohammadi, M., Ebrahimizadeh, W. and Zare, H. (2013). A novel nanobody against urease activity of Helicobacter pylori. International Journal of Infectious Diseases 17(9), e723-e728.

Confirmation of recombinant ureases by protein sequencing Figure 5 shows the results of protein identity based on BLASTp between recombinant urease and H. pylori J99 ureases. These results further confirmed the authenticity of the recombinant ureases expressed by pET32UOA6.

Figure 5: The percentage identity of recombinant ureases against H. pylori J99 ureases, pET32UOA6. CONCLUSION In this study, the availability of a functional replicon carries urease operon has potential benefit related to H. pylori pathogenesis. A through and better understanding of H. pylori pathogenesis process could contribute to the improvement of diagnostic methods. ACKNOWLEDGEMENT

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REFERENCES

Hu, L. T., Foxall, P. A., Russell, R. O. B. E. R. T. and Mobley, H. L. (1992). Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB. Infection and Immunity 60(7), 2657-2666. Kiesslich, R., Goetz, M., Burg, J., Stolte, M., Siegel, E., Maeurer, M. J. and Neurath, M. F. (2005). Diagnosing Helicobacter pylori In Vivo by Confocal Laser Endoscopy. Gastroenterology 128(7), 21192123. Mitchell, H.M., Hazell, S.L., Bohane, T.D., Hu P., Chen, M. and Li, Y.Y. (1999). The prevalence of antibody to cagA in children is not a marker for specific disease. Journal of Pediatric Gastroenterology and Nutrition 28, 71 â&#x20AC;&#x201C; 75. Sasidharan, S., Uyub, A.M. and Azlan, A.A. (2008). Further evidence of ethnic and gender differences for Helicobacter pylori infection among endoscoped patients. Transactions of the Royal Society of Tropical

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Cloning and Expression of the Urease Medicine and Hygiene 102, 12261232. Stephen, F., Altschul, Thomas, L., Madden, Alejandro, A., SchĂ¤ffer, Jinghui Zhang, Zheng Zhang, Webb, M. and David, J. L. (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402. Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences 76(9), 4350-4354.

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Mohamad et al. Uberti, A. F., Olivera-Severo, D., Wassermann, G. E., ScopelGuerra, A., Moraes, J. A., Barcellos-de-Souza, P. and Carlini, C. R. (2013). Pro-inflammatory properties and neutrophil activation by Helicobacter pylori urease. Toxicon 69, 240-249. Voland, P., Zeitner, M., Hafsi, N. and Prinz, C. (2006). Human immune response towards recombinant Helicobacter pylori urease and cellular fractions. Vaccine 24(18), 3832-3839. Zheng, Z., Scott, S., Lukas, W. and Webb, M. (2000). A greedy algorithm for aligning DNA sequences. Journal of Computer Biology 7(1-2), 203-214.

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

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Production of Butter Flavour Concentrate from Butter fat with Lactic Acid Bacteria by Solid Substrate Fermentation Nadaraj Sivan1, Thambirajah, J. J.2 and Guruswamy Prabhakaran*1 1

Department of Biotechnology, AIMST University, Bedong 08100, Kedah Darul Aman, Malaysia; 2Faculty of Business Management, AIMST University, Bedong 08100, Kedah Darul Aman, Malaysia; *corresponding author, e-mail: prabhakaran@amist.edu.my

ABSTRACT Aim: The aim of this study was to investigate the fermentation of butter fat with different lactic acid bacterial strains by solid substrate fermentation (SSF) for the production of butter flavour concentrates. Methodology and results: Lactic acid bacteria (LAB) were isolated from dairy products and environmental samples using Mann Rogosa and Sharpe (MRS) agar. These, together with two reference ATCC lactic acid bacterial strains were evaluated for the production of flavor components which were determined by GC-MS. The SSF was found to be effective in producing sweet and buttery notes within 24 hours of fermentation. Scale up studies with 120 g of butter fat supplemented with 10% galactose enhanced butter flavour production. Butter oil recovered from the fermented samples was subjected to sensory evaluation by 120 volunteers. The butter flavour compounds in butter oil samples were quantitatively analyzed in GC-MS. The untreated butter fat recorded the lowest concentration of diacetyl (211.5 ppm) and acetoin (161.7 ppm) whereas, butter fat supplemented with galactose and fermented showed a significant increase in concentration of acetoin (1321.2 ppm) and diacetyl (511.4 ppm). The formulation of butter powder with maltodextrin was investigated. Conclusion, significance and impact of study: The results obtained from this study will pave the future investigations for development of a microbial process for production of butter flavour concentrate from butter fat. Keywords: Acetoin; Butter fat; Diacetyl; Butter flavor; GC-MS; Lactic acid bacteria; Solid substrate fermentation.

INTRODUCTION Flavour is a combination of taste and aroma. It results from the perception of odor-active volatile compounds. Food flavours are mixtures of natural and/or artificial aromatic compounds. They are designed to impart, modify, or even mask an undesirable flavour. Flavours along with fragrances are highly prized in the global market. Currently there are three known methods of acquiring flavour compounds (Bicas et al., 2010). These include, extraction from pre-existing

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natural sources, synthesis by chemical precursors and by biotechnological routes via de novo or biotransformation. The milk fat component of dairy source largely contributes to the release of flavour. Butter possesses its own distinct flavour. The flavour compounds usually are aldehydes, ketones and lactones of which diacetyl and acetoin play important roles in parting the well-known ‘buttery’ flavour. Lactones play important role in conferring the ‘buttery’ taste and sweet aromas altogether (Hua et al., 2007).

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Microbial Production of Butter Flavour Currently, diacetyl and acetoin, the flavour compounds are chemically synthesised at commercial scale. The inhalation of intense synthetic butter flavour at the source of manufacturing and application results in bronchiolitis obliterans, commonly termed as â&#x20AC;&#x2DC;popcorn lungâ&#x20AC;&#x2122; disease (Morris and Hubbs, 2009). The increasing demand for natural butter flavour has lead to the development of biotechnological processes. However, the fermentation processes which involve lactic acid bacteria in obtaining high yields of desirable enantiomeric butter flavour compounds from butter fat are yet to be established. The development of solid state fermentation techniques for the bioconversion of butter fat and the recovery of butter flavour compounds are important challenges for researchers in this field. Hence, this study was undertaken to optimize the fermentation conditions for the production of butter flavour concentrate from butter fat by lactic acid bacteria in solid substrate fermentation.

Nadaraj et al. MATERIALS AND METHODS The process flowchart in Figure 1 highlights the major stages in the production of butter flavour concentrate. Isolation of Lactic Acid Bacteria (LAB) from various samples Raw milk was purchased from a local market. Soured milk was prepared by allowing the raw milk to sour for a day. Pasteurized milk (Marigold, Dutch Lady), cheese (Emborg, Kraft), unsalted butter (Devondale, Tatura), salted butter (Devondale, Anchor), cultured drink (Solivite, Nutrigen), yoghurt (Dutch Lady) were purchased from TESCO supermarket in Alor Setar, Kedah Darul Aman, Malaysia. Soil, grass and water samples were collected from a nearby cattle farm. Lactic Acid Bacteria (LAB) were isolated from these samples as per the method (Bettache et al., 2012). The isolates were stored in agar slants at 4Â°C. The individual isolates were tested for their ability to ferment butter to produce flavour concentrates.

Figure 1: Flowchart for the production of butter flavour concentrate. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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ATCC strains of Lactic Acid Bacteria Lactobacillus acidophilus (ATCC 314) and Lactobacillus casei (ATCC 393) strains were purchased from ATCC and also evaluated for the production of butter flavour.

Recovery of butter oil Butter oil was extracted from fermented butter fat samples for the sensory evaluation, fatty acid analysis and dertermination of diacetyl and acetoin in the fermented samples.

Pasteurization and sterilization of butter fat The recommended protocol by the International Dairy Federation (Juffs and Deeth, 2007) was used for the pasteurization of butter. Sterilization of butter fat was by autoclaving at 121 ºC at 15 psi, for 15 minutes.

Microbial fermentation of butter fat by Solid Substrate Fermentation (SSF) Ten ml of overnight culture in MRS broth with the isolated LAB bacteria was prepared individually. Six g of butter fat each in glass Perti plates were autoclaved (121 ºC, 15 psi, 15 minutes). The melted butter in the petri dishes was gently swirled and cooled. Individual petri dishes were then inoculated with 2 ml of overnight culture of the Lactic acid bacteria. As a control, an uninoculated petri dish with only the sterilized butter fat was used. At fixed time intervals (24th, 48th and 72th hour) samples were drawn from the petri dishes and evaluated by sensory evaluation. A total of 10 evaluators were selected to provide descriptions of the aroma from the samples such as odourless, sweet, stale, sour, buttery or alcoholic. Butter oil samples were then extracted from the samples after scoring of the aroma description. The samples were stored in the cold room for further analysis.

Standardization of butter oil extraction methods To standardize the butter oil extraction method, three different protocols were studied (Table 1). Based on the total amount of oil extracted, the percentage (%) of recovery was calculated based on the following formula: % recovery = Amount of pure product recovered (g) x 100 / Amount of crude material used (g) Table 1: Different methods for the extraction of butter oil from butter fat. Method Conditions 1 a) Melting in a water bath at 40 ºC (±5ºC) for 10 minutes. b) Centrifugation at 800 rpm for 3 minutes (Chongcharoenyanon et al., 2012). 2 a) Melting in a water bath at 50 ºC (±5ºC) for 8 minutes. b) Centrifugation at 3,700 rpm for 12 minutes(Krause and Gibson, 2008). 3 a) Melting in hot air oven at 70 ºC (±5ºC) for 15 minutes. b) Centrifugation at 3,500 rpm for 5 minutes (Miura et al., 2004).

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Supplementation of butter fat with various carbohydrate sources An experiment was conducted to determine whether supplementation of butter fat with various carbohydrates sources such as galactose, lactose and skim milk may enhance butter flavour production upon solid substrate fermentation. Initial efforts were conducted in petri dishes where 6 g of butter was weighed and supplemented with 10% (w/w) of galactose, lactose and skim milk, individually. The supplemented butter fat was then sterilized and uniformly spread the butter fat in the petri dish. After cooling, each Petri dish was inoculated with 2 ml of overnight cultures of the 44

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Microbial Production of Butter Flavour selected lactic acid bacteria and incubated at 37°C. One petri dish was designated as control. Sensory evaluation of samples was performed at fixed time intervals (24th, 48th and 72th hour) as reported previously. Scale up studies of Solid Substrate Fermentation (SSF) Scales up studies were performed with increased quantities of butter fat. In order to increase the surface area for solid substrate fermentation, experiments were carried out with various amounts of butter fat taken in conical flasks (30 g in 250 ml conical flask, 60 g in 500 ml conical flask and 160 g in 1000 ml conical flask). Overnight cultures were prepared individually and the volume of overnight inoculum used was 10 ml for 30 g, 20 ml for 60 g and 40 ml for 160 g substrates, respectively. These cultures were incubated at 37ºC under anoxic conditions. At specified time intervals of 24th, 48th and 72th hour, samples were acquired for sensory evaluation by 10 random volunteers. Scale up studies of Solid Substrate Fermentation (SSF) with 10% galactose Scale up studies was carried out with 30 g, 60 g and 160 g of butter fat, supplemented with 10% of galactose individually. The flasks were sterilized and inoculated with overnight inoculum respectively. They were incubated and samples were drawn and tested as previously. Sensory evaluation of butter oil samples from different fermented butter samples Sensory evaluation of butter oil samples that were obtained from the fermented butter samples were performed as per the methods by Anvoh et al. (2009). A 5-point hedonic scale was utilized where 1 represents dislike the most, 3 represents neither like nor dislike and 5 represents like the most. Sensory scores were evaluated based on preferences of odour by 120 students of Biotechnology from the Faculty of Applied Sciences, AIMST ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Nadaraj et al. University. Prior to sampling, volunteers were first requested of their health status and free of any respiratory illnesses. During inhalation of aroma, volunteers were required to inhale the samples and suspend breathing for 2-3 seconds and mark their preferences in the flavour survey form provided. Coffee powder was provided as a neutralizer to eliminate traces of previous aromas. Samples indicating high preferences were selected for further analysis by GC-MS (Gas chromatography-mass spectrometry). GC-MS analysis of butter oil for acetoin, diacetyl and fatty acid Volatile compounds such as acetoin and diacetyl are generally analysed by GC-MS (Gokce et al., 2014). Extraction of diacetyl and acetoin from the treated butter oil was performed. Chromatographic analyses of the treated and control butter samples for aroma compounds and fatty acid components were performed using a split less injector system gas chromatograph coupled with a mass spectrometer (SHIMADZU GCMS-QP2010). The carrier gas used was ultra-pure helium with a flow rate of 1.0 mL/min. The injection port was worked at 250°C in split less mode coupled with 1 minute split less time. A 1 µl injection volume was applied for each sample analysis and the syringe was washed with hexane upon completion of injection. Separation was performed using a DB-WAX (60 m x 0.25 mm x 0.15 µm) capillary column with a 0.15 µm stationary film. The oven temperature programme was set as follows: initial temperature 40°C, increased by 7°C min-1 to 200°C and held for 1 minute. Mass spectrometric parameters were set as follows: electron impact ionization with 70 eV energy and 250°C ion source. The aromatic compounds and fatty acid profiles were detected and quantified based on their retention time and peak areas on the chromatogram respectively.

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Microbial Production of Butter Flavour Butter powder formulation with maltodextrin Maltodextrin was used as a carrier material, as it is edible, slightly sweet, and low in cost (Wandrey et al., 2010). For the production of butter oil powder, the extracted butter oil was mixed with maltodextrin with different anti-caking agents and the entire mixture was then treated with liquid nitrogen and mixed evenly using a mixer. The addition of liquid nitrogen was to imitate freeze drying conditions. Formulation of butter powder from carrier material (maltodextrin) and active material (butter oil) was performed individually by using 50 g of maltodextrin powder and varying amounts of butter oil (5 g, 10 g, 15 g, 20 g, 25 g and 30 g). The maltodextrin powder was added into a plastic container, and butter oil of the concerned weight was poured into the container. Simultaneously, the mixture was added with liquid nitrogen (approximately 100 ml) and manually stirred. The prepared powder was then stored in air-tight glass containers and kept at room temperature away from any light source. Anti-caking agents are required in the formulation of powdered food or drug preparations to either stop or postpone occurrences of caking (Lipasek et al., 2011). For this, 50 g of the prepared butter powder was placed in a plastic container and supplemented individually with 2%, 4% and 6% (w/w) anti-caking agent that comprised sodium chloride, sodium silicate, sodium bicarbonate, bentonite, mannitol and potato starch. The mixture was manually stirred with intermittent shaking to enable even dispersion of the anti-caking agent and the powder. The prepared powder was then stored in a glass container that was sealed and stored in a dry area. RESULTS AND DISCUSSION Over the years lactic acid bacteria (LAB) have been used in various fermented food preparations. The benefits conferred by ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Nadaraj et al. LAB are lactic acid production which results in the improvement of flavour, aroma, keeping quality and enhancement in nutritional content (Halรกsz, 2009). Butter is an important flavouring additive and creates the distinctive aroma and taste in bakery, dairy and other food products. Diacetyl and acetoin are two important constituents in butter fat that contribute to unique butter flavour. Natural butter flavour is expensive, hence artificial butter flavour compounds are chemically synthesized from petroleum-derived precursors. Synthetic flavours are produced in bulk at low cost and it is a mixture of racemic compounds. The prolonged exposure to synthetic flavour compounds to the line workers was reported harmful by National Institute for Occupational Safety and Health (NIOSH) (Kreiss, 2007). Biotechnological approaches in producing butter flavour compounds are being investigated as a replacement to chemicalbased processes (Longo and Sanromรกn, 2006). This study was aimed at producing butter flavour concentrate from butterfat by a microbial process. The investigation included isolating LAB strains as well as Lactobacillus acidophilus (ATCC 314) and Lactobacillus casei (ATCC 393) in the fermentation of butter fat by solid substrate fermentation (SSF). After fermentation, removal of solids by centrifugation and recovery of butter oil from the treated butter was performed. The butter oil was then subjected to sensory evaluation and analysed for butter flavour compounds by GC-MS. Isolation of LAB from various samples Raw milk, soured milk, cheese, unsalted butter, salted butter, yoghurt and grass, soil, and water samples from cattle grazing areas were niches of LAB strains. A total of ten colonies were isolated from the different samples.

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Microbial Production of Butter Flavour Pasteurization and sterilization of butter fat Two different methods namely pasteurization and sterilization of butter fat were evaluated. Compared to pasteurization (63ºC±5ºC for 30 minutes), the sterilization of butter fat samples was better. Hence, sterilization was used subsequently. Recovery of butter oil Butter oil was recovered for sensory evaluation, detection and quantification of diacetyl, acetoin and fatty acids in the fermented butter samples which constituted the major butter flavour compounds. Three different methods were adopted for extraction of butter oil from 6 g of butter fat (Figure 2). Percentage recovery of butter oil by different methods On comparative analysis, it was found that the percentage recovery of oil was high (69.67%), when the butter fat was melted at 50 °C for 8 minutes and centrifuged at 3,700 rpm for 12 minutes (Table 2). The oil recovery was recorded low in the other two methods. However, the clarity of butter oil was found to be good in the second method when compared to methods 1 and 3. This may be attributed to high centrifugal forces for efficient separation of denser materials from lighter counterparts (Anlauf, 2007). Solid Substrate Fermentation (SSF) of butter fat in glass petri dishes (Figure 3) and sensory evaluation After 24 hours of incubation, there was a significant improvement in the flavour content in the fermented samples with CFS and CFG strains individually (Table 3). Whereas, with prolonged incubation the off flavour production was recorded. Butter flavour production is based on multi-step reactions which involve lipolytic, proteolytic and glycolytic reactions (Smit et al., 2005). It was ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Nadaraj et al. reported that diacetyl may also contribute to the formation of undesirable, offflavours as observed in spirits manufacture (Krogerus and Gibson, 2013). The fermented butter fat samples were subjected to sensory evaluation by ten volunteers.

Figure 2: Picture showing the recovery of butter oil by using three methods. Table 2: Percentage recovery of butter oil by different methods. Butter oil recovery (g) M* Trial Trial Trial Average Recovery % 1 2 3 1 4.14 4.11 4.12 4.12 68.67 2 4.17 4.19 4.18 4.18 69.67 3 3.79 3.81 3.77 3.79 63.17 Values represent the mean of three replicates; *Method 1: Melting of butter fat at 40 ºC (±5ºC) for 10 minutes and centrifugation at 800 rpm for 3 minutes (Chongcharoenyanon et al., 2012); Method 2: Melting of butter fat at 50 ºC (±5ºC) for 8 minutes and centrifugation at 3,700 rpm for 12 minutes (Krause et al., 2008); Method 3: Melting of butter fat at 70 ºC (±5ºC) for 15 minutes and centrifugation at 3,500 rpm for 5 minutes (Miura et al., 2004). Sensory evaluation of SSF (Solid Substrate Fermentation) samples with various concentrations of butter fat The fermented butter fat samples were recorded with preferred sweet and buttery flavour after 24 hours of incubation with both the isolated strains (CFS and CFG) in Table 4. The increase in butter fat quantity 47

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Figure 3: Solid substrate fermentation of butter fat in glass petri dishes; CFS: bacterial strain isolated from cattle field soil sample.CFG: bacterial strain isolated from cattle field grass sample. (esters, methylketones, lactones, etc.) formation (Smit et al., 2005). Among the 2 Table 3: Sensory evaluation of SSF (Solid strains tested with 160 g butter fat, CFS Substrate Fermentation) fermented butter strain was recorded with increased flavour samplesยง. production. Solid Sensory evaluation Supplementation of butter fat with substrate CFS CFG various carbohydrate sources fermentation strain strain Lactic acid bacteria are able to ferment (SSF) various hexose sugars, from simple (hour) (galactose) to complex (lactose) sugars but 24 Buttery Sweet is species-dependant (Halรกsz, 2009). 48 Sour, Sour, Galactose moieties present with lactose alcoholic alcoholic molecules in milk when metabolized, may 72 Stale, Stale, lead to intense aroma production odourless odourless ยง (Hugenholtz et al., 2002). In order to Scores based on the aroma description by determine if addition of lactose, skim milk 10 volunteers; CFS: bacterial strain and galactose would enhance the butter isolated from cattle field soil sample; flavour production, solid substrate CFG: bacterial strain isolated from cattle fermentation was carried with both the field grass sample. strains. The butter fat (6 g) was supplemented with 10 % (w/w) substrate in the medium may have facilitated the individually and fermented with CFS and volunteers in scoring more for sweet and CFG strains. Table 5 summarizes sensory buttery flavours. The increased amounts of evaluation results of the experiment by raw material (fats and milk sugars) are the intermediates for flavour compounds ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Table 4: Sensory evaluation of SSF samples with varying concentrations of butter fat*. Solid substrate fermentation (SSF) (hour) 24

Butter fat 30 g CFS CFG

Sensory evaluation Butter fat 60 g CFS CFG

Butter fat 160 g CFS CFG

Sweet, Sweet Sweet, Sweet Sweet, Sweet buttery buttery buttery 48 Sour, Sour, Sour, Sour, Sour, Sour, alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic 72 Rancid Rancid Rancid Rancid Rancid Rancid *Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample. Table 5: Sensory evaluation of SSF samples fermented with CFS and CFG strains supplemented with carbohydrate sources Solid substrate fermentation (SSF) (hour) 24

Sensory evaluation CFS strain CFG strain Galactose Lactose Skim Milk Galactose Lactose Skim Milk Sweet, Sour Milky Sweet Sour Milky buttery 48 Sour Sour Milky Sour Sour Milky 72 Stale Sour Sour Stale Sour Sour * Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample. undergraduate students (Figure 4). The sensory evaluation of the samples by students is depicted in Table 6. A sweet, butter flavour production was enhanced with both the CFS and CFG strains in 24 hours of incubation. Upon prolonged incubation (48 and 72 hours), stale and sour odour were recorded. The results indicated that butter fat supplemented with 10 % galactose and fermented specifically with the CFS strain was effective in producing butter oil with intense butter flavour. Butter fat supplemented with skim milk recorded a more pronounced milk flavour, compared to butter flavour. Therefore, scale up studies were carried out with increased amounts of butter fat (30 g, 60 g and 160 g) supplemented with 10 % galactose individually.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Scale up studies of Solid Substrate Fermentation (SSF) with galactose Scale up studies were performed with increased butter fat (30 g, 60 g and 160 g) supplemented with 10% galactose individually. Figure 5, indicates the scale up studies of 160 g butter fat supplemented with 16 g (10%) galactose. After fermentation, the butter oil was extracted and subjected to sensory evaluation. Based on the aroma description (sour, sweet, buttery, milky, stale and rancid). Scoring was done by 10 volunteers. Table 6, indicates the sensory evaluation results of the fermented sample. Sensory evaluation of butter fat samples supplemented with galactose at scale up studies Based on the sensory evaluation, the butter fat samples fermented with strains CFS

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Figure 4: Sensory evaluation carried out by undergraduate students. Table 6: Sensory evaluation of butter fat samples supplemented with 10 % galactose in scale up studies at 30 g, 60 g and 160 g. Solid substrate Sensory evaluation fermentation (SSF) 30 g butter + 60 g butter + 160 g butter + (hour) 3 g galactose 6 g galactose 16 g galactose CFS CFG CFS CFG CFS CFG strain strain strain strain strain strain 24 Sweet, Sweet Sweet, Sweet Sweet, Sweet buttery buttery buttery 48 Sour, Sour, Sour, Sour, Sour, Sour, alcoholic alcoholic alcoholic alcoholic alcoholic alcoholic 72 Rancid Rancid Rancid Stale, Rancid Sour, odourless alcoholic * Scoring based the aroma descriptions by 10 volunteers; CFS: bacterial strain isolated from cattle field soil sample; CFG: bacterial strain isolated from cattle field grass sample. and CFG individually were scored sweet and buttery within 24 hours of incubation (Table 6). Extended incubation period (48 and 72 hours) resulted in formation of undesired, foul (stale or rancid or alcoholic) odours. Therefore, the experiment was repeated and after 24 hours of incubation period, the fermentation was arrested by freezing the samples at -20Â°C. The respective samples were thawed at room temperature. Butter ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

oil was extracted and subjected to sensory evaluation. Sensory evaluation of butter oil samples by 120 students Compared to the control sample (BF), all other samples were preferred (Figure 6). Most individuals (38%) did not approve of sample BF with 53% of the volunteers being unsure of their preference. The least amount (8.3%) of evaluators prefer this 50

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Figure 5: Scale up studies of 160 g butter fat supplemented with 16 g (10%) galactose; 1: BF=Untreated butter fat (160 g) (control); 2: BF+Gal+CFS=Butter fat (160 g) supplemented with galactose (16 g) and fermented with CFS strain; 3: BF+Gal+CFG=Butter fat (160 g) supplemented with galactose (16 g) and fermented with CFG strain. sample on an overall basis. In comparative analysis of unsupplemented butter fat fermented with isolated LAB, sample BF+CFS was more preferred (45%) by students as compared to sample BF+CFG (19.2%). For supplemented butter fat with galactose and fermented with isolated LAB, a large number of evaluators (56.7%) preferred sample BF+Gal+CFS as compared to sample BF+Gal+CFG (45%). Hence, samples BF+CFS and BF+CFS+Gal were selected for further GC-MS analysis of constituents with sample BF serving as the control. Analysis of butter oil volatile constituents It was inferred that the butter fat samples fermented with CFS strain either with or without galactose was most preferred based on the sensory analysis of five different samples by 120 volunteers. Therefore, butter oil samples extracted from BF+CFS (butter fat fermented with strain CFS), BF+Gal+CFS (butter fat ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

supplemented with galactose and fermented with strain CFS) and from BF (untreated butter as control) were analysed for diacetyl and acetoin content by GC-MS method. The results were tabulated. Comparative analysis of diacetyl and acetoin content in the unfermented and fermented butter fat samples with CFS strain Acetoin content was highest (1321.2 ppm) in the butter fat sample fermented with CFS strain (BF+CFS). In contrast, untreated butter fat (BF) reported lowest amount of acetoin (211.5 ppm) amongst all 3 samples (Figure 7). With regards to diacetyl content, butter fat supplemented with galactose and fermented with CFS strain had the highest concentration (511.4 ppm). Lowest content of diacetyl was found to be from untreated butter fat (161.7 ppm). Unlike acetoin, diacetyl strongly contributes to the buttery aroma (Fuquay et al., 2011). Only in cohesion 51

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Figure 6: Sensory evaluation of butter oil samples by 120 participants; BFS: Butter Fat Sample; BF: untreated butter; BF+CFS: butter fermented with strain CFS; BF+CFG: butter fermented with strain CFG; BF+CFS+Gal: butter supplemented with galactose and fermented with strain CFS; BF+CFG+Gal: butter supplemented with galactose and fermented with strain CFG. with diacetyl, does acetoin impart strong, pleasant, mild, overall buttery aroma in addition to toning down diacetyl roughness (Bai et al, 2014). The results of diacetyl and acetoin analysis tallies with the preference of volunteers who preferred supplemented butter fat and fermented with CFS strain the most (56.7%). Untreated butter fat was the least preferred (8.3%) and is reflected by the lowest concentrations of diacetyl and acetoin content. Butter powder formulation Butter powder formulation was made with maltodextrin and to enhance its flow properties anti-caking agents (bentonite, sodium silicate, sodium chloride, potato starch and mannitol) were evaluated (Figure 8). The samples were subjected to sensory evaluation. The recovered butter oil was then formulated to powder form by addition of a carrier material and antiISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

caking agent. This was performed to enhance flow capability and storage property of the butter powder. Standardization of butter oil and maltodextrin ratio for butter concentrate powder formulation It was observed that the use of 25 g butter oil resulted in the formation of minor clumping with maltodextrin (Table 7). Although lower amounts of butter oil did not result in clumping, desired intensity of butter aroma was not imparted. Maltodextrin is a well-known carrier material used for various preparations. It is digestible, lacks artificial colouring or flavour, low in cost and miscible in liquids (Zuidam and Shimoni, 2010). In addition, its good binding and high viscosity capabilities enables spray or freeze drying of active ingredients to be made into powders easily (Akhilesh et al., 2012). From the various anti-caking agents tried, 52

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Figure 7: A comparative analysis of diacetyl and acetoin content in the unfermented and fermented butter fat samples with CFS strain; BF: untreated butter; BF+CFS: butter fermented with strain CFS; BF+Gal+CFS: butter supplemented with galactose and fermented with strain CFS.

Figure 8: Butter powder formulation with different concentrations of butter oil in maltodextrin. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Microbial Production of Butter Flavour Table 7: Standardization of butter oil and maltodextrin ratio for butter concentrate powder formulation. Maltodz Butter Quality attributes (g) oil (g) Texture Aroma 50 5 No clumps Faint 50 10 No clumps Faint 50 15 No clumps Decent 50 20 No clumps Decent 50 25 Less clumps Strong 50 30 Grainy clumps Strong zMaltodextrin

Nadaraj et al. method for optimum recovery of butter oil from the butter fat samples was standardized, which resulted in 69.67 % recovery of butter oil. Upon preliminary screening of the LAB isolates, the CFS and CFG isolates were selected based on the sensory evaluation by 10 volunteers.

none are able to enhance the flow rate of the formulated powder at various concentrations (2%, 4% and 6% w/w). Such findings are in contrast to reports by (Ganesan et al., 2008) who stated application of 2% anticaking agent is adequate for enhancing powder flow rates. A possible reason for failing to achieve the desired powder texture might be due to structural collapse of the sample. This may be attributed to decreased product molecular porosity and overall volume, as a result of improper sample drying and storage (Bhadra et al., 2011).

The solid substrate fermentation condition by the two strains produced sweet and buttery notes within 24 hours. In the final phase, a scale up study of butter fat supplemented with 10% galactose was carried out by SSF. A large scale sensory evaluation was performed with 120 volunteers. The elevated concentration of both diacetyl and acetoin was most preferred (56.7%). The formulation of butter powder with maltodextrin was investigated for its flow properties. Overall, the results obtained from this study will pave way for the future investigations for the development of a microbial process for the production of butter flavour concentrate from butter fat with lactic acid bacteria via solid substrate fermentation.

CONCLUSION

REFERENCES

The demand for natural butter flavours are driven by growing consumer awareness. This has lead to the development of biotechnological processes wherein Lactic acid bacteria (LAB) widely recognized as GRAS (generally regarded as safe) and used for enhancement flavour in fermented food. This research work addressed the application of lactic acid bacteria in obtaining high yields of desirable enantiomeric butter flavour compounds exclusively from butter fat by solid state fermentation. In the first phase, the work focused on the isolation of lactic acid bacteria (LAB) strains from various dairy and environmental samples and screened for the fermentation of butter fat. The pasteurization of unsalted butter fat was ineffective in killing the native microbes. Hence sterilization was adopted. The ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Akhilesh, D., Faishal, G. and Kamath, J.V. (2012). Comparative study of carriers used in proniosomes. Int J Pharm Chem Sci 3, 6–12. Anlauf, H. (2007). Recent developments in centrifuge technology. Sep. Purif. Technol. 58, 242–246. Anvoh, K.Y.B., Bi, A.Z., Gnakri, D. et al. (2009). Production and characterization of juice from mucilage of cocoa beans and its transformation into marmalade. Pak. J. Nutr. 8, 129–133. Bai, J.A. and Ravishankar, R.V. (2014). Beneficial Microbes in Fermented and Functional Foods. CRC Press.

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Microbial Production of Butter Flavour Bettache, G., Fatma, A., Miloud, H. and Mebrouk, K. (2012). Isolation and identification of lactic acid bacteria from Dhan, a traditional butter and their major technological traits. World Appl. Sci. J. 17, 480–488. Bhadra, R., Rosentrater, K.A. and Muthukumarappan, K. (2011). Effects of varying condensed distillers solubles, drying and cooling temperatures on glass transition temperature of distillers dried grains. Can. Biosyst. Eng. 53, 3–9. Bicas, J.L., Silva, J.C., Dionísio, A.P. and Pastore, G.M. (2010). Biotechnological production of bioflavors and functional sugars. Food Sci. Technol. Camp. 30, 07– 18. Chongcharoenyanon, B., Yamashita, N., Igura, N., Noma, S. and Shimoda, M. (2012). Extraction of volatile flavour compounds from butter oil in a low-density polyethylene membrane pouch. Flavour Fragr. J. 27, 367–371. Fuquay, J.W., Fox, P.F. and McSweeney, P.L. (2011). Encyclopedia of Dairy Sciences 2nd Edition, Four-Volume set. Academic Press. Ganesan, V., Rosentrater, K.A., and Muthukumarappan, K. (2008). Flowability and handling characteristics of bulk solids and powders–a review with implications for DDGS. Biosyst. Eng. 101, 425–435. Gokce, R., Akdogan, A., Divriklib, U. and Elci, L. (2014). Simultatenous determination of diacetyl and acetoin in traditional turkish butter

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Nadaraj et al. stored in sheep’s rumen (Karinyagi). Grasas Aceites 65, 10. Halász, A. (2009). Lactic acid bacteria. Food Qual. Stand. 3, 70–82. Hua, D., Ma, C., Song, L., Lin, S., Zhang, Z., Deng, Z. and Xu, P. (2007). Enhanced vanillin production from ferulic acid using adsorbent resin. Appl. Microbiol. Biotechnol. 74, 783–790. Hugenholtz, J., Sybesma, W., Groot, M.N., Wisselink, W., Ladero, V., Burgess, K., van Sinderen, D., Piard, J.-C., Eggink, G., Smid, E.J. et al. (2002). Metabolic engineering of lactic acid bacteria for the production of nutraceuticals, in: Lactic Acid Bacteria: Genetics, Metabolism and Applications. Springer, pp. 217– 235. Juffs, H. and Deeth, H. (2007). Scientific evaluation of pasteurisation for pathogen reduction in milk and milk products. Food Standards Australia New Zealand. Kreiss,

K. (2007). Flavoring-related bronchiolitis obliterans. Curr. Opin. Allergy Clin. Immunol. 7, 162–167.

Krause, A.J., Miracle, R.E., Sanders, T.H., Dean, L.L. and Drake, M.A. (2008). The effect of refrigerated and frozen storage on butter flavor and texture. J. Dairy Sci. 91, 455–465. Krogerus, K. and Gibson, B.R. (2013). 125th Anniversary Review: Diacetyl and its control during brewery fermentation. J. Inst. Brew. 119, 86–97.

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Microbial Production of Butter Flavour Lipasek, R.A., Taylor, L.S. and Mauer, L.J. (2011). Effects of anticaking agents and relative humidity on the physical and chemical stability of powdered vitamin C. J. Food Sci. 76, C1062–C1074. Longo, M.A. and Sanromán, M.A. (2006). Production of food aroma compounds: microbial and enzymatic methodologies. Food Technol. Biotechnol. 44, 335–353. Miura, S., Tanaka, M., Suzuki, A. and Sato, K. (2004). Application of phospholipids extracted from bovine milk to the reconstitution of cream using butter oil. J. Am. Oil Chem. Soc. 81, 97–100. Morris, J.B. and Hubbs, A.F. (2009). Inhalation dosimetry of diacetyl and butyric acid, two components of butter flavoring vapors. Toxicol. Sci. Off. J. Soc. Toxicol. 108, 173– 183. doi:10.1093/toxsci/kfn222.

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Nadaraj et al. Smit, G., Smit, B.A. and Engels, W.J. (2005). Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products. FEMS Microbiol. Rev. 29, 591–610. Wandrey, C., Bartkowiak, A. and Harding, S.E. (2010). Materials for encapsulation, in: Encapsulation Technologies for Active Food Ingredients and Food Processing. Springer, pp. 31–100. Zuidam, N.J. and Shimoni, E. (2010). Overview of microencapsulates for use in food products or processes and methods to make them, in: Encapsulation Technologies for Active Food Ingredients and Food Processing. Springer, pp. 3–29.

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

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Reconfigurable Filter Bank for Accurate Spectral Decomposition of EEG Signals Biju K. S.1, *, Hareeshkumar M.2, Girishkumar C.3, Jibukumar M. G.1 1

Electronics Engineering Division, School of Engineering, Cochin University of Science & Technology, Kochi, 682022, India. 2Electronics and Communication Engineering Department, Government Engineering College, Bartonhill, Thiruvananthapuram, 695035, India. 3 Faculty of Engineering & Computer Technology, AIMST University, Malaysia; *corresponding author, e-mail: bijukarunnya@gmail.com

ABSTRACT Aim: Electroencephalography (EEG) signals contain vital information which is extremely helpful for studying the functionalities and disorders of brain. For detailed analysis, the spectral decomposition of EEG signals are split into different EEG rhythms. Since the different EEG rhythms are of non-uniform bandwidth, existing techniques results in inaccurate decomposition and which in turn inaccurate results. The reconfigurable filter bank proposed can replace the existing methods for an efficient and accurate spectral decomposition of EEG signals. Methodology and results: The structure of the reconfigurable filter bank (RFB) includes a uniform filter bank followed by frequency response masking (FRM) filters. The uses of FRM filters provide a sharp transition bandwidth which optimizes the design. The first masking filter for delta band was designed such that it extracts the 0.5-4 Hz band. The second masking filter for theta band was of extracts 4-8 Hz. The masking filter for alpha band extracted about 8-13 Hz. The beta band was extracted using the masking filter applied to the sub filter of fourth stage extract greater than 14Hz. Analysis of RFB magnitude spectrum of both healthy EEG and seizure EEG signal is carried out. Conclusion: The proposed reconfigurable filter bank is based on frequency response masking technique and it provides an accurate extraction of EEG rhythms. The spectra of each band are very much clear that the extracted rhythms have much less components from the adjacent spectra. Keywords: Frequency response masking; Reconfigurable filter banks; Spectral decomposition.

INTRODUCTION Brain is the most complex part in the human body and therefore studying and analysis of the same for understanding the features, functioning and artifacts are difficult as compared to the other organs and or parts in the body. Brain waves are analyzed for the study of functioning and diagnosis of brain disorders. A number of techniques have ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

been in use for recording these brain signals. Electroencephalogram (EEG) recording is one of the most prominent among them (Teplan, 2002). The brain reacts differently at different stages of time; hence, the brain signals will be different accordingly. Brain signals consist of different frequency components termed as EEG rhythms. For a proper 57

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Spectral Decomposition of EEG Signals analysis, these EEG rhythms have to be extracted separately. For spectral decomposition and analysis lots of techniques like time-frequency analysis have been introduced earlier (Shayan et al., 2014). The various time-frequency analysis techniques in practice include Short Time Fourier Transform (STFT), Wavelet Transform (WT), Wavelet Packet Transform (WPT), Filter Banks, Moving Average filtering, Auto Regressive analysis, Auto Regressive Moving Average model (Saeid and Chambers, 2007). The STFT, WT, WPT, Filter Banks etc. enhance decomposition of signals into sub-bands and simultaneous analysis of the various (wanted) spectral components. Spectral analysis is performed on the signals to extract various key-information (Bhagwat and Vinod, 2013). For the spectral analysis lots and lots of transform techniques and their variants have been proposed and are being used through decades. Since, in particular, EEG is a non-stationary signal, transforms like DFT, FFT etc. cannot describe it completely, and some transform technique localized in time and frequency as well is needed (Bhagwat and Vinod, 2013). The wavelet transform technique is in wide practice for analysis of EEG signal for decomposition into different bands of equal widths (Bhushan, 2013; Rafiee et al., 2011). Wavelet Packets has been found better in the analysis of biological signals. At higher frequencies WT fails to localize time with required accuracy. Discrimination of frequency is sacrificed at higher frequencies for localization of time. WPT generalizes the time-frequency analysis of WT. Filter Bank is also a candidate of sub-band decomposition of EEG signals (Alfred, 1999; Chen et al., 2014; Darak et al., 2011). In all these cases the sub band bandwidths are fixed. This leads to an inaccurate decomposition and analysis and in turn ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. inaccurate results. Use of Reconfigurable Filter Banks helps solving this issue. Various methods have been proposed for achieving the reconfigurability in the cutoff frequency and bandwidths of filters. One approach is to implement a variable cut off digital filter in which the cut off frequency could be controlled through a single parameter which uses the principle of frequency transformation of linear phase FIR filters (Oppenheim et al., 1976). In fractional delay method, each unit in delay operator in fixed coefficient FIR filter is replaced with second order FIR fractional delay structure (Sasikumar et al., 2013). Cut-off frequency is changed by changing the FD value which in turn changes the bandwidth. The frequency response masking (FRM) technique comprises the complete finest transition-band filter using many wide transition-band sub-filters (Lim, 1986; Lim and Boroujeny, 1992). In the modified frequency transformation technique the fixed-coefficient low-pass sub filter in the first stage of a Fast Filter bank (FFB) is replaced by a modified second-order frequency transformation (MFT) based low pass variable digital filter (Filipe et al., 2007). In this paper the design of a reconfigurable filter bank for EEG spectral decomposition is proposed. Here a uniform filter bank is used as a prototype filter bank and FRM Technique is applied for achieving reconfigurability in sub band bandwidths. METHODOLOGY The brain cells transfer the information by means of biochemical reactions across small spaces which are termed as synapses. The nerve cells can be considered as a dipole with its own particular orientation as well as polarity. Such dipoles with identical polarity will receive similar inputs and they add up 58

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Spectral Decomposition of EEG Signals together resulting in potentials. These signals are termed as brain waves and when recorded using Electroencephalogram referred to as EEG signals (Alfred, 1999). EEG rhythms are different phenomena or events in the EEG. The commonly used terms for EEG frequency (đ?&#x2018;&#x201C;) bands are as follows (Saeid and Chambers, 2007).

delta (ď ¤) â&#x2C6;ś 0.5 â&#x2030;¤ f < 4đ??ťđ?&#x2018;§ theta (ď ą) â&#x2C6;ś 4 â&#x2030;¤ f < 8đ??ťđ?&#x2018;§ alpha (Îą) â&#x2C6;ś 8 â&#x2030;¤ f â&#x2030;¤ 13Hz beta(Î˛) â&#x2C6;ś 13Hz ď&#x20AC;ź f In general, EEG signals do not reveal information about any abnormalities or disorders of brain. Time-frequency analysis is required to extract such information, if any, from EEG signals. Spectral decomposition of EEG signals into EEG rhythms have been always a challenge since the EEG rhythms are of low frequency and accurate decomposition is very difficult (Shayan et al., 2014). The non-uniform band widths of the sub bands also add up to the complexity in spectral decomposition. Lots of methods like Wavelet Transform, Wavelet Packet transform, Filter Banks, etc. have been used for the same spectrum (Alfred, 1999; Shayan et al., 2014). The analysis using WT and WPT decomposes the finite energy signal in successive stages each with different scaling factor, Thus it gives the information in both time and frequency domains. But at higher frequencies discrimination of frequencies is sacrificed for localization of time (Alfred, 1999). Wavelet Packet Transform mitigates this problem. But, the bandwidth of packets is multiples of two; hence, it is suitable only for uniform sub band decomposition. The existing methods have not succeeded in nonuniform sub band decomposition of EEG ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. signals. Since each rhythm of EEG represents the activity of brain in different stages of activity accurate spectral decomposition is of great importance (Saeid and Chambers, 2007). So far no work has been done on the design of a method for accurate sub band decomposition. The proposed reconfigurable filter bank is the first attempt of its kind in the scenario of EEG signal processing. In the proposed Reconfigurable Filter Bank, the center frequency and bandwidth of the sub filters can be varied according to the requirements. The design requirements include sub band filters with sharp transition bands and considerably large stop band attenuation. Proposed design The proposed RFB consists mainly of two stages: i) A perfect reconstruction Uniform Filter Bank and ii) Frequency Response Masking based masking filters. In section 3.1, the design and response of Uniform Filter Bank is described. In the following section the principle of Frequency Response Masking and the design steps of FRM masking filters is discussed. Design of perfect reconstruction 16 channel filter bank The filter banks comprise two stages, analysis filter banks and synthesis filter banks filter banks (Vaidyanathan, 1993). Each stage consists of low pass, band pass and high pass filters. In analysis filters the input finite energy signal is applied to M filters which will be decomposed into M uniform width sub-bands. The resulting signals are sub-sampled by a factor N to avoid redundancies. At the synthesis filter bank stage, up sampling is done to recover the sampling rate of input signal. The up sampled signals are applied to synthesis filters which will compose the original signal. If number of filter stages equals the A.

