ABSTRACTS

AsPac J. Mol. 2013 Mol. Biol. Biol.Biotechnol. Biotechnol. Vol. 21 (2/supp), 2013 Vol. 21 (2/Supp) : 1-43

Abstracts of 20th MSMBB Scientific Meeting

ABSTRACTS

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AsPac J. Mol. Biol. Biotechnol. Vol. 21 (2/supp), 2013

Sustaining Malaysia’s Future; MegaScience Strategy Ahmad bin Ibrahim Executive Director, Executive Committee, Akademi Sains Malaysia (ASM) Email:[email protected] Malaysia faces a challenging future. The sectors driving the economy have reached stagnation point. The country’s supply of petroleum is drying up. We are already a net importer and therefore at the mercy of global oil prices. The country’s expansion in palm oil is severely limited by constraints of land, not to mention labour availability. Much of our water sources are seriously polluted. The manufacturing sector is not doing that well either. Global competition in the electric and electronic sector is eroding away our market share. The message is clear: We desperately need to build new sources of growth; otherwise the country’s future is at stake. The MegaScience Framework is a platform initiated by the Academy of Sciences Malaysia to undertake strategic studies to develop effective planning on how to sustain Malaysia’s sterling performance. More implicitly, how can science help to sustain Malaysia’s future? This paper will present the recommendations of phase one of the MegaScience study on water, energy, health, agriculture and biodiversity, with special focus on the role of biotechnology. Session 1: Biomedical and Health Sciences From BRCA to GWAS - Getting the Data to the Clinic in Asia Soo-Hwang Teo


families can be identified, the current utility methods and limitations of genetic testing, the issues surrounding the management of women who are identified as being at high risk of developing cancers, and the implications of therapy. Finally, I will briefly provide an update on our current understanding of other genes and genetic loci identified through genome- wide association studies and discuss whether these this SNP- based testing are is ready for clinical practice. Evolution of The Drug Discovery Biotech Industry in Asia – with Specific Focus on Developments in India. CSN Murthy CEO, Aurigene Discovery Technologies Ltd, India. Over the past 10 years, there has been a rejuvenation of interest in the Drug Discovery space in India. While initial participation of companies in this complex space has been through providing services to pharma companies in the West, there has been a pronounced shift in focus on generating IP and developing drugs either for the global markets, or for licensing the same to Pharma companies for further development by them. This is propelled not only by the value creation opportunity, but also equally by the development of many core competencies needed for drug development in a relatively short time period. Tumor Targeting Delivery of Poorly Water Soluble Drugs by Non-covalent Complexes with Recombinant Human Alpha-Fetoproten Elena Dudich1,2, Lidia Semenkova1, Galina Soboleva2, Igor Dudich1, Eduard Tatulov2,Khalijah Awang3, Noor Hasima Nagoor3. Institute of Engineering Immunology, 2BioSystems Ltd., Lyubuchany, Moscow Region, Chekhov District, Russia 142380; 3Faculty Science, University Malaya, 50603, Kuala Lumpur, Malaysia. E-mail: [email protected] 1

Chief Executive, Cancer Research Initiatives Foundation Adjunct Professor, University Malaya Medical Centre Although Asians account for more than 60% of the world’s population, relatively little is known about the prevalence and penetrance of cancer due to inherited- predisposition genes in Asian populations, particularly in low- and middle- income Asian countries. It is estimated that 10-30% of breast and ovarian cancers may be due to an inherited predisposition, including through inherited mutations in the breast/ovarian cancer genes [BRCA1 and BRCA2], and other candidate high and moderate penetrance genes, including TP53, PALB2 and ATM. In my talk, I will review our molecular understanding of the function of these genes; the risks associated with each gene, and provide an update of what we currently know about the relevance of this information to the Malaysian population. In addition, I will review the ways in which high-risk