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decimation factor it is referred to as critical sub-sampling, since above that factor perfect reconstruction is not possible. The perfect reconstruction filter bank returns the original input signal with time shift and amplitude scaling (Vaidyanathan, 1990). Let c(n) be the input signal and r(n) represents the response, here the signals are related as R0(z2) = 0.5 [A0(z) C(z) + A0(-z) C(-z)] (1) R1(z2) = 0.5 [A1(z) C(z) + A1(-z) C(-z)] (2) C'(z) = [R0 (z2) S0(z) + R1(z2) S1(z)]

(3)

Combining these equations, input-output relation is given by

the

C'(z) = 0.5[A0(z)S0(z) +A1(z)S1(z)]C(z) + 0.5[A0(-z)S0(z)+A1(-z) S1(z)]C(-z) (4) Here A(z) represents the analysis filter bank frequency response and S(z) represents the synthesis filter bank frequency response. The first term and the second term represent transmission of the c(n) through the filters and the aliasing component at the output of the filter bank respectively. Perfect reconstruction is achieved if the transfer function for the aliasing component is zero. Design of masking filters The reconfigurable Filter Bank was obtained as a result of combining the uniform filter bank with masking filters generated using the Frequency Response Masking technique (Mahesh and Vinod, 2011). The masking filters of desired bandwidths are applied at the output of the uniform filter bank sub filters at the desired stages to extract the desired bands. In the masking filter method the spectral analysis of complementary pair filters are masked by the two suitable masking filters. So the desired output is the B.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

combination of the masking filters. This method has very scanty coefficients and very low computation (Lim, 1986). Initially a low pass fir filter is selected which is termed as the modal filter with transfer function Ha(z). Now each delay of this filter is replaced by M delays to get a new filter with transfer function Hb(z) = Ha(zM). If Hb(z) is masked with another filter Hc(z) weâ&#x20AC;&#x2122;ll get a new impulse response Hd(z) with transition width 1/M times that of Ha(z). This method is used to obtain a sharp transition band filter using Frequency response masking. The consequences of the masking technique is the transition width reduce the by a factor of M for every M delay. Which affect width of the pass band reduced by M factor (Sumedh et al., 2013; Lim and Lian, 1993). Hence, it is suitable only for a narrow-band design. Since our application requires narrow band filters only FRM technique is optimum for the achieving reconfigurability in Filter banks for non-uniform sub-band decomposition of EEG signals. Proposed method As initial stage, a 16-channel perfect reconstruction filter bank was designed. For this initially, a two channel perfect reconstruction filter bank was designed. The order chosen was 90. This was chosen as the prototype filter bank for designing the Mchannel Uniform Filter Bank. Then, the tree type 16 channel filter bank was developed. Here, each filter impulse response is convolved with the interpolated impulse response of the filters in previous stage to get the new impulse response. C.

Let â&#x201E;&#x17D;đ??żđ?&#x2018;&#x192; and â&#x201E;&#x17D;Ě&#x2026;đ??żđ?&#x2018;&#x192; are the low pass filter and the complementary filter of the 2 channel perfect reconstruction model filter. Let đ?&#x2018;&#x201D;đ??żđ?&#x2018;&#x192; and đ?&#x2018;&#x201D;Ě&#x2026;đ??żđ?&#x2018;&#x192; are represent their interpolated versions respectively. Then the sub-band filters in the (i+1)th stage of the M stage 60

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Spectral Decomposition of EEG Signals filter bank are given by the following design equations.

Biju et al.

â&#x201E;&#x17D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1)= â&#x201E;&#x17D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;) * đ?&#x2018;&#x201D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;) Ě&#x2026;â&#x201E;&#x17D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1) = â&#x201E;&#x17D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;)* đ?&#x2018;&#x201D;Ě&#x2026;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1)

(5) (6)

single hardware structure can be used for mapping these sub-filters. Hence, just one filter structure is needed and the number of adders and multipliers can be reduced. This reduces the complexity in design.

â&#x201E;&#x17D;đ??ťđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1) = â&#x201E;&#x17D;Ě&#x2026;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;) â&#x2C6;&#x2014; đ?&#x2018;&#x201D;Ě&#x2026;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1)

(7)

RESULTS AND DISCUSSION

Ě&#x2026;â&#x201E;&#x17D;đ??ťđ?&#x2018;&#x192; (đ?&#x2018;&#x2013; + 1) =â&#x201E;&#x17D;Ě&#x2026;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;) â&#x2C6;&#x2014; đ?&#x2018;&#x201D;đ??żđ?&#x2018;&#x192; (đ?&#x2018;&#x2013;)

(8)

The EEG signals were adopted from CHBMIT scalp EEG database prepared and hosted by Children's Hospital Boston (CHB) and the Massachusetts Institute of Technology (MIT). The signals are in the European data format (.edf), which is a standard file format used for the storage and exchange of medical time series. The sampling frequency is 256 Hz and the file includes signals from 16 channels. Another source of database was that from Department of Epileptology, University of Bonn.

The response shows minimum overlap in complementary bands and also the filter bank has got fairly sharp transition band. The response of the 16-channel perfect reconstruction filter bank is shown in the following Figure 1. As the next stage masking filters were designed using FRM technique. This helps in perfect extraction of EEG Rhythms. The cut off frequency and in turn bandwidth of the required masking filters were reconfigured by adjusting the input pass band and stop band frequencies without changing the order. Since the designed sub-filters are identical

The procedure for simulation of the reconfigurable filter bank as follows. The

Figure 1: Magnitude response of 16-channel uniform filter bank. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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input signals read in the .edf format were applied to pre-processing which includes low pass filtering for restricting to 64 Hz followed. The signal was then applied to a notch filter for removing power line components. Pre-processed signal is applied to uniform filter bank. The uniform filter bank designed here is a 16-channel perfect reconstruction filter bank which provides accurate decomposition into bands of width 4 Hz each. Masking filters are applied to sub filters for extracting the desired Rhythms. Masking filters are designed according to the requirements. The first masking filter for delta band was designed such that it extracts the 0.5-4 Hz band. The second masking filter for Theta band was of bandwidth 4 Hz. The masking filter for Alpha band was of frequency width 5 Hz and was applied to the sub filter of third stage. The Beta band was extracted using the masking filter applied to the subfilter of fourth stage of uniform filter bank. The output response and spectrum of uniform Input Signal 500 0 -500

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Figure 2: Response of Uniform Filter Bank. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

2 1 0 2 1 0 4000 2000 0 40 20 0 4000 2000 0

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Figure3: Magnitude spectrum of Uniform Filter Bank response. Filter Bank is shown in the Figure 2 and Figure 3 respectively. From the waveforms it is clear that there is an overlap in the spectra; hence, the obtained result is inaccurate. The adjacent spectral components are present in the desired spectrum which will affect the accurate diagnosis of diseases. Use of Reconfigurable Filter Bank helps to mitigate this problem. The proposed RFB provides accurate extraction of EEG rhythms with high resolution. The waveforms in the Figure 4 and Figure 5 are magnitude spectrum of healthy EEG signal and the abnormal (seizure) EEG signal respectively. From the spectra it is clear that the extracted rhythms have much less components form adjacent spectra. Hence, it will be easier to analyze different bands separately and to determine in which band abnormality occurs. This makes the spectral decomposition analysis for EEG signals and 62

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to detect the seizure activity which is dominating in the sub-bands.

provides considerably narrow transition band width without higher orders. Hence, the circuit complexity is less. Since FRM filters are FIR based, the design retains all the advantages of FIR filters including guaranteed stability, low coefficient sensitivity, free of phase distortion etc.

The main advantage of using FRM filters for reconfiguring bandwidth is that no hardware overhead is required for changing the bandwidth and centre frequency. Only thing required is the change in coefficients. Also it

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1 0 10000 5000 0

Figure 4: RFB Magnitude Spectrum of healthy EEG signal. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Res. Highl. 4Bs (2016)

Spectral Decomposition of EEG Signals

Biju et al.

Figure 5: RFB Magnitude Spectrum of seizure EEG signal. CONCLUSION In this paper design of a reconfigurable filter bank for efficient and accurate spectral decomposition of EEG signals is proposed. The existing techniques employed for spectral decomposition such as Wavelet Transform, Wavelet Packet transform and DFT Filter bank could only extract bands of uniform bandwidth. The proposed RFB based on FRM technique provides accurate ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

extraction of EEG Rhythms. Use of reconfigurable filter bank is the first attempt in EEG rhythm extraction. A perfect reconstruction 2-channel filter bank was used as modal filter bank and an M-channel filter bank was designed using this modal filter bank. To mitigate the problem of varied bandwidth EEG spectral extraction, FRM based filters were applied as masking filters. Use of FRM technique for the generation of masking filters has provided 64

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Spectral Decomposition of EEG Signals sharp transition width narrow band low pass and band pass filters to be used for extracting desired bands. Since the sub filters are identical they can be mapped into a single hardware structure, it reduces the complexity of hardware implementation. Since the reconfigurability is achieved without a change in hardware the technique is found to be optimum for EEG rhythm extraction. REFERENCES Alfred, M. (1999). Signal Analysis: Wavelets, Filter Banks, TimeFrequency Transforms and Applications John Wiley & Sons Ltd. Singapore. pp. 225-245. Bhagwat, S. D. and Vinod, J. (2013). EEG Data Sets Signal Processing Using Wavelet Transforms. International Journal of Innovative Technology and Exploring Engineering 2(6), 108-111. Bhushan, N. P. (2013). A review of ECG monitoring system using Wavelet Transform. International Journal of Innovative Technology and Exploring Engineering 2(4), 334341. Chen, J. Ding, W. and Zhou, J. (2014). Design of Hardware Efficient Modulated Filter Bank for EEG Signals Feature Extraction. In: IEEE Proceeding of 57th International Midwest Symposium on Circuits and Systems. College Station, Texas. pp.793-796. Darak, S. J. Vinod, A. P. and Lai, E. M. K. (2011). A new variable digital filter design based on fractional delay. In: IEEE Proceeding of ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biju et al. International Conference. Acoustics, Speech and Signal Processing, Prague. pp. 1629-1632. Filipe, C.C.B. D. IuriKothe, S. L. N. and Luiz, W. P. B. (2007). High selectivity Filter Banks for spectral analysis of music signals. Advances in Signal Processing 1-13. Lim, Y. C. and Lian, Y. (1993). The optimum design of One- and TwoDimensional FIR filters using the frequency response masking technique. Circuits and Systems II: Analog and Digital Signal Processing 40(2), 88-95. Lim, Y. C. (1986). Frequency-response masking approach for the synthesis of sharp linear phase digital filters. Circuits and Systems 33, 357-364. Lim, Y. C. and Boroujeny, B. F. (1992). Fast filter bank (FFB). Circuits and Systems II: Analog and Digital Signal Processing 39(5), 316–318. Mahesh, R. and Vinod, A. P. (2011). Reconfigurable Low Area Complexity Filter Bank Architecture Based on Frequency Response Masking for Non-uniform Channelization in Software Radio Receivers. Aerospace and Electronic Systems 47(2), 1241-1255. Oppenheim, A. Mechlenbräuker, W. and Mersereau, R. (1976). Variable cutoff linear phase digital filters. Circuits and Systems 23(4), 199– 203. Rafiee, J. Rafiee, M. A. Prause, N. and Shoan, M. P. (2011). Wavelet basis functions in biomedical signal 65

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Spectral Decomposition of EEG Signals processing. Expert Systems with Applications 38(5), 6190-6201. Saeid, S. and Chambers, J. A. (2007). EEG Signal Processing, John Wiley & Sons Ltd. pp.51-75. Sasikumar, G. VudiS. M. and Rittwika, G. (2013). Analysis and simulation of brain signal data by EEG signal processing technique using MATLAB. International Journal of Engineering and Technology 5(3), 2771-76. Shayan, M. Mohamed, M. Martyn, H. Catherine, M. H. and Paul, R. W. (2014). Signal processing techniques applied to human sleep EEG signalsâ&#x20AC;&#x201D;A review. Biomedical Signal Processing and Control 10, 21â&#x20AC;&#x201C;33.

Biju et al. reconfigurable channel filter based on decimation, interpolation and frequency response masking. In: IEEE Proceeding of International Conference on Acoustics, Speech and Signal Processing. Vancouver, Canada. pp. 5583-87. Teplan, M. (2002). Fundamentals of EEG Measurement. Measurement Science Review 2 (2), 1-11. Vaidyanathan, P. P. (1990). Multirate Digital Filters, Filter Banks, Polyphase Networks, and Applications: A Tutorial. Proceedings of the IEEE 78(1), 5693. Vaidyanathan, P. P. (1993). Multirate Systems and Filter Banks. Pearson Education, Inc. New Delhi. pp.188234.

Sumedh, D. Smitha, K. G. and Vinod, A. P. (2013). A low complexity

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Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016), P67-76

Coenzyme Q10 Dietary Supplementation during Antitubercular Therapy Prevents Renal Damage in Rats Udhaya Lavinya B. and Evan Prince Sabina* School of Biosciences and Technology, VIT University, Vellore-632014, Tamilnadu, India; *corresponding author, e-mail: eps674@gmail.com

ABSTRACT Aim: Antitubercular therapy leads to the development of acute renal injury (ARI) in some individuals. Though isoniazid (INH) has been evidenced as an ARI inducing drug, mounting evidences from several studies identify rifampicin (RIF) as the most common ARI inducing antitubercular drug. Current study was carried out to evaluate the nephroprotective effect of the oral supplementation of coenzyme Q10 in INH and RIF treated Wistar albino rats. Methodology and results: Rats were administered with INH and RIF (50 mg/kg b.w. each/day) for 28 days. The effect of concomitant treatment with coenzyme Q10 (10 mg/kg b.w./day) on INH and RIF-induced renal injury was evaluated by estimating the serum levels of renal functional markers such as creatinine, urea, uric acid and acid phosphatase. In addition, the antioxidant profile, levels of non-enzymic antioxidants and lipid peroxidation were assessed in renal homogenates of experimental rats. Histological studies were also performed. The standard hepatoprotective drug silymarin (25 mg/kg b.w./day) was used for the purpose of comparison. The tested parameters of the coenzyme Q10 treated INH and RIFinduced rats were compared with that of the normal control rats and silymarin-treated INH and RIF-induced rats. Coenzyme Q10 significantly reduced the elevated levels of serum renal functional markers in INH and RIF-administered rats. Also, the food supplement was able to restore near normal the antioxidant status and noted to prevent renal damage in experimental rats. Conclusion, significance and impact of study: Current study reveals the potential of coenzyme Q10 in minimizing the renal injury due to antitubercular therapy. Hence, CoQ10 supplementation would be useful to patients on antitubercular regimen. Keywords: Acute renal injury; Coenzyme Q10; Isoniazid; Rifampicin.

INTRODUCTION Oxidative stress plays a major role in the induction and progression of renal failure both acute and chronic. Several conditions like hypertension, diabetes, infection, obstruction in the urinary tract, autoimmune disorders such as lupus erythematosus, genetic disorders such as polycystic kidney disease, drugs such as antibiotics, diuretics and anti-inflammatory drugs induce oxidative stress in renal tissues (Schattner et al., 2000; CrispĂn et ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

al., 2008; Chevalier et al., 2010). Several medications are known inducers of acute interstitial nephritis which includes commonly used non-steroidal antiinflammatory drugs (NSAIDS), interferon and antibiotics such as penicillins, cephalosporins and anti-tubercular drugs such as rifampicin (Rossert, 2001). Studies exploring the sub-cellular mechanisms of renal injury causing AIN have been carried out recently (Mitchell et al., 1977; Servais et al., 2007; Chevalier et al., 2010). It has

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Coenzyme Q10 Dietary Supplementation been proven that interstitial inflammation in renal tissue might be due to the predominant infiltration of local B-cells, eosinophils and mononuclear leucocytes in the interstitium (Heller et al., 2007). In addition, the development of antirifampicin antibodies occurs rarely in patients on antitubercular therapy. Studies have shown that these rifampicindependent antibodies cause acute haemolysis and renal failure (van der Meulen et al., 2009; Beebe et al., 2015). Several case studies have reported the occurrence of acute interstitial tubulopathy in pulmonary tuberculosis patients on antituberculosis regimen (Muthukumar et al., 2002; Rosati et al., 2013). Druginduced ARI is a major adverse effect due to rifampicin though uncommon. Isoniazid (INH) and rifampicin (RIF) coadministration causes injury in the hepatocytes leading to hepatotoxicity (Baskaran and Sabina, 2015). Coenzyme Q10 is a fat soluble vitaminlike compound with significant antioxidant potential. It plays key role in mitochondrial bioenergetics. Though the compound is synthesised within the body, there are certain conditions that lead to low levels of coenzyme Q10 (CoQ10) such as vitamin B deficiency, use of statins for hypercholesterolemia, old age, cardiovascular disorders and oxidative stress (Gempel et al., 2007; Quinzii et al., 2007; Quinzii and Hirano, 2011). Recently, it has been found that this antioxidant also plays significant role in the regulation and alteration of genes involving cell signalling and metabolic pathways (Groneberg et al., 2005). Our previous study showed significant protective effects of CoQ10 against INH and RIF induced hepatotoxicity (Baskaran and Sabina, 2015). Current study was an attempt to evaluate the occurrence of renal injury due to the co-administration of INH and RIF and the role of CoQ10 as a nephroprotective agent in Wistar albino rats. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Udhaya and Sabina MATERIALS AND METHODS Chemicals and drugs Synthetic coenzyme Q10 was purchased from Sigma Aldrich, India. INH and RIF were purchased from Lupin Ltd., Aurangabad, India and the standard hepatoprotective drug silymarin from Quality Pharmaceuticals Ltd., India. INH and RIF were dissolved in normal saline while silymarin was dissolved in sterile distilled water. Coenzyme Q10 was dissolved in 0.2 ml corn oil. All the other chemicals and reagents used were of analytical grade procured from SD Fine Chemicals Pvt. Ltd., Mumbai, India. Animals 30 female Wistar albino rats of body weight 143.26Âą12.81 g were procured from the Animal House, VIT University, Vellore, Tamilnadu. The rats were divided into 5 groups and treated as follows for 28 days: group I was normal control; group II was treated with INH and RIF (50 mg/kg b.w. each/day); group III was INH and RIF-induced rats co-administered with coenzyme Q10 (10 mg/kg b.w./day); group IV was INH and RIF-induced rats coadministered with silymarin (25 mg/kg b.w./day); group V was treated with coenzyme Q10 (10 mg/kg b.w./day) alone. The animals were sacrificed after the study duration using ether anaesthesia. Blood and kidneys were procured for further analysis. Renal homogenates were prepared using 5 % phosphate buffered saline (PBS). Estimation of serum markers of renal function Serum levels of renal functional markers such as creatinine, urea, uric acid and acid phosphatase were estimated using commercial diagnostic kits obtained from AutoSpan Diagnostics Ltd., India. Assessment of antioxidant profile Renal homogenates were used to assess the activities of superoxide dismutase 68

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Coenzyme Q10 Dietary Supplementation (Marklund and Marklund, 1974), catalase (Sinha, 1972), glutathione peroxidase (Rotruck et al., 1973) and Glutathione-Stransferase (Habig et al., 1974); levels of total reduced glutathione (Moron et al., 1979) and lipid peroxidation (Ohkawa et al., 1979). Histopathological analysis A portion of the kidneys were fixed in 10 % formalin after thorough washing with ice-cold 5 % PBS. The tissues were then dehydrated with descending grades of isopropanol. After treating with xylene, the tissues were embedded in molten paraffin wax and tissue sections (5 µm) were cut. The sections were stained with hematoxylin and eosin and examined microscopically for pathological changes.

Udhaya and Sabina Effect of coenzyme Q10 on serum markers of renal function in INH and RIF induced rats INH and RIF induced rats showed significant increase in the levels of urea, creatinine, uric acid and acid phosphatase while concomitant administration of coenzyme Q10 caused significant (P<0.05) reduction in the elevated levels of the aforementioned parameters (Figures 1-4). This particular effect of coenzyme Q10 in minimizing INH and RIF-induced changes in serum markers of renal function was comparable with that of silymarin treated rats. The oxidative stress parted by the toxic metabolites of isoniazid on renal tissue might lead to oxidative cellular damage. In addition, evidence from literature suggest that the generation of anti-rifampicin antibodies causes

RESULTS AND DISCUSSION

Figure 1: Effect of coenzyme Q10 on serum urea in INH and RIF induced rats. Each value represents the Mean±sd of six rats. Comparisons were made as follows: a-group I vs. groups II, III, IV, V; b-group II vs. group III, IV, V. c-group III vs. groups IV, V; d-groups IV vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was calculated by one-way ANOVA followed by the student Newman-keul’s test. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Res. Highl. 4Bs (2016)

Coenzyme Q10 Dietary Supplementation formation of drug-antibody immune complexes which result in glomerular endotheliosis and cell damage thereby leading to tubular injury and reduced renal function (Muthukumar et al., 2002). Acute tubular necrosis and interstitial nephritis

Udhaya and Sabina are adverse effects of RIF reported in tuberculosis patients (van der Meulen et al., 2009). A case study has reported the evidence of acute renal failure (ARF) in a 38 year old tuberculosis patient (Beebe et al., 2015).

Figure 2: Effect of coenzyme Q10 on serum creatinine in INH and RIF induced rats. Each value represents the Mean±sd of six rats. Comparisons were made as follows: a-group I vs. groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was calculated by one-way ANOVA followed by the student Newman-keul’s test.

Figure 3: Effect of coenzyme Q10 on serum uric acid in INH and RIF induced rats. Each value represents the Mean±sd of six rats. Comparisons were made as follows: a-group I vs. groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was calculated by one-way ANOVA followed by the student Newman-keul’s test. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Res. Highl. 4Bs (2016)

Coenzyme Q10 Dietary Supplementation

Udhaya and Sabina

Figure 4: Effect of coenzyme Q10 on serum acid phosphatase in INH and RIF induced rats. Each value represents the MeanÂąsd of six rats. Comparisons were made as follows: a-group I vs. groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was calculated by one-way ANOVA followed by the student Newman-keulâ&#x20AC;&#x2122;s test. Effect of coenzyme Q10 on antioxidant profile in INH and RIF induced rats There was significant (P<0.05) reduction in the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-Stransferase in renal homogenates of INH and RIF induced rats (Table 1). Also, there was significant (P<0.05) reduction in the levels of reduced glutathione and significant (P<0.05) increase in the levels of lipid peroxidation on treatment with INH and RIF. Co-administration of coenzyme Q10 was able to restore these parameters to near normal levels which were compared with that of silymarin treated INH and RIF induced rats. The reduction in the activities of the enzymic and non-enzymic antioxidants and elevated lipid peroxidation levels reflect the extent of oxidative stress in the INH and RIF treated rats. It has been proven that mitochondrial membrane damage induces ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

DNA fragmentation and apoptosis in organs that are vulnerable to oxidative damage. The mechanism that underlies mitochondrial membrane damage is oxidative stress exerted due to the production of ROS. Effect of coenzyme Q10 on renal tissues in INH and RIF induced rats ARI is an uncommon and less known adverse effect arising due to antituberculosis regimen which includes rifampicin (Topping et al., 2012). Case studies in elderly population have shown evidence of ARF (De Vriese et al., 1998; Chang et al., 2014). There are also mounting evidences on the occurrence of ARI in patients undergoing intermittent or interrupted administration of rifampicin giving rise to the development of antirifampicin antibodies (Pereira et al., 1991; Rosati et al., 2013). Histopathological examination of renal tissue architecture in 71

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Coenzyme Q10 Dietary Supplementation

Udhaya and Sabina

the current study reveals damage to glomerular cells and karyolysis in the INH and RIF treated rats. Apart from ARI caused by rifampicin, there could have been significant damage caused due to oxidative stress and depletion of cellular antioxidants parted by the toxic metabolites of isoniazid (Chowdhury et al., 2006). Mitochondrial membrane potential (MMP) collapse causes apoptosis

(Ly et al., 2003). It has been shown that CoQ10 prevents MMP collapse by preventing mitochondrial depolarization and hence may possess antiapoptotic activity (Papucci et al., 2003). This particular activity of CoQ10 along with its antioxidant and free radical scavenging activities might have prevented tubular injury.

Table 1: Effect of concomitant administration of coenzyme Q10 on antioxidant parameters in renal tissue homogenates of INH and RIF induced rats. Parameters

SOD (units/min/mg protein) Catalase (units/min/mg protein) Glutathione peroxidase (μg of GSH utilized/min/ mg protein) Reduced glutathione (nmol/mg protein) Glutathione-Stransferase (nmol of CDNBGSH conjugate formed/min/mg protein) TBARS (mM/TBARS/100 g of wet tissue)

Group 1 (Control)

Group 2 (INH+RIF 50 mg/kg b.w.)

Group 3 (INH+RIF & CoQ10 10mg/kg b.w.)

Group 4 (INH+RIF & silymarin 25 mg/kg b.w.)

Group 5 (CoQ10 10 mg/kg b.w.)

180.14±3.42

95.04±3.40a*

165.11±2.29a*b*

168.05±5.21a*b*

186.03±3.84b*c*d*

60.14±2.85

35.12±2.84a*

55.04±2.00b*

53.05±2.28a*b*c*

64.32±3.13b*c*

38.08±2.28

18.02±2.28a*

32.07±1.42a*b*

32.64±3.49a*b*

37.05±1.42b*c*

46.14±1.45

28.04±2.00a*

42.04±4.00b*

41.14±2.02b*

47.09±2.01b*d*

20.09±1.43

11.142±1.45a*

16.00±1.41b*

16.51±3.78b*

20.01±2.00b*

0.80±0.20

1.80±0.14a*

1.00±0.14b*

1.10±0.14b*

0.70±0.14b*d*

Each value represents the Mean±sd of six rats. Comparisons were made as follows: a-group I vs. groups II,III,IV,V; b-group II vs. group III,IV,V. c-group III vs. groups IV,V; d-groups IV vs. group V. The symbol represents statistical significance at *(p<0.05>. Statistical analysis was calculated by one-way ANOVA followed by the student Newman-keul’s test. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Coenzyme Q10 Dietary Supplementation

Udhaya and Sabina

Figure 5: Effect of CoQ10 on the renal histology of INH and RIF treated rats. Kidney histopathology (haematoxylin and eosin staining): A) normal control rats showing normal histology of glomeruli and renal tubules; B) INH and RIF treated rats showing normal to decreased cellularity of glomerulus, renal tubules showing cell swelling with increase in eosinophilia of the cytoplasm with karyolysis in few of the cells; C) CoQ10 supplemented INH and RIF treated rats showing normal histoarchitecture of renal tissue being maintained D) silymarin administered INH and RIF treated rats showing normal morphology of glomeruli and tubules E) CoQ10 alone treated rats showing normal renal tissue histology. CONCLUSION Current study shows that CoQ10 was able to restore normal antioxidant status in INH ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

and RIF treated rats. The determination of serum renal function markers and kidney histopathological alterations reveal that CoQ10 minimizes damage to renal tissue 73

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Coenzyme Q10 Dietary Supplementation due to INH and RIF. Free radical scavenging property of CoQ10 might have reduced the extent of damage to cellular membranes and macromolecules thereby rendering protection against oxidative stress. In addition, the anti-apoptotic property of CoQ10 might have minimized cell death due to mitochondrial membrane depolarization thereby reducing injury to tubular interstitium which might have aided the maintenance of renal histoarchitecture. Further studies would explore the molecular mechanisms of the effect of CoQ10 in INH and RIF induced renal injury. REFERENCES Baskaran, U.L. and Sabina, E.P. (2015). The food supplement coenzyme Q10 and suppression of antitubercular drug-induced hepatic injury in rats: the role of antioxidant defence system, antiinflammatory cytokine IL-10. Cell Biology and Toxicology 1–9. Beebe, A., Seaworth, B. and Patil, N. (2015). Rifampicin-induced nephrotoxicity in a tuberculosis patient. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 1, 13–15. Chang, C.-H., Chen, Y.-F., Wu, V.-C., Shu, C.-C., Lee, C.-H., Wang, J.Y., Lee, L.-N. and Yu, C.-J. (2014). Acute kidney injury due to anti-tuberculosis drugs: a five-year experience in an aging population. BMC Infectious Diseases 14, 23. Chevalier, R.L., Thornhill, B.A., Forbes, M.S. and Kiley, S.C. (2010). Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy. Pediatric Nephrology (Berlin, Germany) 25, 687–697.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Udhaya and Sabina Chowdhury, A., Santra, A., Bhattacharjee, K., Ghatak, S., Saha, D.R. and Dhali, G.K. (2006). Mitochondrial oxidative stress and permeability transition in Isoniazid and Rifampicin induced liver injury in mice. Journal of Hepatology 45, 117–126. Crispín, J.C., Oukka, M., Bayliss, G., Cohen, R.A., Beek, C.A.V., Stillman, I.E., Kyttaris, V.C., Juang, Y.-T. and Tsokos, G.C. (2008). Expanded Double Negative T Cells in Patients with Systemic Lupus Erythematosus Produce IL17 and Infiltrate the Kidneys. The Journal of Immunology 181, 8761– 8766. De

Vriese, A.S., Robbrecht, D.L., Vanholder, R.C., Vogelaers, D.P. and Lameire, N.H. (1998). Rifampicin-associated acute renal failure: pathophysiologic, immunologic, and clinical features. American Journal of Kidney Diseases: The Official Journal of the National Kidney Foundation 31, 108–115.

Gempel, K., Topaloglu, H., Talim, B., Schneiderat, P., Schoser, B.G.H., Hans, V.H., Pálmafy, B., Kale, G., Tokatli, A., Quinzii, C. et al. (2007). The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electrontransferring-flavoprotein dehydrogenase (ETFDH) gene. Brain 130, 2037–2044. Groneberg, D.A., Kindermann, B., Althammer, M., Klapper, M., Vormann, J., Littarru, G.P. and Döring, F. (2005). Coenzyme Q10 affects expression of genes involved in cell signalling, metabolism and transport in human CaCo-2 cells. The International 74

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Coenzyme Q10 Dietary Supplementation Journal of Biochemistry & Cell Biology 37, 1208–1218. Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974). Glutathione STransferases the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139. Heller, F., Lindenmeyer, M.T., Cohen, C.D., Brandt, U., Draganovici, D., Fischereder, M., Kretzler, M., Anders, H.-J., Sitter, T., Mosberger, I., et al. (2007). The Contribution of B Cells to Renal Interstitial Inflammation. The American Journal of Pathology 170, 457–468. Ly, J.D., Grubb, D.R. and Lawen, A. (2003). The mitochondrial membrane potential (Δψm) in apoptosis; an update. Apoptosis 8, 115–128. Marklund, S. and Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry / FEBS 47, 469–474. Mitchell, J.R., McMurtry, R.J., Statham, C.N. and Nelson, S.D. (1977). Molecular basis for several drug-induced nephropathies. The American Journal of Medicine 62, 518–526. Moron, M.S., Depierre, J.W. and Mannervik, B. (1979). Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta (BBA) - General Subjects 582, 67– 78.

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Udhaya and Sabina Muthukumar, T., Jayakumar, M., Fernando, E.M. and Muthusethupathi, M.A. (2002). Acute renal failure due to rifampicin: a study of 25 patients. American Journal of Kidney Diseases: The Official Journal of the National Kidney Foundation 40, 690–696. Ohkawa, H., Ohishi, N. and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Papucci, L., Schiavone, N., Witort, E., Donnini, M., Lapucci, A., Tempestini, A., Formigli, L., Zecchi-Orlandini, S., Orlandini, G., Carella, G., et al. (2003). Coenzyme q10 prevents apoptosis by inhibiting mitochondrial depolarization independently of its free radical scavenging property. The Journal of Biological Chemistry 278, 28220–28228. Pereira, A., Sanz, C., Cervantes, F. and Castillo, R. (1991). Immune hemolytic anemia and renal failure associated with rifampicindependent antibodies with anti-I specificity. Annals of Hematology 63, 56–58. Quinzii, C.M., DiMauro, S. and Hirano, M. (2007). Human Coenzyme Q10 Deficiency. Neurochemical Research 32, 723–727. Quinzii, C.M. and Hirano, M. (2011). Primary and secondary CoQ10 deficiencies in humans. Biofactors (Oxford, England) 37, 361–365. Rosati, S., Cherubini, C., Iacomi, F., Giannakakis, K., Vincenzi, L., Ippolito, G. and Palmieri, F. (2013). Acute rifampicin75

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Coenzyme Q10 Dietary Supplementation associated interstitial tubulopathy in a patient with pulmonary tuberculosis: a case report. Journal of Medical Case Reports 7, 106. Rossert, J. (2001). Drug-induced acute interstitial nephritis. Kidney International 60, 804–817. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G. and Hoekstra, W.G. (1973). Selenium: biochemical role as a component of glutathione peroxidase. Science (New York, N.Y.) 179, 588–590. Schattner, A., Sokolovskaya, N. and Cohen, J. (2000). Fatal hepatitis and renal failure during treatment with nimesulide. Journal of Internal Medicine 247, 153–155.

Udhaya and Sabina Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms and potential approaches to modulation. Apoptosis 13, 11–32. Sinha, A.K. (1972). Colorimetric assay of catalase. Analytical Biochemistry 47, 389–394. Topping, K., Nair, B., Ahmed, A. and Woywodt, A. (2012). Tuberculosis, acute kidney injury and pancreatitis––what is the underlying cause?. Clinical Kidney Journal 5, 364–365. Van der Meulen, J., de Jong, G.M. and Westenend, P.J. (2009). Acute interstitial nephritis during rifampicin therapy can be a paradoxical response: a case report. Cases Journal 2, 6643.

Servais, H., Ortiz, A., Devuyst, O., Denamur, S., Tulkens, P.M. and Mingeot-Leclercq, M.-P. (2007).

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Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016),P77-83

Safe Water as the Key to Food Safety and Global Health Md. Latiful Bari* Center for advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh; *corresponding author, e-mail: latiful@du.ac.bd

ABSTRACT Access to clean water is the inborn rights for everyone in the world and is a prerequisite for safe food production and health improvement. However, till today, water scarcity or lack of safe drinking water is one of the world's leading problems affecting more than 1.1 billion people globally, meaning that one in every six people lacks access to safe drinking water. By 2025, 1.8 billion people will be living in countries or regions with absolute water scarcity. The clean water scarcity usually exists in developing countries due to 1) lack of capacity, 2) community capacity and engagement, 3) technological capacity, 4) institutional capacity and 5) inadequate financial support. The new global ﬁnancing initiative within the water and sanitation sector is highly required before being advocating food safety issues in these countries. In addition, moral, civil, political and economic investment needs to bring adequate sanitation for the global population to improve health and welfare. Keywords: Accessibility; Food safety; Global health; Health improvement; Safe water.

CHALLENGES RELATED TO WATER Access to safe drinking water is the fundamental need for healthy human life and water has been shown linked to the development of all areas including health, nature, urbanization, industrialization, energy production, food security, equality and other aspects of community development (Colwell et al., 2003; Huq et al., 2010). In all these areas, not only water but also clean, pure, safe and quality of water are required to ensure proper development. According to United Nations (UN), to meet up daily necessities like drinking, cooking, and personal hygiene, approximately 20 to 40 liters of clean water is required by each individual. In today’s world, scarcity of safe water is a burning issue; especially in developing countries ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

where nearly 1.0 billion people lack fresh water for daily uses (Aho et al., 2012). Although, approximately 71% of earth’s surface is covered with water and more than 97% of the earth’s water is salty sea water, while 2% is stored in glaciers, ice caps, and snowy mountain ranges (Yongabi, 2010); thus, less than 1.0% of the earth’s water is surface water, of lakes, rivers, streams, ponds (0.022%) and groundwater (0.397%) available for humans for their daily needs (Yongabi, 2010). However, all these accessible surface water is not necessarily safe for drinking, and depending on the location and sources, clean water is exposed to a variety of contaminants, many arising from human and animal wastes, debris from homes, chemicals from industries, fertilizers and pesticides from agricultural lands, and 77

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Safe Water as the Key to Health environmental pollutants as well as other contributing factors (Figure 1) (Zeenath et al., 2015). In addition, insanitary practices of people have greatly contributed to the deterioration of the quality of surface water sources (Zaman et al, 2013). On the other hand, groundwater is the most important source of water supply in most of the countries including Bangladesh.

Bari programme (2014), about 28 million Bangladeshis, or just over 20% of the population, are living in harsh conditions in receiving clean water, and safe clean water supply situation is going to become worst in coming decades to meet the need of predicted 200 million people by 2020 (Silvia et al., 2011). CHALLENGE RELATED CLIMATE CHANGE

Figure 1: Important drivers that impacted health. Physically groundwater is clear, colorless with little or no suspended solids and has a relatively constant temperature. Groundwater is also free from diseaseproducing micro-organisms which are normally present in large numbers in surface waters (Bari and Yeasmin, 2014). However, the groundwater for drinking purposes has become a problem because of: 1) the presence of arsenic, 2) excessive dissolved iron, 3) salinity in the shallow aquifers in the coastal areas, 4) lowering of groundwater level (water table), and 5) rock/stony layers in hilly areas (USEPA, 2015). Among these problems arsenic in groundwater has become a great concern for water supply in Bangladesh. According to a study by the World Bank's water and sanitation ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Climate change can impact human health and wellbeing through multiple avenues and at varying scales. Effects of climate change could include more frequent and intense rainfall events, leading to increased overland and shallow sub surface flow which can mobilize pathogens and other contaminants. Increased frequency and magnitude of flood events impacts not only availability of clean water, but chemical storage and sewage facilities, compromising quality. Links between weather events and waterborne illness have been identified as water temperatures, increased precipitation intensity and longer periods of low flows will exacerbate many forms of water pollution, with impacts on ecosystems and human health as shown in Figure 2. The clean water scarcity usually exists in developing countries due to: 1) lack of capacity, 2) community capacity and engagement, 3) technological capacity, 4) institutional capacity, and 5) inadequate financial support. New global financing initiative within the water and sanitation sector is highly required before being advocating food safety issues in these countries (UNU-INWEH, 2010). As the accessibility of clean drinking water is directly related to, poverty, social and economic development, and human health; thus, the benefits of water and sanitation program can be made in terms of improved

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Safe Water as the Key to Health

Bari

Figure 2: Interrelationships between major global environmental changes (Source: McMichael et al., 2003). contamination, 2) finding more multistate well-being of people and communities, outbreaks, 3) new and emerging bacteria, reduction in public health costs, and toxins and antibiotic resistance, 4) catalysis for local economic growth. Such unexpected sources of foodborne illness, beneďŹ ts accrue in perpetuity and can such as ice cream and raw sprouted nut potentially improve food safety and human butter etc. The food safety challenges exist health. in both developed and developing countries and differ by region, due to differences in income level, diets, local conditions, and CHALLENGES RELATED TO FOOD government infrastructures. Figure 3 shows SAFETY number of foodborne cases per year in The battle against foodborne diseases is percentage of population in different facing new challenges due to the countries. There are four broad uses of water globalization of the food market, climate in every food production: 1) primary change and changing patterns of human production (e.g. farming), 2) cleaning and consumption as fresh and minimally sanitation, 3) as an ingredient or component processed foods are currently preferred (Bari of an ingredient, and 4) processing and Ukuku, 2016). Furthermore, food safety operations (e.g. heating, refrigeration etc.). challenges will continue to arise in unpredictable ways, largely due to: 1) Adequate safe water supplies directly affect changes in the environment leading to food the safety of food; therefore, food handlers

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Safe Water as the Key to Health

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Figure: 3: Foodborne cases per year in percentages of population in different countries. should follow good-sense of practices when considering the source, treatment and intended use of water in food production to ensure the quality and safety of the food products. Food safety concerns in developing countries typically include: 1) the inappropriate use of agricultural chemicals, 2) the use of untreated or partially treated wastewater, 3) the use of sewage or animal manure on crops, 4) the absence of food inspection, including meat inspection, 4) a lack of infrastructure, such as adequate refrigeration, and finally 5) poor hygiene, including a lack of clean water supplies. However, food safety concerns in developed countries are mainly associated with: 1) ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

changes in animal husbandry practices, 2) changes in agronomic process, 3) changes in lifestyle and consumer demands, 4) increase in susceptible populations, 5) increased travel, and 6) increasing international food trade etc., (Bari and Ukuku, 2016). Thus, it appears that each developing countries limitations are more basic in nature, which are recoverable than the developed countries limitation that are non-recoverable. Therefore, greater emphasis on capacity building needs to be built internally to a country and through South-South collaborations and partnerships, rather than depending on consultants from developed countries. Capacity is a ďŹ&#x201A;exible concept and encompasses the public sector, academia; community based organizations, private

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sector, which ranges from the individual to institutions to society as a whole. CREATING FOOD SAFETY CULTURE People’s attitudes, choices and behaviors are some of the most important factors influencing the overall safety of a food. A culture of food safety required to be built on a set of shared values that operators and their staff follow to produce and provide food in the safest manner (Figure 4). Maintaining a food safety culture means that operators and staff know the risks associated with the products or meals they produce, know why managing the risks is important, and effectively manage those risks in a demonstrable way. In an organization with a good food safety culture, individuals are expected to enact practices that represent the shared value system and point out where others may fail. By using a variety of tools, consequences and incentives, businesses can demonstrate to their staff and customers that they are aware of current food safety issues, that they can learn from others’ mistakes, and that food safety is important within the organization (Jack et al., 2012). Thus, creating a culture of food safety requires application of the best science with the best management and communication systems, including compelling, rapid, relevant, reliable and repeated food safety messages using multiple media. Training employees on food safety practices has been shown to be one of the most important programs that food service establishments can implement. However, numerous study reports suggested that traditional approaches used to educate and train employees (such as Serve Safe) may not be particularly effective and new behavior-based approaches that include food safety education as part of the culture of the organization needs to be developed.

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Figure 4: Building a food safety culture.

CONCLUSION It is very clear that water-related diseases are responsible for a significant proportion of the global health burden. On the other hand, it is equally clear that adequate sanitation measures have not been managed to keep up with population growth. Sanitation is not only critical for dignity and health but it is the most basic form of source water protection also. This realization is not new, and focus on clean water alone does not necessarily result in improved access to sanitation. Thus, each gap needs to be taken into account and adequate investment in terms of education, capacity and financing to be done to improve the food safety and sanitation status. A strong moral, civil, and political will is essential to bring in an adequate sanitation for the global population to improve health and welfare. REFERENCES Aho, I. M. and Lagasi, J. E. (2012). A new water treatment system using Moringa oleifera seed. American Journal of Scientific and Industrial Research Science Huβ 3 (6), 487492.

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Safe Water as the Key to Health Colwell, R. R., Huq, A., Islam, M. S., Aziz, K. M. A., Yunus, M., Khan, N. H., Mahmud, A., Sack, R. B., Nair, G. B., Chakraborty, J., Sack, D. A. and Russek-Cohen, E. (2003). Reduction of cholera in Bangladeshi villages by simple filtration. Proceedings of the National Academy of Sciences 100, 1051-1055. Bari M. L. and D. O Ukuku (2016). Foodborne Pathogen and Food Safety (Eds) Md. Latiful Bari and Dike O Ukuku, Published by CRC Press ( Taylor and Francis Group), 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487, USA. pp 1-37. Bari, M.L. and Yeasmin, S., 2014. Water Quality Assessment: Modern Microbiological Techniques. In: Batt, C.A., Tortorello, M.L. (Eds.), Encyclopedia of Food Microbiology, vol 3. Elsevier Ltd, Academic Press, pp. 755–765. Huq, A., Yunus, M., Sohel, S.S., Bhuiya, A., Emch, M., Luby, S. P., RussekCohen, E., Nair, G. B., Sack, R. B. and Colwell, R. R. (2010). Simple sari cloth filtration of water is sustainable and continues to protect villagers from cholera in Matlab, Bangladesh. mBio 1(1), 1 e0003410.

Bari Githeko, A., Scheraga, J.D. and Woodrow, A. (Eds.). (2003). Climate Change and Human Health: Risks and Responses. WHO, WMO and UNEP. Available from: http://www.who.int/globalchange/pu blications/climchange.pdf (Accessed June 9, 2016) Silvia, B., Elena, B., Sara, B., Osvaldo, C., Franca, P. and Elisabetta, C. (2011). Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water. Environmental Monitoring and Assessment 177, 493–503. UNU-INWEH (2010). Sanitation as a Key to Global Health: Voices from the Field. United Nations University Institute for Water, Environment and Health. pp 2-45. USEPA. (2015). Drinking Water Contaminants, http://www.epa.gov/dwstandards regulation (Accessed on April 19, 2016). [WHO] World Health Organization. (2001). Water Quality: Guidelines, Standards and Health. Chapter 6 and 13. Edited by L. Fewtrell and J. Bartram. Published by IWA Publishing, London, UK. ISBN 1 900222 28 0.

Jack, A., Neal, M. B. and Henroid, D. (2012). Assessing Factors Contributing to Food Safety Culture in Retail Food Establishments. Food Protection Trends 32(8), 468–476.

Yongabi, K. A. (2010). Biocoagulants for water and wastewater purification: A review. International Review of Chemical Engineering 2(3), 444458.

McMichael, A.J., Campbell-Lendrum, D.H., Corvolan, C., Ebi, K.L.,

Zaman, S., Yeasmin, S., Inatsu, Y., Ananchaipattana, C. and Bari, M.

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Safe Water as the Key to Health L. (2013). Low-Cost Sustainable Technologies for the Production of Clean Drinking Waterâ&#x20AC;&#x201D;A Review. Journal of Environmental Protection 5, 42-53. Zeenath, F., Md. Harunur, R., Chowdhury, M. A. Z., Alam, Md.

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Bari K., Rahman, Md. A., Uddin, M. A. and Bari, M.L. (2015). Assessment of Heavy Metals, Minerals and Pesticides in different Branded Drinking Bottled Water of Bangladesh and their impact on human health. Online publication (25 Jan 2015).

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

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Short Communication The Kratom Plant [Mitragyna speciosa (Korth.)] Paradox: Beneficial or Detrimental? Low J.W.1, Leela A.*1 and Bhore S.J.2 1

Faculty of Medicine, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah, Malaysia. 2Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: drleelaa@yahoo.com, Ph No: +604-429 8063

The aim of this study was to assess the level of knowledge and practices of some selected/vulnerable residents from ‘Tanjung Dawai’ village (Kedah, Malaysia) about Kratom plant [Mitragyna speciosa (Korth.)]. Several drug addicts from ‘Tanjung Dawai’ village as well as from adjacent areas come to specific places of ‘Tanjung Dawai’ village on a regular basis to buy Kratom drinks and or products. We have visited this village to collect the first-hand primary information about traditional use of Kratom plant. We selected some residents from ‘Tanjung Dawai’ village as well as some individuals those have Kratom plant in their backyard and interviewed them. We found that some consume it intermittently or daily for strength and energy. They felt they could work better after consuming Kratom. Some subjects highlighted that small amounts of Kratom boost their strength and energy, while others claimed that Kratom is useful for lowering blood pressure, treating diarrhea, and as anxiolytic, antidiabetic. However, now Kratom usage is causing concern as it emerges as a potential drug of abuse. Although Kratom has been associated with drug abuse and as a dangerous drug in recent years; it possesses certain beneficial properties as claimed by the villagers. The systematic pharmacological study needs to be done to understand the beneficial properties of this plant. Hence, further research is required to explore the potential applications of this plant. Keywords: Drugs; Health; Kratom, Malaysia; Mitragyna speciose; Tanjung Dawai.

Kratom plant [Mitragyna speciosa (Korth.)] is indigenous to Thailand and Southeast Asia. This plant is also known as Biak or Ketum in Malaysia. It is a member of the Rubiaceae family, and grows in tropical and subtropical regions of Asia (Puff et al., 2005; Hassan et al., 2013). It is well distributed in Southeast Asia, especially Malaysia in general and the northern region bordering Thailand in particular (Hassan et al., 2013; Suwanlert et al., 1975).

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Kratom leaves (Figure 1) are known to produce complex stimulant and opioid-like analgesic effects (Adkins et al., 2011; Babu et al., 2008). In Malaysia, Kratom has been used to starve off fatigue by enhancing tolerance for hard work and to manage pain, diarrhea, and opioid withdrawal (Hassan et al., 2013). Recently, Kratom has become widely available in the Malaysia, as people are allowed to keep the plants for individual’s personal use. In Malaysia, commercial planting of this plant is banned 84

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Kratom Plant: Beneficial or Detrimental?

Low et al.

Figure 1: Morphological features of a leaf, inflorescence and fruit of Kratom plant [Mitragyna speciosa (Korth.)] collected at Tanjung Dawai, Kedah, Malaysia. A) Morphology of a leaf; B) a snap of an inflorescence, and C) morphology of an immature fruit. and its cultivation is considered illegal. Analyses of medical literature indicate that individuals in Malaysia, especially in the north peninsular Malaysia are increasingly using Kratom. Kratom contains pharmacologically active constituents, most notably mitragynine and 7-hydroxymitragynine. Kratom possesses dose-dependent pharmacological effects, with stimulant effects in lower doses and opiate-like effects in higher doses (Babu et al., 2008). Thus, the potential for youngstersâ&#x20AC;&#x2122; addiction and adverse health

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consequences are becoming an important issue for health authorities of Malaysia. There were studies performed to observe the effect of mitragynine several years ago (Grewal, 1932; Macko et al., 1981). The acute toxicities studies in rates include increasing blood pressure, hepatotoxicity, and nephrotoxicity (Harizal et al., 2010). The recent research findings of Kamal et al. (2012) suggest that in very high doses, Kratom did not cause death and or any significant toxicity. In humans, its adverse effects include dry mouth, changes in

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Kratom Plant: Beneficial or Detrimental? urination, nausea, vomiting, anorexia, weight loss, constipation, nystagmus, tremor and seizures (Babu et al., 2008; Boyer et al., 2008; Ulbricht et al., 2013; Nelsen et al., 2010; Trakulsrichai et al., 2013). Primary hypothyroidism (Sheleg and Collins, 2011) and intrahepatic cholestasis (Kapp et al., 2011) are also reported in several cases related to Kratom. For chronic toxicity, impairment of cognitive behavioral function is found in mice fed with mitragynine for a long period (Apryani et al., 2010). However, in a study of chronic Kratom administration showed that it does not significantly impair social functioning of users in a natural context in Malaysia (Singh et al., 2014). In humans, anorexia, weight loss, hyperpigmentation over skin and cheeks on face, and psychosis are also described in chronic abusers (Babu et al., 2008). Kratom is well known and consumed as a norm in the northern peninsular region of Malaysia. Based on prior reports which suggest that several drug addicts and some villagers from ‘Tanjung Dawai’ village as well as youngsters from adjacent areas come here [at ‘Tanjung Dawai’ village] on a regular basis for Kratom drinks and or products. We visited the village to collect primary information. We carefully selected some residents from ‘Tanjung Dawai’ village and also those grew them in their backyard and interviewed them. An interview with the village head was also carried out and comments were noted. The information we received from villagers suggest that the majority of Kratom users are adult males, mostly those are manual laborers. Addicts consume Kratom drinks to enhance their physical endurance, boost their energy, productivity, elicit euphoric effects, and to improve their sexual ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Low et al. performance. Some of them use it as a replacement for expensive drugs if they do not have the cash to buy drugs. From interviewed villagers, we gathered that Kratom is also used as a folk medicine by to treat several diseases or as an opioid alternative by chewing fresh leaves and brewing as tea or concoction. Some villagers claim that Kratom is useful for lowering the blood pressure, treating diarrhea, and as anxiolytic and antidiabetic. Previously, Kratom was not considered to be a drug of abuse, but as a supplement for better health; because, it is known to help in lowering the blood pressure, as an antidiarrheal, anxiolytic, antidiabetic and antinociceptive (Hassan et al., 2013; Suwanlert, 1975). Villagers’ strongly believe that Kratom is a powerful antidiarrheal property. An antidiarrheal property of plant was confirmed when there was an outbreak of food poisoning after a wedding reception, where majority of those who had consumed Kratom drink did not suffer from food poisoning while others who did not were affected. Currently, Kratom is causing concern as it appears as a potential drug of abuse (Hassan et al., 2013). It was noted by Boyer et al. (2008) and Vicknasingam et al. (2010) that Kratom is often used as opioid replacement to alleviate opioid withdrawal symptoms and it is an easily accessible and serves as an economical alternative to other opiodreplacement medications. Kratom is often used as a drug of abuse alone or in combination with other substances, has been shown to cause dependence and withdrawal symptoms following repeated consumption (Hassan et al., 2013; Babu et al., 2008; Vicknasingam et al., 2010; McWhirter and 86

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Kratom Plant: Beneficial or Detrimental? Morris, 2010; Saingam et al., 2013; Saingam et al., 2014; Singh et al., 2014). However, based on the animal studies, Kratom is only slightly toxic (Macko et al., 1981). In mice, repeated 7hydroxymitragynine administrations elicit tolerance, cross-tolerance to morphine, and naloxone-precipitated withdrawal symptoms (Matsumoto et al., 2005). Few poisoning cases related to Kratom consumption has been reported in the past (Kapp et al., 2011; Neerman et al., 2012; Tungtananuwat and Lawanprasert, 2010). However, people including youngsters are using it; hence, its (Kratom) usage is causing concern as it emerges as a potential drug of abuse. Most recently, Malaysian police have seized 2.8 tonne of Ketum powder worth MYR 235,000 which also indicates that Kratom (Ketum) is smuggled for the international market (Star, 2016). In a conclusion, although Kratom has been associated with drug abuse and as a dangerous drug in recent years, it possesses certain beneficial properties. Currently, we do not have enough scientific information to weigh the health benefits of this medicinally important plant. The systematic pharmacological study needs to be done in order to understand the beneficial properties of this plant. Hence, further research is required to explore the potential applications of this plant. REFERENCES Adkins, J.E., Boyer, E.W. and McCurdy, C.R. (2011). Mitragyna speciosa, a psychoactive tree from Southeast Asia with opioid activity. Curr Top Med Chem. 11(9), 1165-75.

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Low et al. Apryani, E., Hidayat, M.T., Moklas, M.A., Fakurazi, S. and Idayu, N.F. (2010). Effects of mitragynine from Mitragyna speciosa Korth leaves on working memory. J Ethnopharmacol. 129(3), 357–360. Babu, K.M., McCurdy, C.R. and Boyer, E.W. (2008). Opioid receptors and legal highs: Salvia divinorum and Kratom. Clin Toxicol (Phila) 46(2), 146–152. Boyer, E.W., Babu, K.M., Adkins, J.E., McCurdy, C.R. and Halpern, J.H. (2008). Self-treatment of opioid withdrawal using Kratom (Mitragynia speciosa korth). Addiction 103(6), 1048–1050. Grewal, K.S. (1932). Observations on the pharmacology of mitragynine. J Pharmacol Expr Ther. 46(3), 251– 271.

Harizal, S.N., Mansor, S.M., Hasnan, J., Tharakan, J.K. and Abdullah, J. (2010). Acute toxicity study of the standardized methanolic extract of Mitragyna speciosa Korth in rodent. J Ethnopharmacol. 131(2), 404–409. Hassan, Z., Muzaimi, M., Navaratnam, V., et al. (2013). From Kratom to mitragynine and its derivatives: physiological and behavioural effects related to use, abuse, and addiction. Neurosci Biobehav Rev. 37(2), 138–151. Kamal, M.S.A., Ghazali, A.R., Yahya, N.A., Wasiman, M.I. and Ismail Z. 87

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Kratom Plant: Beneficial or Detrimental? (2012). Acute toxicity study of standardized Mitragyna speciosa Korth aqueous extract in Sprague Dawley rats. J Plant Stud. 1(2), 120– 129. Kapp, F.G., Maurer, H.H., Auwärter, V., Winkelman, M. and HermannsClausen, M. (2011). Intrahepatic cholestasis following abuse of powdered Kratom (Mitragyna speciosa). J Med Toxicol. 7(3), 22731.

Low et al. exposure. J Med Toxicol. 6(4), 424– 426. Puff,

C., Chayamarit, K. and Chamchumroon, V. (2005). Rubiaceae of Thailand; a pictorial guide to indigeneous and cultivated genera Bangkok: Forest Herbarium, National Park, Wildlife and Plant Conservation Department.

Macko, E., Weisbach, J.A. and Douglas, B. (1981). Some observations on the pharmacology of mitragynine. Arch Int Pharmacodyn Ther. 145–161.

Saingam, D., Assanangkornchai, S., Geater, A.F. and Balthip, Q. (2013). Pattern and consequences of krathom (Mitragyna speciosa Korth.) use among male villagers in southern Thailand: a qualitative study. Int J Drug Policy. 24(4), 351–358.

Matsumoto, K., Horie, S., Takayama, H., Ishikawa, H., Aimi, N., Ponglux, D., Murayama, T. and Watanabe. K. (2005). Antinociception, tolerance and withdrawal symptoms induced by 7-hydroxymitragynine, an alkaloid from the Thai medicinal herb Mitragyna speciosa. Life Sci. 78(1), 2-7.

Saingam, D., Assanangkornchai, S., Geater, A.F. and Lerkiatbundit, S. (2014). Validation of Krathom (Mitragyna speciosa Korth.) Dependence Scale (KDS): a dependence screen for internationally emerging psychoactive substance. Subst Abus. 35(3), 276–283.

McWhirter, L. and Morris, S. (2010). A case report of inpatient detoxification after Kratom (Mitragyna speciosa) dependence. Eur Addict Res. 16(4), 229–231.

Sheleg, S.V. and Collins, G.B. (2011). A coincidence of addiction to “Kratom” and severe primary hypothyroidism. J Addict Med. 5(4):300–301.

Neerman, M.F., Frost, R.E. and Deking, J. (2012). A drug fatality involving Kratom. J Forensic Sci. 58(S1), S278-S279.

Singh, D., Müller, C.P. and Vicknasingam, B.K. (2014). Kratom (Mitragyna speciosa) dependence, withdrawal symptoms and craving in regular users. Drug Alcohol Depend. 139, 132–137.

Nelsen, J.L., Lapoint, J., Hodgman, M.J. and Aldous, K.M. (2010). Seizure and coma following Kratom (Mitragynina speciosa Korth) ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Star (Newspaper), March 11 2016: “Cops smash ketum plants”. 88

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Kratom Plant: Beneficial or Detrimental? Suwanlert S. (1975). A study of Kratom eaters in Thailand. Bull Narc. 27(3), 21–27. Trakulsrichai, S., Tongpo, A., Sriapha, C., et al. (2103). Kratom abuse in Ramathibodi Poison Center, Thailand: A five-year experience. J Psychoactive Drugs. 45(5), 404–408. Tungtananuwat, W. and Lawanprasert, S. (2010). Fatal 4x100: Homemade Kratom juice cocktail. J Health Res. 24(1), 43-7.

Low et al. Windsor, R.C. (2013). An evidencebased systematic review of Kratom (Mitragyna speciosa) by the Natural Standard Research Collaboration. J Diet Suppl. 10(2), 152–170. Vicknasingam, B., Narayanan, S., Beng, G.T. and Mansor, S.M. (2010). The informal use of ketum (Mitragyna speciosa) for opioid withdrawal in the northern states of peninsular Malaysia and implications for drug substitution therapy. Int J Drug Policy. 21(4), 283–288.

Ulbricht, C., Costa, D., Dao, J., Isaac, R., LeBlanc, Y.C., Rhoades, J.,

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Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

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Abstracts (Keynote and Plenary Talks) Moving the Regional Biotechnology and Bioeconomy Forward YBhg. Dato' Prof. Dr. Asma Binti Ismail* Director General, Department of Higher Education Ministry of Higher Education Malaysia; *corresponding author, e-mail: asma@mohe.gov.my, / asmainformm@yahoo.com, Ph No: +603 88706381.

Abstract: Asian countries are blessed with plenty of natural resources that can be used systematically in boosting region’s bioeconomy significantly. In line with the international agenda of the sustainable development, each Asian country can and should take initiatives to boost the bioeconomical growth. In fact, many Asian countries are aggressively taking steps to grab the opportunities to enhance their biotechnology sectors in order to boost the national GDP. By realizing the applications of bio-based innovative technologies and its economic benefits to the society, Malaysia has taken several initiatives including ‘Bioeconomy Transformation Programme (BTP)’. In fact, Malaysia is the 2nd in Asia and 1st in Southeast Asia to announce a bioeconomy initiative as platform for bioeconomy development. The Organisation for Economic Co-operation and Development (OECD) estimates clearly suggest that bioeconomy is going to contribute a global average of 2.7% to GDP by 2030. It appears that bioeconomy is the way forward to address regional and global sustainability challenges. In Asia and beyond, we need to focus on renewable resources (energy, chemical and material products), enhance agriculture-based industry (production of high-value end products using innovative technologies), use innovative healthcare products and services (cheaper and more accessible). The coherent and supportive policy framework will be the key for Asian countries for the development of their Bioeconomy. In my keynote address, I will highlight my perspectives on the opportunities and challenges in boosting bioeconomy in Asia region and beyond. Keywords: Asia; Bioeconomy; Biotechnology; Health Care; Renewable Resources.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Nanotechnology Oriented Biosensors and Biomedical Application Tamiya E.* Nanobio Engineering and Biosensor Lab. Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; *corresponding author, e-mail: tamiya@ap.eng.osaka-u.ac.jp

Abstract: Nanostructured metals have been studied for the localized surface plasmon resonance (LSPR) and electrochemical biosensors. Photonic plasmon spectra are caused by the refractive index variations that result from the binding of molecules to the metal nanostructures. There are optically detectable parameters in biophotonics and biosensor devices. We have studied several types of nanostructures, (1) gold-capped nanostructure connecting with the core of silica nanoparticle capped by deposited gold film, (2) golddeposited porous anodic alumina layer chip, (3) gold nanoparticles onto silicon oxide /silicon interferrometric multilayer and (4) nano-pillar structures by nanoimprinted polymer materials and sputtering a thin gold layer on them. The bio-sensing of these nanostructures have been examined by monitoring the biomolecular interactions in various flexible formats. Antibodyantigen and DNA hybridization reactions were performed to detect various biomarkers, with the detection limit of picogram levels. The multi array format was constructed by a core-shell structured nanoparticle layer, which provided 300â&#x2C6;&#x2019;1000 spots on the sensing surface. A microfluidic biochip based on PDMS was useful for real-time analysis, rapid detection. DNA amplification process, and monoclonal antibody production from hybridoma cell library can be monitored. Electrochemistry measurements connecting to LSPR chips were successfully exploited in a simultaneous detectable scheme. The binding of melittin to lipid membrane was measured using localized surface plasmon resonance, and the permeability of the lipid membrane was then assessed electrochemically as a function of melittin with the purpose of seeking a novel, sensitive detection system for peptide toxins. These nanoporous structures were transferred to the cyclo-olefin polymer film surface from the porous mold by a thermal nanoimprinting process. A plasmonic substrate was fabricated by sputtering a thin layer of gold onto this nanopillar polymer structure and the refractive index response in a variety of media was evaluated. Finally, the biosensing capacity of this novel plasmonic substrate was verified by analysis of human immunoglobulin With the advantages of mass production with consistent reproducibility stemming from the nanoimprint fabrication process, our goldcapped polymeric pillars are ready for the transition from academic interest into commercialization systems for practical use in diagnostic applications. Surface Enhanced Raman Scattering (SERS) was also discussed with gold and silver nanoparticles interacting with bio-molecules. Gold nanoparticles were successfully delivered into single cells. Spatiotemporal measurements of SERS fingerprints suggested the dynamic molecular interactions and transformations taking place at different locations with time in cardiomyocytes. Keywords: Antibody; Biosensors; DNA; Innovation; Nanotechnology; PCR.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Current Progress in Cholera Diagnostics Chan Yean Yean1 and Manickam Ravichandran*2 1

Department of Medical Microbiology and Parasitology School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kelantan, Malaysia. 2AIMST University, Bedong, Semeling, Kedah, Malaysia; *corresponding author, e-mail: ravichandran@aimst.edu.my, Ph. No.:+60 4 429 8103.

Abstract: Cholera is a diarrhoeal disease caused by a Vibrio cholerae. The diagnosis of this disease is by the conventional culture method and more recently by various PCR based molecular diagnostic tests. The main limitation of these tools is that it requires skilled personnel and its reagents have to be transported in cold storage. To overcome these problem, our research team have developed simple platform technologies i.e., a). thermostabilization of PCR reagents and b). biosensor method. A thermostabilized PCR kit was developed for the detection of V. cholerae. This kit detects all serogroups (O1, O139 and non O1, non O139) based on lolB gene and rfb gene, biotypes (classical and EI Tor) identification that is coded by tcpA gene, virulence genes namely ace, zot and ctxA genes and tetA antibiotic (tetracycline) resistant determinant. The kit can be performed by simply adding water and bacterial lysate to the kit. This kit does not require multiple pipetting steps and cold chain for the transport. The second series of platform technology developed by our research team is called biosensors. It is an enzyme-based amperometric electrochemical biosensor assay to detect PCR product on a screen-printed carbon electrode (SPCE). The methods we have developed are multiplex genosensor and magentosensor. The main advantage of these methods is that it can detect PCR product without utilizing agarose gel electrophoresis. Hence a combination of cold chain free formulation of molecular diagnostic test with field adaptable biosensor detection method will be very useful for the detection of cholera in resourcelimited settings. Keywords: Biosensors; Cholera; Diagnostics; DNA; PCR; Vibrio cholera.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Supercomputing in Biotechnology: Making Sense of Big Data Bent Petersen* Center for Biological Sequence Analysis, Institute of Systems Biology, Technical University of Denmark, Dk-2800 Kongens Lyngby, Denmark; *corresponding author, e-mail: bent@cbs.dtu.dk, Ph No: +45 20 84 74 92.

Abstract: Currently, genomic and metagenomic experiments are generating massive amounts of data. Due to modern day technological advances, it has become increasingly affordable to produce sequencing data, which becomes very cost effective even when compared to storage, management, and analytical expenses. Simultaneously, the importance of biotechnology towards the production of safer, healthier and a sustainable food source is constantly increasing. How can the abundance of sequencing data benefit a food product? In this talk, I will briefly touch upon two projects that exemplify this point of view. In the first project, genomics was the game changer towards establishing a modern fermentation process for carmine production â&#x20AC;&#x201C; a widely used natural red dye found in pastries, confections, cosmetics and a plethora of other products. In the other project, metagenomics played a key role towards the identification of new bacteria and yeast species that can be utilized as starter cultures in wine and silage production. Both projects faced the modern challenge of accumulating massive amounts of data where state-of-the-art supercomputers were crucial in order to deliver the anticipated results. I will also give a short introduction to other exciting projects where big data and supercomputers played a central role. Keywords: Big data; ; Biotechnology; Genomics; Metagenomics; Supercomputers.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Molecular Approaches to Fundamental Studies on Biomarkers and Development of Sustainable Rapid Nano-biodiagnostics to Enteric Diseases for Low Resources Settings Phua K. K* and Asma Ismail Enteric Diseases Research Cluster, Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia (USM), 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail: kkphua7@gmail.com

Abstract: The Enteric Diseases Research Cluster is a multi-disciplinary project that harness the expertise from various fields of fundamental research and applied sciences to embark on Translational Research to develop innovative diagnostics for enteric diseases in low resources settings. By means of computer modelling, molecular docking and X-ray crystallographic techniques, coupled with various technology platforms, including non-PCR and lateral flow technolgy, sustainable diagnostics that fulfill WHO’s ASSURED criteria were developed and evaluated. Talents from Engineering, Chemistry and Physics enabled indigeous materials and reagents, such as nano-gold particles, recombinant enzymes, nitrocellulose membrane and synthetic peptides to be manufactured, which enabled these diagnostics to be produced at competitive prices for developing countries. Partnerships forged with foreign research institutions, such as the National Synchrotron Radiation Research Center, Taiwan; Sanger Institute, United Kingdom; King Saud University, enable high-impact factor publications to be enable Universiti Sains Malaysia (USM) to remain relevant and referred. With a budget of RM4.8 million, and a total of 10 principle investigators; 58 publications in citation-indexed journals, 66 cumulative Impact Factor and 107 citations; a book on “Sustainable Diagnostics for Low Resource Areas”; 3 patent filings; 22 awards; over 70 conference presentations; and provided opportunities for 1 Post-doctoral trainee, 25 Masters and Doctoral postgraduate students. But perhaps our most significant achievement is in providing impactful solutions for the community and local government agencies to help reduce the burden of Enteric Fever in the state of Kelantan. Industrial engagement, with manufacturing companies, such as Reszon Diagnostics and Malaysian Biotechnology corporation, will help realise the RMK10 agenda, ie. to provide health and wealth for the nation. Keywords: Biomarkers; Diagnostics, DNA; Health; PCR; Protein.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Bio-Applications of Innovative Nano-materials T. Theivasanthi* International Research Center, Kalasalingam University, Krishnankoil– 626126, India; *corresponding author, e-mail: ttheivasanthi@gmail.com, Ph No: +91-4563-258084 / +919524818862, 9344643384.

Abstract: This lecture will deliver nanoscience & nanotechnology based research activities. Sensitive nanomaterials like graphene, graphite, carbon nanotubes and metal nanoparticles are used to prepare electrochemical biosensors. Graphene has exquisite sensitivity to environmental changes. This property, combined with other characteristics such as optical transparency and excellent electrical / electronic properties provides the bio/inorganic interface required for sensors. Recent, innovations such as “World’s first plants materials based superparamagnetic particles” – named “Santhi Particles” and superparamagnetic plants materials like turmeric (Curcuma longa) & coconut shell (Cocos nucifera) are useful in cancer hyperthermia which will be discussed. Superparamagnetism improves the accuracy of spintronic sensors because a small sensed field is sufficient to order the spins in a superparamagnetic material. Such improved and accurate sensors are useful in various biomedical applications. Vegetable powder (Abelmoschusesculentus) for diabetes, nanoparticles for treatment of cancer, diabetes, psoriasis and “World’s first plants materials Nanoparticles” (Andrographispaniculata) for EBOLA, DENGUE, HIV & H1N1 virus infections, nanostructured / smart materials for Bio-sensors (like Amaranthus), Surface Enhanced Bio-materials, Bio-nanomaterials, Bio-polymers will also be discussed. This work also deals with societal impacted innovative research works like agricultural products like nanofertilisers (produced from Jack fruit seeds) etc. Various Technologies / Advanced Materials like low cost / mass production of Graphene, polymers, Synthesis, characterizations of metal nanopowders, metal oxide nanopowders, polymer-metal nanocomposites and their novel biotechnological applications will be explored. Large surface area, high surface‐area‐ to‐volume ratio and compatibility with flexible substrates of these materials make them as unique candidate for various applications. Surface Plasmon Resonance (SPR) of nanometallic surfaces is the incoming light results in a collective oscillation of the electrons at the metal’s surface. It has many promising applications which can be exploited to transmit optical signals, to interact with bio-molecules. Influences of nanomaterials in applications like sensor, imaging and biomedicine will be discussed. In addition, “Research Motivation” lecture will be presented to motivate students/ researchers to concentrate more on / towards research. Keywords: Biosensors; EBOLA; H1N1; HIV; Innovation; Nanomaterial; Technology.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Aptasensors: Bench to Bedside and Beyond Subash C.B. Gopinath* Institute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis (UniMAP), 01000 Kangar, Perlis, Malaysia; *corresponding author, e-mail: subash@unimap.edu.my, Ph No: (+60) 125565881. Abstract: Aptamers are single-stranded nucleic acids, so-called ‘artificial antibodies’, identified from the randomized combinatorial library against the target by the process called ‘SELEX’ (Systematic Evolution of Ligands by EXponential enrichment). Target can have any sizes from small molecules to the whole cell, attests the versatility of aptamers to bind a wide range of targets. Aptamers showed properties that are analogous to antibodies with high specificity and affinity to their target molecules. Aptamers have several advantages over antibodies, such as they are easy to prepare, cheaper, have no batch variations, are easy to modify, stable and most importantly, non-immunogenic. Because of these positive characteristics, aptamers are incorporated in different fields, and most attractive in applications involving therapeutics and diagnoses (theranostics). Aptamer-mediated biosensors (Aptasensors) have been attested with high-performance with different interdisciplinary sciences, making roads from laboratory to Industry and bridging the gaps. With either aptamers alone or complementing with antibodies, several high sensitive, portable sensors have been demonstrated for use in ‘bedside analysis’. Moreover, aptamers are more amenable to chemical modifications, making them capable of utilization with the most developed sensors. The development of more sensitive aptasensors could be useful and important for medical diagnosis, identification of pathogens for the quality control of consumable items, and surveillance of emerging diseases. In fact, aptasensors have already shown efficacy in the detection of life threatening diseases caused by early stage of viral and bacterial infections. Further, success in aptasensor development was driven in most cases by bottom-up and top-down approaches with inter- and multi-disciplinary strategies. Commercialization evidences a complete platform for aptasensor research and development. Current researches are expanding towards high-throughput screening and drug discovery process for next-generation sensing, using the aptamer as the probe. In this talk, will discuss about progression in the development of aptasensors, as bench to bedside and beyond. Keywords: Aptamer; Aptasensor; Bedside analysis; Diagnosis; SELEX.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Recent Progress in the Production of Biodegradable Plastics from Palm Oil in Malaysia Sudesh K.* School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia; *corresponding author, e-mail: ksudesh@usm.my, Ph No: (+6)04-6534367

Abstract: Background: Palm oil is known as an essential vegetable oil commodity. It is a renewable resource which provides a wide array of applications. Studies have shown that palm oil is an efficient feedstock for the production of biodegradable plastics, namely polyhydroxyalkanoate (PHA) via microbial fermentation. PHA is accumulated as water insoluble storage polyester in the cell cytoplasm of bacteria. For the past three decades, PHA has been the subject of intense investigation due to its thermoplastic properties as well as being biodegradable and biocompatible.Therefore, PHA has a high potential in substituting some petrochemical plastics as a renewable and sustainable material. Bacterial strains of Cupriavidusnecator H16 are reported to be cultivated to more than 100 g/L dry cell weight containing up to 80 wt% bioplastics using palm oil or used cooking oil as the carbon source. However, successful commercialization of PHA is currently hindered by its high cost compared to existing polymers in the market. Recovery and purification process of PHA from bacterial cells are mainly responsible for costly production of PHA. Methods: A novel biological extraction method has been developed by feeding freeze-dried cells containing PHA granules to animal models. The resulting whitish fecal matter was collected and subjected to GC, GPC, DSC, TGA, rheology and protein analyses. Results: GC analysis revealed that the fecal pellets contained up to more than 90 wt% of PHA. Further increase to 95% PHA can be achieved by washing treatments with detergents. DSC, GPC and TGA analyses proved that the PHA granules recovered using this method had similar properties when compared to PHA extracted with chloroform. However, there was obvious differences in rheological properties between the PHA obtained from both methods. Biologically extracted PHA granules contained traces of proteins, which may be the reason for differences in rheological properties. Conclusion: This simple biological extraction method is scalable and can be incorporated into some existing animal production systems. Keywords: Biodegradable; Biological extraction; Palm oil; Polyhydroxyalkanoate.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Recent Advances in Biosensors Based on Enzyme Inhibition Aziz Amine* Faculty of Sciences and Techniques, Hassan II University of Casablanca, Morocco; *corresponding author, e-mail: azizamine@yahoo.fr

Abstract: Background: Biosensors based on enzyme inhibition represent a cost-effective device for fast screening of inhibitors. They could be used as alarm systems for environmental monitoring and as a complementary technique to traditional methods. In the present conference we would like to underpin the recent advances in biosensors based on enzyme inhibition field, focusing on: the investigation of a new theoretical approach in order to easily understand the type of inhibition and calculate the kinetic parameters; the evaluation of the performances of the biosensor in the case of reversible and irreversible inhibition, in terms of time of analysis, detection limit, matrix effect the use of nanomaterials; the development of biosensors based on enzyme inhibition embedded in labs on a chip; The applications of biosensors based on enzyme inhibition in real samples. Methods: In the case of reversible inhibition, the most frequent procedure is based on the measurement of enzyme activity in absence (I0) and in presence of inhibitor (I1). The decrease of enzymatic activity can be evaluated by measuring the degree of inhibition as follows: ((I0 â&#x20AC;&#x201C; I1)/I0) Ă&#x2014; 100. After inhibition, the biosensor is washed with buffer, and the enzyme activity is again evaluated. If the enzyme restores the initial activity, then the inhibition is reversible, otherwise the inhibition is irreversible. Furthermore, in the case of irrreversible inhibition, an incubation time is required. Results: Experimental results of enzymatic inhibition by pesticides aflatoxins, cyanide, sulfide, methylmercury, nerve agents, and various other drugs and toxins will be presented. These inhibitors were usually detected at levels of ppb. Conclusion: The diagnosis of type of inhibition greatly improves sensitivity by a judicious choice of the enzyme concentration and incubation time. The use of nanomaterials in the preparation of the inhibitive biosensors improves some analytical features including sensitivity and stability. Keywords: Analytical applications; Enzymatic biosensors; Inhibition; Nanomaterials.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Genomics of the Endangered Orang Asli: Disease Susceptibility and Sustainability Mohd Zaki Salleh* and LRGS Orang Asli team Integrative Pharmacogenomics Institute (iPROMISE , Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia; *corresponding author; e-mail: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258 4652

Abstract: Different groups of indigenous people had populated Southeast Asian regions via different waves of migration. As such, the indigenous people have been the subjects for studies (1) to track patterns of migration of modern human; (2) to understand the mechanisms of evolution and natural selection; and (3) evolution and mechanism of diseases. Despite the developmental programmes runned by the government, most of the Orang Asli in Malaysia are still plagued with negative pressures in both the socio economics and health aspects. Some of the sub-tribes of the Orang Asli such as the Kanaq, Che Wong and Lanoh are left with less than 500 surviving individuals. Factors such as capital domain and human resourse domain that may contribute to these observations are continuously being researched by many. To understand the problems better, we integrated the omics and anthropological approaches in a multipronged research in order tofind solutions to improve the situation.Orang Asli from different geographical areas in Peninsular were recruited. Medical examinations, biochemical and metabolomics analysis were conducted. The DNAs of the Orang Asli were sequenced using second generation technologies. Only reads with a Q score of 30 and above (>Q30) were included in the analysis.The variants were uncovered using the GATK Best Practices workflow for variant discovery. The genomics structures of the Orang Asli were successfully mapped. Genetic variants, both existing and novel ones were detected. Genetic variants that predispose them to higher risks towards diseases were determined. Correlations of the genomics risks with the biochemical profiles and metabotypes provide an in depth understanding on the disease susceptibilities of the Orang Asli. This study also provide a wealth of data on the whole genome sequences of the Orang Asli which can be continuously mined by researchers for advancement of knowledge. There is still much to be done with the metadata generated to protect and enhance the health and welfare of the Orang Asli communities in the country, especially those in the minority groups. The assistance provided should gear towards improving control over preventable diseases and physical conditions that cause the population to be endangered. Keywords: DNA; Genetics; Genomics; Medication; Orang asli; Sustainability.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Highly Sensitive Detection of DNA Hybridization and Immunoassay Based on Nanomaterials Werasak Surareungchai* King Mongkutâ&#x20AC;&#x2122;s University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand; *corresponding author, e-mail: werasak.sur@kmutt.ac.th

Abstract: High specific and sensitive detections can be achieved via labeling techniques in DNA hybridization and antibody-antigen interaction. Labels based on nanoscale materials open a new opportunity over the traditional methods - in terms of greater reporting signal per binding event. We have been able to lower the limit of detection of DNA hybridization and Ab-Ag binding from fM to aM, and fg, respectively. In addition, some possibilities on highthroughput simultaneous assays have been attempted and reported. The talk will describe some our approaches engineered either electrochemical or optical labels using nanomaterials such as carbon nanotubes, graphene, and metal nanoparticles. Last, the talk will also present some real foodborne pathogen applications. Keywords: Biodiagnotics; Biosensors; DNA; Immunoassay; Nanomaterial.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

100

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Fungal Secondary Metabolites - A Pharmaceutical Chemist Perspective J.-F. F. Weber* Microbial Metabolites Laboratory, Atta-ur-Rahman Institute for Natural Product Discovery, Aras 9, Bangunan FF3 Fakulti Farmasi, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail: jffweber@puncakalam.uitm.edu.my Abstract: Drug discovery (DD) by the “big pharmas” went down to a dangerous low level that could be explained by a general natural tendency of staying confined into one’s comfort zone. DD from natural products (NP) is and shall remain a primary source of inspiration for pharmaceutical chemists, even though the challenges are nowadays much greater than they used to be. DD is akin to a number game: how many compounds should a good library include and how fast can we get that library? Microorganisms are the main suppliers of new drug leads. Yet, DD from microbial NPs is hampered by two issues: the low available biomass and the variable phenotypic expression of biosynthetic genes. At Atta-ur-Rahman Institute for Natural Product Discovery, UiTM, we have developed a unique protocol that we named MECSUS (Microtiter plate, Elicitors in Combination, Solid phase extraction, UHPLC, Statistical analysis) aimed at addressing the above issues and amenable to automation. Some aspect of this protocol will be presented together with some results accumulated along the development path. Keywords: Bioactive Phytochemicals.

compounds;

Biodiagnotics;

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Biosynthesis;

Drugs;

Fungi;

101

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Foot-and-Mouth Disease: Current Scenario in Asia and Bangladesh M Anwar Hossain* Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh; *corresponding author, e-mail: hossaina@univdhaka.edu

Abstract: Background: Foot-and-mouth disease virus is a positive stand RNA virus (FMDV, family Picornaviridae, genus Aphthovirus) causes an acute vesicular disease of bovid wild and domesticated ruminants. Foot-and-mouth disease virus (FMDV) comprises of 7 antigenically distinct serotypes (Type O, A, Asia1, C and SAT1-3) that do not provide cross-protection against one another. FMD is a pandemic disease accounting for a global loss of 6.5-21 billion USD per annum. Methods: Categorizing, estimation and demography of FMD occurrence in Asia continent is analyzed. Comparative genomic and phylogeography of the FMDV in Asia is discussed. Results: Three serotypes of FMDV are circulating in Asian territory including mainland Southeast Asia, South Asia and Middle East with predominance of type O, whereas Serotype A and Asia1 are found to be confined in certain geographical region. Cattle is the most susceptible toward FMD, whereas Pig serves as mixing vessel that may boosts the emergence and re-emergence episode of several lineages/genotypes. Whole Genome and phylogeography analysis revealed that transboundary movement of FMDVs are responsible for spreading of this disease in Asian regions. In 2013-2015, Saudi Arabia experienced the emergence of Ind-2001 lineage under Middle East South Asia (ME-SA) topotype of FMDV type O and Genotype VII of FMDV type A which are normally endemic in the Indian subcontinent. Intrusion of type SAT1-3 in Arabian Peninsula occurs due to transboundary animal movement from FMDV enzootic African countries. Conclusion: Transboundary movement of FMDV, inappropriate vaccination and inadequate awareness are the main reason of FMD spread in most of the Asian Countries. Keywords: Asia; Bangaladesh; Foot-and-mouth disease; RNA; Serotypes; Virus.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

102

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Abstracts (Oral Presentations) Paper-based visual detection of Salmonella bacteria using Isothermal DNA amplification and magnetic beads aggregation Wei, S.X. Sharmili Roy S., Rahman, I.A., and Ahmed, M.U.* Biosensors and biotechnology laboratory, Chemical Science programme, Faculty of Science, Universiti Brunei Darussalam; *corresponding author, e-mail: minhaz.ahmed@ubd.edu.bn, Ph. No.: +673 888 4752

Abstract: Background: The aim of this study was to develop a sequence-specific and simple method for detection of Salmonella bacteria using isothermal loop mediated amplification (LAMP) and MB-mediated aggregation. Salmonella is a food-borne bacterial that can affect human and animals alike. According to World Health Organization (WHO), the disease, salmonellosis, affects tens of millions of people worldwide every year which can cause hundred thousand deaths. Following LAMP amplification, the magnetic beads (MBs) were added to the LAMP products. This produced aggregates that showed up as dark spots on paper surface. By contrast, when there was the absence of LAMP products, stable aggregation did not form. This method can detect bacteria DNA at picogram level in 25 th minutes. Methods: Salmonella bacteria was used here as a model organism.The magnetic beads were used as an alternative method for detection of DNA. WhatmanÂŽpapers were used as a sample carrier for the aggregation of magnetic beads with DNA. In the presence of a magnetic field, the MBs form aggregates (LAMP-MBs) with the long DNA strands produced by the LAMP process, and these aggregates are stable when the magnetic field is removed. By contrast, LAMP-MBs with short strands of DNA will lose their aggregated form and be resuspended in the solution when the magnetic field is removed.The aggregation of LAMPMBs complex was measured and analysed using ImageJ software. Isothermal amplicons were also tested concurrently with agarose gel electrophoresis after staining with DNA intercalators. Results: Using this method,1 pg/ÂľL of Salmonella DNA was detected on paper for the first time. On the other hand, this faster and sophisticated instrument free protocol allowed LAMP products detection starting from 20th min of amplification.The LAMP-MBs took around 5 minutes to detect on to the paper which is obviously efficient compared with other conventional methods. For example, our result shows better resolution compared to agarose gel electrophoresis. For the selectivity test, Listeria innocua, Staphyloccous aureus, Bacillus subtilis microorganisms were also tested and found no cross-reactivity with these organisms. Conclusion: It can be concluded that LAMP- MBs technique was a sensitive, specific, cost-effective and time efficient method. It only took a total of 25 minutes to obtain the result which can be detected visually. This technique could potentially be employed in health and environmental screening with the development of paper based microdevices. Keywords: Magnetic beads; Loop-mediated amplification (LAMP); Paper; Salmonella.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