Plant-derived chemotherapeutic drugs are widely used in medicine for combined cancer therapy. These compounds belong to the class of flavones, isoflavones, flavonoids, etc., which are highly hydrophobic and are insoluble in water solutions. This leads to the significant difficulties upon administration of these drugs in to patients in injectable form. Interactions of pharmaceutical drugs with serum proteins are an important issue in drug delivery. We have studied the drug-binding capability of recombinant human alpha-fetoprotein (rhAFP) that is produced in the yeast S. cerevisiae, and anti-tumor efficacy of these non-covalent rhAFP/drug bioconjugates with various water-insoluble tumoricidal plant-derived


compounds: paclitaxel, curcumin, genistein and retinoic acid. Non-covalent complex formation of rhAFP with paclitaxel, curcumin and RA was approved by adiabatic scanning microcalorimetry technique, allowing the detecting of changes in the thermodynamic parameters of the AFP macromolecule following complex formation. Our experimental data demonstrated that small lipophilic drugs can interact with the hydrophobic binding sites of the rhAFP molecule by forming high- affinity non-covalent complexes, allowing drug solubilization and its targeting targeted delivery to cancer cells while avoiding normal cells. Tumor- targeting of the rhAFP/drug complexes was provided by their interaction with specific membrane AFP-receptors, which are expressed on the surface of cancer cells but are not available on the normal cells. Our results indicated that drug complex formation with rhAFP markedly increases increased water solubility, bioavailability and efficacy of the drug/rhAFP non-covalent complexes against resistant human breast cancer MCF-7, glioma C6 and human hepatocarcinoma HepG2 cell lines in vitro as compared to the efficacy of standalone drug compounds standalone. It was also shown that rhAFP/drug complexes do not penetrate into normal cells, allowing a significant decrease of in unspecific toxicity. We propose here a novel approach for targeting combined anti-cancer biochemotherapy with combination of rhAFP and suitable tumoricidal plant-derived compounds, providing targeted tumor-specific drug delivery and enhanced therapeutic efficacy. Natural Compounds: The Screening and Development of Anti-Cancer Drugs Noor Hasima Nagoor Pitchai Institute of Biological Science, Division of Genetics & Molecular Biology, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. To date, the majority of new anti-cancer drugs have been generated from natural products (secondary metabolites) and from compounds derived from natural products. Despite Malaysia being one of the richest countries in terms of untapped biodiversity resources, their progressions through the drug development pipeline from upstream screening to downstream clinical trials has been hampered by both financial limitations and confound research methods. Here, the necessary non-clinical steps and experimental examples required to bring natural small molecules and biologics to potential anti-cancer clinical trial candidates will be elaborated. This includes bioassay-guided screening methods for hits and leads, reporting cytotoxicity values and cutoffs, structure-activity relationships (SAR), mode-of-actions (MOAs), various assays for screening anti-cancer prop-


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erties including metastasis, angiogenesis, invasion, apoptosis and cell cycle arrest, multi-drug combination pharmacokinetics and dosages, as well as design and analyses of in vivo tumor xenograft models. Experimental examples involving 1’-acetoxychavicol acetate (ACA), which is a plant phenylpropanoid extracted from a Malaysian wild ginger, will be elaborated. ACA has been shown to induce cytototoxic and apoptotic effects on various epithelial-based cancer cell types with significant inhibition of NF-KB activation. Its in vivo efficacy has also been experimentally validated in nude mice xenografted models with tumor regression effects surpassing anti-neoplastic commercial drugs such CDDP and paclitaxel. Additionally, impediments and solutions in pre-clinically developing ACA such as its aqueous solubility, in vivo stability and target specificity are also discussed. Session 2: Agriculture From Omics to the Field: An Oil Palm Perspective David R. Appleton Biotechnology and Breeding, Sime Darby Research Sdn. Bhd., 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh Silikon, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia. Crop yield is a complicated trait involving many genes across many biological pathways. Oil palm yield is no different, as evidenced by the lack of observed simple mendelian heritable patterns in overall fruit bunch or oil yields. However, several intermediate traits that contribute to yield do display higher degree of heritability. This ultimately complicates the breeding selection process and in particular, makes the application of individual or combinations of genetic markers (such as QTLs and SNPs) difficult if used in isolation. Biochemical “omics” technologies can complement the genetic marker discovery process through the identification of important gene and gene variants for intermediate phenotypes leading to important agronomic traits such as oil yield. In many cases, the biochemical information obtained provides insights into the regulation of key biological pathways involved in lipid biosynthesis in fruit and may lead to the identification of intermediate phenotypes that are more closely regulated by genetics. Omics tools were used to study oil palm mesocarp during critical stages of fruit development in comparatively high and low yielding oil palm samples, resulting in the identification of gene regions that are associated with higher oil yield. In addition to global regulation differences of primary metabolites such as amino acids and nucleosides being observed in high-yielding palms, interesting divergence of flux in carbon utilization