103

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Development of a Reverse Hybridization Assay (RHA) for Simultaneous Identification of Salmonella Serotypes Causing Enteric Fever Carlos S., Huda H. A., Goay Y. X., Phua K. K.* Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia (USM), 11800 Penang, Malaysia; *corresponding author, e-mail: kkphua7@gmail.com

Abstract: Background: Enteric fever is a fatal systemic infection caused by four humanadapted pathogens, Salmonella Typhi and Salmonella Paratyphi A, B and C. Multiplex PCR has increased the molecular detection capacity for diagnosis of enteric fever as multiple DNA targets can be detected in a single test. This study reports the development of a reverse hybridization assay (RHA) for detection of the 4 Salmonella serotypes using multiplex PCR. Interpretation of RHA results was easy as the appearance of black dot(s) on the test strip, visualized using naked eyes and without the use of any carcinogenic chemicals or specialized equipment. Unlike other PCR-based diagnostic assays, this assay can differentiate several Salmonella serotypes more effectively in terms of cost and time. Methods: A multiplex PCR assay with biotinylated primers specific for S. Typhi, S. Paratyphi A, B and C, and a PanSalmonella gene (invA) as PCR amplification control was developed and optimized. The RHA strip was developed with a blue line to mark the orientation of the strip, and five DNA probes which capture specific biotinylated-PCR products for the 4 Salmonella serotypes and 1 Pan-Salmonella control. 125 strains of S. Typhi, S. Paratyphi A, B or C, and 42 Salmonella and non-Salmonella bacteria were tested using the RHA test. Results: The assay was found to be 100% specific (42/42) and 100% sensitive (125/125). Conclusion: A highly sensitive and specific RHA has been developed and is now ready for clinical field trials to ascertain its diagnostic sensitivity and specificity. Keywords: Enteric fever; Diagnosis; Multiplex PCR; Reverse hybridization assay; Salmonella.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

104

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Decrypting the Evolutionary Path of Antimicrobial Resistance of Acinetobacter baumannii via Next-Gen Sequencing Mohamad Izwan Ismail, Lee Lian Shien, Teh Lay Kek, and Mohd Zaki Salleh* Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM) Puncak Alam, Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail: zakisalleh.mzs@gmail.com, Ph No: +603-3258 4685

Abstract: Background: Ever since the conception of the first antimicrobial, the threat of resistance has become an ongoing problem. Despite the copious amounts of research on the matter, antimicrobial resistance has yet to be eradicated. Despite the discovery of the key mutations in pathogens in response to exposure to antimicrobials, the step-by-step process of this adaptation has yet to be clearly understood. Common nosocomial pathogens such as A. baumannii have been widely reported to develop such resistances at an alarming rate, adding pressure to the need to understand their adaptive evolution process. Here, we aim to generate four drug-resistant variants of a susceptible A. baumannii strain and track its mutation development against ciprofloxacin, erythromycin, meropenem and imipenem. Methods: A. baumannii was isolated from the blood of a septicemic patient hospitalized at a local hospital. After confirmation of antimicrobial susceptibility toward the four target drugs, the isolate was cultured and divided into four subcultures and subjected to daily exposure to ciprofloxacin, erythromycin, meropenem and imipenem, separately. The antimicrobial concentrations were steadily increased by two-folds until MIC-level, and high-level resistances were acquired. The whole-genome of each isogenic variant and the susceptible parent were then sequenced using the Illumina GAIIx sequencer. Variant analysis of the sequencing output was done using CLC Bio, and primers were designed using PerlPrimer. PCR, gel electrophoresis and amplicon sequencing were carried out on the selected samples in each timeline of the isogenic variants. Results: Each isogenic variant was confirmed to carry different combinations of mutations; AbRC (gyrA and yihG), AbRE (bvgS, srrA, ftsI and ribonuclease I), AbRM (epsL, mexB and atpD), and AbRI (ftsI and acrB). The following mutation timeline analysis revealed variations in early-, middle-, and late-stages of the resistance induction, leading to a final stable resistance. Furthermore, isogenic variant AbRI was identified to carry two mutations in a single ftsI gene, occurring at two varying time points throughout the resistance induction process. Conclusion: The chronology of mutations was suggested to be influenced by both the duration and concentration of antimicrobials that the A. baumannii is exposed to. As such, these two parameters need to be taken into account when tackling the issue of antimicrobial resistance. Keywords: Acinetobacter baumannii; Antimicrobial; Resistance; Sequencing; Timeline; Whole-genome. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

105

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Isoluminol-functionalized gold nanoparticles and graphene oxide nanoribbons composite for development of enzyme-based electrochemiluminescence biosensors Nur Syakimah Ismail1* and Eiichi Tamiya2 1

School of Microelectronic Engineering, Kampus Pauh Putra, Universiti Malaysia Perlis, 02600 Arau, Perlis, Malaysia; 2Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1Yamadaoka, Suita, 565-0871 Osaka, Japan; *corresponding author, e-mail: syakimah@unimap.edu.my

Abstract: Background: Electrochemiluminescence (ECL) has become an important detection method due to its simplicity, rapidity and high sensitivity. N-(aminobutyl) -N(ethylisoluminol) (ABEI) is luminol derivatives, which widely use luminophore to generate ECL emission. Enhanced ECL emission can be obtained by using gold nanoparticles (AuNPs) that facilitate the generation of oxidative radicals and fast electron transfer due to catalytic surface effects. Previously, we have synthesized AuNPs coated with ABEI (ABEIAuNP) that demonstrated ECL intensity enhancement by graphene oxide nanoribbon (GONR) as functional supporting matrix on disposable screen printed electrode (SPE). Herein, we utilized ABEI-AuNP-GONR/SPE in development of enzyme-based ECL biosensors. Methods: GONRs and ABEI-AuNPs were synthesized through the longitudinal unzipping of MWCNTs and seed growth method, respectively. SPE was used for all electrochemical measurements that contained a three-electrode system; working electrode, counter electrodes, and the Ag/AgCl reference electrode. GONRs (1 mg/mL) were mixed with as-prepared ABEI-AuNP in the ratio of 1:3 before casted on SPE working electrode and dried at room temperature overnight. All electrochemical measurements were performed with a USB-powered handheld potentiostat BDTminiSTAT100 while ECL intensities were recorded by Hamamtsu Photon Detection Unit. Results: Firstly, glucose oxidase enzymes catalyzed production of hydrogen peroxide that subsequently turns to superoxide radicals to promote ECL enhancement. Secondly, urease enzyme used to catalyze the decomposition of urea into ammonia, which consequently increases the solution pH. Therefore, ECL intensity increases proportionally with urea concentration. Conclusion: We have successfully developed ECL glucose and urea sensors by using ABEI-AuNP-GONR/SPE. They provide sensitive detection paving the way for future development of portable ECL biosensors. Keywords: Electrochemiluminescence; Gold nanoparticle; Graphene oxide nanoribbon; Luminol.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

106

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Disposable Screen-Printed Electrodes Modified With Nanoparticles for Sucrose Sensor Detpisuttitham W.a, Rijiravanich P.b,c*, and Surareungchai W.d a

Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150 Thailand; bBiochemical Engineering and Pilot Plant Research and Development Unit, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand; cNational Center for Genetic Engineering and Biotechnology, NSTDA, Khlong Luang, Pathum Thani 12120, Thailand; dSchool of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand; *corresponding author, e-mail: patsamon.rij@biotec.or.th, Ph No: +662-4707475.

Abstract: Background: The measurement of sucrose is important not only in industrial analysis but also in clinical analysis. Sucrose detection can be accomplished by detecting its oxidation at high pH, with no need for complex and time consuming sample preparation. However the oxidation products can gradually passivate the electrode surface, causing a loss of analyte signal. Methods: Here, the disposable screen-printed carbon electrodes modified with Au-SiO2 nanoparticles (Au-SiO2/SPEC) were developed for amplifying electrochemical signal in nonenzymatic electrochemical sucrose sensors. Each Au-SiO2 nanoparticle consists of an amine functionalized silica particle (SiO2), coated sequentially with polyelectrolyte and gold nanoparticles. After casting Au-SiO2 on the surface of the carbon working electrode, the electrochemical characterization of Au-SiO2/SPCE was investigated by voltammetry and amperometry. Results: Au-SiO2/SPEC Provide highly catalytic oxidation peak of sucrose at potential above 50mV in a NaOH electrolyte. Then the amount of Au-SiO2 casting, concentration of electrolyte, and potential were optimized to establish high and reliable responses for sucrose detection. The characterization of this sensor have also been described. Conclusion: Our disposable Au-SiO2/SPEC was successfully fabricated for sucrose sensor determination. This sucrose sensor can be directed towards applications in biofuel and food industries and clinical fields. Keywords: Electrochemical sucrose sensor; Gold nanoparticles (AuNPs); Screen printed carbon electrodes.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

107

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Analysis of Chalcone-Flavanone Isomerase (CHI) Gene cDNA Isolated from American oil-palm (Elaeis oleifera) Mesocarp Tissue cDNA Library Yu Chuen Leow1, Amelia Kassim2, Subhash J Bhore1, 2 * 1

Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong Semeling Road, Semeling 08100, Kedah, Malaysia. 2 Department of Molecular Biology, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia; *corresponding author, e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com

Abstract: Background: The nutritional quality of the American oil-palm (Elaeis oleifera) mesocarp oil is superior to that of African oil-palm (Elaeis guineensis Jacq. Tenera) mesocarp oil. Therefore, it is important to identify the genetic features for its superior value. This could be achieved through characterization of oil-palm genes. Hence, we constructed a cDNA library and generated expressed sequence tags (ESTs) from the mesocarp tissue of the American oil-palm. The primary analysis turned our attention to the Elaeis oleifera chalconeflavanone isomerase (EoCHI) enzyme encoding cDNA. This EoCHI is an important enzyme. Therefore, to understand more about it this study was undertaken. The objective of this study was to elucidate EoCHI gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. Methods: Positive and negative strands of the EoCHI cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. The EoCHI cDNA and deduced protein sequence was analysed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of EoCHI protein were predicted by using the PHYRE automatic fold recognition server. Results: The sequencing results analysis showed that EoCHI cDNA is 942 bp in length. Its open reading frame (ORF) encodes for a protein that contains 234 amino acids. The analysis results showed the presence of chalcone superfamily domain in the protein sequence. The multiple sequence alignment of selected CHI amino acid sequences from other plant species with E. oleifera CHI using ClustalW and its phylogenetic analysis suggest that CHI from Elaeis guineensis and Phoenix dactylifera are the most closely related with EoCHI. Conclusion: The annotation of EoCHI showed that 942 bp long EoCHI cDNA encodes for 234 amino acid long protein that contains conserved domains required for biological functions of CHI. The predicted deduced EoCHI protein's 3D structure is predicted. However, further study is required to validate the predicted structure. Keywords: African oil-palm; American oil-palm; Chalcone-flavanone isomerase; Edible oil; Palm oil

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

108

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Non-Protein coding RNA genes as novel diagnostic markers to detect pathogenic bacteria Suresh C.V.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, Bedong 08100, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph No: 044298000-8165

Abstract: Background: Small non protein-coding RNAs (npcRNAs) have emerged as the important regulators of many cellular pathways in bacteria. The specificity of the npcRNA varies from species to phylum level. High accuracy in the detection of specific pathogen is very important in clinical diagnosis. We used npcRNA genes as diagnostic markers for various pathogenic bacteria detection. Methods: We used various computational tools to identify specificity of the npcRNAgenes in Salmonella typhi, Vibrio cholerae, Shigella flexneria and Staphylococcus aureus. Using specific primers, we performed PCR to amplify the target npcRNA genes from aforementioned bacteria. Specificity of the target was checked using various Gram positive and Gram negative bacteria. Sensitivity was recorded with serially diluted genomicDNA and bacteria separately. Results: The results confirmed that the genes StyR-36, VrrA, CssrB and Sau-02 are very specific to Salmonella typhi, Vibrio cholerae, Shigella flexneri and Staphylococcus aureus respectively. The sensitivity limit of detection using genomic DNA of these bacteria attained was in the range of 30fg to 10pg. The sensitivity of detection using whole bacteria obtained was in the range of 7-15 CFU/ml. Conclusion: This investigation reveals that npcRNA genes serve as excellent molecular biomarkers for the effective diagnosis of bacterial infections. The results of the present study support the use of npcRNA genes in other molecular-based diagnostic methods, such as nucleic acid sequence-based amplification (NASBA). Also, npcRNAs are acquiescent for use in real-time detection of live bacterial species via RT-qPCR diagnostics. Keywords: Pathogenic bacteria; Diagnostic marker; Non-protein coding RNA; Specificity and sensitivity.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

109

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Herbal Based Stabilizers of Native and Misfolded State of Nuclear Corepressor (N-CoR) Matiullah Khan1*, Thunga Pandurangan2, Sridevi Visvanathan3, Sam Annie Jeyachristy3, Mahes Maheswaram4 1

Department of Pathology, 2Surgery and Biochemistry3, Faculty of Medicine: Department of Biotechnology4, Faculty of Applied Sciences, AIMST University, Semeling, Bedong 08100, Kedah, Malaysia; *corresponding author: Matiullah Khan, e-mail: khanmmd@gmail.com, Ph No: +60125855284

Abstract: Background: Nuclear receptor co-repressor (N-CoR) is a generic co-repressor essential for the suppression of cellular self-renewal potentials during cell maturation. Finding from our lab suggest that heterogeneous oncogenic insults can induce nearly homogenous change in N-CoR conformation through phosphorylation, which ultimately leads to its misfolding and premature loss in wide variety of human cancers. The misfolded N-CoR acquires pleotropic properties due to the highly transitional nature of its misfolded state and triggers malignant growth and transformation through a variety of unique "loss” and “gain” of function mechanisms. The aim of this project was to investigate the role of genistein and curcumin, two pleotropic medicinal compounds linked to the regulation of protein misfolding pathway, on the conformation and function of native and misfolded NCoR protein as well as fate of tumour cells harbouring the misfolded N-CoR protein. Methods: We analysed the fate of tumour cells along with the conformational dynamics of native and misfolded N-CoR protein in tumour cells treated with genistein and curcumin, the herbal based stabilizers of native and misfolded state of N-CoR protein. Results: Genistein promoted differentiation of tumour cells through the stabilization of native N-CoR state while curcumin promoted apoptosis by stabilizing the misfolded state of N-CoR. Conclusion: Our finding illustrate how small molecule mediated stabilization of native and misfolded N-CoR state could be exploited for the design and development of highly selective anti-cancer therapeutic approach. Keywords: Curcumin; Genistein; Misfolded protein; N-CoR.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

110

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Hepatoprotective effect of methanol extract of Polygonum minus leaves in carbon tetrachloride-induced liver damage in rats Manoontri T, Joe L.S, Tanusha S. B. M, Parasuraman S, *Christapher P.V Faculty of Pharmacy, AIMST University, Semeling 08100, Malaysia; *corresponding author, e-mail: christapher@aimst.edu.my, Ph No: +60 44298000 (extn: 1029)

Abstract: Background: Hepatotoxicity, damage to liver cells caused by various chemicals and drugs, is a serious health risk. Treatment options are inadequate and novel cost effective and low toxic drugs are need of the hour. Polygonum minus (Family: Polygonaceae) is a commonly available food additive plant in Malaysia. Different parts of this plant are used in the Malay folk medicine; to relive pain, as health supplement and to get rid of dandruff. Pharmacological studies and clinical trials have reported its various effects including antioxidant, anti-inflammatory, analgesic, antiulcer, sexual well-being and cognitive improvement functions. The present study evaluates the hepatoprotective potential of methanol extract of leaves of P. minus on chemical-induced liver injury. Method: P. minus leaves were shade dried, powdered, and macerated in methanol for 5 days, filtered and evaporated to obtain dry methanol extract (MEPM). The standard drug and MEPM treated groups of animals were administered with sylimarin (50 mg/kg) or MEPM (100 mg/kg or 200 mg/kg) for 14 days. All the animals were administered carbon tetrachloride (1:1 v/v in olive oil, 2 ml/kg, intraperitoneally; on day 6, 9 and 12) except normal control group to induce liver toxicity. Body weight, biochemical parameters and histopathology studies were conducted. Results: Body weight of animals did not show significant variation in any treated group, compared with normal control, but was markedly decreased in carbon tetrachloride control group. The biochemical analysis showed that the higher dose of MEPM exhibited significant hepatoprotective activity. The histopathology study also confirmed the hepatoprotective effect. Conclusion: Methanol extract of P. minus possesses significant hepatoprotective activity and the activity is increased with dose. More detailed studies are required to establish its hepatoprotective efficiency. Keywords: Carbon tetrachloride (CCl4); Hepatoprotective effect; Polygonum minus; Sylimarin.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

111

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail, Pomacea maculata Guruswamy Prabhakaran1*, Thambirajah, J.J2, Subhash Janardhan Bhore1 and Manikam Ravichandran1 1

Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling Campus, 08100 Bedong, Kedah Darul Aman, Malaysia. 2 Faculty of Business Management, AIMST University, Semeling Campus, 08100 Bedong, Kedah Darul Aman, Malaysia; *corresponding author, e-mail: prabhakaran@aimst.edu.my

Abstract: Background: The Golden Apple Snail (GAS), Pomacea maculata, is a major rice pest in Southeast Asia. The cost of synthetic molluscicides, their toxicity to non-target organisms and buildup of snail resistance have given new impetus to study on plant molluscicides. Most research efforts have focused on individual plant extract for molluscicidal properties, and are not proved entirely effective. Selective consortium of potent molluscicidal compounds from various plants might be an effective alternative. Six different plants extracts viz; Nerium indicum, Azadirachta indica, Nicotiana tabacum, Piper nigrum, Pongamia pinnata and Zingiber officinale were evaluated individually as well as in selective combinations on golden apple snails’ mortality in laboratory conditions. Methods: The dried and coarse plant material (100 g) was macerated in 1000 ml of 95% ethanol at room temperature (25± 2°C) for 3 days. The filtrate was concentrated to dryness with a rotary evaporator at 60° C. Individual extracts were then combined at 1:1 to 1:1:1 ratio by w/w and diluted to different concentrations with distilled water. Groups of 10 juvenile snails were placed in plastic containers with 1000 ml of plant extract suspension and set in triplicate at room temperature. The number of dead snails was recorded after 24 hours, 48 hours and after 72 hours. Results: Combining extracts of two or three plant extracts of Nerium indicum, Nicotiana tabacum, Piper nigrum and Azadirachta indica cause 73 to 96 % mortality of GAS. The poly extracts were more effective at Lethal Concentration (LC90) of 177 to 191 mg/l on GAS in comparison to individual extracts. The synergistic effect of the combined extracts resulted in around 45 % reduction in LC90 values of the individual extracts. Conclusion: This study demonstrated that combined crude extracts of Nerium indicum, Nicotiana tabacum, Piper nigrum and Azadirachta indica are effective for sustainable control of GAS. Keywords: Azadirachta indica; Combined plant extracts; Golden apple snail; Nerium indicum, Nicotiana tabacum; Piper nigrum; Plant molluscicides; Pomacea maculate.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

112

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Design and Characterization in Time of an On-off DNA Biosensor Guajardo-Yévenes C. F.1,4*, Issaragkul P.2,4, Sae-Tang C.2,4 and Surareungchai W.3,4 1

Pilot Plant, Development and Training Institute 2 Department of Chemistry, Faculty of Science 3 School of Bioresources and Technology 4 King Mongkut's University of Technology Thonbury, Bangkok, Thailand; *corresponding author, e-mail: cristian.gua@kmutt.ac.th, Ph. No: (+66) 2470 7562

Abstract: Background: Naked-eye detection and detection time are important characteristics for biosensors. Also their ease-of-use is improved if the output is clearly “off” in absence and clearly “on” in presence of analyte. In this research an on-off DNA biosensor was designed and characterized in time. This biosensor produces a fixed readable output, for small or large concentrations of analyte, by means of a catalytic amplification mechanism. Methods: The catalytic amplification was based on DNA strand displacement and toehold exchange (seesaw gate motif). Here an analyte strand displaces the output from a doublestranded gate:output complex, later a fuel strand displaces and releases the analyte from the gate:analyte complex formed previously, completing a catalytic cycle. Simulations were performed to get approximate detection times. Finally, preliminary experiments were conducted using fluorescent reporter probes, in order to measure detection times. Results: Simulations indicate that detection times are in the order of hours for low nM range of analyte, whereas they can go down to minutes for hundreds of nM. In both cases the output signal reached the same readable level. Likewise, experiments indicate that detection times decrease with increasing concentration of analyte, however it is not as evident as in the simulations. The fluorescent output reached only similar readable levels for different concentrations of analyte. Conclusion: Characterization through simulations and experiments show that readable outputs can be obtained, even when analyte concentration decreases. Detection times are also reduced as analyte concentration increases, however there is clear difference between detection times obtained from simulations and experiments. Keywords: Chemical reaction network; DNA Biosensor; DNA strand displacement; Toehold exchange.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

113

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Optimization of PCR for Rapid Detection of CTX-M Gene in ESBL Producing Klebsiella pneumoniae Clinical Isolates Rasheeda B1*, Neelam Z1, Imran A2, Shagufta N1 1 2

Department of Biotechnology, Lahore College for Women University Lahore, Pakistan. Quality Operational Laboratory, University of Veterinary and Animal Sciences Lahore, Pakistan; *corresponding author, e-mail: rashidasbs@yahoo.com, Ph No: 099203024680187

Abstract: Background: The emergence of drug resistance bacteria are the burning issue of modern medical science associated with the infectious diseases. The detection of antibacterial resistant genes is important for successful treatment against infectious disease by using the effective antibiotics. Among many methods the PCR is most precise and acceptable procedure for the detection of different antibacterial genes. This study was designed to check the antibiotic resistance pattern and frequency of CTX-M in Extended spectrum beta lactamase producing Klebsiella pneumoniae clinical isolates from different region of Punjab, Pakistan. Methods: Two hundred isolates of Klebsiella pneumoniae isolates were obtained from different clinical samples. Blood and Mac-Conkey Agar were used to isolate and identify bacterial microorganisms while Muller Hinton Agar was used to evaluate the antimicrobial susceptibility against different antibiotics as per CLSI 2012 guidelines. ESBL producing bacteria were screened by Double Disk Synergy and Combination Disk test. For the molecular detection of the resistant gene (CTX-M) PCR was performed. Results: 49% of the total isolates showed beta lactamase activity and resistant to multiple antibiotic including Cephalosporin, Aztreonam, Sulphamethoxazole/Trimethoprim, Ciprofloxan, Doxycyclin. Imipenam and Amikacin were observed to be least resistant in ESBL producing isolates as 13% and 12% respectively. CTX-M gene was detected in 94% of ESBL isolates. Conclusions: Based on the finding of this study it is suggested that prevalence of CTX-M gene (95%) is very high among ESBL producing isolates. Therefore PCR based method may help clinicians for rapid detection and treatment of patients by choosing right medication against the resistant bacteria as early as possible. Keywords: Antibiotic resistance; CTX-M; ESBL bacteria; Klebsiella pneumonia.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

114

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Umami Tasting Detection Based Electrochemical Sensor Promphan R.a, Chaiboon T.b, Lertanantawong B.b,* and Surareungchai W.c a

Department of Chemical Engineering, King Mongkut's University of Technology Thonburi, ThungKhru, Bangkok 10140, Thailand. b Pilot Plant Development and Training Institute,King Mongkut's University of Technology Thonburi, Bang KhunThian, Bangkok 10150, Thailand. c School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang KhunThian, Bangkok 10150, Thailand; *corresponding author, e-mail: benchaporn.ler@kmutt.ac.th, Ph No: +662-4707475

Abstract: Background: Umami taste is elicited from small amino acid, mainly glutamic acid. Glutamic acid can be found in a variety of natural materials such as plant, meat and vegetable root. Umami is the fifth flavor which has a characteristic taste and as a stimulant the appetite. Methods: In this work, we developed an electrochemical glutamate sensorbased enzymatic assay. Glutamate Dehydrogenase (GLDH) is aspecific receptor for glutamic acid sample. The enzymatic reaction of GLDH and glutamic acid in the presence of NAD+at carbon screen printed electrode (CSPE) produces the product and NADH.NADH was undergo oxidation at the CSPE and the current was detected by differential pulse voltammetric technique (DPV). Results: The optimal amount of enzyme and NADH suitable for the measurement of glutamate are at 50 units of the enzyme GLDH with 6 mM NAD+ in 0.1 M phosphate buffer in the presence of 0.1 M potassium chloride, respectively. The oxidation peak potential of NADH was found at 0.50 â&#x20AC;&#x201C; 0.66 V vs.Ag/AgCl. The linearity of this umami sensor is in the range of 0.1 mM to 1000mM and the limit of detection is at 0.61mM. Conclusion: We have reported a method to detect umami by combining an electrochemical technique with enzymatic assay. Our sensors are simple with high specificity and sensitivity so we believe that the glutamic acid sensor may be a good alternative for testing umami in food sample. Keywords: Electrochemistry; Enzyme biosensors; Glutamate dehydrogenase; Umami

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

115

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Detection of Salmonella enterica serovar Typhi Form Water Samples and Its Association with Geographical Clustering of Enteric Fever Ahmad Filza Ismail2, Mohd Irwan Maarof2, Salwani Mohd Harish1, Phua Kia Kien1, Fauziah Mohd Nor3, Hani Mat Hussin4, Ismail Aziah1* 1

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia; 2Department of Community Medicine, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; 3 Communicable Disease Control Unit, Kelantan State Health Department, 15200 Kota Bharu, Kelantan Malaysia; 4Public Health Laboratory, 16450 Kota Bharu Kelantan, Malaysia; *corresponding author, e-mail: aziahismail@usm.my, Ph No: +60 9 767 2426

Abstract: Background: Typhoid fever infection caused by Salmonella enterica serovar Typhi remains as a significant cause of morbidity and mortality of children and adults in underdeveloped and developing countries. The aim of the study is to detect the presence of S. Typhi in water samples and the spatial distribution of enteric fever cases and its water source positivity. Methods: A cross sectional study was conducted among all positive enteric fever cases from January 2012 to December 2013 in five districts (Pasir Puteh, Bachok, Kota Bharu, Pasir Mas and Tumpat) in Kelantan. The isolation of S. Typhi and polymerase chain reaction were carried out to detect the bacteria in water as well as stool samples of food handlers. Genotyping was carried out using PFGE and genome sequencing from two S. Typhi were performed from the isolates from human and water samples. All data of coordinate of cases and positive water samples were analyzed using ArcGISÂŽ 10.2 and ArcMapÂŽ 10.2 GIS software. Results: The data showed 72 confirmed enteric fever cases in five districts in Kelantan from January 2012 to December 2013. Eight positive water samples were detected by culture method and PCR. The point pattern analysis showed that the distribution of enteric fever cases was clustered with the NNI value less than 1 (NNI = 0.59; z score = - 6.61: CI = 99%) whereas for positive water sample, it was random with NNI more than 1 (NNI = 1.042; z score = 0.228). Six of the cases (8.3%) lived within 500 meters, 19 (26.4%) within 1000 meters and 59 (81.9%) within 3000 meter from the river. All eight (100%) of positive water sample sources were within 3000 meter from river. There was one positive water source that has 17 (23.6%) of positive enteric fever cases lived within 500 meters around it, 17 (23.6%) within 1000 meters and 23 (31.9%) within 3000 meters. Genotyping of S. Typhi of showed similar pattern when analyzed using PFGE. Next generation sequencing showed both S. Typhi are highly homology. Conclusion: Enteric fever in five districts in Kelantan was distributed in cluster form. Majority of the cases stayed near the river and near the source of positive water sample area. It is suggested that there is a correlation between positivity of water samples and typhoid cases. Keywords: Geospatial analysis; S. enterica subsp. enterica ser. Typhi; Typhoid.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

116

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

The Fabrication of Membrane-Based Pneumatic Microvalves in Microfluidic System Suppaso L.1, Boonpakdee D.1, Ngamchana S.2*, Guajardo-Yévenes C. F.3 and Surareungchai W.4 1

Department of physics, Faculty of Science, KMUTT, Bangkok 10150 Thailand. Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology at King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok 10150 Thailand. 3 Pilot Plant Development and Training Institute, KMUTT, Bangkok 10150 Thailand. 4 School of Bioresources and Technology, KMUTT, Bangkok 10150 Thailand; *corresponding author, e-mail: sirimarn.nga@kmutt.ac.th, Ph No: (+66) 24707561

Abstract: Background: Membrane-based pneumatic microvalves have been fabricated to control the flow of a solution in microfluidic channels. Methods: Two PDMS layers on glass substrate were fabricated by soft lithography technique. The top layer of PDMS was designed to bear the flow channels for a solution. The bottom layer corresponded to pneumatic channels for microvalve control. The PDMS membrane was placed in between these two layers to form the microvalves. The air pressure, which was generated from solenoid valves, has been controlled by LABVIEW in order to close and open microvalves automatically. To get accurate thickness of channel and membrane, the optimization of spin-coater speed was performed for a positive photoresist (AZP4620), a negative photoresist (SU8-50) and PDMS coatings on silicon substrate. Results: After the microfluidic channel with membrane-based pneumatic microvalves was fabricated, the flow control of solutions has been demonstrated in an automated and programmable fashion. The thickness profile for speeds within the range of 1000 – 4000 rpm was obtained for AZP4620 and SU8-50, and ranged between at 16.67 – 6.25 µm and 172.42 – 31.08 µm respectively. The thickness profile of PDMS for speeds within the range of 500 – 6000 rpm, and different curing temperatures 60, 80 and 100 °C, was obtained in order to fabricate films with thickness in the micrometer range. Conclusion: The fabrication of this prototype of membrane-based pneumatic microvalves and their automatic control by software programming could be demonstrated. This will be applied for sensing and microfluidic manipulation in the future. Keywords: Microvalve; Microfluidics; Membrane-based pneumatic microvalves; PDMS

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

117

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Role of Outermember Proteins (OMP) and Lipopolysaccharides (LPS) in Antibody Response Against Pasteurella multocida type B-2 in Bovines Imran A1*, Anika K2, Jawad N2, Waseem S3, Rasheeda B4 1

Quality Operational Laboratory (QOL), University of Veterinary and Animal Sciences (UVAS) Lahore Pakistan. 2Department of Microbiology, University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan. 3Institute of Biochemistry and Biotechnology (IBBT), University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan. 4Department of Biotechnology, Lahore College for Women University (LCWU) Lahore, Pakistan; *corresponding author, e-mail: imran.altaf@uvas.edu.pk, Ph No: 0092-3074464628

Abstract: Background: The activation of cellular and humoral immunity depend upon nature of antigens. Complex proteins like bacterial outer membrane proteins (OMP) usually successfully activate both wings whereas antigens like bacterial lipopolysaccharides (LPS) usually elicit T-independent immunityi.e. homural immunity without the activation of cellular immune wing. Hemorrhagic septicemia is highly contagious bacterial disease of bovine cause by gram negative bacteria Pasteurella multocida (PM). Both LPS and OMP play important role in the pathogenic potential of PM. The present study was under taken to evaluate the comparative immunologic behavior of both the important molecules of pasteurella multocida alone and in combination in bovine calves in field conditions. Methods: Pasteurella multocida was isolated, purified and identified from an outbreak by mean of culture and biochemical methods. The pathogenicity of the confirmed isolates was done in rabbits on the principles of Kochâ&#x20AC;&#x2122;spostulates. For vaccine preparation dry mass was estimated by filter method and vaccine was alum gel precipitated Complement fixation test (CFT) was used for the antibody against Outer membrane protein (OMP) and LPS separately. Results: The results showed that the antibody titer against OMP and LPS in whole culture vaccine is significantly higher than the respective tested vaccines.These results concluded that OMP no doubt is an active T-dependent immunogenic molecule but it immunogensity increases many times when combined with LPS in whole culture vaccine. Conclusion: Lipopolysaccharides (LPS) in combination with outer membrane proteins (OMP) synergistically boost up the humoral immune response in vaccinated animal. Keywords: Complement fixation test (CFT); Lipopolysaccharides (LPS); Outer membrane proteins (OMP); Pasteurella multocida (PM).

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

118

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Pro-oxidant Properties Monowar T.1,2, *, Md. Sayedur Rahman2, Bhore S. J.2 1

Unit of Microbiology, Faculty of Medicine, AIMST University, 08100 Bedong, Kedah Darul Aman, Malaysia. 2Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah Darul Aman, Malaysia; *corresponding author, e-mail: tahminamonowar@aimst.edu.my, Ph No.: +60−4−4293006.

Abstract: Background: Endophytes are known to produce various secondary bioactive compounds which may be used therapeutically as antimicrobial, antiviral, anticancer, antioxidants, antidiabetic, and immunosuppressant agents. The objective of this study was to evaluate antioxidant properties of five novel endophytic bacterial strains which were isolated in our laboratory. Methods: Five endophytic bacterial isolates namely, Acinetobacter baumannii, Bacillus subtilis, Enterobacter hormaechei, Klebsiella pneumoniae and Pantoea ananatis were cultivated in nutrient broth separately at 37°C, 180 rpm for 24 hours. Crude extracts were prepared from the broth using three different solvents such as chloroform, diethyl ether and ethyl acetate. Total antioxidant capacity (TAC), total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging activity and metal chelating assay were performed in triplicates. Results: TAC, TPC and TFC were found in the range of 175.71±7.05 to 761.32±6.01 g Ascorbic Acid Equivalent/mg extract, 254.44±5.36 to 1451.67±27.54 g Gallic Acid Equivalent/mg extract, and 12.22±9.62 to 615.00±30.05 g Rutin Equivalent/mg extract, respectively. Crude extracts of A. baumannii, B. subtilis and E. hormaechei exhibited pro-oxidant properties at lower concentrations while those of the remaining two strains showed antioxidant properties to some extent. Crude extracts of B. subtilis and P. ananatis were found as good metal chelators. Conclusion: Crude extracts of K. pneumoniae and P. ananatis showed antioxidant properties. But, crude extracts of A. baumannii, B. subtilis and E. hormaechei exhibited prooxidant properties at lower concentrations. Further studies in this respect are highly warranted to explore their pharmacological activities based on their antioxidant and prooxidant properties. Keywords: Antioxidants; Endophytic bacteria; DPPH; Metal chelating assay; Pro-oxidants; Total antioxidant capacity.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

119

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Sensitivity Analysis of Graphene Based Surface Plasmon Resonance Biosensor Toloue H.1,2*, Centeno A.1, Tamiya E.2, Kuwano N.1 1

Malaysia-Japan International Institute of Technology (MJIIT), University Teknologi Malaysia, Kuala Lumpur, Malaysia; 2Department of Applied Physics, Graduate school of Engineering, Osaka University, Osaka, Japan; *corresponding author, e-mail: h.toloue@gmail.com, Ph No: (+60) 173002072.

Abstract: Background: In a conventional surface plasmon resonance sensor a thin metal layer is sandwiched between two dielectrics. Noble metals such as gold (Au) or silver (Ag) are used as the metallic films since they lead to SPR at visible light frequencies. The use of both Au and Ag in a biosensor is limited because of their weak biomolecule adsorption, restricting the sensitivity of the sensor. Functionalization of metal film with biomolecular recognition elements (BRE) is a way to improve sensitivity for surface plasmon resonance biosensor. Methods: In this paper, the effect of variation in thickness of graphene layer as a BRE on reflection curve and sensitivity of surface plasmon resonance biosensor is numerically presented. The method is based on transfer matrix for N-layer Fresnel equation in Kretschmann configuration. The electromagnetic field in SPR condition analyzed using COMSOL Multiphysics. Results: The change in the minimum reflection in regard to the number of extra graphene layer on varied metal films is demonstrated. The result illustrated by coating a graphene layer on metal surface, the sensitivity improved as compared to that of conventional sensor. This is due to a better adsorption efficiency of graphene and greater change in refractive index of sensing medium near the sensor surface after biomolecule adsorption. Conclusion: The model suggested that graphene is a promising material as a BRE to increase total sensitivity with the number of graphene layers used. Keywords: Biosensor; COMSOL; Graphene; SPR.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

120

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Ameliorative Effect of Curcumin on Olanzapine Induced Obesity in Sprague Dawley Rats Parasuraman. S.*, Khor. M. Z., Ee Wen. L. Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Malaysia; *corresponding author, e-mail: parasuraman@aimst.edu.my, Ph No: (+60) 103895096.

Abstract: Background: Antipsychotic drugs are very much essential in the treatment and management of various mental illnesses such as schizophrenia and other psychoses. Although they have many beneficial effects, they are also not devoid of serious side effects. The management of antipsychotics (olanzapine) induced weight gain has very limited options. The effect of natural food antioxidant on weight gain is known, but the effect of the same on drug induced weight gain remains unclear. Hence the present study was planned to evaluate the effect of curcumin on olanzapine induced obesity in rats. Methods: Sprague-Dawley (SD) rats were used for experiments. The animals were divided into six different groups viz., normal control, olanzapine control, betahistine (10 mg/kg), curcumin 50, 100 and 200 mg/kg treated groups. Except the normal control group, all other animals were administered with olanzapine 4 mg/kg intraperitoneally to induce obesity. The drugs were administered once daily, per oral for 28 days. During the experiment, body weight changes and behavior alterations were monitored at regular intervals. At the end of the experiment, blood sample were collected from all the experimental animals for biochemical analysis. Part of the liver and kidney tissues were excised from the sacrificed animals and preserved in neutral formalin for histopathological studies. Results: Curcumin showed a significant reduction in olanzapine induced body weight gain on the rats and improved the locomotor effects. The effect of curcumin on olanzapine induced body weight gain is not comparable with that of betahistine. Olanzapine treated animals showed significant increase in levels of AST, creatinine, TC, TG, LDL, HDL, VLDL and AD levels compared with control group, whereas the animals treated with curcumin 200 mg/kg prevented the olanzapineâ&#x20AC;&#x2122;s induced changes in biochemical parameters. In histopathological analysis, olanzapine treated animals showed mild degeneration of hepatocytes in liver and moderate to severe tubular cell degeneration in kidneys, whereas curcumin 200 mg/kg prevented the olanzapine inducted tubular cell degeneration in kidneys. Conclusion: This study has shown metabolic alteration effect of curcumin on olanzapine treated SD rats. Keywords: Betahistine; Curcumin; Obesity; Olanzapine.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

121

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of Sudan I contamination in chili Phanthong C.1 and Khownarumit P.2* 1

Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology at King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok 10150, Thailand; e-mail: chatuporn.pha@biotec.or.th; 2Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand; *corresponding author, e-mail: porntip.tas@kmutt.ac.th, Ph No:+662 470-7475.

Abstract: Background: Sudan I is a cancer-causing chemical used in chili and cosmetic to give a strong color. Sudan I is a category 3 carcinogen previously, now banned, used to color certain foodstuffs. Hence, the sensing of Sudan I is of interest. Methods: Screen-printed electrodes (SPCEs) were modified by nanoparticles: graphene oxide (GO), silicon dioxide (SiO2), and magnetic iron oxide (Fe3O4). The electrochemical characteristics of the SPCEs were studied for the irreversible electrochemical oxidation of sudan I. Results: The standard rate constants (ks(cm/s)) for the reaction were determined using linear sweep voltammetry. The values of ks were found to be 0.009(±0.00051), 0.01(±0.00084), and 0.0006(±0.00081) cm/s; for GO/SPCE, SiO2/SCPE, and Fe3O4/SPCE, respectively. The total active area (A(cm2)) was determined from the Anson equation using 20 M. Sudan I and assuming a diffusion coefficient of 3.41 × 10-5 cm2/s. Surface coverages ( (mol/cm2)) were also determined from the Anson equation, using 0.1 mM. potassium hexacyanoferrate (III) and assuming a diffusion coefficient of 7.6 × 10-6 cm2/s. A was found to be 1.11× 10-5, 5.12 × 106 , and 6.04 × 10-6 cm2 from which  was found to be 5.43 × 10-6, 7.26 × 10-7, and 4.00 × 10-7 mol/cm2 for GO/SPcCE, SiO2/SPCE, and Fe3O4/SPCE, respectively. Calibration curves for the analytical determination of Sudan I by the modified SPCEs are presented. Conclusion: The GO/SPCE showed the highest A and  values, which resulted in a higher sensitivity than the SiO2/SPCE, and Fe3O4/SPCE. Keyword: ; Active area; Graphene oxide; Magnetic iron oxide; Sudan I; Silicon dioxide; Surface coverage.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

122

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Bioengineering of Tacca integrifolia (Bat flower): Effects of Hormones on in vitro Rooting and Production of Taccalonolides Fatimah A.L.1, 2, Teh L.K.2, Asmah A.1 and Salleh M.Z.2,* 1

Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia; 2 Integrative Pharmacogenomics Institute (iPROMISE) , Universiti Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail: zakisalleh.mzs@gmail.com / zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258 4652.

Abstract: Background: Bat flower plant (Tacca integrifolia Ker Gawl) is a rare plant species that is often collected for its medicinal value. However, its distribution is limited due to poor germination of seed and short period of seed viability. The objective of this study was to develop an in vitro rooting system for T. integrifolia from in vitro seedlings. Methods: Murashige and Skoog (MS) basal medium was used for the growth of seedlings. Seeds were sown on the MS basal medium whilst shoots from the in vitro germinated seedlings were excised and cultured on MS medium containing three different hormones {1Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA)} with concentrations of 0.25, 0.5, 1.0, 2.0 and 4.0 mg/L, respectively. After 12 weeks, the characteristics of the newly produced roots were observed, measured and analysed statistically. Metabolites produced from the treatments were profiled using LCMS Q-TOF. Results: The MS basal medium produced plantlets with the highest number of leaves, shoots and roots compared to the other three rooting hormones. However, MS basal medium supplemented with 1.0 mg/L IBA produced the longest roots. Interestingly, Taccalonolides A and B were detected in plantlets grown on MS basal medium. On the other hand, Taccalonolides E, N and Z were detected in plantlets grown on MS medium supplemented with 0.25 mg/L IAA, 1.0 mg/L NAA and 1.0 mg/L IBA, respectively. Conclusion: Tacca integrifolia were successfully grown using tissue culture techniques at laboratory scale and the Taccalonolides A, B, E, N and Z were detected using LCMS Q-TOF. Keywords: Bioengineering; In vitro; MS medium; Tacca integrifolia; Taccalonolides.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

123

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Isolation, characterization and potential application of bacteriophages for phage therapy Bhandare, S.G†.; Barrow, P.A. and Atterbury, R.J.* University of Nottingham, School of Veterinary Medicine and Science, Sutton Bonington Campus, Sutton Bonington, Loughborough LE12 5RD, United Kingdom; † Faculty of Veterinary Medicine, Universiti Malaysia Kelantan, Locked Bag 36, Pengkalan Chepa, 16100 Kota Bharu, Kelantan, Malaysia; e-mail: sudhakar@umk.edu.my, Ph No: +609-7717325; *corresponding author, e-mail: robert.atterbury@nottingham.ac.uk

Abstract: Background: The recent surge in bacteriophage (bacterial viruses) research is indicative of their huge potential utility for various applications which includes phage therapy for bacterial pathogens, rapid detection of bacteria and for other biotechnological purposes. The bacteriophages can be used as natural bio-control, bio-sanitation and bio-preservation agents in the food processing environment, pre-harvest application in animals prior to slaughter and in rapid diagnostics. Bacteriophages specific to Vibrio cholerae O1 were characterized for potential application in phage therapy. Methods: Sewage samples were collected on two occasions from Severn Trent Sewage Works, Raynesway, Derby, UK, a local wastewater treatment plant. The protocol used was modified from Van Twest and Kropinski, (2009). Two phages (Φ2 and Φ3) were obtained from Public Health England (PHE), London. The phages were characterized biologically (Lytic spectra and One step growth curve); physically (Electron Microscopy) and Genomically (PFGE and Restriction analysis). Results: Bacteriophages could not be isolated from the sewage samples from the local wastewater treatment plant. The two phages obtained from PHE, London; Φ2 and Φ3 could lyse 4.3% and 62.6% of the total 91 Vibrio cholerae strains, respectively. The latent period and burst size of Φ2 were 14 ± 1.6 m and 06 ± 01 PFU/cell; while that of Φ3 were 13 ± 4.1 m and 54 ± 26 PFU/cell, respectively. Electron microscopy revealed that Φ2 was of the Myoviridae family while Φ3 was of the Siphoviridae family. Phage Φ2 had a genome of less than 48.5 kb; while Φ3 had a larger genome of 114 kb. Restriction analysis could differentiate these phages. Conclusions: The non-isolation of Vibrio cholerae O1 specific phages from the UK environment is likely to be due to cholera being non-existent in that country. The phages characterized have potential to be phage therapy candidates. Keywords: Bacteriophages; Biocontrol; Genome; Phage therapy; Vibrio cholera.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

124

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Eco-friendly Biosynthesis of Atrocarpus altilis Mediated Silver Nanoparticles - Characterization and Evaluation of its Antimicrobial and Antioxidant Potential Ravichandran V.1*, Vasanthi S.2, Shalini S.1, Syed Adnan A.S.3and Haris R.4 1

Faculty of Pharmacy, AIMST University, Kedah, Malaysia; 2Faculty of Engineering, University of Nottingham, Semenyih, Selangor, Malaysia; 3Faculty of Pharmacy, Universiti Teknologi MARA, Selangor, Malaysia; 4SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India; *corresponding author, e-mail: sameshyaravi@gmail.com, Ph No: +60164581626

Abstract: Background: The biological entity is gaining significance in biosynthesis of silver nanoparticles as a result of their potential applications in nanomedicine and material engineering. Numerous approaches are used to prepare the nanoscale silver particles such as electrochemical, sonochemical and microwave-assisted process, but many of these strategies are having difficulty in purification, used hazardous chemicals and need high energy. To toggle over these difficulties, recently the eco-friendly approaches are established by using biological principles. In the present study we aimed to use plant materials for nanoparticles synthesis; hence, it does not need any elaborate processes such as compound purification steps and the microbial cell cultures maintenance. Methods: Silver nanoparticles (AgNPs) were synthesized by biological reduction of silver nitrate with aqueous extract of Breadfruit (Artocarpus altilis) leaves. Results: Synthesized colloidal BAgNPs were confirmed spectrophotometrically at 432 nm and the various reaction conditions were optimized. The SEM, TEM and DLS analysis confirmed that the average particle size of 34 nm, 38 nm, and 162.3 d.nm, respectively. Nature and presence of silver were confirmed by XRD and EDX. Further, FT-IR spectra of the synthesized AgNPs authorized the presence of phenols, proteins and flavonoids within the plant extract which can be accountable for the reduction and stabilizing the nanoparticles by capping. The AgNPs showed moderate antimicrobial actions than A. altilis leaf extract indicating its antimicrobial value. The DPPH assay results indicated the good antioxidant activities of AgNPs. Conclusion: The present study revealed the ecofriendly biosynthesis of AgNPs and the safer use of it in the field of biomedicine, water treatment/purification, and nanobiotechnology. Keywords: Antimicrobial; Antioxidant; Artocarpus altilis; Biosynthesis; Colloid; Silver nanoparticles.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

125

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Mutiplex Isothermal Amplification for Detection of Melioidosis Jilien Michelle Wong Tzelinga, Chan Yean Yeana,b* a

Department of Microbiology & Parasitology, School of Medical Science, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan. b Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail: jilienwong@hotmail.com / yeancyn@yahoo.com, Ph No: +609 7676258.

Abstract: Background: Burkholderia pseudomallei is a causative agent of melioidosis, causing potentially fatal disease of humans and animals in the tropics. Laborious and time consuming laboratory diagnostic may delay the treatment and results in high mortality rate. This study was conducted to develop a multiplex isothermal amplification assay (MIA) for rapid and sensitive identification of B. pseudomallei. An internal amplification control (IAC) was included for assay reliability purpose. Methods: A duplex isothermal amplification assay incorporating an IAC were performed with sets of loop-mediated isothermal amplification (LAMP) primers, which were designed to specifically identify the conserved region of B. pseudomallei. The sensitivity of the assay was performed by defining the limit of detection (LOD) of the optimized multiplex LAMP assay with serially diluted B. pseudomallei genomic DNA. Clinical isolates of B. pseudomallei, B. cepacia, B. thailandensis, and other bacteria were used to evaluate the analytical specificity of the assay. Results: The multiplex LAMP assay was found to be highly specific when tested with other similar bacteria. The assay of serially diluted gave a limit of detection (LOD) of B. pseudomallei genomic DNA 100 fg/ul. Conclusion: This MIA assay demonstrating promising results which can be used as method to detect melioidosis in near future for better and prompt therapeutic approach. Keywords: Burkholderia pseudomallei; Melioidosis; Multiplex loop-mediated isothermal amplification (M-LAMP); Rapid diagnostic.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

126

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves Nebulization and Phononic Crystal Structures Mohd H. Ismail* School of Microelectronic Engineering, Universiti Malaysia Perlis, Malaysia. Division of Biomedical Engineering, School of Engineering, University of Glasgow, United Kingdom; *corresponding author, e-mail: hafizismail@unimap.edu.my, Ph No: +60172386048.

Abstract: Background: Effective pulmonary therapeutic delivery requires a device which delivers sufficient medication to the site of action with minimal wastage. The effective delivery of the medication to the targeted area in body depends crucially on the droplet size distribution. A new platform which utilizes the surface acoustic waves (SAWs) and phononic crystal (PnC) technologies has been developed to generate monodispersed droplet size within the respirable fraction (between 1 Âľm to 5 Âľm). In this paper, the nebulized droplet size distributions of deionized water and budesonide generated by commercialized nebulizers are compared with the ones generated by the SAWs and PnC. Methods: The piezoelectric SAW substrate and the silicon PnC superstrate were fabricated using the standard microelectronic fabrication process. To perform nebulization of the deionized water and budesonide suspension for inhalation, a high frequency electrical signal was supplied to the electrodes using the signal generator and amplifier. The electrical signal generated mechanical oscillations on the LiNbO3, which subsequently produced a surface acoustic wave. The mean diameters of nebulized droplet sizes were measured using Malvern Spraytec. Results: A higher respirable fraction of droplet has been successfully obtained from nebulization performed on the PnC superstrate at low input power as compared to directly on SAW substrate and the commercialized nebulizers. Conclusion: The efficient nebulization on silicon PnC coupled to SAW propagating on a piezoelectric substrate has been successfully demonstrated. The advancement of SAW technology offers opportunities for the development of nebulizers as efficient and portable pulmonary therapeutic delivery platform. Keywords: Phononic crystal structure; Pulmonary therapeutic delivery; Surface acoustic waves nebulization.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

127

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Development of a Novel Duplex PCR Assay for Specific Detection of Salmonella enterica subspecies enterica serovar Typhi Based on SingleGene Target Goay Y. X.a, Carlos S.a, Suresh V. C.b, Zaidah A. R.c, and Phua K. K.a*. a

Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia (USM), 16150 Kubang Kerian, Kelantan, Malaysia; b Faculty of Applied Sciences, Asian Institute of Medicine, Science & Technology (AIMST), Jalan Bedong-Semeling, 08100 Bedong, Kedah; cDepartment of Medical Microbiology and Parasitology, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail: kkphua7@gmail.com

Abstract: Background: Typhoid fever, caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) remains major public health concern worldwide. Rapid and accurate detection is essential for surveillance, treatment and control of the disease. In this study, a duplex PCR assay, which is faster, more sensitive and specific than traditional biochemical and serotyping method, was developed for diagnosis of typhoid fever. Methods: Primers targeting STY0307 gene were designed and used in the PCR assay. Primers targeting 16S rRNA gene was included to serve as an internal control. The PCR assay was optimized using Taguchi method. The analytical sensitivity and specificity of the optimized PCR assay were determined using DNA obtained from; 1) ATCC 7251 S. Typhi reference strain, 2) 38 different PFGE-typed S. Typhi local strains, and 3) 72 strains from other enteric pathogens. The diagnostic sensitivity and specificity of the assay was further evaluated using a total of 120 human clinical specimens collected from Hospital Universiti Sains Malaysia (HUSM). Results: The assay was found to be 100% sensitive and specific in detecting 39/39 S. Typhi strains and 0/72 strains from other enteric pathogens. The assay limit of detection was as low as 1.5 x 105 cfu/ml of bacteria count or 1.28 pg of purified DNA. The sensitivity of the PCR assay using spiked stool samples was found to be 1.5 x 104 cfu/ml. Evaluation of the PCR assay using 120 human clinical specimens showed 100% diagnostic specificity and sensitivity. Conclusion: A highly sensitive and specific duplex PCR assay has been developed using single-gene target, STY0307, for the detection of S. Typhi, and was found to be suitable for diagnosis of typhoid fever in clinical settings. Keywords: Diagnostic; Duplex PCR; S. Typhi; Typhoid fever; Single-gene target; STY0307.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

128

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Assessment of Biodiesel Properties From the FAME Composition of a Malaysian Rhodophyte (Kappaphycus sp.) Md. Sayedur Rahman, and Kathiresan V. Sathasivam* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah Darul Aman, Malaysia; *corresponding author, e-mail: skathir@aimst.edu.my

Abstract: Background: Biodiesel derived from renewable lipid feedstocks is largely used in diesel engine. Third generation biodiesel is known to produce from oil of algal biomass and is considered as a very promising fuel. The objective of the present study was to evaluate some physico-chemical properties of biodiesel produced from a Malaysian Rhodophyte, Kappaphycus sp. using its fatty acid methyl esters (FAMEs) composition. Methods: Lipid was extracted from the dried Kappaphycus sp. collected from Sabah, Malaysia, which was converted into FAMEs using base-catalyzed transesterification process. The FAMEs were analyzed by GC-FID (Agilent 7890A, USA). Some characteristic biodiesel properties were calculated from the FAMEs composition using reference standard mathematical models. Results: Total lipid contents in the dried biomass of Kappaphycus sp. was found as 15.66 mg/g dw whereas biodiesel yield was recorded as 91.09% of lipid weight. The percentage of saturated, mono- and poly-unsaturated fatty acids in FAMEs was recorded as 85.98, 8.39 and 5.63 wt.%, respectively. Comparative study showed that biodiesel produced from Kappaphycus sp. is fairly better than those produced from some land plants as well as microalgae as it satisfies almost all the quality standards defined by the American, European and Malaysian biodiesel quality standards under study. Conclusion: Kappaphycus sp. can be a promising source of lipid feedstock for biodiesel production. But, the low lipid content of the species is a limiting factor to bring forth economic feasibility of biodiesel development. We, therefore, emphasize biotechnological interventions through genetic engineering for enhancing the lipid content and quality in the seaweed species. Keywords: Biodiesel; Biodiesel quality standards; Fatty acid methyl esters; GC-FID; Kappaphycus sp.; Lipid.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

129

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Generation of RNA Aptamers Against Mycobacterium tuberculosis Secretory Protein ESAT-6 - a Preliminary Study Bakhtiar Bukari, Citartan Marimuthu and Thean Hock Tang* Infectomics Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Kepala Batas, Pulau Pinang, 13200, Malaysia; *corresponding author, e-mail: tangth@amdi.usm.edu.my Abstract: Background: ESAT-6 is a secretory protein produced by Mycobacterium tuberculosis and is released in early stages of infection. It forms a heterodimer complex with another protein called CFP-10 and has been implicated with Mycobacterium sp. pathogenicity. Aptamers are chemical ligands made up of short nucleotides sequences that are able to bind to target proteins with high affinity and specificity. They are developed using a process called Systemic Evolution of Ligands via Exponential Enrichment (SELEX). Due to their chemical stability and high specificity against the target, aptamers have the potential to become very useful biological tools. Objective: The objective of this study is to develop RNA aptamers that bind specifically to ESAT-6 protein. Methodology: Eleven SELEX cycles were carried out using the N40-randomised RNA pool. Stringency of the binding reaction in each SELEX cycles was increased gradually by varying the amounts of protein, RNA pool and the competitor. The resulting RNA pool from the 11th cycle of SELEX was subjected to filter binding assay to assess its binding against ESAT-6 protein. Results: RNA pool was successfully derived from SELEX cycle 11. Filter Binding assay against the target protein at 800 and 1600 nM confirmed that binding enrichment of the RNA pool has occurred. Conclusion: Filter Binding assay suggested the presence of potential binders in the RNA pool. Further SELEX cycles will be carried out to improve the binding enrichment of the RNA pool and for sequence deconvolution. Sequencing will be carried out to identify the putative aptamer. Keywords: Aptamer; ESAT-6; Filter binding assay; M. tuberculosis; SELEX.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

130

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

An Expression Analysis of Salmonella Pathogenicity Island (SPI)-Derived Non-Protein Coding RNAs in S. Typhi Biofilm formation Kogaan Anbalagan1, Suresh V. Chinni2, Phua Kia Kien1* 1

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, USM, Pulau Pinang, Malaysia; 2Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100, Bedong, Kedah, Malaysia; *corresponding author, email: kkphua7@gmail.com, Ph No: +6046534853

Abstract: Background: Chronic infection with Salmonella Typhi (S. Typhi) is associated with long-term localization of the bacteria in the gallbladder in the form of biofilm. Recent studies have demonstrated that genes in the Salmonella Pathogenicity Island (SPI) region of the bacteria and many non-protein coding RNAs (npcRNAs) play a crucial role in bacterial stress response. Therefore, expression analysis of SPI-derived npcRNAs in S. Typhi will give an idea of their role in biofilm formation. Methods: S. Typhi biofilm was cultured in 6-well tissue culture plates by incubating at 37Ë&#x161;C in a shaker at 350 rpm. Total RNA from 3 different stages of S. Typhi development (planktonic, intermediate and biofilm) were extracted and resolved on Urea-PAGE gel, and then transferred onto the nylon membrane. Northern blot hybridization was performed using DIG-labeled specific probes to verify the expression of the SPI-derived npcRNAs. Results: Expression of five npcRNAs, i.e. StyR-327, StyR-9, StyR-143, StyR-161, and StyR-381, were detected. StyR-161 was equally expressed in all 3 stages of S. Typhi development, suggesting a house-keeping function for this npcRNA. StyR9 and StyR-381 were marginally down-regulated in the biofilm stage compared to the planktonic stage. However, StyR-143 and StyR-327 was clearly up-regulated in the intermediate and biofilm cells compared to the planktonic cells; indicating a possible role of this npcRNA in biofilm adaptation. Conclusion: In conclusion, expression of 5 SPI-derived npcRNAs were verified in S. Typhi cells during normal and biofilm conditions. StyR-143 was significantly up-regulated with biofilm formation, and may be related to bacterial pathogenesis. Further studies need to be carried out to identify the mRNA target and its regulatory mechanism. Keywords: Biofilm; npcRNA; Pathogenesis; S. Typhi.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

131

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood for Personalized Medicine Khor S. M.1*, Ang S. H.1, Rambeli M.1, Thevarajah T. M.2, Alias Y.1 1

Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia; 2Department of Pathology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; *corresponding author, e-mail: naomikhor@um.edu.my, Ph No: +603-79677022/Ext: 2520

Abstract: Background: We describe a gold nanoparticle-based sandwich immunoassay for the dual detection and measurement of hemoglobin A1c (HbA1c) and total hemoglobin in the whole blood (without pretreatment) in a single step for personalized medicine. Methods: The optimized antibody-functionalized gold nanoparticles immunoreact simultaneously with HbA1c and total hemoglobin to form a sandwich at distinctive test lines to transduce visible signals. The applicability of this method as a personal management tool was demonstrated by establishing a calibration curve to relate % HbA1c, a useful value for Type 2 diabetes management, to the signal ratio of captured HbA1c to all other forms of hemoglobin. Results: The platform showed excellent selectivity (100%) toward HbA1c at distinctive test lines when challenged with HbA0, glycated HbA0 and HbA2. The reproducibility of the measurement was good (6.02%) owing to the dual measurement of HbA1c and total hemoglobin. A blood sample stability test revealed that the quantitative measurement of % HbA1c was consistent and no false-positive results were detected. Also, this method distinguished the blood sample with elevated HbF from the normal samples and the variants. Conclusion: The findings of this study highlight the potential of a lateral flow immunosensor as a simple, inexpensive, consistent, and convenient strategy for the dual measurement of HbA1c and total Hb to provide useful % HbA1c values for better on-site diabetes care. Keywords: Colloidal gold-based immunochromatographic assay; Hemoglobin A1c; Lateral flow immunosensor; Single-step dual measurement; Total haemoglobin; Type 2 Diabetes Mellitus.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

132

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Development of Rapid Diagnostic Detection for Salmonella enterica Subspecies enterica Serovar Paratyphi A using Cross Priming Amplification Roziana, M.H. 1,2, Tan S.C. 1, Ismail, A. 1 and Aziah, I*1 1

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Health Campus, Kelantan, Malaysia; 2Universiti Teknologi MARA, Perlis, Malaysia; *corresponding author, e-mail: aziahismail@usm.my, Ph. No: +609-7672426

Abstract: Background: Paratyphoid fever is a systemic infection caused by Salmonella enterica subspecies enterica serovar Paratyphi A contributing one case in every four cases of typhoid. An isothermal amplification, cross priming amplification (CPA), is amplified using several primers by DNA polymerase with displacement activity at a constant temperature was developed to overcome the limitation of the current PCR test. As a point of care, CPA is developed for rapid detection of bacteria suitable for resource-limited settings with the basic requirement of simple heating block or waterbath for the amplification. This study aims to establish an in-house CPA combined with lateral flow assay (LFA) for the detection of S. Paratyphi A (SPA). Methods: In-house CPA for S. Paratyphi A was developed three pairs of primers (displacement, cross and detector) from six locations of an intergenic region between SSPA1723a and SSPA1724 of S. Paratyphi A genome sequence. The forward and reverse detector primers were labeled with biotin and FAM at 5â&#x20AC;&#x2122;end respectively. Optimisation of the CPA-SPA components was performed using Taguchi method. The assay was further tested with few parameters such as primer concentration, temperature and incubation time. Analytical sensitivity of CPA was performed at DNA and bacterial level. Diagnostic sensitivity and specificity of CPA-SPA were tested with 30 isolates of each S. Paratyphi A, S. Typhi, other Salmonella serovars, and other bacterial strains. CPA-SPA products were detected via 2% agarose gel electrophoresis (AGE) and compared with LFA. Results: The optimum condition of CPA-SPA was at 63Â°C for 60 minutes. The limit of detection of the CPA-SPA was 10 fg of the pure genomic DNA. CPA-SPA was sensitive up to 103 CFU/ml and 101 CFU/ml via AGE and LFA respectively. CPA-SPA was positive for all 30 S. Paratyphi A and negative for other 90 bacterial strains. Conclusion: The present study demonstrated high sensitivity and specificity of CPA-SPA. Rapid, sensitive and specific detection of CPA-SPA was successfully developed. The findings suggested that the in-house CPA-LFA has great potential for detection of S. Paratyphi A in resource-limited settings. Keywords: Cross-priming amplification; Enteric fever; Isothermal; S. Paratyphi A.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

133

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Conversion of Rice Husks to Polyhydroxyalkanoate (PHA) Heng K.S.1, Adam F.2, Sudesh, K.1* 1

School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia; School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia; *corresponding author, e-mail: ksudesh@usm.my, Ph. No: +604 6534367

Abstract: Background: Sugars obtained from the hydrolysis of rice husks have the potential to be used as an economical feedstock for the production of polyhydroxyalkanoates (PHA), a biodegradable polymer produced intracellularly many types of bacteria. Methods: The rice husks were first pretreated with a combination of alkali and physical methods and then hydrolyzed enzymatically under conditions that were optimized previously. Characterization of the sugars in the hydrolysate was performed to determine the sugar composition. The hydrolysate was fed to two strains, Burkholderia cepacia USM (JCM 15050) and Cupriavidus necator NSDG-GG, an engineered strain of Cupriavidus necator H16, to evaluate their PHA production. Results: Based on high performance anion exchange chromatography (HPAEC) analysis, glucose and xylose were the main sugars present in the hydrolysate, with low amounts of arabinose. B. cepacia USM utilized the hydrolysate more efficiently compared to C. necator NSDG-GG, with a maximum cell dry weight (CDW) of 4.9 g/L and 40 wt % PHA at shake-flask scale. The CDW and PHA content of the B. cepacia USM cultivated in a 5-L fermentor after 36 hours of fermentation were 7.80 g/L and 50% respectively. The decrease in total phenolics at the end of fermentation suggested that B. cepacia USM was able to metabolize phenolic compounds. Conclusion: These results indicate that rice husks can be used as carbon sources for PHA production, thus adding value to this agricultural by-product. Keywords: Biosynthesis; Hydrolysis; Polyhydroxyalkanoate; Rice husks; Sugars.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

134

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Salmonella typhimurium Detection Based on Electrochemical Immunoassay using Methylene blue/MWNTs/Magnetic Particle Ngoensawat U.a, Rijiravanich P.* b, c, Surareungchai W.a and Somasundrum M. b, c a

School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand. b Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand. c National Center Biological Engineering Graduate Program, King Mongkut's University of Technology Thonburi, Bangmod, Bangkok 10140, Thailand; *corresponding author, e-mail: patsamon.rij@biotec.or.th, Ph No: (+66)2-4707475

Abstract: Background: Electrochemical sensors are an attractive technology for food borne pathogen detection, offering sensitivity, selectivity, fast response, low cost, amenability to mass production and the possibility of miniaturization. To achieve high sensitivity and selectivity, nanomaterials have often been integrated into detection platforms. Due to possessing, high surface-to-volume ratios, nanomaterials have the advantage of being able to carry high amounts of redox mediator and can often be surface-modified with biomolecules. This has made nanomaterials highly attractive as electrochemical labels. Methods: We report an electrochemical immunoassay using modified magnetic beads as electrochemical labels. The labels are prepared by modifying magnetic beads with methylene blue (MB)functionalized multiwall carbon nanotube (MWNTs). The outermost layer of the beads is coated with an antibody specific to Salmonella typhimurium. After binding, the target cells can be easily separated from the solution by applying a magnetic field. The label-cell conjugated is deposited on an anti-Salmonella Typhimurium-modified screen-printed electrode for sandwich immunoassay. The signal from direct reduction of MB is recorded by differential pulse voltammetry (DPV). Results: Each electrochemical label was found to carry 2.35x108 molecules of MB, as determined by DPV. This is a 1000 fold increase in the MB loadings of labels used in previous work. The peak reduction current was observed at 0.264V which is similar to MB in solution. The label signal was significantly different in the absence of the cells. Conclusion: Magnetic beads were successfully modified with MBloaded carbon nanotubes. The resulting labels showed a high current signal for MB reduction. The magnetic beads could be used to capture and separate target cells from solution, enabling Salmonella typhimurium detection. Keywords: Electrochemical immunosassay; Magnetic separation; MWNTs-methylene blue; Salmonella typhimurium.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

135

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Electrochemical Characterisation and Determination of Mycobacterium tuberculosis by Voltammetry at Polymer Nanocomposite modified Platform Himkusha Thakur1, Navpreet Kaur1, Dipti Sareen1, Priyanka2, Nirmal Prabhakar*1 1

Department of Biochemistry, Panjab University, Chandigarh, India. Institute of Nano Science & Technology, Mohali, India; *corresponding author, e-mail: nirmalprabhakar@pu.ac.in

Abstract: Background: Tuberculosis, caused by Mycobacterium tuberculosis, produces illhealth among millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide.The conventional TB detection methods are highly time consuming, laborious and less result oriented, that can be overcome by the use of biosensors. We have developed a novel bioelectrode for the detection of active tuberculosis using polymer nano-composite platform. Methods: PEDOT, a polymer, exhibits relatively high electrical conductivity and is very stable even during the electrochemical changes, while CNT gives advantages like small size with larger surface; high sensitivity and fast response. Biotinylatedaptamerhas been successfully immobilised onto streptavidin coated PEDOTCNT matrix. Characterisation studies like FT-IR, FE-SEM and electrochemical characterisation (DPV) studies were done to confirm step-wise fabrication of the aptaelectrode. Different parameters involving the aptamer concentration, binding time and response time with the target have been optimised. The electrochemical response study of aptaelectrode treated with a wide range of target protein was done by DPV. Results: The electrochemical response study of aptaelectrode treated with a wide range of target protein was done by DPV. There was increase in current response with increase of target concentration as the 3-D complex formed improved the electron flow as comparison to sole aptamer immobilisation. Conclusion: The detection limit of the fabricated bio-electrode has been reported to be 0.01ng/ml. The aptaelectrode showed more than half a month stability along with good regeneration and repeatibilty, thus establishing its potential in the field of diagnosis in the future. Keywords: Aptaelectrode; Biotin-Streptavidin interaction; CNT; DPV; PEDOT.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

136

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Abstracts (Poster Presentations) Cloning, Over-expression, and Purification of Hfq Protein from Klebsiella pneumoniae Devarubini K.1, Kishan S.1, Suresh V.C.1* 1

AIMST University, Department of Biotechnology, Faculty of Applied Sciences, Bedong, 08100, Kedah, Malaysia, *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165.

Abstract: Background: Hfq is a RNA chaperone present in Klebsiella pneumonia and many other bacteria. It plays a vital role in virulence of the bacteria. Hfq exhibits its function by binding with npcRNA which will be needed for the trans-acting npcRNA, mRNA interaction. To gain further insights on the target bacterial npcRNAs that interact with this RNA chaperone, highly pure Hfq protein is needed. Methods: Hfq gene from Klebsiella pneumonia was amplified and cloned into pET-28b+ vector. Ligated mixture was transformed into TOP-10 cells. The transformed bacterial colony with recombinant plasmid was screened by antibacterial (Kanamycin) selection and confirmed by PCR methods. The recombinant plasmid was transformed into E.coli BL21 and induced the expression with IPTG. The over expressed Hfq protein was purified using Ni-NTA affinity chromatography. Results: Hfq gene had been successfully amplified, digested with appropriate restriction enzyme and cloned in to pET-28b+ vector. Recombinant plasmid was successfully transformed into TOP10 and BL21. A highly purified Hfq protein was obtained and confirmed by SDS PAGE analysis. The best elution of the Hfq protein was actualized using 1 M of imidazole. Conclusion: In this study we successfully purified Hfq protein from Klebsiella pneumonia for further RNA binding study to select the bacterial npcRNA that binds to the protein for further identification and characterization studies. .Keywords: Hfq recombinant protein; Klebsiella pneumonia; Ni-NTA.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

137

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

In vitro Anti-oxidant Assay, HPLC Profiling of Polyphenolic Compounds, AAS and FTIR Spectrum of Malaysian Solanum torvum Swartz fruit Sathyanarayana N.a, *, Sunitha P.b, Suresh V.C.c, Sreeramanan S.d., Marimuthu K.c, and Xavier R.c a

Unit of Anatomy, b Unit of Physiology, Faculty of Medicine, c Faculty of applied sciences, Department of Biotechnology, AIMST University, Semeling, Bedong, Malaysia, d School of Biological Sciences, UniversitiSains Malaysia, Penang, Malaysia; *corresponding author, email: drsatyanarayana.aimst@gmail.com, Ph No: +6 010-5600574

Abstract: Background: Solanum torvum Swartz, a medicinal plant of Solanaceae family and is used in traditional systems of medicine. Since, the plant is widely used as a medicinal plant among the Malaysian population it is important to understand the phytochemical content. The aim of this study is to determine the phytochemical content and antioxidant potential of the fruits of Solanum torvum. Methods: The phytochemical analysis was carried out following standard protocol with the aqueous, ethanolic and methanolic extracts of S. torvum fruit. Anti-oxidant potential of S. torvum fruit was evaluated by DPPH, FRAP and HPLC methods. Elemental and functional group analysis were done by Atomic Absorption Spectrophotometry (AAS) and Fourier Transform Infrared Spectrophotometry (FTIR) methods respectively. Results: Quantitative assessment of total phenols and flavonoid content, DPPH and FRAP assay was done in the ethanolic extracts of S. torvum. Qualitative analysis of each extract showed the presence of reducing sugars, saponins, alkaloids, phenols and flavonoids except anthraquinones. Quantitative determination of total phenols and flavonoids showed 16.4 mg GAE/g and 2.8 mg QE/g respectively. In DPPH radical scavenging assay, the IC50 value of the extract was found to be 1.62 mg/ml and the FRAP value was found to be 470 mg FeSO4 E/g. The High Performance Liquid Chromatography (HPLC) analysis revealed presence of polyphenolic compounds such as gallic acid, rutin, quercetin and ascorbic acid. Elemental determination by AAS showed the presence of essential elements such as Calcium (Ca), Copper (Cu), Iron (Fe), Manganese (Mn), Lead (Pb), Zinc (Zn), Nickel (Ni), Magnesium (Mg), and Sodium (Na). FTIR results showed the presence stretching vibrations of OH groups in phenyl, CH2 asymmetric stretch of methyl groups, C-O stretching vibrations ring of phenyls, CH bending vibration. With this it is concluded that S. torvum fruits are a rich source of antioxidants. Conclusion: S. torvuma wild Malaysian medicinal plant fruit ethanolic extract possesses good amount of phenols and flavonoids responsible for the antioxidant property. Keywords: AAS; Antioxidant property; FTIR; HPLC; Polyphenolic compounds.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

138

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Phytochemical Analysis and Antioxidant Activity of Malaysian Medicinal Plant Abroma augustum Leaf Extract Sunitha P.1, *, Sathyanarayana N.1, Suresh V.C.2, Sreeramanan S.3, Sam A.1 and Xavier R.2 1

Faculty of Medicine, AIMST University, Semeling, Bedong, Malaysia Faculty of Applied Sciences, Department of Biotechnology, AIMST University, Semeling, Bedong, Malaysia 3 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia; *corresponding author, e-mail: drsunitha33@gmail.com, Ph No: +6016-4542155

Abstract: Background: Antioxidants from plant materials terminate the action of free radicals thereby protecting the body from various diseases. Phenolic compounds from medicinal plants possess strong antioxidant activity and may help to protect the cells against the oxidative damage caused by free-radicals. Hence, the present study is aimed at to evaluate the phytochemical content, antioxidant activity, functional group and elemental analysis of the leaves of A. augustum of Malaysian origin. The investigation on the phytochemical analysis of A. augustum is the pioneering study and the results will form the basis for explaining the medicinal properties of this plant. Methods: Antioxidant potential and polyphenolic compounds of A. augustum were evaluated by DPPH, FRAP assays and HPLCPDA method respectively. Elemental analysis and Functional group analysis were done by an Atomic Absorption Spectrophotometry (AAS) and Fourier Transform Infrared Spectrophotometry (FTIR) methods. Results: Qualitative analysis of each extract showed the presence of reducing sugars, alkaloids, tannins, phenols and flavonoids. Quantitative analysis of total phenolic and flavonoid content showed 15.76 mg/g GAE and 8.6 mg/g QE of A. augustum leaf extract respectively. IC50 value of the extract was found as 790Âľg/ml by DPPH free radical scavenging assay. In the FRAP assay, the ethanolic leaf extract showed 367.6 mg/g FeSO4 equivalants. High performance liquid chromatography (HPLC) of the leaf extract revealed the presence of polyphenolic compounds such as gallic acid, quercetin and ascorbic acid. FTIR results showed the presence stretching vibrations of OH groups in phenyl, C-H asymmetric stretch of methyl groups, C=O stretching vibrations ring of phenyl, CH bending vibration. Presence of essential elements was detected by AAS. Conclusion: Abroma augustum is a rich source of polyphenolic compounds (phenols and flavonoids) and antioxidants which might be responsible for the unexplored medicinal properties of A. augustum. Keywords: AAS; A. augustum; Antioxidant potential; FTRI; HPLC-PDA; Polyphenolic compounds.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

139

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Reduced Reproductive Function up to Three Generations of Rats Due to Paternal Heroin Addiction M. Z.Farah Naquiah 1, R. J. James 1,2 *, M. I. Mohd Hafidz 2, M. Z Salleh 1,2, L.S. Lee 1, S. Suratman 2, L. K. Teh 1,2 * 1

Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3, Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia. 2 Faculty of Pharmacy, Universiti Teknologi MARA Selangor, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor. Malaysia; *corresponding author, e-mail: tehlaykek2016@gmail.com, and ritchjj@yahoo.fr

Abstract: Background: Little is known about the long-term consequences of heroin addiction on either the user or his future offsprings. In this study, we look into the effects of paternal heroin exposure on the reproductive capacity and the weights of their progenies up to three generations. Methods: Male Sprague Dawley rats (6-weeks-old) were divided into: (1) heroin addiction induced rats (F0-H) and (2) control group treated with saline solution (F0C). Heroin or saline solution was administered intraperitoneally twice-daily for fourteen days with increasing dosage regimen. The dosage regime started with the lowest dose of 3 mg/kg per day of heroin followed by 1.5 mg/kg increments per day to a final dose of 13.5 mg/kg per day for 14 days. Results: Administration of heroin in the sires resulted in a statistically significant smaller number of litters with a 40% reduction in the size. However, this pattern was not observed in the second and third generations. Weights of the litters were measured on day 21 and day 90 (adult). There was no significant difference in the body weights of the rats when compared between the control and heroin treated groups. However, on day 90, the F0H group had a lower average of weight when compared to the control group. This pattern was also seen in the F1 and F2 generations but not in the F3 generation. Conclusion: Our results suggest that paternal addiction to heroin caused reduction in the size of the litters and lower body weight which is transmitted up to two generations. Keywords: Adolescent; Transgenerational; .

Heroin

addiction;

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Opiates;

Opioid;

Paternal

addiction;

140

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Optimization of Cryopreservation Using Different Cryoprotective Agents and Differential Temperatures on Freeze Dried Probiotics Hassan Pyar1, 2 and K.K Peh*3 1

Faculty of Pharmacy, AIMST University, Semeling, 08100, Bedong, Kedah Darul Aman, Malaysia; 2Hadramout University of Science and Technology, Mukalla, Hadramout, Yemen; 3 School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; *corresponding author, e-mail: kkpeh@usm.my, Ph No: +604-6533888

Abstract: Background: The use of probiotics in biotechnological applications has steadily increased over the past decades. However, these beneficial microbes were losing viability or activity during the preservation process. To avoid cellular damage during cryopreservation and subsequent thawing, a wide array of cryoprotective agents has been applied. Methods: Investigation was done on the viability and stability of probitic and the effect of different cryoprotective agents (namely, sodium chloride, sucrose, dextran, sorbitol, monosodium glutamate, glycerol, skim milk and skim milk with malt extract) with modified De-Man Rogosa Sharpe (MMRS) medium were examined. Results: Commercial De-Man Rogosa Sharpe (MRS) medium was proved to be more expensive than the modified MRS medium with relatively low yield of probitic. Significantly high viable counts were achieved with monosodium glutamate, skim milk and skim milk with malt extract, with optimum concentration at 0.3% w/v. There was a reduction in cell viability at concentration above 0.5% w/v, which could be attributed to cell shrinkage associated with osmotic pressure changes inside the cells. Probitic Lactobacillus species was found to be stable at room temperature (28Â°C) for eight weeks. A significant growth of probiotics was produced from skim milk. Conclusion: modified MRS medium with skim milk is suggested for the remarkable growth and yield of Probitic lactobacilli. Keywords: Cryoprotective agents; De-Man Rogosa Sharpe (MRS); Probiotics.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

141

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Development of a Reusable Electrochemical Immunosensor for Direct Detection of Small Organic Molecules Khoo M. M.1, Yatimah A.1, and Khor S. M.1* 1

Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia; *corresponding author, e-mail: naomikhor@um.edu.my, Ph No: +603-79677022/Ext: 2520

Abstract: Background: In order to reduce costs and waste produced, an electrochemical immunosensor, which able to perform multiple measurements without sensor detection ability loss, has attracted the interest of many researchers. Reusability of immunosensor depends on the rapid reversible interaction between antibody and antigen. Dissociation of antibodies from the immunosensor surface gives the detection signal for target analyte. Exposure of the used immunosensor surface to antibody will give a new detective immunosensor surface. The use of electrode polarization can help to regenerate immunosensor surface and also eliminate the non-specific protein absorption to immunosensor surface. In this study, the main objective was to develop a reusable and nonfouling surface for electrochemical biosensor applications. Methods: A combination of aryl diazonium salt with zwitterions such as sulfanilic acid and (4aminophenyl)trimethylammonium were deposited onto the electrode surface. These zwitterions molecules are chemically stable, less subjected to oxidation and low impedance. Besides, gold nanoparticles were employed to lower the impedance and increase the sensor detection area. For regeneration, a constant potential of -800 mV was applied to the immunosensor interface for 10 min to remove the surface-bound antibodies. Results: For optimization, the electrode polarization of -800 mV at the 10-min interval time was recognized as the optimum condition to yield the highest sensor sensitivity.The antigenantibody binding was found to be reversible with the aid of the electrode polarization, after five replicate measurements (with an RSD of 9.14%) performed within the same day at ambient temperature (25ยบC). The good blocking of Fe(CN)63-/4- shown in cyclic voltammograms indicated that the immunosensor surface was not physically damaged after multiple times of surface regeneration. Besides, the lowest detection limit was improved from 100 ng mL-1 to 1 ng mL-1 with the aid of electrode polarization. Conclusion: This biosensor interfacial design is suitable to be used for repeated electrochemical immunosensor use. Keywords: Electrochemicalimmunosensor; Protein-surface interactions; Reversible affinity interactions; Reusable biosensor; Surface regeneration.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

142

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Over-expression and Purification of an RNA Chaperone, Hfq Protein of Proteus mirabilis Kishan S.1, Citartan M.2, Prabu S.2, Tang T.H.2, Suresh C.V.1* 1

Faculty of Applied Sciences, Department of Biotechnology, AIMST University, 08100 Bedong, Kedah, Malaysia. 2 Advanced Medical & Dental Institute, Universiti Sains Malaysia, 13200 Bertam, Kepala Batas, Penang, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Hfq, an RNA chaperone plays a vital role in virulence of various bacteria including P. mirabilis. Hfq exhibits its function by binding with npcRNA which will be needed for the trans-acting npcRNA, mRNA interaction. To gain further insights of the regulating npcRNAs that interacts with this RNA chaperone, pure form of Hfq protein and a standardized Hfq-npcRNA binding protocol need to be established. Methods: Hfq gene from P. mirabilis was amplified and cloned in to pET-28b(+) vector and then transformed into TOP10 cells. The bacterial with recombinant plasmid was screened by antibacterial selection and confirmed by sequencing. The recombinant plasmid was transformed into E. coli BL21 and induced the expression with IPTG. The over expressed Hfq protein was purified using Ni-NTA affinity chromatography. Total RNA from P. mirabilis was extracted using TRIzol and verified by BioAnalyzer. A protocol was standardized for Hfq-npcRNA binding using Nitrocellulose membrane affinity. Results: Hfq gene had been successfully cloned into pET28b(+) vector and transformed into TOP10 and BL21. A highly purified Hfq protein was obtained and confirmed by SDS PAGE. The good intact of total RNA was extracted from P. mirabilis. The first trial of the recombinant protein Hfq and total RNA binding assay was carried out and the protein bound RNAs were precipitated and sent for Next-Generation Sequencing. Conclusion: In this study we successfully purified Hfq protein from P. mirabilis. Hfq bound RNAs were sent for Illumina sequencing to identify the npcRNAs and their partners involved in Hfq mediated trans-gene regulation. Keywords: Hfq; Nitrocellulose membrane; P. mirabilis; Recombinant protein.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

143

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Peritoneal Mast Cell Stabilization and Toxicological Properties of the Ethanolic Extract of Solanum trilobatum Linn. Lee Yu Ren, Stephanie Wong Kah Yee, Bobby Lau Chik Chuon, Christapher Parayil Varghese, Subramani Parasuraman* Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Malaysia; *corresponding author, e-mail: parasuraman@aimst.edu.my, Ph No: (+60) 103895096

Abstract: Background: Solanum trilobatum Linn., (Solanaceae) is known as thoodhuvalai in Tamil. The leaf of S. trilobatum is commonly used as a food supplement by South Indians, the aqueous extract from same plants has been used for the treatment of respiratory illness. In pre-clinical studies, the extract of S. trilobatum showed hepatoprotective, antimicrobial, larvicidal and anticancer activities. In few clinical studies, the extract of S. trilobatum showed bronchiolitic effect. The antihistaminic effect and toxicity profile of S. trilobatum remain unclear, hence the present study has been planned to carry out the peritoneal mast cell stabilization activity and chronic toxic effect of ethanolic extract of S. trilobatum (EEST) in Sprague Dawley (SD) rats. Methods: The SD rat intestinal mesentery was used to study the peritoneal mast cell stabilization of EEST. The rat intestinal mesentery was exposed to 50, 100, 200, 300, 400 and 600 mg/ml of EEST and the peritoneal mast cell stabilization activity was compared with that of chlorpheniramine. In chronic toxicity testing, rats were treated once daily with 100, 200 and 400 mg/kg of EEST for 30 days. During the study, animalsâ&#x20AC;&#x2122; behaviour and biochemical alterations were observed at regular intervals. At the end of the study, rats were sacrificed and their organs were collected for histopathological analysis and part of brain was preserved at-80Â°C for dopamine assay. Result: EEST showed significant antihistaminic activity at the dose of 300 mg/ml onwards and the effect was comparable with that of standard. In chronic toxicity testing, EEST significantly reduced the immunization time, locomotor activity and increased the dopamine (results were not significant) levels in brain tissue. In histopathological analysis, EEST showed marked changes in brain, liver and kidney. Conclusion: EEST showed significant antihistaminic activity at 300 mg/ml onwards and had mild to moderate toxic effect at 200 mg/kg onwards. Keywords: Antihistamine; Solanum trilobatum; Toxicology.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

144

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Understanding the Host-Pathogen Interaction in Klebsiella pneumoniae Infected Rat Model via Metabolomics Approaches Mohd Izwan Mohamad Yusof1,2, Mohd Salleh Rofiee2, Teh Lay Kek2, Mohd Zaki Salleh*2 1

Integrative Pharmacogenomics Institute (iPROMISE), Level 7, FF3 Building, Universiti Teknologi MARA Selangor Branch, Puncak Alam Campus, 42300 Puncak Alam, Selangor, Malaysia. 2 Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, 42300 Puncak Alam, Selangor, Malaysia; *corresponding author, e-mail: tehlaykek@yahoo.com, Ph No: (+60) 332584652