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during glycolysis was also evident. Sequence comparisons of these genes indicated evolutionary importance to oil production and allowed the prioritization of markers for population genotyping. In the future, focused biochemical studies should be able to probe the many different physiological processes contributing to yield and other important traits and increase our understanding on how we can optimize these to increase productivity of oil palm through marker assisted breeding and/or larger scale genomic selection processes. Freshwater Prawns: A Candidate for Aquaculture - Mining of Markers for Aid in Selection, Adaptation and Survivability. Subha Bhassu Genetics and Molecular Biology Div., Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia and Centre for Research in Biotechnology for Agriculture, CEBAR, University of Malaya, Kuala Lumpur, Malaysia. Email:[email protected] Macrobrachium rosenbergii(de Mann, 1879), known as the Malaysian Giant Freshwater Prawn, is listed as one of the most commercially important crustacean species worldwide and this is reflected by the phenomenal increase in its production for human consumption. In response to the Malaysian government’s demand in establishing food self-sufficiency and as well as reducing the food import bills, the issues faced by the local prawn aquaculture industry should be addressed urgently through a systematic and efficient genetic improvement program. The control of diseases is one of the most important aspects in aquaculture, and like other aquaculture products, the giant freshwater prawns are prone to diseases. For one to understand the problem of these diseases, its genome and its adaptation to the environment needs to be addressed carefully. We would like to present a series of hypotheses of the possible adaptations of these prawns undergo that leads to genetic polymorphism in these prawns. Outcome of the work might explain whether it is natural selection or adaptation that has played an important role in the survivability of these prawns over the past decade. Functional validation of candidate genes identified from rice quantitative trait loci (QTL) for environmental stresses. Slamet-Loedin, Inez H1. Gamuyao R2. Heuer, Sigrid2. Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines 2 Current address: Australian Centre for Plant Functional Genomics. Adelaide. Autralia. 1,2


Phosphorus (P) and water are indispensable in agricultural production systems. A major quantitative trait locus (QTL) that enhances P uptake, Pup1, was identified in the traditional aus-type rice variety Kasalath about a decade ago; however, the mechanism of one of candidate genes underlying the QTL was only recently revealed. Genetic transformation platform we developed for indica rice with the efficiency of 30-50% allows functional validation of candidate genes in a precise manner. Agrobacteriummediated transformation was routinely applied using immature embryo at the International Rice Research InstituteGenetic Transformation Laboratory. Over-expression of candidate genes in the popular recipient parents by constitutive promoter, in combination with silencing or down regulation of the genes using micro-RNA technology or RNA-i in the donor parent, and expression of fusion of native promoter with GUS-A marker gene are typically conducted to study and reveal the major gene/s underlying the major QTLs. Phenotypic studies comparing the transgenic rice and the near isogenic QTL lines with control plants are undertaken to show the role of the candidate gene for identified target trait. These approaches were applied to elucidate the function of OsPSTOL1 for phosphorous uptake. Session 3: Bioprocess Technology and Bioproducts Sustainable Bioenergy, Biofuels and Biomaterials from Palm Biomass Mohd Ali Hassan1 and Yoshihito Shirai2 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 2 Department of Biological Functions and Engineering, Graduate School of Life Sciences and System Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu 808-0196, Japan. 1