Abstract: Background: Bacteremia can be defined as the presence of viable bacteria in the bloodstream which can lead to multiple organ failures if managed incorrectly. To better understand the interaction between pathogen-host metabolic response, we investigated the metabolic consequences of a Klebsiella pneumoniae infection in vivo via metabolomics approaches. Methods: K. pneumoniae was intravenously injected into rats, and serum samples were collected at three different time points (0 hours (pre-infection), 2 hours after infection (early infection) and 192 hours after infection (post-infection). Results: Fifteen (15) metabolites were characterized as potential biomarkers related to K. pneumoniae infection. The identified potential biomarkers were derived from nine (9) pathways which were found significantly perturbed during K. pneumoniae infection. Tryptophan metabolism was the most prominently influenced in K. pneumoniae-induced bacteremia according to the metabolic pathway analysis (MetPA), suggesting that significant modulation of immune system activity occurred during early infection of K. pneumoniae. In addition, we also capture several metabolites that represent the host is in oxidative stress, inflammation, and high energy demand state during early infection of K. pneumoniae. Conclusion: Our findings provide a novel perspective on the metabolites signatures together with perturbed pathways in related to bacteremia, which provided us with new insights into the pathogenesis of bacteremia, and the discovery of targets for clinical diagnostic and treatment. Keywords: Bacteremia; Klebsiella pneumoniae; Metabolomics.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

145

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

New PDE4 Inhibitors: Design, ADMET and Docking Studies on Chalcones and Flavones for Anti-Inflammatory Activities Idris M. H. M.1,2, Siti Norhidayu Mohd Amin1,2, Siti Norhidayah Mohd Amin1,2, Manikandan Selvaraj1,2, Mohd Zaki Salleh1,2 and Teh Lay Kek*1,2 1

Abstract: Background: Phosphodiesterase type 4 (PDE4) regulates cyclic adenosine monophosphate (cAMP) which acts as intracellular secondary messenger by hydrolysis. Selective inhibition of PDE4 therefore elevates cAMP which then downregulate inflammation. Chalcones and flavones with anti-inflammatory properties which inhibit PDE4 are potential lead compounds as anti-inflammatory and analgesic agents. Methods: Computational studies were undertaken to test the inhibitory scaffold of 18 synthesized chalcones and flavones on PDE4. Structures of chalcones and flavones were drawn using Marvin Sketch 16.2.8. Protein crystal structure with rolipram (PDB ID: 1OYN) was retrieved from Protein Database Bank (PDB), US. Docking study was performed using Glide 6.9. ADMET properties of the compounds were calculated using QikProp and ACD/I-Lab. Results: The docking result of chalcones showed that the binding energies were in the range of -4.258 kcal/mol to -10.209 kcal/mol. For flavones, the range of binding energies were 5.282 kcal/mol to -7.552 kcal/mol. However, five (5) out of the fifteen (15) chalcones and one (1) of the three (3) flavones showed good binding pose as compared to rolipram. For druglikeness properties, ten (10) chalcones and three (3) flavones fulfilled the “Lipinski’s Rule of Five” criteria while four (4) chalcones and one (1) flavone were predicted to cause genotoxicity. In addition, eleven (11) chalcones and three (3) flavones were predicted to have good ADMET properties. Conclusion: Based on the binding energies, chalcones and flavones are potentially new PDE4 inhibitors. Keywords: Anti-inflammatory; Cyclic adenosine monophosphate; In silico.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

146

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Effect of Telfaira occidentalis in Mice Fed Aflatoxin Contaminated Feed Ndanusa Abdullahi Hassan1*, T.A Gbodi2, Rohini Karunakaran1, Uma Sankar A1, Khin Mar Aye1, Ahmad Alhaji2, Umar Muazu2 1

Unit of Biochemistry, Faculty of Medicine, AIMST University, Malaysia. Department of Biochemistry, Ibrahim Badamasi Babangida University, Lapia, Niger state, Nigeria; *corresponding author, e-mail: ndanusabb@gmail.com, Ph No: 0149321340

Abstract: Background: Aflatoxin is potent hepatotoxic and hepatocarcinogenic agents. This hepatotoxicity is thought to be mediated by its ability to generate reactive oxygen species and cause peroxidative damage. This study investigated possible effect of pumpkin (Telfaira occidentalis) in ameliorating the toxic effect of aflatoxin using animal model. Methods: Twenty albino mice were procured, grouped into four of five groups each and allowed to acclimatize for one week. Aflatoxigenic Aspergillus flavus, cultured groundnut cake was used for oral aflatoxin exposure. Group 1 served as the control and fed commercial feed. Group 2 received 2.5g of aflatoxin contaminated diet and commercial feed. Group 3 received 2.5g+0.1g of T.occidentalis leaves powder+ commercial feed. Group 4 received 2.5g+0.2g of T.occidentalis leaves powder+ commercial feed. Group 5 received only 0.1g of T.occidentalis leaves powder+ commercial feed. The diet was administered daily for the period of 3weeks. Blood glucose test was carried out at the end of each week using acute check active glucometer. At the end of the experiment blood samples were collected from the mice through ocular puncture into clean containers. The samples were analyzed for liver function test (Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Total protein, Albumin, Bilirubin.) and renal function test (RFTs) (Urea, Sodium, Potassium, Chloride, creatinine). Results & Conclusion: From the results, there was significant (p<0.05) decrease in blood glucose levels of all the groups of animals after week 2 and week 3 as compared to the control group 1. There was significant difference in renal function test values of urea, sodium, potassium and creatinine in all the groups and there was no significant difference between group 2, 3, 4 and 5 of chloride as compared to the control group 1. Also there was significant difference between AST, ALT, TP, ALB, and BL. The significant changes in the above mentioned parameters were likely to be due to toxic effect of aflatoxin contaminated feed. From the findings of these studies, T. occidentalis may have a potential to reduce the toxic effect of aflatoxin in diet. Keywords: Aflatoxin; Antioxidant; Aspergillus flavus; Telfairia occidentalis.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

147

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Accuracy of Rapid Point-of-Care Diagnostic Tests for Acquired Immune Deficiency Syndrome - A Systematic Review and Meta-analysis Paramasivam R.1,*, Veeramachineni A.K.1, Janarthanan P.1, Sathasivam T.2, and Langford S.J.1 1

School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia. 2 School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia; *corresponding author, e-mail: rpar21@student.monash.edu.my, Ph No: (+60)1111887169

Abstract: Background: Rapid point-of-care tests provide a feasible diagnostic strategy for HIV detection in low resource areas. However, rapid assays are relatively fallacious than conventional ELISA and Western blot technique. We performed a systematic review to assess the diagnostic accuracy of multiple point-of-care rapid assay platforms for HIV detection according to standard methods and summarized test performance using metaanalysis. Methods: A computer-aided search of MEDLINE (1950-March 2016), EBSCO (1966-June 2016), OVID database (1966 to January 2016), and EMBASE (1974- January 2016) was performed for relevant publications. Reports meeting inclusion criteria of rapid assays performed with serum, oral and urine samples for antigen and/or antibody detection of HIV was assessed for methodological quality by using the QUADAS 2. Meta-DiSc, a Windows-based, user-friendly, freely available software was used to perform and validate diagnostic meta-analysis. Results: Out of 2724 citations which were identified and screened from four databases, 86 rapid assays which met the inclusion criteria were included in the study. Four themes were identifies 1) serological assays yield a higher pooled sensitivity and specificity than urine or oral saliva-based assays, 2) antibody + antigen detecting combination assays are relatively better than antigen or antibody detection assay, 3) Multi-antigen assays yield a better pooled sensitivity and specificity than single-antigen or whole cell antigen assays and 4) HIV 1/2 detecting combination assays are relatively better than HIV 1 and HIV 2 detection assays. Conclusion: Most reported studies are conducted on small sample number which misleads the diagnostic accuracy of the assay, this is overcome by understanding the pooled sensitivity and specificity using meta-analysis of the reported studies. It is clear that serological assays that can detect both HIV 1/2 antibody and antigen will yield higher pooled sensitivity and specificity.

Keywords: Acquired immune deficiency syndrome; Meta-analysis; Meta-disc; Rapid assay.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

148

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Biocontrol of Macergen Infestation on Plants using Bacteriophage Cocktail Sasireigga J.*, Ravichandran M., and Kurunathan S. Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: sasi7278@gmail.com, Ph No: +60194451285.

Background : Macergens is a term used to describe bacteria capable of releasing pectic enzymes that degrades the plant cell wall which leads to maceration, a symptom associated to soft rot disease. Among these macergens, Dickeya chrysanthemi has been reported to cause stem rot disease in several important crops such as potato, tomato, lettuces and papaya. As bacteriophages are well known potent biocontrol agents, we were interested in isolating and examining the efficiency of novel bacteriophages in controlling the progression of soft-rot disease in plants infected by D. chrysanthemi. This has been our interest as there has been limited studies on isolation of bacteriophage and its application against D. chrysanthemi infected plants. Methods : Bacteriophages were isolated from soil and water sample by primary enrichment method. Host range of bacteriophages was assessed using spot tests. Biocontrol test was performed on three weeks old young seedlings of chill, papaya and tomato. The young seedlings were inoculated by syringe infiltration with D. chrysanthemi bacterial cell suspensions after wounding the stems with sterilized scalpers. Bacteriophage cocktail were applied on the young seedlings by spraying method after infected with D. chrysanthemi. Results: Five lytic bacteriophages were isolated against D. chrysanthemi. All the five bacteriophages showed high specificity against D. chrysanthemi. Interestingly, stem rot disease caused by D. chrysanthemi on these plants were greatly reduced by cocktail of bacteriophages within 24 h. It was also observed that the growth of plants treated with bacteriophage cocktail after D. chrysanthemi infection were similar with untreated sample and it had no stem rot symtoms were recorded. Conclusion: This study had successfully isolated bacteriophages that have the potential to be used as a biocontrol agent to prevent stem rot disease caused by D. chrysanthemi. Key words: Bacteriophages; Biocontrol; Dickeya chrysanthemi; Macergen.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

149

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Oral Bacterial Diversity Study in Malay Ethnic Group in Malaysia Kah Man Woh1, Kameswari K.2, Sivakumar P.2, Lay Kek Teh3, Moh Zaki Salleh3, Subhash J Bhore*1 1

Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong Semeling Road, Semeling 08100, Kedah, Malaysia; 2Faculty of Dentistry, AIMST University, Bedong Semeling Road, Semeling 08100, Kedah, Malaysia; 3Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Puncak Alam, Selangor DE, Malaysia; *corresponding author, e-mail: subhash@aimst.edu.my, subhashbhore@gmail.com, Ph No: (+60) 4 429 8176

Abstract: Background: Human body contain several indigenous microorganisms that vary at different anatomical sites. Human oral cavity is one of the most diverse sites of microorganisms. It is estimated that approximately 500 to 700 different bacterial species might be existing in human oral cavity. Malaysia is a multi-racial country with different lifestyles, cultures and eating habits. In Malaysia, Malay is a major ethnic group and limited information is available about this communityâ&#x20AC;&#x2122;s oral bacterial diversity. Therefore, this research project was undertaken. The objective of this research project was to elucidate the oral bacterial diversity among healthy subjects of Malay community. Methods: Based on consent, eleven (11) healthy subjects were selected randomly from Malay community. From oral cavity of selected subjects, saliva and sub-gingival plaques samples were collected. Separately, saliva and sub-gingival plaques samples were pooled together and bacterial DNA samples were prepared using kit. Metagenomics approach was used to elucidate DNA sequence based identities of the bacteria using Illumina, a next generation sequencing (NGS) platform. Results: The primary analysis of results indicates that 441 types of bacteria were present in the saliva samples of Malay subjects. The analysis of the data generated from subgingival plaques samples suggest that 325 types of bacteria were embedded in the subgingival plaque samples. Data analysis suggests that 166 bacterial species were common in saliva and sub-gingival plaque samples. Conclusion: Based on primary analysis of the results data, we conclude that about 600 different types of bacteria are harboured in the oral cavity of the healthy subjects from the Malay community of Malaysia. Our research findings clearly elucidate the oral bacterial diversity and these finding could serve as foundation for the further study in understanding the connection between oral bacterial diversity and the oral health or overall health of the individuals. Keywords: 16S rRNA; Bacteria; Human oral cavity; Illumina; Malay ethnic group; Ribosomal DNA; Saliva; Sub-gingival plaque.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

150

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Isolation and Characterization of Seven Lytic Bacteriophages As Candidates for Phage Therapy Sanirbandha C., Vickneswaran N.M., Sivachandran P., Ravichandran M., Lee, S.Y.* Department of Biotechnology, Faculty of Applied Sciences, AIMST, University, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: su_yin@aimst.edu.my, Ph. No: +604-4298177 Abstract: Background: Bacteriophages are bacteria-speciďŹ c viruses that can infect and destroy their host bacteria. Phage therapy has been used to combat bacterial infections. In order to understand the mechanism in which bacteriophage uses its lytic enzymes to kill its host, we have isolated and characterized 7 novel bacteriophages from various environmental samples. Methods: Water and soil samples from different environmental sources were used to isolate bacteriophages. The samples were processed through membrane filtration and eventually spotted on a range of bacterial strains to identify the possible host. After enrichment with the bacterial host for 72 hours, the bacteriophage was recovered by membrane filtration. Morphological characterization of each bacteriophage was performed using transmission electron microscopy (TEM) to determine the morphology and the type of nuclear material was also determined by S1 nuclease and DNase digestion. Results: Seven different bacteriophages were isolated from the environmental samples. The phages were found to be specific against Salmonella paratyphi A (SPA-S), Salmonella paratyphi B (SPBS and SPB-V), Salmonella paratyphi C (SPC-S and SPC-V), Salmonella typhi (ST-S) and Citrobacter freundii (CF-S). Nuclease digestion revealed that SPA-S, SPC-S, ST-S, CF-S, SPB-V, SPC-V were double-stranded DNA phages, while SPB-S was a single-stranded DNA phage. TEM analysis showed that the six double-stranded DNA phages had T4 or Îť phagelike structure with non-enveloped contractile tail, while the single-stranded DNA phage (SPB-S) was seen to be non-enveloped rod-shaped in structure. Conclusion: The results of this study paves the way for further studies into whole genome sequencing of the bacteriophages and to understand the function of specific genes, such as lysin that can be used for phage therapy in the future. Keywords: Bacteriophage; Enrichment; Environmental samples, Isolation; TEM.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

151

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Transcriptome Analysis for the Identification of Novel ncRNAs in Acinetobacter baumannii Saw H. S., Sumitha S., Kishan Raj S., Xavier R., Suresh V.C.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong Semeling Road, Semeling 08100, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Multidrug-resistant Acinetobacter baumannii is recognized to be among the most difficult antimicrobial-resistant Gram-negative bacilli to control and treat. Study of ncRNA of A. baumannii would shed light on the unexplored regulation in understanding the pathogenicity of this organism. In general, recent reports indicated that ncRNA plays important role in regulation of metabolic pathways, gene expression and pathogenicity of several bacteria. Methods: In our study, we used A. baumannii transcriptome data from NCBI to identify the novel ncRNA candidates. In parallel, we also performed genome wise search for ncRNA genes using computational prediction software nocoRNAc. The transcriptome data of A. baumannii was analyzed through the genome viewer Artemis to screen the un-annotated transcripts, which were further analyzed for the absence of ORF and having no hit in Rfam and Genbank database. The RPKM value and read count of the transcripts were also calculated by creating ncRNA candidates gff file. Results: Total 637 ncRNA transcripts are predicted by nocoRNAc. Transcriptome data disclosed 50 possible ncRNA candidates. Among these, 5 could be possible novel protein coding genes as they are possessing possible novel ORFs. Finally 13 were identified as the potential ncRNA candidates, which are further validating their expression by Northern blot analysis. Conclusion: In this study we identified 13 novel ncRNAs in A. baumannii and validation of their expression is under progress. This could serve as foundation for further understanding of the gene regulation in this bacteria. Keywords: Acinetobacter baumannii; Bioinformatics; Non-coding RNA; Transcriptome.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

152

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Computational Modelling of the Newly Synthesized Chalcone Derivatives in Inhibiting 5-lipoxygenase Siti Norhidayah M.A., Teh L.K., Manikandan S. and Mohd Zaki S.* Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, email: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 32584652.

Abstract: Background: 5-lipoxygenase (5-LOX) is an enzyme that is involved in inflammation. Thus, inhibiting this enzyme will reduce undesired inflammatory reaction. Currently, the commercially available 5-LOX inhibitor is Zileuton, but it is less widely prescribed due to its side effects. Therefore, this study aims to design new alternative 5-LOX inhibitor using molecular docking approach. Methods: 5-LOX crystal structure was retrieved from Protein Database Bank. The structures of the compounds which are derivatives of chalcones synthesised in-house were drawn using Marvin Sketch. The active site of 5-LOX enzyme was predicted using SiteMap. GOLD and AutoDock softwares were used to investigate the parameters of the complex of synthetic chalcones with 5-LOX enzyme. The docking results were compared with the standard reference ligand (Zileuton). Results: 10 (ten) out of 15 (fifteen) synthetic chalcones had higher fitness score and lower binding energy compared to the standard reference ligand when docked with 5-LOX. The fitness scores shown by GOLD ranged from 54.19 to 66.21. On the other hand, the binding energies of the compounds as shown by AutoDock were within the range of -8.98 to -5.76 kcal/mol. Conclusion: Ten newly synthesised derivatives of the chalcones have potential as 5-LOX inhibitor. The in vitro and in vivo studies elucidating the mechanism of the compounds are currently being carried out. Keywords: Bioinformatics; In vitro; Lipoxygenase chalcone; Molecular docking.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

153

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Computational Design of Flavone and Chalcone Derivatives as Cyclooxygenase-2 (COX-2) Inhibitor Siti Norhidayu M.A., Teh L.K., Manikandan S. and Salleh, M.Z.* Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia; *corresponding author, email: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 32584652

Abstract: Background: Cyclooxygenase-2 (COX-2) is an enzyme responsible for the conversion of prostaglandin H2 to prostanoids; which are the crucial mediators of inflammation. Inhibition of the COX-2 enzyme is reduced or inhibited the proinflammatory enzyme activity. We aim to evaluate the inhibitory efficiency of chalcone and flavone derivatives for COX-2 and to identify compounds that are selective to COX-2. Methods: In the present work, we evaluated the interaction of compounds with COX-1 (PDB ID: 1Q4G) and COX-2 (PDB ID: 3LN1) using GLIDE and GOLD modelling software. The flavone and chalcone derivatives were synthesized, and the structures of the compounds were drawn using Schrodinger. Structure of the new compounds was verfied for their similarity with other existing compounds in ChemSpider database. Before proceeding to molecular docking, Qikprop were used to screen the compounds for their ADME properties, and compounds were excluded if it violates more than one Lipinski's rule of five. Results: Most of the compounds have shown pi interactions with TYR385, TYR355, TRP387, and ARG120 of COX-2. 8-iodo-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (F3) was found to have interaction with COX-2 only. Conclusion: We conclude that F3 could be a potent antiinflammatory compound based on the molecular interaction studies. Keywords: Chalcone; Cyclooxygenase; Flavone; Molecular docking.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

154

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Identification of Novel npcRNA Candidates in Klebsiella pneumoniae Sridevi V., Sumitha S., Kishan S. and Suresh CV.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, Bedong 08100, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph No: +604 42988165

Abstract: Background: Small non protein-coding RNAs (npcRNAs) have emerged as the important regulators of many cellular pathways in bacteria. These npcRNAs regulate the virulence of bacteria by interfering with the target mRNAs involved in bacterial pathophysiology. The Klebsiella pneumoniae, a gram negative bacterium, is associated with various infectious diseases in human, including pneumonia and urinary tract infections. Due to the emergence of antibiotic resistance strains, there is an increasing need to identify novel npcRNAs involved in virulence and antibiotic resistance of Klebsiella pneumoniae. Methods: We used various computational tools to identify novel npcRNA candidates from the transcriptome of Klebsiella pneumoniae HS11286. The intergenic (un-annotated) transcripts were selected by viewing transcriptome bam file on Artemis. The most possible npcRNA candidates were identified by further screening for the absence of ORF and no hit in Rfam database. Total RNA of bacteria during different growth phases and stress conditions were extracted using Trizol reagent and transferred to the membrane for Northern blot hybridization. Results: A total of 238 intergenic transcripts were selected from the transcriptome of Klebsiella pneumoniae. By using ORF finder, 137 of these are possessing possible ORF and could be potential novel protein coding genes. Interestingly, 14 out of these remaining 101 transcripts were found in Rfam database as known npcRNA in other bacteria. So, finally we have identified 87 most potential npcRNA candidates in Klebsiella pneumoniae. Total RNA extracted was highly intact and was confirmed by Bioanalyzer. Conclusion: Totally 87 potential npcRNAs were identified and confirmation of the expression of these npcRNAs during different stress conditions is in progress. This novel npcRNA candidates can fill the gaps in further understanding of the regulation of virulence of the organism. Keywords: Klebsiella; Non-coding RNA; npcRNA; Trascriptome.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

155

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Arduino Microcontroller Based Heart Rate Monitor using Fingertip Sensors Srilahari Namani1,*, Manickam Ramasamy1, Sunitha Namani2, Satyanarayana Namani2 1 2

Faculty of Engineering and Computer Technology, AIMST University, Malaysia. Faculty of Medicine, AIMST University, Malaysia; *corresponding author, e-mail: srilahari4u@gmail.com, Ph No: +60164375166

Abstract: Background: The main aim of this project is to develop a portable device which helps to record heart rate of a person. The heart rate also referred as pulse rate, has been recognized as the most important vital parameter which helps the individual as well as doctor to spot out developing health problems. Using Heart Rate Monitor (HRM) is a more accurate way to monitor heart rate than manually taking your pulse at carotid and radial pulse. A Heart Rate Monitor (HRM) detects the electronic signal of heart and automatically computes the heart rate in BPM. Method: It is a portable heart rate device developed using Arduino microcontroller and infrared sensors to detect the heartbeat. It is the non-invasive method of determining heart rate. Transmittance and Reflectance are two basic types of photoplethysmography (PPG). Reflectance (PPG) is used here.An Infrared Light Emitting Diode is used as a transmitter and a Phototransistor is used as receiver. The Infrared (IR) Sensors use principle of reflectance plethysmography (PPG) to sense the pulse signal from finger tip. The light source and the light detector are both placed on the same side of a body part. The light is emitted into the tissue and the reflected light is measured by the detector. As the light doesnâ&#x20AC;&#x2122;t have to penetrate the body, the reflectance PPG can be applied to any parts of human body.The sensor output is read by the arduino board, computes the Beats per Minute (BPM) and display the instantaneous heart rate on LCD display module. Results: Heart beat waveform was observed in Oscilloscope. It was able to record heart rate of different people and display on LCD module. Conclusion: Arduino Based heart monitor is able to detect Heart rate of a person. It is able to measure heart rate of the person and display BPM on LCD display module. Keywords: Arduino microcontroller; BP; Heart Rate Monitor (HRM); Health; Infrared sensors.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

156

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Transcriptome Analysis of Proteus mirabilis during Oxidative Stress Adaptation Sumitha S.1, Kishan S.1, Yukgehnaish K.1, Laurence J.C.2, Xavier R.1, Suresh C.V.*1 1

AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; 2Malaysian Genomics Resource Centre Berhad, Mid Valley City, 59200 Kuala Lumpur, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Bacteria are well known for their fast adaptation to the stress condition. Bacteria undergo some changes in the gene expression which enable us to understand their adaptation to the stress condition. A transcriptome study is conducted to compare the differential gene expression of ncRNA and mRNAs between normal and oxidative stress condition of P. mirabilis. This study elucidates novel ncRNAs and also enables us to understand the adaptation of P. mirabilis during stress. Methods: P. mirabilis cultured in Luria-Bertani broth and the total RNA was extracted during exponential and oxidative stress. These total RNA were sequenced via Illumina HiSeq 2000 platform. The fastQ format transcriptome sequences were analysed using bioinformatics software tools such as Trimmomatic and Bowtie2. Un-annotated intergenic regions were screened for the possible novel ncRNA candidates using Artemis. Differential expression of mRNAs and ncRNAs was analysed using HTSeq and DESeq software. Results: A total of 207 possible ncRNA candidates were identified. By verifying the annotation in other bacteria and Rfam database, 52 were selected as potential ncRNAs. Interestingly, 26 ncRNAs are up-regulated while other 26 ncRNAs are down-regulated during oxidative stress condition. Out of 3460 genes, 1693 and 1688 showed significantly up and down regulations respectively. Most of the phage protein and dimethyl sulfoxide reductase genes are up-regulated. The genes coding for respiratory nitrate reductase, tetrathionate reductase, flagellar related proteins, tryptophan synthase and alkyl hydroperoxide reductase were shown to be down-regulated during oxidative stress. Conclusion: This transcriptome analysis data reveals both protein coding and non- coding genes are playing a major role in bacterial oxidative stress adaptation. A total of 26 novel and potential ncRNAs have been identified in P. mirabilis. Keywords: Differential expression; ncRNA; Oxidative stress; P. mirabilis; Transcriptome.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

157

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Safety and antiobesity effect of Garcinia atroviridis Suriati Mohd Nasir1, Teh Lay Kek1,2, Mohd Zaki Salleh*1,2 1

Integrative Pharmacogenomic Institute (iPROMISE), Level 7, FF3 Building, Universiti Teknologi MARA (UiTM) Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia. 2Faculty of Pharmacy Universiti Teknologi MARA (UiTM) Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; *corresponding author, e-mail: zakisalleh@puncakalam.uitm.edu.my, Ph No: (+603) 3258 4652

Abstract: Background: Obesity is a global health problem affecting both males and females. Many attempt to reduce body weight by different approaches, including the consumption of natural product with antiobesity effect. Garcinia atroviridis (GA) was claimed to have antiobesity properties due to the presence of hydroxycitric acid (HCA).The aim of this study is to elucidate safety andantiobesity effect of methanolic extract of Garcinia atroviridis fruit. Methods: Acute and sub-acute toxicity study was conducted according to OECD 407 and 423 guidelines, respectively. Forty-eight(48) female sprague-dawley rats were divided into six groups (n=8). Obese rat model was developed using high fat diet (60% fat, Research diet, USA). Then the treatment was given orally until significant reduction in body weight gain was observed. Results: No toxicity effect was observed in the animalfollowing the treatment. Histological study revealed no lesions or pathological changes in the organs of either sex of the GA treated rats compared to control groups. Obese rats treated with GA showed significant reduction in body weight gain. Conclusion: We conclude that GA is a potential antiobesity agent. Keywords: Antiobesity; Diet; Fat; Health; Oil; Rats.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

158

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Development of Ochratoxin A Detection Based on Electrochemical Sensor by Using Au-ball Labels Suttiporn. P.*1, Patsamon. R2 and Werasak. S3 1

Food Technology, School of Agro-Industry, Mae Fah Luang University,333 Moo 1 Thasud Muang, Chiang Rai, Thailand; 2National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park Phahonyothib Road Klong 1 Klong Luang, Pathumthani, Thailand; 3School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 83 Moo 8 Thakham Bangkhuntein, Bangkok, Thailand; *corresponding author, e-mail: suttiporn.pin@mfu.ac.th, Ph No: (+66)923808281

Abstract: Background: There are increasing international attention to the problem of ochratoxin A (OTA) contamination in coffee. This toxin represents a risk for human and animal health when ingested through contaminated food.Therefore, developing a highly sensitive and rapid method for OTA detection is necessary. Methods: In this study, an electrochemical peptidesensor for OTA detection was developed using numerous number of Au particles coated on the surface of silica particle (Au-ball). Moreover, this assay was performed in 384 well plate, so the multiple detections was done. Results: The synthesized silica particle was spherical in shape and size was 275±17 nm. After coated with Au layer, size of Au-ball was 280±14 nn. Au particles loaded can be taken up resulting in approx. 1 x 107 Au3+ molecules per silica particle. Moreover, the optimization conditions were studied. The limit of detection of this assay showed as low as 2 ppb. Conclusion: This platform showed rapid, sensitive and specific to ochratoxin A detection. In spite of these advantages, this Au-ball based peptidesensors is suitable to use in the detection of ochratoxin A contaminated in coffee or seed industry. Keywords: Anodic stripping voltammetry; Biosensor; Ochratoxin A; Peptide.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

159

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Effect of Different Diets on the Growth and Survival of Silver Arowana (Osteoglossum bichirrosum) Tan W.S, Sivachandran P., Kurunathan S., Marimuthu K.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: aquamuthu2k@gmail.com, Ph. No: +60164723672

Abstract: Background: Silver arowana, (Osteoglossum bichirrosum) is a bony-tongue fish native to South America and is one among the highly demanded ornamental fish species in Asian countries. The present study investigated the effect of different diets on the growth and survival of silver arowana. Methods: The experimental set up consists of 12 glass aquarium tanks and each tank was bifurcated into three compartments and stocked with one fish for each compartment. The initial length and weight were 9.0 ± 0.26 mm and 4.05 ± 0.30 g, respectively. The fish were fed with four different types of diets namely, fish pellet, mealworms, frozen bloodworms and mosquito fish for six months. Total weight and total length of the fish and growth and survival was monitored each month. The weight gain and specific growth rate (SGR) were calculated. Results: The study results found that, the highest weight gain (56.335 ± 9.003 g) and SGR (1.471) was observed in fish fed with fish pellets when compared to all the diets tested. Significantly lower weight gain (16.062 ±1.808) and growth performance was observed in fish fed with bloodworms. Conclusion: The present study is the first kind of its type and evaluated the growth performance of silver arowana fed with four diets under laboratory conditions. Results obtained suggest that artificial pelleted feed is the best for larval rearing of silver arowana. Keywords: Feed; Growth; Osteoglossum bichirrosum; Silver arowana.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

160

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Effect of Aqueous and Methanol Extracts of Polygonum minus Leaves on Drug-Induced Hepatotoxicity in Rats Thanapakiam G, Lim S.Y, Grace K.Y.X, Loo S.W, Amutha V. V, Pavitra L, Neoh H. F, Ahmad H.R, *Christapher P.V Faculty of Pharmacy, AIMST University, Semeling 08100, Malaysia; *corresponding author, e-mail: christapher@aimst.edu.my, Ph No: +60 44298000 (extn: 1029)

Abstract: Background: Polygonum minus, a commonly available plant in Southeast Asia. This plant is traditionally used for ailments such as pain relief, as a tonic after child birth and to remove dandruff. Presence of high content of flavonoids and phenolic compounds is responsible for its excellent antioxidant activity. The reported pharmacological studies include anti-inflammatory, analgesic, antiulcer and cognitive improvement effects. Our study aimed to evaluate the hepatoprotective activity of aqueous and methanol extract of P. minus leaves in paracetamol-induced hepatotoxicity in rat models. Method: Dried leaves of P. minus were powdered and macerated for 5 days using distilled water and methanol, separately, filtered and evaporated to obtain dry crude extracts. Hepatic cell damage was induced in Sprague Dawley rats by the administration of paracetamol (750 mg/kg; paracetamol at every 72 h for 10 days) for all groups except the normal control (1 ml of CMC was administered). All the treated groups were administered with either standard or extract according to the treatment protocol. Parameters such as body weight, biochemical and histopathology studies were studied. Results: Animalsâ&#x20AC;&#x2122; body weights did not show significant variation in any treated group, compared with normal control, but was significantly decreased in paracetamol control group. The biochemical analysis showed that the higher dose of both extracts treated groups exhibited significant variations from the paracetamol control group. The histopathology study also confirmed the hepatoprotective effects of both extracts. Conclusion: Aqueous and methanolic P. minus extracts possess significant hepatoprotective activity. More detailed studies are required to establish its hepatoprotective efficiency. Keywords: Hepatoprotective effect; Liver damage; Paracetamol; Polygonum minus.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

161

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Identification of Novel Non-protein Coding RNAs (ncRNAs) in Staphylococcus haemolyticus Biofilm Thurga Devi N., Saw H.S., Kishan S., Sumitha S., Xavier R., Suresh V. C.* AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Staphylococcus haemolyticus is an opportunistic nosocomial bacterial pathogen often cause wide range of infections as simple as acne to serious infections like endocarditis, peritonitis and UTI. Its ability to form biofilms especially on medical devices is a major virulence factor associated with S. haemolyticus. Non-coding RNAs, the newly emerged class of RNAs, having increasing evidence on their role in the regulation of majority of the bacterial pathways including virulence and biofilm formation. This study elucidates novel ncRNAs and also enables us to understand the adaptation of S. haemolyticus to the different environment. Methods: S. haemolyticus was cultured in specialized media to form biofilm. Total RNA was extracted and sequenced using Illumina platform. The transcriptome sequences were aligned with reference genome of S. haemolyticus using Bowtie2. Using genome viewer, un-annotated transcripts were selected and performed sequence search similarity in other organisms using Blastn and Rfam database respectively. The total RNA extracted from S. haemolyticus grown under different stress conditions, resolved on denaturing gel and trans-blot to nylon membrane for Northern hybridization. Results: A total of 147 possible ncRNA candidates identified and after filtering through blastn, ORF search and Rfam, 68 were selected as most potential novel ncRNAs. 13 ncRNAs with the highest RPKM value has been chosen for Northern hybridization. Interestingly 64 of these 68 ncRNAs are specific to S. haemolyticus. Conclusion: This is the pioneering study reveals 68 novel ncRNAs in S. haemolyticus whose expression is under the progress of validating by Northern blot hybridization. Further research need to be done to deeply understand the possible function of ncRNA of S. haemolyticus. Keywords: Biofilm; ncRNA; Staphylococcus haemolyticus; Transcriptome.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

162

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Application of a Novel Lytic Bacteriophage Strain as Biocontrol Agent for Water Sanitization Vickneswaran N.M., Sanirbandha C., Sivachandran P., Ravichandran M., Lee, S.Y.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: su_yin@aimst.edu.my, Ph. No: +604-4298177

Abstract: Background: Bacteriophages are viruses that are parasitic on bacteria, and they have been considered as one of the agents for treating bacterial infections. Bacteriophages have a special adaptation to lyse the specific host cell by using the lysin enzyme. The aim of this study is to investigate the use of bacteriophages as biocontrol agent for water sanitization. Methods: Bacteriophages were isolated from various environmental samples by filtration using 0.22Âľm and 0.45Âľm membrane filters. Spot test on different bacterial lawn cultures was used to detect presence of life phages and determine host specificity. Morphological characterization was done by using transmission electron microscopy (TEM). The biocontrol study was done by inoculating the bacteriophage in pond water and LB culture (as control), followed by incubation for 5 days. Each day, the phage titer (PFU/ml) and the bacteria titer (CFU/ml) were enumerated to determine the effectiveness of the bacteriophage as biocontrol agent. Results: Three novel strains of lytic bacteriophages were isolated, which was specific towards Salmonella paratyphi a, Salmonella enteritidis and Salmonella typhimurium. TEM analysis revealed that the unique structure of protein coat of this strain as icosahedral head with longitudinal tail that is from the myoviridae family. The biocontrol studies with the 3 phages showed that the bacterial titer reduced after 20 hours post-inoculation, while the phage titer continued to increase until 100 hours post-inoculation in both pond water and LB control media. The inverse relationship of the bacterial and phage titres showed that the phages were able to control the bacterial growth and subsequently multiply. Conclusion: The study showed that the isolated 3 strains of bacteriophage have the potential to be used as effective biocontrol agent for sanitization of water and infection control. Keywords: Bacteriophage; Biocontrol agent; Lysin; Plaque assay.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

163

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Phytochemical Analysis and Pharmacological Screening (Dopamine level) of the Ethanolic Extract of Solanum trilobatum Linn. Stephanie Wong Kah Yee, Bobby Lau Chik Chuon, Lee Yu Ren, Christapher Parayil Varghese, Subramani Parasuraman* Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Malaysia; *corresponding author, e-mail: parasuraman@aimst.edu.my, Ph No: (+60) 103895096

Abstract: Background: Solanum trilobatum Linn., (Solanaceae) is known as thoodhuvalai in Tamil. The leaves of S. trilobatum are most widely used as food supplement in southern part of Indian subcontinent and also this plant was traditionally used for the treatment of respiratory illness. In pre-clinical studies, the extract of S. trilobatum showed favorable antimicrobial, hepatoprotective and anticancer activities. The complete phytochemical profile and dopaminergic activity of the S. trilobatum remain unclear, hence the present study was planned to carry out the Gas Chromatography Mass Spectrometry (GC-MS) analysis and dopaminergic activity of ethanolic extract of S. trilobatum (EEST). Methods: The phytochemical analysis was carried out using chemical and instrumental (GC-MS) analytical methods. The dopaminergic activity of EEST was determined in Sprague Dawley (SD) rats. The rats were divided into two groups each of five animals. Rats were treated with vehicle and EEST at 400 mg/kg, once daily for 30 days. During the study, body weight variations and behavioral alterations were monitored. At the end of the study, rats were sacrificed, brain was collected and weighed. The brain homogenate was used for the estimation of dopamine levels using UV-visible spectrophotometer. Results: The phytochemical analysis showed the presence of carbohydrates, saponins, flavonoids, alkaloids, tannins and phenolic compounds, and GC-MS analysis showed the presence of 44 fragmented compounds which included epoxylinalol, himachalol, illudol, epibuphanamine, baimuxinal and edulan IV. On chronic administration, EEST increased the dopamine (*P<0.05) levels in brain tissue and not having any significant effect on immunization time, locomotor activity when compared with the control. Conclusion: EEST has significantly increased the dopamine levels in rat brain and doesnâ&#x20AC;&#x2122;t alter the behaviour of the animals. Keywords: Dopamine; GC-MS analysis; Solanum trilobatum.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

164

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Computational Analysis of Common Bean (Phaseolus vulgaris L.) Sadenosyl-L-methionine dependent methyltransferase gene cDNA Isolated from Bean-pod-tissue cDNA Library Cheah V.S.1, Amelia K.2, and Bhore S.J.*1, 2 1

Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling 08100, Kedah, Malaysia; 2Department of Molecular Biology, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka; *corresponding author, e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com, Ph No: +604 429 8176.

Abstract: Background: Common bean (Phaseolus vulgaris L.) is one of the legumes cultivated in many countries. Common beans are important in diet as a source of proteins especially for the poor people; because, it is inexpensive as compare to other sources of proteins. In order to study the expressed genes, transcriptomics study was initiated and about 6000 ESTs were generated. While processing data, we identified a complimentary DNA (cDNA) clone of S-adenosyl-L-Methionine-dependent Methyltransferase (PvSAM MTase) gene and to understand more about it this study was undertaken. The objective of this study was to annotate PvSAM MTase cDNA and deduced amino acid sequence using computational tools. Methods: Both strands of PvSAM MTase cDNA clone were sequenced using M13 forward and M13 reverse primer to reveal the nucleotide sequence. Online bioinformatics tools were used for analysis and annotation of both cDNA and deduced protein sequences. Sequence comparison was carried out using Blastp whereas split tree program was used to construct a phylogenetic tree. The secondary and tertiary structure was predicted using PHYRE automatic fold recognition server. Results: The cDNA and deduced protein sequence analysis showed that PvSAM MTase gene cDNA is 1312 bp in length and its open reading frame (ORF) encodes for 344 amino acids residues. The phylogenetic analysis suggest that PvSAM MTase protein is closely related to its counterpart from Vigna radiata L. The PvSAM MTase protein structure was successfully predicted and will be discussed. Conclusion: The cDNA was annotated and primary analysis of the PvSAM MTase deduced protein sequence suggested that it contains catalytic sites essential for its function. Further study is required to validate the predicted structure of PvSAM MTase. Keywords: cDNA; Common bean; Phaseolus vulgaris L.; Protein structure prediction; SAM MTase.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

165

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Biochemical Changes in African catfish, Clarias gariepinus Exposed to Buprofezin Gobinath R., Marimuthu K., Xavier R., Suresh C.V.* AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165 Abstract: Background: Buprofezin, is an insecticide widely used in Malaysia for paddy cultivation. The study was carried out to investigate the biochemical changes in a freshwater African catďŹ sh, Clarias gariepinus exposed to different concentrations of buprofezin under laboratory conditions. The biochemical markers represent the most sensitive and relatively early events of pollutant damage. Thus, it is imperative to study the impacts of pollutants in aquatic organisms and delineate the mechanisms of pollutant action, and possible ways to mitigate the adverse effects. Methods: The 96 h LC50 value of buprofezin to C. gariepinus was estimated using probit analysis method. The experimental fishes were exposed to sublethal concentrations of the LC50 values of buprofezin at 0.15 mg/L, 0.30 mg/L and 0.60 mg/L for a period of 8 days. At the end of the 2nd, 4th and 8th day of the exposure periods, the fish blood sample was withdrawn and the concentrations of metabolites (glucose and total protein) and enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and alkaline phosphatase [ALP]) in all experimental fishes were determined. Results: The fish exposed to sub-lethal concentration of buprofezin showed several abnormal behaviours which including restlessness, loss of equilibrium, increased opercular activities, strong spasm, paralysis and sudden erratic movements. Biochemical analysis showed that, on the 2nd, 4th and 8th day of exposure, the amount of aspartate transaminase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT) and total protein levels were significantly declined. The increased amount of total glucose was observed in all the higher concentrations of buprofezin exposed fishes compared to control group. Conclusion: The present results elucidate that, the insecticide, buprofezin exposure had induced biochemical alterations in fishes. These biochemical parameters offer a rapid and sensitive means of monitoring towards the impact of buprofezin in fish model organisms. Keywords: African catfish; Biochemical parameters; Buprofezin; Clarias gariepinus; Sub letahl toxicity.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

166

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Haematological Changes in African catfish, Clarias gariepinus exposed to Buprofezin Gobinath R., Marimuthu K., Xavier R., Suresh C.V.* AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165 Abstract: Background: The study was carried out to investigate the haematological changes in a freshwater African catďŹ sh, Clarias gariepinus exposed to buprofezin under laboratory conditions. Buprofezin, is an insecticide widely used in Malaysia for paddy cultivation. Effects of different pesticides on hematological parameters of several fish species have been reported and most of the studies described on the effects of pesticides on the biochemical and physiological changes, but very little attention has been paid to the hematological modulation induce by buprofezin. Methods: The 96 h LC50 value of buprofezin to C. gariepinus was estimated using probit analysis method. The experimental fishes were exposed to sub-lethal concentrations of the LC50 buprofezin at 0.15 mg/L, 0.30 mg/L and 0.60 mg/L for a period of 8 days. At the end of the 2nd, 4th and 8th day of the exposure periods, fish blood sample was withdrawn and haematological parameters were determined. The cells with two nuclei were considered as binuclei (BN). The blood parameters white blood cell (WBC), neutrophil (NUE), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), red blood cell distribution width (RDW) and platelets (PLT) were analyzed by "Cell-Dyn Haemotology Analyzer". Results: Haematological changes have been observed in buprofezin treated fish in which, the amount of white blood cell, neutrophil, lymphocyte, monocyte, eosinophil, basophil, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red cell distribution width (RDW) and platelet were decreased from control to higher concentration of buprofezin exposed for the periods of 8 days. Amount of lymphocyte was increased from control to higher concentration of this treatment for the period of 8 days. These alterations have been attributed to direct responses of structural damage to RBC membranes resulting in haemolysis and impairment in haemoglobin synthesis induced by buprofezin exposure. Conclusion: The present study results showed that, the sub-lethal effect of buprofezin on adult catfish C. gariepinus, by measuring a set of haematological parameters. This study therefore gives an insight and baseline information about the toxicity of buprofezin in fish model organisms, African catfish, Clarias gariepinus. Keywords: African catfish; Buprofezin; Clarias gariepinus; Haematological parameters.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

167

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Identification of Novel Non-coding RNA (ncRNA) from Tannerella forsythia Kavithannjali K.1, Saw H.S. 1, Sumitha S. 1, Jawahar D.2, Suresh C.V1.* 1

AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; 2AIMST University, Faculty of Dentistry, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +6044298165

Abstract: Background: Tannerella forsythia is an anaerobic, Gram-negative bacteria of the Cytophaga Bacteroidetes family. It has been strongly implicated in the onset of periodontitis. T. forsythia is associated more frequently in higher levels with various forms of the diseases, including gingivitis, chronic and aggressive periodontitis, than normal. Thus, it is important to identify the virulence functions of the organism. One of the way to understand the virulence of T. forsythia through identification of ncRNAs as they play a significant role in regulation of fundamental bacterial adaptive processes including bacterial virulence. Methods: nocoRNAc software was used to identify the ncRNA genes in T. forsythia. Artemis viewer was used to select the predicted ncRNA genes at intergenic region of T. forsythia. The obtained candidates were screened with blastN and Rfam. Results: NocoRNAc predicted 429 ncRNA candidates in T. forsythia. Among these, 82 are derived from intergenic regions of T. forsythia. Further sequence similarity search against the annotated Genbank and Rfam databases showed 343 are annotated as protein coding genes and 80 as known ncRNA genes respectively. Finally 80 are identified as most possible ncRNA genes in T. forsythia. Interestingly, 53 of these are highly specific to T. forsythia. Conclusion: The study predicts 80 ncRNAs in T. forsythia which need to be further confirmed for their expression. The function and regulatory roles of these ncRNAs will be a great insight in understanding the virulence of T. forsythia. Keywords: DNA; ncRNA; nocoRNAc; Periodontitis; Tannerella forsythia.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

168

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Microbial Load, Antimicrobial Sensitivity and Plasmid Profiles of Vibrio cholerae in Fruit Juice Md. Mehdi Hasan2, 5, Sharmin Islam*1., Rayhan Ahmed 2, 5 ., Md. Rowfur Rahman 2., Md. Shahidul Islam 3 and Md. Masudur Rahman Khalil 1,4. 1

Center for Communicable Diseases, International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka-1212, Bangladesh. 2Department of Microbiology, Gono Bishwabidyalay (University), Dhaka-1344, Bangladesh. 3Department of Microbiology, Prime Asia University, Dhaka-1213, Bangladesh. 4Department of Microbiology and Biotechnology, Jagannath University, Dhaka-1100, Bangladesh. 5Department of Microbiology, faculty of medicine, AIMST University, Bedong-08100, Kedah, Malaysia; *corresponding author, email: mukta.sharmin@gmail.com, Ph No: +8801710365279

Abstract: Background: Vibrio cholerae is the causative agents of cholera, an acute dehydrating diarrhea. The research report was designed for isolation and identification of V. cholerae from juices. A total of forty samples of different fruit juices such as orange, papaya, grape, malta, sugarcane , lemon, aloe vera and isupgul juice were collected from different areas of Dhaka city. Methods: The samples were subjected to inoculate into TCBS agar media upon dilution for the isolation of V.cholerae, followed by biochemical identification of the bacteria. Different biochemical tests were performed such as oxidase, TSI, MR-VP, Citrate, Motility etc. And finally for plasmid isolation agarose gel-electrophoresis has been done. Results: The total viable count was varying from 7.73 x104-7.62 x108 cfu/ml where the highest count observed in sugarcane juice and lowest count in orange juice. V. cholerae was present in 20% of samples where the 75% of them were detected from sugarcane juice which was also contaminated with high bacterial and fungal load. The occurrence of drug resistance was also determined in the 8 isolated V. cholerae. With all the investigation it is bearing on mind that Vibrio cholerae cause severe diarrhoea and occurance of Vibrio cholerae in juice may cause a serious health hazards. Conclusion: About 40% isolates were resistant to tetracycline and neomycin and 80% isolates were resistant to amikacin. Plasmid profiling of eight isolates also performed, where most of the strain harbor two plasmids. All the isolates were resistant to polymixin B, and sensitive to gentamycin and ciprofloxacin. Keywords: Bangladesh; Coliform; E. coli; Fruit juice; Plasmid; Vibrio cholerae.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

169

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Isolation of Arsenite Resistant Bacteria from Ground Water and Soil of Dhaka, Bangladesh Rayhan Ahmed2,3, Ezazul Haque 2,3, Md. Jewel Rana2 and Taslin Jahan Mou1* 1

Dept. of Microbiology, Faculty of Biological Science, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh. 2Dept. of Microbiology, Faculty of Health and Medical Science, Gono University, Savar, Dhaka-1340, Bangladesh, 3Unit of Microbiology, Faculty of Medicine, AIMST University, Semeling, Bedong-08100, Kedah, Malaysia; *corresponding author, e-mail: moumicro@juniv.edu, Ph No: +88 01717562770

Abstract: Background: Worldwide more than 100 million people have been chronically exposed to arsenic by either drinking high level arsenic containing water or through soil. Arsenic resistant bacteria can tolerate high level of arsenic by the mechanism of energydependent efflux of either arsenate or arsenite from the cell mediated via the ars operon. The objective of the study was isolation and characterization of arsenite resistant bacteria from arsenic prone area in Bangladesh. Methods: Total of 6 samples (three each for groundwater and soil) were collected in May, 2014 from different places of Dohar, Dhaka, Bangladesh. 100 mL of each groundwater sample and 1 g of each of the soil samples were diluted with distilled water and 10-2 dilution of water and 10-3 dilution of soil sample inoculated to the 300 mL of minimal salt medium (MSM) supplemented with sodium arsenite. Arsenic tolerance of the bacteria was determined at 2,3,5,8 & 10 mM of sodium arsenite concentration respectively on minimal salt media. Bacterial strains were presumptively identified according to “Bergey’s manual of systemic bacteriology”. Results: A total of 60 bacterial isolates were found among which E.coli, Bacillus and Pseudomonas were presumptively prevalent according to the biochemical tests. 63% and 55% of the isolates were resistant against 8mM and 10mM respectively. Conclusion: From the study it can be concluded that arsenite resistant bacteria are prevalent in the water and soil sample of Dohar, Dhaka which are tolerant to high level of arsenic. Further investigation is required for molecular analysis and screening of the arsenite oxidizing bacteria. Keywords: Arsenite resistance; Bangladesh; Soil; Water.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

170

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Compared to Serum Media, Serum-Free Medium Enhanced the Generation of Mesenchymal Stem Cells Derived from Full-Term Human Amniotic Fluid Ghoraishizadeh P.1, Rohayu I.M.R.1, Ramasamy R.2, Singh G.3, Tan B.C.4, Rejali Z.5, Rosli R.1,6 Nordin N.1,6 and Thilakavathy K1,6* 1

Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 3 Stempeutics Research Sdn Bhd, Technology Park Malaysia, 57000 Bukit Jalil, Kuala Lumpur, Malaysia; 4Britannia Women and Children Specialist Centre, Selangor, Malaysia; 5 Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 6Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; *corresponding author, e-mail: thilathy@upm.edu.my, Ph No: +603-89472652

Abstract: Background: Mesenchymal stem cells (MSCs) are multipotent stem cells that are abundantly found in human amniotic fluid and possess therapeutic potential. DMEM supplemented with fetal bovine serum is the standard culture medium used to generate the human amniotic fluid-derived MSCs (hAF-MSCs). However, culturing of these cells in serum and xeno contained medium has challenged their usage in clinical applications. Hence, we aimed to use serum media and MesencultTM-XF xeno- and serum-free medium (XSFM) to generate, characterize and compare the properties of full-term hAF-MSCs. Methods: Amniotic fluid cells obtained from caesarean sections were cultured and propagated in XSFM, DMEM low glucose supplemented with 15% fetal bovine serum (DMEM-FBS), and 15% human serum (DMEM-HS). Immunophenotyping, differentiation and CFU-F assays were carried out to characterize the hAF-MSCs. Results: Spindle-shaped fibroblast-like cells were observed at passage 0 in all culture media used. These cells proliferated beyond passage 7 in XSFM medium and up to passage 3 in DMEM-FBS, however, failed to proliferate beyond passage 0 in DMEM-HS. Immunophenotyping analysis revealed that the cells generated in XSFM and DMEM-FBS expressed MSCs markers. hAFMSCs generated in XSFM were able to differentiate into adipocytes, osteoblasts and chondrocytes, however in DMEM-FBS, they did not differentiate into chondrocytes. Population doubling time and CFU-F of hAF-MSCs generated in XSFM was 14 times shorter and 11 folds higher, respectively, compared to DMEM-FBS. Conclusions: XSFM medium was found to be better in culturing and generating the hAF-MSCs isolated from full-term pregnancy compared to DMEM-FBS and DMEM-HS, suggesting its potential therapeutic usage in clinical applications. Keywords: Fetal bovine serum; Full-term pregnancy; Human amniotic fluid; Human serum; Mesenchymal stem cell; Xeno-and serum-free.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

171

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Hybrid Assembly of Salmonella enterica subsp. enterica ser. Typhi Isolates PM016/13 and B/SF/13/03/195 from a Typhoid Outbreak in Pasir Mas, Kelantan in 2013 Salwani Muhamad Harish1, Nazalan Najimuddin2, Ismail Aziah1* 1

Institute for Research in Molecular Medicine (INFORMM), Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. 2 School of Biological Science, Main Campus, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia; *corresponding author, e-mail: aziahismail@gmail.com

Abstract: Background: The ability to sequence the entire genome of individual bacteria helps to improve understanding of the organization and evolution of the organism at the molecular level. The generation of complete genome sequences provides a blueprint that facilitates the genetic characteristics of pathogens and their hosts. In this study, two isolates of S. enterica subsp. enterica ser. Typhi PM016/13 from untreated well water and also B/SF/13/03/195 from food handler were sequenced using next generation sequencing platforms provided by Pacific Biosciences and Illumina. Methods: The two isolates were assembled using the method of hybrid assembly. De novo assemblies of PacBio reads from both isolates were generated using PacBioâ&#x20AC;&#x2122;s SMRT Portal (v2.3.0) and hierarchical genome assembly process. Illumina reads were first screened by FastQC to identify low quality reads and adapters. The low quality reads and adapters were trimmed off the reads using NGS QC Toolkit before mapped onto PacBio assembled contigs using SSPACE Standard. The contigs obtained from the mapping were aligned according to S. enterica subsp. enterica ser. Typhi CT18 genome (GenBank accession number: NC_003198) using CONTIGuator2 to determine their correct order and orientation. The gaps from the scaffolds obtained were filled by Gapfiller. Results: The hybrid assembly resulted in one contig for both isolates meaning a complete genome of both isolates were obtained from this method. Isolates PM016/13 was assembled with 4,803,901 bp in size while isolates B/SF/13/03/195 was assembled with 4,801,617 bp in size. The GC content of both isolates was 52.00%. Conclusion: Hybrid assembly of the isolates had successfully assembled the reads into complete genome. The combination of short read Illumina and long read PacBio can give better accuracy to the assembly since the use of single molecule third generation sequencing data only give low accuracy, which causes inherent errors in the sequenced DNA. Using solely second generation sequencing data on the other hand lead to incomplete assembly of important part of a genome. Combination of both sequencing data can overcome these limitations. Keywords: Genomics; Hybrid assembly; Illumine; Next generation sequencing; Pacific biosciences; S. enterica subsp. enterica ser.; Typhi; Typhoid.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

172

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Identification and Characterization of Novel Non-protein Coding RNAs in Salmonella serovar Typhi Biofilm Satisvar S.1, Kogaan .A.2, Phua K.K. 2, Suresh C.V.1* 1

AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia. 2 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, USM, Pulau Pinang, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Salmonella enterica serovar Typhi (S. Typhi) is the sole cause of typhoid fever, which is common among travellers to third world countries. The battle against typhoid fever is intensified due to the ability of S. Typhi to form biofilm in the gallbladder. The biofilm provides a rudimentary protection against commonly used antibiotics. Further understanding of the role of non-coding RNA (ncRNA) in its formation will aid physicians and scientists alike to counter biofilm formation. Methods: Total RNA was extracted from various growth phases of S. Typhi including biofilm formation. The transcriptome sequences of S. Typhi were aligned with the reference genome S. Typhi Ty2 using Bowtie and analyzed using genome viewer. Online ORF finder was used to detect the presence of possible ORFs. The candidates were then cross-checked with known ncRNAs in Rfam database. The total RNA was resolved on denaturing gel and transferred to a nylon membrane using semi-dry transfer protocol. Results: A total of 754 possible ncRNA transcripts were obtained from the intergenic regions of S. Typhi. These were screened for their annotation as protein coding genes or ncRNA genes in other organisms using Blastn/BlastP and Rfam search, respectively. Nine potentially novel ncRNAs were found in S. Typhi biofilm. Interestingly, one of these novel ncRNA gene was located adjacent to topoisomerase gene and having the conservation of STAXI motif which may interact with the anti-restriction enzyme proteins. Conclusion: In summary, we have identified 9 novel ncRNAs from S. Typhi biofilm. Current work is in progress to analyze the variations and specificity of their expression during biofilm formation using Northern-blot. This study could help better understand the mechanism of biofilm formation in S. Typhi which is worldwide becoming more resistant to antibiotics. Keywords: Biofilm; Non-coding RNA; S. Typhi; Transcriptomics.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

173

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Identification of Novel Non-coding RNA (ncRNA) from Bacillus thuringiensis Shereenrani S., Saw H.S., Sumitha S., Maheswaran S., Xavier R., Suresh C.V.* AIMST University, Department of Biotechnology, Faculty of Applied Sciences, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: cvsureshgupta@gmail.com, Ph. No: +604-4298165

Abstract: Background: Bacillus thuringiensis (Bt) is a Gram-positive, rod-shaped, sporeforming bacterium. It grows at body temperature and produces a different shaped crystal proteins. It used to fend off insects, predators, and other pathogens. Bt toxin is speciesspecific and different shape and activity depends on unknown regulatory pathways. The objective of this study is to identify the possible ncRNA candidates in Bt which can fill the gaps of understand the gene regulation. Methods: Bt transcriptome data was downloaded from DNA Data Bank of Japan (DDBJ) database. FastQC analysis was performed to view the quality of the transcriptome. Trimmomatic is done to remove the adapter sequences. The sequences were then aligned to Bt genome using Bowtie2. the un-annotated transcripts were selected using genome viewer Artemis. These possible ncRNAs were filtered further using blastN, ORF Finder and Rfam to exclude the annotated genes. ncRNA genes also were predicted computationally using nocoRNAc software. Results: A total of 418 candidates were identified as un-annotated transcripts. Finally 12 candidates were selected as potential ncRNA candidates after filtering through ORF Finder, blastN and Rfam. Interestingly, all these 12 candidates are genus specific to the Bacillus sp. In parallel, 1454 ncRNA candidates were predicted by nocoRNAc. Seven of these 12 candidates were overlapping with nocoRNAc prediction list. Conclusion: The preliminary study reveals 12 novel ncRNAs from Bt transcriptome. However, need to confirm their expression again by real-time PCR. Keywords: Bacillus thuringiensis; ncRNA; nocoRNAc; Transcriptome.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

174

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Morphological and Differential Protein Expression Analysis of Placentas from Term and Spontaneous Preterm Labor with Intact Membrane Tan N.J.1, Daim L.D.J.2, Jamil A.A.M.3, Mohtarrudin N.4 and Thilakavathy K.1,5* 1

Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2Sime Darby Technology Centre Sdn. Bhd, 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh Silikon, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia; 3Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 4Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 5Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia;*corresponding author, e-mail: thilathy@upm.edu.my, Ph No: +60389472652

Abstract: Background: Spontaneous preterm labor with intact membrane (sPTL-IM) accounts for approximately 45% of the total preterm delivery worldwide. Many major pregnancy complications had been association with placenta abnormalities. Hence, evaluating the histology and protein profiles of the placenta will provide us better understanding on the cause of sPTL-IM, which will help in improving the current diagnosis, prognosis, treatment and decision making. Therefore, this study aimed to determine the term and sPTL-IM placentasâ&#x20AC;&#x2122; histopathology, and differentially expressed proteins in fetal site of the placenta cotyledon. Methods: Term and sPTL-IM placentas were obtained from Hospital Serdang, Malaysia. The whole thickness of placenta disc, umbilical cord and membrane were formalin fixed, paraffin-embedded, stained with hematoxylin and eosin (H&E) stain, and slides were viewed under the light microscope. Subsequently, to study the differential protein expression, fetal site of the placentas tissues were pulverized and proteins were extracted using DNase/Lithium chloride-dense sucrose homogenization coupled dichloromethanemethanol precipitation. Extracted proteins were separated on 2D-gel electrophoresis and visualized using silver stain. Results: The H&E appearance of the whole thickness of placental disc including decidua and chorion, distal and proximal site of the umbilical cords and the membrane tissues showed no difference for both term and preterm delivery placentas. Interestingly, 7 protein spots found in 2D-gel were found differentially expressed between normal and sPTL-IM placentas at the fetal site. Conclusion: Although there is no difference morphologically between term and sPTL-IM placentas, but some proteins were differentially expressed, suggesting molecular investigation might benefit in future clinical advancement. Keywords: 2D-gel electrophoresis; Histology; Protein profile; Placenta; Spontaneous preterm labor with intact membrane.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

175

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Expression of Salmonella Typhi Ty21 TolC Protein in Different Host Cells Teh Boon Auna, Amy Amilda Anthonyb, Ismail Aziahb, Asma Ismaila, Eugene Boon Beng Onga, and Phua Kia Kien*a a

Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia. b Institute for Research in Molecular Medicine, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; *corresponding author, e-mail: kkphua@usm.my

Abstract: Background: Salmonella enterica serovar Typhi 50 kDa outer-membrane protein was reported to be antigenic. Here we investigated the effects of recombinant (r) protein expression in different host cells on the antigenicity of S. Typhi TolC protein, a component of the OMP. Modification and over-expression in E. coli could render the antigenicity of rTolC useless. Epitopes on rTolC appear to be conformational as native TolC reacted with typhoid patient serum while denatured TolC had much lower reactivity. Methods: Bacteria strains used as host cells in this experiment were S. Typhi Ty21a, TolC deletion mutan, E. coli DH5a, and E. coli Lemo21. Recombinant TolC was cloned into pBAD Myc-His A plasmid and the protein was subsequently overexpressed in S. Typhi Ty21a and E. coli. Recombinant TolC was then purified and analyzed by MALDI-TOF/TOF. ELISA was used to measure the antibody reactivity with rTolC antigens expressed in the different host cells. Results: The ELISA results demonstrated that antibodies were reactive with rTolC expressed in S. Typhi with a significant difference in the mean OD value between typhoid sera (OD405 = 3.15, n=2) and normal sera (OD405 = 1.19, n=2). However, for rTolC expressed in E. coli, no significant difference in the mean OD was observed between normal sera (OD405 = 0.30, n=2) and typhoid sera (OD405 = 0.32, n=2), whereas for rTolC purified under denaturing condition a slight difference in the mean OD values was found between pooled typhoid sera (OD405 = 0.23 ) and pooled normal sera (OD405 = 0.11, n=2) while for rTolC expressed in E. coli host cells, a significant difference in OD was observed between pooled typhoid sera (OD405 = 0.25) and pooled normal sera as controls (OD405 = 0.15). Thus, the ELISA results showed that the rTolC was highly reactive with typhoid sera. Conclusion: This study we showed that recombinant TolC from S. Typhi TolC deletion mutant had the potential to be used as the antigen for development of a rapid ELISA diagnostic test for typhoid fever. Also found was that rTolC purified from S. Typhi host cells were more antigenic compared with rTolC purified from E. coli host cells. Future studies include determining the analytical sensitivity and specificity of the antigens on various diagnostic platforms for diagnosis of typhoid fever. Keywords: Antigen; ELISA; Salmonella; TolC; Typhoid.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

176

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Stemness of Spontaneously Transformed Murine Bone Marrow Mesenchymal Stem Cells is Maintained Upon Prolonged In Vitro Expansion Lye K.L.1, Vidyadaran S.2,3, Nordin N.1,2 and Thilakavathy K.1,2* 1

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2 Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 3Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; *corresponding author, e-mail: thilathy@upm.edu.my, Ph No: +603-89472652

Abstract: Background: Mesenchymal stem cells (MSCs) are a unique population of stem cells that possess immunomodulatory properties, making them one of the ideal candidates for regenerative medicine. MSCs are also known to suppress or activate cancer formation. In this study, we aimed to investigate the spontaneous transformation of murine bone marrow MSCs (mBM-MSCs) and evaluate their stemness characteristic. Methods: mBM-MSCs were isolated from the femur of C57BL/6 mice and cultured in DMEM high glucose supplemented with 15% fetal bovine serum and 1% antibiotic/antimycotic. Immunophenotyping and differentiation assays were carried out to characterize the stemness of the isolated MSCs population. Chromosome analysis and selected oncogenes expressions analysis were performed to confirm the occurrence of spontaneous transformation in these cells. Results: Homogeneous population of mBM-MSCs with spindle shaped fibroblast-like morphology was successfully generated from passage 5 onwards. Variations in chromosome numbers were observed at passage 20 and beyond, indicating spontaneous transformation. This was further supported by the over-expression of c-Myc, MDM2, and Fos oncogenes as compared to the earlier passages. However, these transformed cells were still expressing positive MSCs markers and able to differentiate into adipocytes and osteoblasts, denoting the stemness. Conclusion: This study clearly indicates that mBM-MSCs undergo spontaneous transformation at molecular level but still maintain their stemness in prolonged culture. This study also suggests that early passage MSCs to be used in research studies, and molecular characterization should be made obligatory prior to their application in regenerative medicine and therapy. Keywords: Bone marrow; Chromosomes; Mesenchymal stem cells; Oncogenes; Spontaneous transformation.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

177

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

16S Ribosomal DNA Sequence Based Identification of Bacterial Endophytes Isolated from Seeds of Starfruit (Averrhoa carambola L.) Narmataa M. and Bhore S. J.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: subhashbhore@gmail.com / subhash@aimst.edu.my

Abstract: Background: Starfruit plant (Averrhoa carambola L.) is cultivated in tropics for its fruits (Starfruit). Fruits are sold, imported and exported extensively in South East Asia region and beyond. However, this plant is poorly studied at molecular level as well as for its endophytes. Endophytes are the microbes (usually, bacteria and fungi) that stay inside plant body without causing any visible symptoms of disease. Endophytes can be used to promote plant growth and development and have a great potential in agriculture sector and other sectors of biotechnology. The objective of this study was to isolate and identify bacterial endophytes associated with seeds of Starfruits. Methods: Starfruits (Clone B10) were purchased in local market of Sungai Petani, Kedah, Malaysia. Surface sterilisation of fruits was done by following a standard method and extracted seeds were macerated and plated on sterile Luria-Bertani Agar. Plates were incubation at 37Â°C for period for 36 hours and bacterial endophytes were isolated. 16S ribosomal DNA fragments were amplified using genomic DNA from pure cultures of respective isolated bacterial endophytes. Isolates were identified based on sequence of amplified 16S ribosomal DNA. Results: A total of five endophytic bacterial isolates (EBIs) were obtained from the seeds of the Starfruit. The sequences of amplified 16S ribosomal DNA were analysed using nucleotide database of National Centre for Biotechnology Information (NCBI). Analysis revealed the identity of the isolated EBIs as Bacillus anthracis, Bacillus cereus, and Bacillus toyonensis. The nucleotide sequence data of 16S ribosomal RNA gene (partial sequences) fragments was submitted to nucleotide sequences database of NCBI. The annotated 16S rRNA gene fragment nucleotide sequences of all five isolates has been submitted to the nucleotide database at GenBank/DDBJ/EMBL under accession numbers: KT861455 - KT861459. Conclusions: In conclusion, Starfruit (Clone B10) seeds do possess bacterial endophytes. This study reveals the presence of Bacillus anthracis, Bacillus cereus, and Bacillus toyonensis in Starfruit seeds. Further study is required to find out the benefits of bacterial endophytes to the Starfruit plant. Keywords: 16S Rrna; Averrhoa carambola L.; Endophytes; Seeds; Starfruit.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

178

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

An Alternative Method of Screening the M2/ANXA5 Haplotype for Repeated Pregnancy Loss 1

Kai-Cheen Ang*, 2Sushilnathan Kathirgamanathan, 3Yan-Yeow Lee, 4Anna Liza binti Roslani, 3Krishna Kumar, 5Bavanandan Naidu, 2Ridzuan bin Abdullah, 6Nadja Bogdanova, 1 Narazah Mohd Yusoff, 7Wan Zaidah Abdullah, 6Arseni Markoff, 1Thean-Hock Tang* 1

Advanced Medical and Dental Institute, University Sains Malaysia, Bertam, Penang, Malaysia. 2Department of Obstetrics and Gynaecology, Hospital Sultan Abdul Halim, Sungai Petani, Malaysia. 3Department of Obstetrics and Gynaecology, Hospital Tuanku Jaafar, Seremban, Malaysia. 4Department of Obstetrics and Gynaecology, Hospital Tengku Ampuan Afzan, Kuantan, Malaysia. 5Department of Obstetrics and Gynaecology, Hospital Sultanah Bahiyah, Alor Setar, Malaysia. 6Institute of Human Genetics, University of Muenster, Muenster, Germany. 7Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; *corresponding author, e-mail: jocelyn85ang@yahoo.com, Ph No: (+60) 174059103

Abstract: Background: Repeated pregnancy loss (RPL) has an adverse psychosocial impact in women and their families, resulting in depression or anxiety for the next pregnancy. Recently, M2/ANXA5 haplotype, a variant in the proximal core promoter region of the annexin A5 (ANXA5) gene, was suggested as a risk factor in thrombophilia-associated RPL impeding embryonic anticoagulation. M2/ANXA5 haplotype is suggested as the third hereditary predisposition gene for thrombophilia-associated RPL. We developed and validated of an Allelic specific PCR (AsPCR) for the detection of M2/ANXA5 haplotype. The AsPCR is simple, sensitive, less time consuming and cheap assay. Methods: Allele specific PCR for M2/ANXA5 haplotype is designed based on the concept of nested PCR by using a common primers and allele specific primers sets. All the primers, DNA, MgCl2 and Taq were adjusted to an optimum amount until the specific bands were produced. The annealing temperature was optimized using a gradient PCR to determine the optimum annealing temperature at which all the primers react to anneal with the template DNA at their optimum conditions. A total of 105 blood samples were collected for genotyping with AS PCR and also validated with DNA sequencing. Results: The AsPCR was successfully discriminated the M2 and â&#x20AC;&#x2DC;normalâ&#x20AC;&#x2122; haplotypes. Sequencing results confirmed and validated the AsPCR result with 100% reliability. Conclusions: AsPCR was developed for the detection M2/ANXA5 haplotype. This assay provides an easier, faster and more cost effective method for screening the M2/ANXA5 haplotype. Keywords: Allelic specific PCR; M2/ANXA5 haplotype; Repeated pregnancy loss.

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

179

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology

Res. Highl. 4Bs (2016)

Computational Analysis of Class IV Chitinase Gene cDNA Isolated from American Oil Palm (Elaeis oleifera) Fruit Mesocarp Tissue cDNA Library Vengadan S.1, Amelia K.2 and Subhash J. Bhore*1, 2 1

Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, Bedong, 08100, Kedah Darul Aman, Malaysia; 2Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia; *corresponding author, e-mail: subhashbhore@gmail.com / subhash@aimst.edu.my, Ph No: +604-429 8176

Abstract: Background: Elaeis is a genus of palms which contains two Oil-palm species. The African Oil-palm, Elaeis guineensis Jacq (variety Tenera) is native to west and southwest Africa and cultivated widely in tropics because of its high yield of oil. The American Oilpalm, Elaeis oleifera is native to tropical central and South America and it is used locally for oil production. However, it is not cultivated on a commercial scale to produce palm oil because of its low yield. In order to study the genes expressed in fruits of the American Oilpalm, about 3200 expressed sequence tags (ESTs) were generated and while processing the ESTs, class IV Chitinase gene cDNA was identified. The objective of this study was to analyze and annotate Elaeis oleifera class IV Chitinase (Cht) full-length cDNA and its deduced protein sequence. Methods: The cDNA sequence and its deduced protein sequence was analyzed and annotated using online bioinformatics tools including JustBio. The multiple sequence alignment (MSA) and the phylogenetic analysis of Elaeis oleifera class IV chitinase protein was carried out by using CLUSTALW and treeviewX. Secondary structures and tertiary (3D) structure of class IV Chitinase protein was predicted by using Phyre 2 server. Results: In total, 3258 ESTs (cDNA) clones have been isolated from mesocarp cDNA library by using random method of gene isolation. Class IV chitinase protein cDNA gene was found when the generated ESTs were processed and analysed. Result analysis suggest that class IV chitinase protein cDNA sequence is 1049 bp in length. The open reading frame analysis suggest that it encodes for a protein which contains 268 amino acids. Phylogenetic analysis indicated that Elaeis oleifera class IV chitinase protein is closely related to its counterpart from African Oil-palm. The 3D structure prediction work is in progress and will be reported in due time. Conclusion: The Elaeis oleifera class IV chitinase protein cDNA clone sequence and deduced protein sequence is successfully annotated. Further research is required to understand the role of class IV chitinase in the Oil-palm biology. These primary research findings will be presented in the conference. Keywords: American oil-palm; BLAST; Class IV chitinase; Elaeis oleifera; Oil.

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Res. Highl. 4Bs (2016)

In Silico Analysis of Omega-6 Fatty Acid Desaturase Gene cDNA Isolated from Common Bean (Phaseolus vulgaris L.) Pod Tissue cDNA Library Teh S.Y.1, Amelia K.2 and Subhash J. Bhore*1, 2 1

Abstract: Background: Common beans (Phaseolus vulgaris L.) are widely consumed worldwide because of its high protein content. However, to meet the demand of growing population, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated which we have deposited in GenBank. Omega-6 fatty acid desaturase (O6FAD) encoding gene cDNA was one of the clones among generated ESTs. The O6FAD is an important enzyme in fatty acid biosynthesis pathway; hence, to understand more about it this study was undertaken. The objective of this study was to elucidate P. vulgaris L. O6FAD (PvO6FAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. Methods: Positive and negative strands of the PvO6FAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvO6FAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvO6FAD protein were predicted by using the PHYRE automatic fold recognition server. Results: The sequencing results analysis showed that PvO6FAD cDNA is 1470 bp in length and it's open reading frame (ORF) encodes for a protein that contains 383 amino acids. Deduced protein sequence analysis showed the presence of essential domains of the enzyme. Protein structure prediction is in progress and will be reported in due time. Conclusions: The 1470 bp long PvO6FAD cDNA encodes for 383 amino acid long protein that contains conserved domains required for biological functions of O6FAD. Further research is needed to predict and annotate the PvO6FAD protein's 3D structure as well as to validate it. These primary research findings will be presented in the conference. Keywords: cDNA; Omega-6 fatty acids; Omega-6 fatty acid desaturase; Phaseolus vulgaris; Proteins.

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Res. Highl. 4Bs (2016)

Computational Analysis of Common Bean (Phaseolus vulgaris L.) Palmitoyltransferase Gene cDNA Isolated from Bean Pod Tissue cDNA Library Archana.E.1, Amelia K.2 and Subhash J. Bhore*1, 2 1

Abstract: Background: The common bean (Phaseolus vulgaris L.) is a main grain legume consumed worldwide for its edible seeds and pods. Common beans are a rich and affordable source of proteins especially for people from low income category. To meet the demand of growing population, we need to increase its yield. As a part of Phaseomics (an international consortium) consortium, work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated and during analysis of data we identified a cDNA clone of palmitoyltransferase (PvPT) gene and to understand more about it this study was conducted. The objective of this study was to annotate PvPT cDNA and deduce amino acid sequence using computational tools. Methods: Online bioinformatics tools were used for analysis and annotation of both cDNA and deduced protein sequences. Blastp was used to compare the sequence and the phylogenetic tree was constructed using Treeview after sequence alignment is done in ClustalW. The secondary structure and tertiary (3D) structure was predicted using PHYRE automatic fold recognition server. Results: Based on complimentary DNA and deduced protein sequence analysis, PvPT gene cDNA is 1420 bp in length and its open reading frame (ORF) encodes for 343 amino acids residues. The predicted tertiary (3D) structure of PvPT protein shows that it is analogous to protein S-acyl transferase (PAT). Conclusions: We have successfully annotated the cDNA and deduced protein sequence of PvPT. Although 3D structure is predicted for the protein, further research is needed to annotate the predicted structure by docking study and predicted structure needs to be validated. Keywords: cDNA; Common beans; ESTs; mRNA; Phaseolus vulgaris L., Protein structure.

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Res. Highl. 4Bs (2016)

Annotation of Elaeis oleifera CAP cDNA and its Deduced Amino Acid Sequence using Computational Tools Jihan M.R.1, Amelia K.2 and Subhash J. Bhore*1, 2 1

Abstract: Background: American Oil-palm (Elaeis oleifera) is an important oil producing crop; but, it yields relatively less palm oil as compared to African Oil-palm, Elaeis guineensis Jacq. Hence, Oil-palm plantation companies and farmers prefer African Oil-palm over American Oil-palm for commercial plantation. In order to study the gene expression in developing fruit tissue, transcriptomics study was initiated and about 3250 ESTs were generated. While processing data, we identified a cDNA clone of cytosolic ascorbate peroxidase (CAP) gene and to understand more about this gene this study was undertaken. The main objective of this study was to annotate E. oleifera CAP cDNA and its deduced amino acid sequence using computational tools. Methods: Both strands of CAP cDNA clone were sequenced using M13 forward and reverse primer to elucidate the nucleotide sequence. Online bioinformatics tools were used for analysis and annotation of both cDNA and deduced protein sequence. Sequence comparison was carried out using Blastp whereas split tree program was used to construct a phylogenetic tree. The secondary structure and tertiary (3D) structure of protein was predicted using PHYRE automatic fold recognition server. Results: Complimentary DNA and deduced protein sequence analysis showed that E. oleifera CAP gene cDNA is 1101 bp in length and its open reading frame (ORF) encodes for 249 amino acid residues. The predicted 3D structure of CAP protein shows that it is analogous to ascorbate peroxidase. Conclusions: E. oleifera CAP cDNA is 1101 bp in length and its ORF encodes for 249 amino acid residues. The primary analysis of cDNA and deduced protein sequence suggest that it contains catalytic sites essential for the enzymes function. Further study is required to annotate the predicted 3D structure of the protein and to validate the predicted structure. Keywords: American palm oil; Ascorbate peroxidase; cDNA; Cytosolic ascorbate peroxidase; DNA; mRNA.

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Res. Highl. 4Bs (2016)

Comparison of Methods for Pure Quality RNA Isolation from Polysaccharide Rich Averrhoa carambola L. Fruits (Starfruits) Narmataa M. and Bhore S. J.* Department of Biotechnology, Faculty of Applied Sciences, AIMST University, BedongSemeling Road, 08100 Bedong, Kedah, Malaysia; *corresponding author, e-mail: subhashbhore@gmail.com / subhash@aimst.edu.my, Ph No: +604-429 8176

Abstract: Background: Starfruit plants (family: Oxalidaceae) are cultivated commercially for its fruits in several countries, especially in tropical belt. This plant species belongs to Averrhoa genus and A. carambola and A. blimbi species are cultivated widely. Starfruit is fleshy, juicy, slightly tart, acidic and sweet in taste. Traditionally, Starfruit is used in Ayurvedic and Traditional Chinese Medicines (TCM) preparations to relieve ailments such as chronic headache, fever, cough, gastro-enteritis, ringworm infections, and skin inflammations. Fruits are good source of antioxidants. However, this fruit contains high oxalate which gives adverse effect to high uremic patients because of their limited ability to excrete waste substances from bloodstream. The main objective of our research project is to study the transcriptomics of the Starfruit. However, we are reporting the comparative analysis of RNA extraction methods to obtain high yield and pure quality of RNA from starfruits. Methods: Starfruits (Clone B10) were purchased in local market of Sungai Petani, Kedah, Malaysia. The fruits were washed thoroughly to remove dirt from the fruit surface and cut into small pieces; seeds and ridges were removed and Starfruit tissue samples were instantly freezed using liquid nitrogen and then stored at −80 °C. The samples were used for RNA extraction using three different methods. The qualitative and quantitative analysis of isolated total RNA samples was carried out by using agarose gel-electrophoresis and spectrophotometry, respectively. Results: Based on the qualitative and quantitative analysis of isolated total RNA samples, ‘Phenol Chloroform’ based method of total RNA extraction appears more effective in comparison to ‘TriZol Reagent’ and ‘Chloroform with LiCl Precipitation’ based methods. The Nanodrop® based quantitative analysis of isolated total RNA samples clearly suggest that ‘Phenol Chloroform’ based method of total RNA extraction gives maximum yield of pure quality total RNA. Conclusions: For the extraction of the pure quality total RNA from polysaccharide rich Starfruit samples, the ‘Phenol chloroform’ based method is the most efficient method. Keywords: Averrhoa carambola; Polysaccharides; RNA quality; Starfruit; Total RNA extraction.

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Appendices Appendix I: Brief Biography of Keynote and Plenary Speakers who delivered their talk in 3rdRC4Bs-2016. Biography of YBhg. Dato' Professor Dr. Asma Binti Ismail (Keynote Speaker 1)

Asma Ismail is currently the first woman appointed as the Director General of Higher Education, Ministry of Higher Education, Malaysia. She graduated from the University of Nevada, Reno, USA with distinction in biology, received her MA in Microbiology from Indiana University, Bloomington, USA and obtained her PhD in the field of Cellular and Molecular Biology at the University of Nevada, Reno, USA in 1986. Her areas of expertise include Medical Microbiology, Clinical Microbiology and Medical Biotechnology. Prof. Asma started her career as a lecturer in the Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia (USM) in 1986. She was a visiting scientist at University of Tokyo in 1989 and a visiting fellow at the Medical College, St Bartholomew’s Hospital in London in 1992. She was promoted to Associate Professor in 1993 and served as Deputy Dean of Administration in 1994. She was promoted to Professor in 2000 and became Deputy Dean of Research in the same year. In 2001, she became the Director for the Centre for Medical Innovations and Technology Development, USM. In 2003, she was appointed as the Founding Director, Institute for Research in Molecular Medicine (INFORMM), the first multi-disciplinary cluster based research institute for Universiti Sains Malaysia. In May, 2008 she became the first woman in USM to hold the post of Deputy Vice-Chancellor (Research and innovation). In December 2012, she became the first woman to hold the Vice-Chancellor’s post at Universiti Sains Islam Malaysia and the fifth woman to be appointed as Vice-Chancellor of a public university. Prof. Asma’s research interest is in the scientific discovery of biomarkers and the development of rapid diagnostics for infectious diseases especially for typhoid and paratyphoid fever. Prof Asma is especially interested in the development of technology platforms for diagnostics in low resource settings. Her efforts in this area was given due recognition when she was conferred the Honorary Doctor of Science from University of Glasgow, Scotland, United Kingdom in June 2013. Indiana University, Bloomington, USA has also honored her work in development of rapid diagnostics for typhoid by conferring her the Indiana University’s Thomas Hart Benton Mural Medallion for her contributions in medical diagnostics which was awarded on 23rd May of 2015.

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 Prof Asma is among the countryâ&#x20AC;&#x2122;s research leaders. She is actively involved in research having attained more than RM19 million (USD 5.6 million) worth of grants within the last 5 years, presented 340 papers and received more than 184 invitations as a plenary or keynote speaker at national and international levels to share her scientific findings and experience in the commercialization of R&D products. Prof Asmaâ&#x20AC;&#x2122;s R&D achievements and research impact at national and international levels have led to more than 132 scientific publications, 30 patents filed world wide of which 13 have been granted, and received more than 162 awards and recognitions at national and international levels. The prestigious awards received include the National Young Scientist Award in 1991, Malaysian Toray Science and Technology Award for outstanding contribution in science in 2002, National Inventor Award in 2003, National Innovation Award, 2006 and the National Academic Award for Product and Commercialization in 2007. She was conferred Top Research Scientist Malaysia in 2012 by Academy of Sciences Malaysia. For her significant contribution in science, she was made a Fellow of the Academy of Sciences Malaysia in 2003 and served as its council member from 2007-2011. In 2010, she was made a member of the Third World Academy of Sciences (TWAS). In 2012, she became the first woman elected as the Vice-President for Academy of Sciences Malaysia for a period of 4 years. Based on her expertise, she served as the WHO temporary Advisor for vaccine and diarrheal diseases since 2002. At the National level, she is the founding chairperson for the establishment and implementation of the National Academic Award (Anugerah Akademik Negara), a job that she maintained for seven years to ensure the successful set up, branding and positioning of the award. She was also entrusted to chair the Implementation of Innovative Human Capital for the Ministry of Higher Education that developed action plans for to develop innovative talent from cradle to career. She also chairs the Cluster Development Committee for the Nobel Laureate grants in Physiology or Medicine under the Academy of Sciences Malaysia and the Fundamental Research Grant Scheme (medicine) for the Ministry of Higher Education. She is a committee member for the Establishment of Research Universities in Malaysia and is also a member of the Assessment Committee for Research Universities by the Ministry of Higher Education that led to the landmark establishment of Research universities in Malaysia. She also serves as the chair for the development and direction of R&D towards implementation of the national Strategic Planning for Higher Education. She also helped determine the direction in Biotechnology by being a member of the Cluster Working group on Healthcare biotechnology, Member of the Penang Biotech, Pharma and Neutraceutical Advisory panel for Invest Penang and member for the National Strategic Planning and Advisory for Biotechnology, Ministry of Higher Education. Prof Asma played a key role as she co-helmed the development of the landmark Malaysian Education Blueprint (Higher Education) 2013-2025. She is one of the authors as well as Deputy to the blueprint project taskforce. Prof Asma is responsible for making strategic decisions on policy directions, coordinating the blueprint development process, manage key resources of input, engaging a broad range of stakeholders and is one of the two people who has to sign-off the document. She is currently overseeing the implementation of the Blueprint including the soon to be launched University Transformation Programme. As a researcher, Prof Asma is passionate about the idea of developing indigenous health technologies and innovations that can generate wealth for the country and improve the quality ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 of life of society at large. She is committed to the development of rapid and affordable diagnostics for infectious diseases as a means for ensuring availability and accessibility to quality healthcare especially for the people from the underdeveloped countries. To drive this passion, she specializes in the area of proteomics and its application in the rapid diagnosis of infectious diseases, especially typhoid fever. Her studies on specific biomarkers led to the discovery of an antigenically specific 50kDa of Salmonella Typhi. Prof Asma is one of the scientists credited with the translation of the scientific discovery into four rapid diagnostic kits for typhoid that have been successfully commercialized globally to more than 18 countries since 1994. Commercialization of TYPHIDOT, a rapid diagnostic test to diagnose for acute typhoid, has generated sales, publications, improved the quality of healthcare especially among the poor, generated more than 500 jobs worldwide and supported growth of the local industries in Malaysia. In partnership with Malaysian Technology Development Corporation, she has helped to create a startup biotech company pioneering in Biodiagnostics for the country. TYPHIDOT became the flagship product of the company which generated at least RM 16 million in gross sales and helped to diagnose more than 2 million cases.