This paper addresses the development of bioenergy, biofuels and biomaterials within the context of a palm biomass refinery by integrating the oil extraction and biomass utilization in palm oil mills in Malaysia. The current practice in the palm oil industry is to optimise oil palm growth for maximum oil yield in fresh fruit bunches at the plantations, and improve mill operations for maximum extraction and recovery of palm oil and palm kernel oil. Much of the huge biomass generated either at the plantations (oil palm frond) and at the mills (empty fruit bunch, mesocarp fiber and palm kernel shell) are not efficiently utilized. In particular, palm oil mill effluent (POME), which is the wastewater stream in palm oil mills, is treated mainly to remove its high biological



oxygen demand (BOD) in order to meet discharge standards prior to disposal. The most common treatment system presently employed for POME is the anaerobic ponding system, whereby the biogas produced is released into the atmosphere, causing environmental pollution due to the greenhouse effect. However, with biogas capture projects as suggested in the Economic Transformation Program under Palm Oil NKEA EPP5, the scrubbed biogas can be fed into a gas engine to generate 1MW of green renewable electricity for grid connection, or alternatively used in the mill such that the solid biomass can be released and used for other value-added products. The anaerobic sludge can be co-composted with various palm biomasses to produce organic compost.Other appropriate biomass processing technologies include the use of biosugars from oil palm fronds as fermentation feedstock, biochar production from solid biomass residues, energy efficiency and water recycling. With these integrated technologies in place, the palm oil mills can further improve their operations towards a biorefinery, achieving a more sustainable palm oil industry in the near future. Integrative operation in downstream application of aqueous two-phase systems

processing:

Ling Tau Chuan Institute of Biological Sciences, University of Malaya (UM)

Faculty

of

Science,

Aqueous two-phase systems (ATPS) can be exploited for the direct capture of target bioproducts from particulate-containing feedstock without prior complicated solid/liquid separation steps such as centrifugation and filtration. Several successful downstream processing operations exploiting ATPS have been described in the literature for various fermentation broths and cell homogenates. In this paper, the ATPS extractive bioconversion of cyclodextrins (CDs) with crude Bacillus cereus cyclodextrin glycosyltransferase (CGTase) using sago starch as the substrate is investigated. Enzyme Catalysis in a Non-Conventional Reaction System M Suffian M Annuar Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. E-mail: [email protected] Enzymes have been proven to be efficient and environmental-friendly catalysts in vitro under mild aqueous conditions. However, as long as the enzymes are restricted to their gentle, natural reaction milieu, the scope of industrial bioconversion is necessarily limited

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by a number of considerations. This is true especially for chemicals and polymers. For instance, many such compounds are water insoluble and may complicate the mass transfer process. An aqueous environment also frequently gives rise to unwanted side reactions. In addition, thermodynamic equilibria of many desired reactions are unfavorable in water. Product recovery is often difficult from aqueous medium. Lipase (triacylglycerol acylhydrolases EC 3.1.1.3) is widely used in the processing of food, pharmaceuticals, leather, textiles, cosmetics, detergents and paper. Lipase-catalyzed reactions in environments perceived as biologically harsh and potentially destructive, for example reaction under high pressure-high temperature generated by acoustic cavitation, in organic solvents and their binary mixture as media, and in bulk ionic liquids, will be discussed. The enzymatic synthesis of high molecular weight biopolymers is made possible by intense acoustic mixing effects overcoming elevated viscosity due to polymerization. This would not have been possible under a conventional mixing system. Efficient enzymatic esterification of bulky biopolymers is demonstrated in solvents and their binary mixtures. This model system led to the synthesis of a macromer that became the building block of smart hydrogels displaying pH responsiveness and shape memory. These non-conventional reaction systems are adaptable to existing infrastructure for bulk-synthesizing of biomaterials with potential niche applications. Construction of a Reactive Oxygen Species (ROS) Biosensor for Non-Invasive in vivo ROS Detection in Plants Say Chong WONG, 1Chong Siang TEE, 2Chai Ling HO, Hann Ling WONG