Biography of Professor Dr. Eiichi Tamiya (Keynote Speaker 2)

Eiichi

Tamiya is Professor, Department of Applied physics, Graduate School of Engineering, Osaka University. He received Ph.D. in Science and Engineering, Graduated School, Tokyo Institute of Technology in 1985. He has been Assistant Professor at Tokyo Institute of Technology from1985 to 1987, Lecturer at Tokyo Institute of Technology from 1987 to 1988 and Associate Professor at Tokyo University from 1988 to 1993 and Professor at Japan Advanced Institute of Science and Technology from 1993 to 2007. He has been Professor at Osaka University from 1993 until now. His research topics have been (1) Biochips and Biosensors, (2) Nanotechnology based bioscience and bioengineering, (3) Biomass energy conversion systems, (4) POC (point-of-care) biosensors for medical diagnosis, food safety and environmental protection, (5) Cell based chips for tissue and stem cell engineering.

Biography of Professor Ravichandran Manickam (Plenary Speaker 1)

Professor

M. Ravichandran obtained his M.Sc.-Medical Microbiology from Christian Medical College, Vellore in 1991 and gained his PhD degree from Anna University, Chennai, India in 1997. Currently his research interests are cholera vaccine, molecular diagnostics and phage therapy. He has constructed several cholera vaccine candidates for O139 Vibrio cholerae and developed several simple cold chain free molecular diagnostic kits. He has 59 publications in international journals (Cumulative impact factor 119), supervised 36 postgraduate students, filed 8 patents and commercialized 3 products on diagnostics. He is an ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 associate member of Academy of Sciences Malaysia (ASM) and Expert panel member for R, D & C grants- Sciencefund, Technofund, Community Innovation Fund (CIF) and Enterprise Innovation Fund (EIF), and Innovation (MOSTI); He was the Cluster Working Group (CWG) Committee member on Human Capital Development, Malaysian Biotechnology Corporation and the Committee member on ‘TOP RESEARCH SCIENTISTS MALAYSIA’(TRSM) of Academy of Sciences Malaysia (ASM), Biotechnology Road map of the Kedah state was formulated under his supervision. He is currently the Chief Executive and Vice Chancellor of AIMST University, Kedah, Malaysia.

Biography of Dr. Bent Petersen (Plenary Speaker 2)

Bent

Petersen is an Associate Professor at the

Department of Systems Biology, Technical University of Denmark. He has 8 years of experience within Bioinformatics, Computational biology, DNA Sequencing, NGS data analysis, Metagenomics, Protein structure prediction and Genome assembly of a variety of organisms, such as bacteria, insects, mammals, parasites, complex organisms and metagenomics samples. Currently, he is working within the area of Metagenomics with special focus on Next Generation Sequencing technologies. He has made a significant contribution in exciting large variety of exciting projects - spanning from ancient horses to modern breeds, extinct animals, malaria parasites, and metagenomics samples from exotic places. The results have been published in highimpact journals such as Science, Nature, Cell and PNAS. In 2014, he started his own company together with his colleagues, Bison-SeqTech, which offers fast and highly professional bioinformatics services that are tailored to the customers’ needs. While providing easily affordable access to a top 100 High-Performance Computer Systems BisonSeqTech is not hindered by traditional cluster and cloud limitations, such as insufficient memory, processing capabilities, and disk space. He is also a passionate blogger and science enthusiast. Read more at www.bpetersen.dk and www.bison-seqtech.dk

Biography of Professor Dr. Prakash P. Kumar (Plenary Speaker 3)

Professor

Prakash Kumar’s early education was from India, and PhD (1988) from the University of Calgary, Canada. He joined the National University of Singapore (NUS) in 1989, where he currently serves as a full professor at the Department of Biological Sciences. He is a prominent scientist in the fields of plant molecular developmental biology and biotechnology. He has supervised 25 PhD students at NUS. He is Editor of four international journals, namely, BMC Plant Biology, Frontiers in Plant Science, Plant Cell Reports and Plant ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 Biotechnology Reports. Several of his 90 scientific papers are published in prestigious journals. In addition to a mangrove salinity tolerance work, he leads a major rice research program funded by the National Research Foundation, Singapore, in collaboration with TLL Singapore and IRRI Philippines. He has held several department and University-level senior administrative positions at the NUS and serves on National and International committees, including, Genetic Modification Advisory Committee, Singapore. He is the Elected Secretary/Treasurer of Plant Biotechnology Section (2014 to 2016), Society for In Vitro Biology, USA.

Biography of Professor Dr. Phua K.K. (Plenary Speaker 4)

Professor

Phua K.K. pursued his undergraduate and

postgraduate studies at the University of Liverpool, United Kingdom, where he obtained his PhD in Immunology from the Medical School in 1986. Upon his return to Malaysia, he joined the School of Medical Sciences, Universiti Sains Malaysia (USM) and was appointed lecturer at the Department of Chemical Pathology in 1986, Senior lecturer at the Department of Immunology in 1990, and Associate Professor of Immunology in 1994. In 2004, Dr. Phua joint the Institute for Reseach in Molecular Medicine (INFORMM), and in 2008 he was appointed Acting Deputy Director for Research and Commercialization. In 2010 he was appoint Deputy Director for Quality and Sustainability. In 2012, he was transferred to Chancellory USM to resume the post of Director for Industry and Community Network. In 2013, he was appointed Research Cluster Head. Today, he is the Director for the Division of Institutional Development, USM. Dr. Phua has held several academic administrative posts at the School of Medical Sciences, USM, including Program Coordinator for Diploma in Medical Laboratory Technology (MLT) course, Block Coordinator for undergraduate Medical Doctor (MD) and Postgraduate Masters in Medicine (M.Med) courses. Being a pioneer in Computer-Aided Instruction (CAI), and Chairman of the first committee for implementation of CAI in the medical curriculum, he has written many CAI programs for MLT, MD and M.Med courses for the Medical School undergraduate and postgraduate programs. He has published many articles on the development of CAI programs for teaching, and have shared his insights in designing and integrating Interactive multimedia (IM) for teaching and administrative purposes in local and international conferences, for which he was awarded the prestigous Best Technology Using Educator ISTE (1994), â&#x20AC;&#x2DC;APPLE Distinguished Educatorâ&#x20AC;&#x2122; award (2008) award by the Vice President, Education Division, Apple Inc. In scholarly pursuit of excellence in teaching, Dr. Phua had secured international grants from Apple Inc. and IBM Inc. and published his research findings in peer-reviewed journals.

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 Dr. Phua has been actively involved in research and have secured many grants for research in Immunology, Biotechnology, ICT, Microbiology and recently Community-engagement work in support of USM’s APEX agenda for sustainability. These have yielded many publications and learning resource materials, such as ‘Handbooks’, Student Study aids, PBL and CAI materials, that are relevant to Malaysia’s health problems. Eg., his research in viral hepatitis not only brought in grants from IDRC but also helped developed the National Immunization Policy EPI for Hepatitis B immunization and mandatory Hepatitis C blood screening in all blood banks in Malaysia. He has also received many awards including the “Young Scientist of the Nation Award” (Medical Sector) by the Malaysian Government in 1992, Best Invention Product 1993 (Research Exhibition, SIRIM, MOSTE), PTJ Excellence Service Awards (1994, 2003, 2006), USM’s Quality Award 2000 for development of the Postgraduate Information System, Best Oral Fee Paper Presentation awards for Basic Sciences (2005) and Medical Education (1993) at the National Conference in Medical Sciences, many Gold awards for Innovative and Creative Circles 2007 (ICC, National Productivity Corp. Malaysia), AKSA 2002 Public Services Sector Quality Innovation Award MAMPU, Gold medal from the Malaysian Invention and Design Society (MINDS) 23rd International Invention, Innovation & Technology Exhibition (ITEX) 2008, Bronze award (Salon International Des Inventions Geneve, 2009), Gold medal ITEX 2012, MPC ICC Anugerah Inovasi Terbaik, Malaysian Productivity Corporation (MPC) (Public University category), 2012. In addition Dr. Phua has received several USM Sanggar Sanjung awards for achievements in Quality, Publications and Research products. As Head of the Enteric Diseases Research Cluster at USM, Prof Phua has worked with Prof Asma the Founding Director of INFORMM, to promote Typhoid Fever research to the next level with the help of 10 researchers and 25 postgraduate students. Amongsts the successes of the Research Cluster include 58 publications in Citation Indexed Journals, Cumulative impact factor of 70, 3 patent filings, and 22 awards for diagnostic products. The group has helped to significantly reduce the incidence of Typhoid fever in the state of Kelantan. Todate, Prof Phua has trained over 15 M.Sc. & Ph.D. students and many M.Med students, and published over 100 papers and hold 2 IPRs. Amongst his quality pursuits include serving as Chairman, Secretary, and Management Representative for ISO 9001:2000, ISO 9001:2008 and ISO 17205 implementation in USM, and have achieved the necessary accreditations from IRCA, Moody Inc. and GCP to support these efforts.

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Biography of Dr.T.Theivasanthi (Plenary Speaker 5)

Dr.

(Ms) T.Theivasanthi is a Physicist (with nanomaterials / nanotechnology interests) working as Assistant Professor of Physics in International Research Centre of Kalasalingam University, Krishnankoil. She has completed Ph.D. (Synthesis and Characterisations of Metal Nanopowders) during 2014. She has 12 years teaching experience, published many research articles / books and h-index 8. She is a life member of The Indian Science Congress Association and Magnetics Society of India. She has achieved some awards/ honours/ recognitions including - Madurai Women Achievers Award - 2013, Motivational Award - 2015 and 2015 Women’s Day Award of St.Annes College. She serves, as Editorial Board Member in Nanoscience and Nanotechnology (ISSN:21632588). Her publications have been cited 211 times which shows the high impact / value. Her nanotechnology based innovations “World’s first plants materials based superparamagnetic particles” – named “Santhi Particles” (has been honoured as World Record in LIMCA Book of Records-2015 and also received award from Kalasalingam University– http://goo.gl/1F5Ru2 -Entry in GUINNESS World Record Book is under process), "The World’s lowest priced graphene" & "World’s Smallest Particles of Vegetables" (have been recognized as World Records by Limca Book of Records – 2016) - http://goo.gl/QbbdFo , http://goo.gl/jGATFk superparamagnetic nanoparticles of graphene, lead (http://goo.gl/ftS0hr), semiconducting Pb Nanoparticles, agricultural nanofertilisers and nanoparticles for treatment of diabetes, psoriasis and EBOLA, DENGUE, HIV & H1N1 virus infection. News about her research – Santhi Particles in The Economic Times & The Telegraph. She has been invited to deliver lectures (more than 35) in many International / National scientific conferences and foreign countries (China & Malaysia). She was an invited speaker, for the 7th Bangalore INDIA NANO organized by the Govt. of Karnataka. She delivered an EXPERT TALK in 2nd National Seminar on “Nanotechnology: New Materials & Applications” at ITM Universe, Vadodara, Gujarat (www.prlog.org/12442698), National conference (NCIEST-2015) in Ramco Institute of Technology, Rajapalayam, Advances in Computational Material Science (ACMS 2015) in Central University of Tamilnadu (http://goo.gl/SxlmmD ). She was an Invited Speaker for PLENARY Session and Co-Chairman for Oral Presentation in 2nd International Conference on Nanotechnology (ICNT - 2015) & Indo-USA Joint Symposium Organized by Indian Institute of Chemical Engineers (Haldia Regional Centre) at Haldia Institute of Technology, Haldia (Kolkatta). She has been invited to deliver talk in Central University of Tamilnadu, Thiruvarur, FDP at Anna University, Trichy and National Institute of Technology, Rourkela. She is delivering Motiva tional Lectures for women, students and staff of institutes / Multi-national corporate companies. Prime Minister Office / Dept.of Biotechnology (Govt. of India) shows interests to develop her inventions related to nano-biotechnology for the benefits of public.

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Biography of Dr. Subash C.B. Gopinath (Plenary Speaker 6)

Dr. Subash Gopinath has earned his doctorate degree from the University of Madras. Then he was working in the Institute of Academia Sinica, Taiwan (1999-2003). Following this he received “Japan Society for the Promotion of Science-Fellow” award to advance his research in National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan and continued as Senior Researcher (2003-2014). Recently, he moved to the Institute of Nano Electronic Engineering, Universiti Malaysia Perlis (UniMAP). Dr. Gopinath’s main expertise includes biosensors, aptamer technology and diagnosis of infectious diseases. In the past 15 years of scientific career, he produced 9 patents including an immune-aptamer that has been successfully commercialized internationally by Wako Chemicals, Japan. His research works (total of 120) were published in refereed scientific journals and he has 9 book chapters. His research is frequently referred (by fellow scientific communities) with total citations of 2000 and H-index 24. The outcome of his research and academic recognition includes the appointment as Editorial and Advisory member of scientific journals, including Plos One and BioMed Research International. He was awarded the Excellent Academic Award by Optical Society of Singapore, and Distinguished Visitor, Brain Gain Malaysia by MOSTI.

Biography of Professor Dr. K. Sudesh (Plenary Speaker 7)

Professor Sudesh’s main research interest is in the design and synthesis of biodegradable polyhydroxyalkanoates (PHAs) using microbial systems. He started research in this area in 1992 and obtained his Masters in Biotechnology from University Malaya. Then, he continued his research for his PhD, which was sponsored by Japanese Government (Monbusho). The research was conducted at RIKEN Institute, Japan under the supervision of Prof Y. Doi. He obtained his PhD in 1999 and then continued as a Special Postdoctoral Researcher at RIKEN. He returned to Malaysia under the Brain Gain program and joined the School of Biological Sciences, Universiti Sains Malaysia as a lecturer in Nov., 2001 and became a full professor in 2011.

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 He has significantly contributed to the research and development of biodegradable plastics in Malaysia from palm oil products. In addition to the numerous scientific publications in both local and international journals, he has 5 granted patents, two of which has been successfully licensed.

Biography of Professor Aziz Amine (Plenary Speaker 8)

Professor

Aziz Amine, has been Head of Department of Chemical Engineering and Environment of the Hassan II University of Casablanca during the period 1999-2003. Professor Amine’s research over the last 25 years has focused on sensors and biosensors and there use in Analytical Chemistry. He is author of more than 110 papers and has served as coordinator of several national and international research projects. He is a reviewer for several scientific international journals. He is one of the Editors of the International Journal “Biosensors and Bioelectronics” published by Elsevier with Impact Factor 6.5. Chairman of the International Workshop “Biosensors for Food Safety and Environmental Monitoring” organized every two years in Morocco (www.biocap.ma). He was invited speaker in several congresses. He has more than 3400 citations in Scopus and has an h-index of 32.

Biography of Professor Dr. Quamrul Hasan (Plenary Speaker 9)

Professor Dr.

Quamrul Hasan has a Ph.D. in Biotechnology

from Kyoto University, Japan. Prior to joining at Universiti Utara Malaysia (UUM) as a full professor in August 2014 he was managing his own firm-Bioinnovare Co., Ltd., an international business development consulting company, based in Kobe, Japan which he founded in 2009. Prior to that he was a Professor at Japan Advanced Institute of Science and Technology (JAIST), a national postgraduate university, in Ishikawa, Japan (1997-2005). He also worked for Procter & Gamble Company, as an R&D Scientist and Manager (19942001).

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 Prof. Dr. Quamrul Hasan established a non-profit organization –Japan Halal Research Institute (JAHARI) in Hyogo, Japan in 2014 and he became the founder Chairman of this organization. A Japanese national, he had been living in Kobe, Japan with his family since 1994. Some of the key achievements of Prof. Dr. Quamrul Hasan are: 1. Professional biotechnologist with more than twenty five years of experiences in research and management (in Japan and USA) 2. Extensively experienced in both the Western and Japanese (multi-cultural) business settings. 3. Invented, co-developed and successfully launched a health-care consumer product, from original idea and laboratory test to prototyping and field-testing (FebrezeAllergen Reducer has been globally marketed by Procter & Gamble since 2004) 4. Recipient of Procter & Gamble Innovation Award 5. Published more than 50 patents and articles. At UUM, currently Prof. Quamrul Hasan is also the Director of an international research center collaborating with the universities and companies in Japan, which were initiated by him.

Biography of Professor Dr. Mohd Zaki Salleh (Plenary Speaker 10)

Professor

Dato’ Dr. Mohd Zaki Salleh started his academic career at the Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia in 1995 at the age of 40 years old after obtaining PhD from Universiti Sains Malaysia under the supervision of Prof. Dato’ Dr. Asma Ismail and Prof. Dr. Zainul F Zainuddin. Prior to that, he was a marketing executive at an American Firm, a Bank Officer at Chung Khiaw Bank Ltd (Singapore Incorporated) and a Credit Officer at Mayban Finance Bhd. In 2004, he left Institute for Research In Molecular Medicine (INFORMM), Universiti Sains Malaysia to join UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia. In 2005, he joined Malaysian BioDiagnostics Research Sdn Bhd (MBDr) as the Technical and Business Development Director. His passion in academic and research prompted him to join the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) in 2007. In 2008, together with Prof Teh Lay Kek, they started the Pharmacogenomics Centre (PROMISE). In September 2013, the centre was upgraded to an institute and was named as Integrative Pharmacogenomics Institute (iPROMISE) and he was appointed as the founding director of the institute. Their research areas include pharmacogenomics, genomics, proteomics, metabolomics and epigenetics. They had managed to develop and innovate a few PCR based genotyping kits to identify patients of different genetic backgrounds for CYPs, MDR1 and VKORC1 to help make therapy more personalized for patients. Some of these kits have been made available to hospitals. They are also in the forefront in Malaysia for ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 whole genome sequencing and metabolomics studies. They had performed whole genome sequencing of a few Malay Human Genomes, some subtribes of the Orang Asli of Malaysia, some pathogens and more genomes are in the pipeline. In metabolomics, they look for potential biomarkers in cancers and responses to drugs using LCMS-QTOF. From the research in natural products, a few compounds were isolated and are being optimized using in silico models to produce drugs for anti-inflammatory disorders. They hope to generate high quality research knowledge and information in genome sequences and useful metabolomics and proteomics markers for better patients’ management in Malaysia. They also provide services and consultations in whole genome sequencing and metabolomics related studies to the local scientists. iPROMISE had established some collaborations in research which include the local universities, Malaysian Ministry of Health, Malaysian Bureau of Pharmacy, Agilent Technologies, CSIR-IGIB India and the National University of Singapore. In 2014, Prof. Zaki was appointed as an Adjunct Professor to the Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research (CSIR), India. Together with a few other researchers, the Malaysian Metabolomics Society (MyMs) was established in 2011 and Prof Zaki was appointed as the founding president.

Biography of Dr. Werasak S. (Plenary Speaker 11)

Dr. Werasak Surareungchai received his bachelor degree in Chemistry and Biology from Silpakorn University, Thailand and his Ph.D. from Cranfield University, UK in 1997. He is now an associate professor at KMUTT. He chairs the Sensor research lab and the Nano research cluster at KMUTT. He is the founder of a spinoff company, Quasense which is commercializing custom-made screen printed electrodes and electrochemical devices. His research interests are biosensors, electroanalytical chemistry and bionanotechnology.

Biography of Professor J.-F. F. Weber (Plenary Speaker 12)  Natural product chemist: Pharmacy degree (Reims, France, 1981), Master in Pharmacochemistry of Natural Products (Paris, France, 1984), PhD in Phytochemistry under the supervision of Prof. Bruneton (Angers, France, 1988).  1991: First academic appointment at U. of Bordeaux, Faculty of Pharmacy, Laboratory of Pharmacognosy; main research theme: polyphenols and tannins from grape vine and other plants. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

195

Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016 

Associate Prof. at the National University of Malaysia – UKM, Pharmacy Department (1997); main research theme: oligostilbenes (polyphenols) from Malaysian timber trees

 Professor at the MARA University of Technology – UiTM, Faculty of Pharmacy (2003) o Conceived and set up Atta-ur-Rahman Institute for Natural Product Discovery o Head of the Microbial Metabolite Lab ( 20 members) o Main research themes:  oligostilbenes (polyphenols) from Malaysian timber trees  drug discovery from bioactive fungal secondary metabolites   40 research papers in peer-reviewed journals, 10 PhD graduates, 2 post-docs. Currently supervisor or co-supervisor of  10 MSc and PhD students by research only.

Biography of Dr. Latiful Bari (Plenary Speaker 13)

Dr.

Latiful Bari completed his Ph.D. in Veterinary public health in 2001 from Osaka Prefecture University, Japan and M.Sc in Microbiology in 1994 from the University of Dhaka. He did his post-doctoral work under JSPS fellowship at National Food Research Institute, Tsukuba, Japan and thereafter continued his research work on food safety and hygiene and the improvement of food safety status in different countries. His research work produces more than 100 peer reviewed publications in Journals, books-chapters, booklets etc. Many of his articles are very popular in the field of food protection. Dr. Bari, joined at the University of Dhaka in June 2010 and working on the same field and trying to improve the hygiene and food safety status in Bangladesh. Dr. Bari’s current research including intervention strategies for controlling foodborne pathogens and develop strategies for performing the technology in commercial scale: Microbiological safety of minimally processed foods; fresh produce, meat and meat products etc. Develop predictive modeling for agricultural products and used in microbial risk assessments. Emerging technologies in food preservation, Processes to reduce pathogen contamination in foods, Food irradiation, chemicals and biochemical’s used in food preservation and application of combined factors technology in the shelf life and safety of minimally processed foods including fruits and vegetables. His recent research develop lot of bio based products for water treatment, soil fertility improvement using microbiological model, natural de-coloring agents and so on. Besides his research, In addition to his research, he has social connection worldwide to the scientific community, and he is serving as general secretary for Asian Food Safety and Security Association (AFSA). Dr. Bari provides laboratory training to the scientists, students, working people, and govt. employee, on microbial detection and intervention procedure in food. ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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Keynote and plenary speakers who delivered their talk in 3rdRC4Bs-2016

Biography of Prof. M. Anwar Hossain (Plenary Speaker 14)

Professor M. Anwar Hossain awarded PhD in Pharmaceutical Analytical Chemistry 1991 from University of Tokyo, Japan, and B.Sc (Hons) & M.Sc in Biochemistry and Molecular Biology in 1981 & 1983 from the University of Dhaka. He did his post-doctoral work in industry Suntory Crop., Osaka, Japan and in academia UMDNJ, Robert-Wood Johnson Medical University, NJ, USA. Dr. Hossain joined as faculty member in the department of Biochemistry and Molecular Biology in 1984 and currently serving as a Professor since 1997 in Department of Microbiology, University of Dhaka. He was Chairman of Microbiology Department from 20092012 and 1997. Dr. Hossain served as Visiting Professor, Department of Cultural Fisheries, Faculty of Agriculture, Kochi University, Japan and RheinWaal University, Germany. Dr. Hossain served or has been serving in the highest authoritative bodies like Senate, Syndicate, Regent bodies, Academic Council and Board of Advance studies in more than six national or private universities in Bangladesh. He has been serving as National Expert member in National Task Force on National Biotechnology for Bangladesh since 2009 to date â&#x20AC;&#x201C;the committee which is chaired by the Prime Minister, Government of the Peopleâ&#x20AC;&#x2122;s Republic of Bangladesh. Dr. Hossain had served as Board Governors Member Bangladesh Council for Scientific and Industrial Research, (BCSIR), (S&IT Minister Nominee) & Council Member, BCSIR, 2009-2015. His research work produced more than 120 peer reviewed publications in Journals, bookschapters, proceedings and abstract in international conferences. Dr. Hossain awarded international bodies fellowships Common Wealth, Monbusho, USESCO, FOBMB, Fida Foundation etc. Dr. Hossain awarded best researcher award in 2011 by University Grants Commissions of Bangladesh. Currently, Dr. Hossain and his team have been working in Foot-and Mouth Disease Virus and vaccine development; Antibiotics resistant mechanism and pollutions; Poultry Salmonella and route of transmission of pathogens and resistant bacteria by migratory birds; Arsenic microbiomes and bioremediations; and bioinformatics. In addition to his research, he has social connection worldwide to the scientific community, and he is serving as Vice-President and also founder of Asian Food Safety and Security Association (AFSA). He had served as secretary general and president of Bangladesh Society of Microbiologists; and also organized two international conferences one in 2010 and other in 2015. He is/was also member of large numbers of national and international professional organization like American Society of Microbiology, NY academy of Science, USA and The Society for Neuroscience, USA.

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Scientific programme of the 3rdRC4Bs-2016

Appendix II: A copy of scientific programme of the 3rdRC4Bs-2016.

Day 1: 20/04/2016 (Wednesday) Preconference Workshop Programme

Time 08.00-09.00

Registration Workshop-1: Drug-Protein Interaction and Personalized Medicine. Speaker: Ms. Monisha Hajra, ScientiaBio, Bangalore, India

09.00-17.00

Time

Workshop-2: Basic Statistics using Microsoft Excel and SPSSâ&#x20AC;&#x2122; & â&#x20AC;&#x2DC;Basic Concept of Effective Scientific Communications. Speakers: Snr. Assoc. Prof. Dr. K. Marimuthu and Snr. Assoc. Prof. Dr. P. Balakumar, AIMST University, Malaysia Registration of Participants (Main Conference) Programme

15.00-19.00

Registration

15.15-19.00

Check-in (Pre-booked participants only)

09.00 - 10.45

Venue Medical Building, AIMST University Guest House / Apartment / Hostels of AIMST University

Day 2: 21/04/2016 (Thursday) Programme

Time 07.30 - 08.45

Venue Medical/ Dental Building Ground Floor Computer Lab, Medical Building, AIMST University Third Floor Computer Lab, Medical Building, AIMST University

Registration

Venue Great Hall

Opening Ceremony Welcome Address Snr. Assoc. Prof. Dr. Subhash J. Bhore Chairman, 3rd Regional Conference on Biosensors, Biodiagnostics, Biochips and Biotechnology 2016 Felicitation Speech Snr. Prof. Dr. M. Ravichandran Vice Chancellor and Chief Executive, AIMST University, Malaysia Introduction of Keynote Speaker Prof. Dr. Mohd. Baidi bin Bahari Deputy Vice Chancellor (Research and Innovation), AIMST University, Malaysia Officiating and Keynote Address YBhg. Dato' Prof. Dr. Asma Binti Ismail Director General, Department of Higher Education, Ministry of Higher Education, Malaysia Keynote Address Talk Title: Moving the Regional Biotechnology and Bioeconomy Forward

10.45-11.00 ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Tea break

Great Hall

Foyer

198

Scientific programme of the 3rdRC4Bs-2016 Session: 1 Chair: Prof. Dr. K. Sudesh, University Sains Malaysia, Malaysia Co-chair: Prof. Dr. Mohd. Baidi Bahari, AIMST University, Malaysia Current Progress in Cholera Diagnostics 11.00-11.30

P1 Snr. Prof. Dr. M. Ravichandran, AIMST University, Malaysia

Great Hall

Supercomputing in Biotechnology: making sense of big data 11.30-12.00

12.30-12.40

Associate Prof. Bent Petersen, Technical University of Denmark, Denmark Relevance of Biotechnological Applications for Global Food Security and Sustainability P3 Prof. Prakash P. Kumar, National University of Singapore, Singapore Summary and conclusion of the session

12.45-14.00

Lunch

Cafeteria, 1st Floor

13.15-14.00

Poster evaluation

Medical Building

12.00-12.30

Great Hall

Session 2 Session 2A Chair: Prof. M Anwar Hossain, University of Dhaka, Bangladesh Co-chair: Dr. Lee S. Y., FAS, AIMST University, Malaysia Molecular Approaches to Fundamental Studies on Biomarkers and Development of Sustainable Rapid Nano-biodiagnostics to Enteric Diseases for Low Resources Settings 14.00-14.30 P4 Prof. Dr. Phua K. K, Universiti Sains Malaysia (USM), Malaysia

14.30 -14.45

OP1

Paper-based visual detection of Salmonella bacteria using Isothermal DNA amplification and magnetic beads aggregation Dr. Ahmed M.U., Universiti Brunei Darussalam, Brunei

14.45 -15.00

OP2

15.00 -15.15

OP3

Development of a Reverse Hybridization Assay (RHA) for Simultaneous Identification of Salmonella Serotypes Causing Enteric Fever Carlos S., Universiti Sains Malaysia (USM), Malaysia Decrypting the Evolutionary Path of Antimicrobial Resistance of Acinetobacter baumannii via Next-Gen Sequencing Mohamad Izwan Ismail, Universiti Teknologi MARA (UiTM) Puncak Alam, Malaysia

15.15- 15.25

Summary and conclusion

15.25-16.00

Tea Break

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Lecture Theatre- 1, Medical building

Medical building

199

Scientific programme of the 3rdRC4Bs-2016 Session 2B Chair: Assoc. Prof. Dr. S. Kathiresan, AIMST University, Malaysia Co-chair: Snr. Assoc. Prof. Dr. V. Ravichandran, AIMST University, Malaysia Bio-Applications of Innovative Nano-materials 14.00-14.30 P5 Dr. T. Theivasanthi, Kalasalingam University, India Isoluminol-functionalized gold nanoparticles and graphene oxide nanoribbons composite for development of enzyme-based 14.30 -14.45 OP4 electrochemiluminescence biosensors Nur Syakimah Ismail, Universiti Malaysia Perlis, Malaysia

14.45 -15.00

OP5

15.00 -15.15

OP6

Disposable Screen-Printed Electrodes Modified With Nanoparticles for Sucrose Sensor Detpisuttitham W., King Mongkut's University of Technology, Thailand Cloning and expression of the urease operon from Helicobacter pylori J99

Lecture Theatre- 2, Medical building

Mohamad CWSR, Universiti Malaysia Perlis, Malaysia. 15.15- 15.25

Summary and conclusion

15.25-16.00

Tea Break

Medical building Session 2C

Chair: Assoc. Prof. Dr. Bent Petersen, Technical University of Denmark, Denmark Co-chair: Dr. Ramesh Kumaresan, AIMST University, Malaysia

14.00-14.30

14.30 -14.45

14.45 -15.00

15.00 -15.15

15.15- 15.25

15.25-16.00

OP7

Aptasensors: Bench to Bedside and Beyond Dr. Gopinath S.C.B, Universiti (UniMAP),Malaysia

Malaysia

Perlis

Analysis of chalcone-flavanone isomerase (CHI) gene cDNA isolated from American oil-palm (Elaeis oleifera) mesocarp tissue cDNA library Yu Chuen Leow, AIMST University, Malaysia

OP8

OP9

Lecture Theatre- 3, Medical building

Non-Protein coding RNA genes as novel diagnostic markers to detect pathogenic bacteria Suresh C.V., AIMST University, Malaysia Herbal based stabilisers of native and misfolded state of nuclear co-repressor (N-CoR) Matiullah Khan, AIMST University, Malaysia

Lecture Theatre- 3, Medical building

Summary and conclusion

Medical building Tea Break

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Scientific programme of the 3rdRC4Bs-2016 Session 3 Session 3A Chair: Prof. Dr. Tang Thean Hock, University Sains Malaysia Co-chair: Dr. Yu Chye Wah, AIMST University, Malaysia

16.00-16.30

16.30 -16.45

OP10

16.45 -17.00

OP11

17.00 -17.15

OP12

Recent progress in the production of biodegradable plastics from palm oil in Malaysia Sudesh K., Universiti Sains Malaysia, Malaysia Hepatoprotective effect of methanol extract of Polygonum minus leaves in carbon tetrachloride-induced liver damage in rats Christapher V, AIMST University, Malaysia Molluscicidal Effect of Poly herbal Extracts on Golden Apple Snail, Pomacea maculata Guruswamy Prabhakaran, AIMST University, Malaysia. Production of Butter Flavour Concentrate from Butter fat with Lactic Acid Bacteria by Solid Substrate Fermentation Thambirajah J.J, Department of Biotechnology, AIMST University, Malaysia.

17.15- 17.30

Summary and conclusion

17.30-18.00

Poster evaluation

Lecture Theatre-1, Medical building

Medical Building Session 3B

Chair: Assoc. Prof. Dr. Chan Yean Yean, University Sains Malaysia Co-chair: Dr. Sawri Rajan, AIMST University, Malaysia

16.00-16.30

Recent advances in biosensors based on enzyme inhibition Prof. Aziz Amine, Hassan II University of Casablanca, Morocco Design and characterization in time of an on-off DNA biosensor

16.30 -16.45

16.45 -17.00

17.00 -17.15

17.15- 17.30 17.30-18.00

OP13

OP14

OP15

Guajardo-YĂŠvenes C. F, King Mongkut's University of Technology Thonbury, Bangkok, Thailand. Optimization of PCR for rapid detection of CTX-M gene in ESBL producing Klebsiella pneumoniae clinical isolates Rasheeda B, Lahore College for Women University Lahore, Pakistan

Lecture Theatre-2, Medical building

Coenzyme Q10 dietary supplementation during antitubercular therapy prevents renal damage in rats Evan Prince Sabina, School of Biosciences and Technology, VIT University, Vellore, India

Summary and conclusion

Poster evaluation

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Medical Building

201

Scientific programme of the 3rdRC4Bs-2016 Session 3C Chair: Dr. Gopinath S.C.B, Universiti Malaysia Perlis (UniMAP),Malaysia Co-chair: Dr. Subhash J. Bhore, AIMST University, Malaysia

16.00-16.30

16.30 -16.45

OP16

Nano- and Bio-technological Advancement to assist in the Determination of Halal Products Prof. Quamrul Hasan, Universiti Utara Malaysia (UUM), Malaysia & Chairman, Japan Halal Research Institute for Products and Services (JAHARI), Japan

Lecture Theatre- 3, Medical building

Umami Tasting Detection Based Electrochemical Sensor

17.15- 17.30

Chaiboon T, King Mongkut's University of Technology Thonburi, Bang KhunThian, Bangkok 10150, Thailand. Detection of Salmonella enterica serovar Typhi Form Water Samples and Its Association with Geographical Clustering of OP17 Enteric Fever Ismail Aziah, Universiti Sains Malaysia, Malaysia The fabrication of membrane-based pneumatic microvalves in microfluidic system OP18 Ngamchana S, King Mongkutâ&#x20AC;&#x2122;s University of Technology Thonburi (KMUTT), Bangkok 10150 Thailand. Summary and conclusion

17.30-18.00

Poster evaluation

19.00-22.30

Cultural Programme and Conference Dinner

16.45 -17.00

17.00 -17.15

Lecture Theatre- 3, Medical building

Medical Building Great Hall

Day 3: 22/04/2016 09.00-09.10

09.10-09.55

09.55-10.10

Introduction of Key note Speaker by Snr. Prof. M. Ravichandran, VC & CEO, AIMST University, Malaysia Keynote Address Tamiya E., Osaka University, Japan Nanotechnology oriented biosensors and biomedical application Tamiya E., Professor, Department of Applied Physics, Graduate School of Engineering, Osaka University, Japan Tea Break

Lecture Theatre- 1, Medical building

Session 4 Session 4A Chair: Snr. Assoc. Prof. Dr. P. Balakumar, AIMST University, Malaysia Co-chair: Dr. Annie Jeyachristy, AIMST University, Malaysia

10.10-10.40

10.40 -10.55

P10

OP19

Genomics of the endangered Orang Asli: disease susceptibility and sustainability Datoâ&#x20AC;&#x2122; Prof. Dr. Mohd Zaki Salleh., UiTM, Malaysia Role of Outer member proteins (OMP) and lipopolysaccharides (LPS) in antibody response against Pasteurella multocida type B2 in bovines Imran A, University of Veterinary and Animal Sciences (UVAS),Pakistan

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Lecture Theatre- 1, Medical building

202

Scientific programme of the 3rdRC4Bs-2016

11.40- 11.55 12.00-14.30

Exploration of Novel Endophytic Bacterial Isolates for Their Antioxidant and Pro-oxidant Properties Monowar T, AIMST University, Kedah, Malaysia Sensitivity analysis of graphene based surface plasmon resonance biosensor OP21 Toloue H., University Teknologi Malaysia, Kuala Lumpur, Malaysia Ameliorative Effect of Curcumin on Olanzapine Induced Obesity OP22 in Sprague Dawley Rats Parasuraman. S, AIMST University, Malaysia Summary and conclusion Lunch

13.10-14.30

Poster evaluation

10.55 -11.10

11.10 -11.25

11.25 -11.40

OP20

Cafeteria Medical building

Session 4B Chair: Prof. Md. Latiful Bari, University of Dhaka Co-chair: Dr. Suresh C.V., AIMST University, Malaysia

11.40- 11.55 12.00-14.30

Highly Sensitive Detection of DNA Hybridization and Immunoassay Based on Nanomaterials P11 Werasak Surareungchai, King Mongkutâ&#x20AC;&#x2122;s University of Technology Thonburi, Thailand Study of Nanoparticle-Modified Screen-Printed Electrodes for detection of Sudan I contamination in chili OP23 Phanthong C, King Mongkutâ&#x20AC;&#x2122;s University of Technology Thonburi (KMUTT), Thailand. Bioengineering of Tacca integrifolia (Bat flower): effects of hormones on in vitro rooting and production of Taccalonolides OP24 Fatimah A.L, UniversitiTeknologi MARA, Shah Alam, Selangor,Malaysia Isolation, characterization and potential application of OP25 bacteriophages for phage therapy Bhandare, S.G, Universiti Malaysia Kelantan, Kelantan, Malaysia Reconfigurable Filter Bank for Accurate Spectral Decomposition OP26 of EEG Signals Girish Kumar C., AIMST University, Malaysia Summary and conclusion Lunch

13.10-14.30

Poster evaluation

10.10-10.40

10.40 -10.55

10.55 -11.10

11.10 -11.25

11.25 -11.40

Lecture Theatre-2, Medical building

Cafeteria

Medical building Session 4C

Chair: Prof. Dr. Pandurangan, AIMST University, Malaysia Co-chair: Dr. Leela A. J, AIMST University, Malaysia

10.10-10.40

P12

P12 - Fungal Secondary Metabolites - A Pharmaceutical Chemist Perspective Prof. Dr. J.-F. F. Weber, Universiti Teknologi MARA, Kampus Puncak Alam, Malaysia

ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

Lecture Theatre-3, Medical building

203

Scientific programme of the 3rdRC4Bs-2016

10.40 -10.55

OP27

10.55 -11.10

OP28

11.10 -11.25

OP29

11.25-11.40

OP30

11.40-11.55

OP31

Eco-friendly Biosynthesis of Atrocarpusaltilis Mediated Silver Nanoparticles - Characterization and Evaluation of its Antimicrobial and Antioxidant Potential Dr. Ravichandran V, AIMST University, Malaysia Mutiplex Isothermal Amplification for Detection of Melioidosis Jilien M. W. T, School of Medical Science, Universiti Sains Malaysia, 16150 KubangKerian, Kelantan. Effective Pulmonary Therapeutic Delivery via Surface Acoustic Waves Nebulization and Phononic Crystal Structures Mohd H. Ismail, School of Microelectronic Engineering, Universiti Malaysia Perlis, Malaysia. Development of a Novel Duplex PCR Assay for Specific Detection of Salmonella enterica subspecies enterica serovar Typhi Based on Single-Gene Target Goay Y. X., INFORMM, Health Campus, Universiti Sains Malaysia, Kelantan, Malaysia.

Lecture Theatre-3, Medical building

Assessment of Biodiesel Properties From the FAME Composition of a Malaysian Rhodophyte (Kappaphycus sp.) Md. Sayedur Rahman, Aimst University

11.55- 12.10 12.10-14.30

Summary and conclusion Lunch

13.10-14.30

Poster evaluation

Cafeteria Medical building Session 5 Session 5A

Chair: Snr. Assoc. Prof. Dr. K. Marimuthu, AIMST University, Malaysia Co-chair: Dr. Kazi A Selim, AIMST University, Malaysia 14.30-15.00

P13

15.00 -15.30

P14

15.30 -15.45

OP32

15.45 -16.00

OP33

16.00- 16.15

Safe Water as the Key to Food Safety & Global Health Associate Prof. Md. Latiful Bari, University of Dhaka, Bangladesh Foot-and-Mouth Disease: Current Scenario in Asia and Bangladesh Prof. M Anwar Hossain, University of Dhaka, Bangladesh Generation of RNA aptamers against Mycobacterium tuberculosis secretory protein ESAT-6 - a preliminary study

Lecture Theatre-1, Medical building

Bakhtiar B, AMDI, Universiti Sains Malaysia, Kepala Batas, Malaysia.

An Expression Analysis of Salmonella Pathogenicity Island (SPI)Derived Non-Protein Coding RNAs in S. Typhi Biofilm formation Anbalagan, INFORMM, Universiti Sains Malaysia, Malaysia.

Lecture Theatre-1, Medical building

Summary and conclusion

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204

Scientific programme of the 3rdRC4Bs-2016 Session 5B Chair: Snr. Prof. Dr. M. Ravichandran, AIMST University, Malaysia Co-chair: Assoc. Prof. Dr. Matiullah Khan, AIMST University, Malaysia

14.30-14.45

OP34

14.45 -15.00

OP35

15.00 -15.15

15.15 -15.30

15.30- 15.45 15.45- 16.00

Quantitative, Single-Step Measurement of Hemoglobin A1c in Whole Blood for Personalized Medicine Khor S. M., University of Malaya (UM), Malaysia Development of rapid diagnostic detection for Salmonella enterica subspecies entericaserovar Paratyphi A using cross priming amplification Roziana, M.H, INFORMM, Universiti Sains Malaysia, , Kelantan, Malaysia Conversion of rice husks to polyhydroxyalkanoate (PHA)

OP36

Heng K.S, Universiti Sains Malaysia, Penang, Malaysia

Lecture Theatre-3, Medical building

Salmonella typhimurium Detection based on Electrochemical Immunoassay using Methylene blue/MWNTs/Magnetic Particle OP37 Ngoensawat, U, King Mongkut's University of Technology, Bangkok 10150, Thailand. Electrochemical Characterisation and Determination of Mycobacterium Tuberculosis by Voltammetry at Polymer OP38 Nanocomposite modified Platform Himkusha Thakur, Panjab University, Chandigarh, India Summary and conclusion Valedictory function

15.45-16.15

16.15- 17.00

  

Welcome Prizes and awards Vote of thanks

Lecture Theatre- 1, Medical building

Tea Break

Note: K, Keynote address; P, plenary talk; OP, oral presentation

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ISBN: 978-983-43522-8-8, e-ISBN: 978-983-43522-7-1

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About Editor Subhash Bhore, PhD: Subhash completed his BSc (Botany) and MSc (Botany) degree education at University of Pune, India. Immediately after completing his MSc (May 1996), he got an opportunity to work at ‘Biochemical Engineering Department’ and ‘Plant Tissue Culture Pilot Plant’ of the National Chemical Laboratory, Pune, India. In June 2000, he received a Doctoral Fellowship (GRA) to pursue a PhD Degree in Molecular Genetics at the National University of Malaysia (UKM). In 2004, he was appointed as Senior Research Officer at Melaka Institute of Biotechnology (MIB), a research wing of Melaka Biotechnology Corporation, Malaysia. Based on his performance, in April 2005, he was promoted as ‘Principal Investigator & Head of R&D Department’ at MIB, Malaysia. In 2008, he was invited by the AIMST University as a ‘Visiting Faculty’ for their Department of Biotechnology and now serving as a Senior Associate Professor. In 2009, he was nominated for the AASIO (Association of Agricultural Scientists of Indian Origin) Young Scientist Award. He has published more than 45 peer-reviewed articles, 4 books, and submitted more than 11,900 DNA sequences in Gene Bank, and got more than 10 awards/fellowships. As of May 2016, he has supervised more than 67 students including postgraduates, undergraduates and industrial trainees. He is actively involved in research as well as teaching and advising of postgraduate and undergraduate students. You may contact him using email, subhash@aimst.edu.my or subhashbhore@gmail.com

Research Highlights in 4Bs Biosensors, Biodiagnostics, Biochips and Biotechnology Published by AIMST University