1 1

Faculty of Science, UTAR, Faculty of Biotechnology and Biomolecular Sciences, UPM

1 2

Reactive oxygen species (ROS), such as superoxide anions, singlet oxygen, hydrogen peroxide and hydroxyl radicals, is well-known for its devastating effect on biological molecules. Despite of its toxicity, it has been reported that ROS also serves as a signalling molecule that regulates biochemical and physiological processes in plants, such as programmed cells death (PCD), stomatal closure, defence mechanisms, plant growth regulator signalling and gene expression regulation. In various studies of the ROS-related signalling pathway, methods involving fluorescent chemicals, e.g. 2’,7’-dichlorofluorescein (DCF), dihydrocalcein, luminol or their variants were used. ROS is also monitored indirectly by measuring the expression levels of ROS-responsive genes. However, this method is tedious and time-consuming. Although reporter systems such as the luciferase reporter system are used, it involves the substrate luciferin, which is costly and infeasible. In this study, we were interested in the detection of intracellular ROS

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production through the expression of green fluorescent protein (GFP) driven by the ROS-inducible promoter of Zat12 gene. Using Gateway® Cloning Technology, we had cloned the Zat12promoter (proZat12) from Arabidopsis thaliana genomic DNA and subcloned into the Agrobacterium binary vector pGWB4, thereby placing the promoter at the 5’ upstream of the sGFP gene. Putative clones were screened using colony PCR. The sequence and orientation of the cloned fragments were confirmed using DNA sequencing. Recombinant pGWB4 carrying the Zat12promoter was used to transform into Agrobacterium tumefaciens. This construct (proZat12::sGFP) will be tested for potential in detecting the presence of ROS by transiently co-expressing proZat12::sGFP and the ROS-inducing gene OsRbohB in Nicotiana benthamiana. OsRbohB encodes for the catalytic subunit of NADPH oxidase, which is responsible for intracellular ROS production. Hypothetically, in the presence of hydrogen peroxide and transiently-expressed ROS-inducing genes, sGFP expression will be induced, thus producing green fluorescence at 509 nm with excitation at 489 nm, and detecting ROS in vivo. Expression of the Bacterial yoeBspn Chromosomal Toxin Gene Causes Cell Death in the Model Plant Arabidopsis thaliana Fauziah Abu Bakar1, Yeo Chew Chieng2 and Jennifer Ann Harikrishna1 Centre for Research in Biotechnology for Agriculture (CEBAR) and Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur; 2 Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Kampus Gong Badak, 21300 Kuala Terengganu. Email: [email protected] 1

Bacterial toxin-antitoxin (TA) systems usually comprise of a pair of genes located in the chromosome or in some plasmids of several bacterial species, and encode a stable toxin and its cognate labile antitoxin. Chromosomally-encoded TA systems have been shown to be involved in bacterial stress responses and activation of the toxins which usually leads to cell death or dormancy. The yefM-yoeB TA locus from the Gram-positive bacterium Streptococcus pneumoniae has been shown to be a functional TA system with over expression of the YoeB toxin leading to cell death in S. pneumoniae as well as in E. coli. In other bacteria, homologous YoeB toxins have been proven to be endoribonucleases and their activity was neutralized by tight binding with their cognate YefM antitoxins. Some of these bacterial toxins have been found to be functional in eukaryotic cells, such as yeast, zebrafish and human cell lines.However, no such work has been reported in any plant system. In this study, an inducible expression system was used to introduce the bacterial yoeB toxin gene


into Arabidopsis thaliana as a model plant. The results indicated that expression of the YoeB-GFP fusion protein was lethal in a high proportion of T2 transgenic Arabidopsis. Quantitative RT-PCR demonstrated that the expression of the yoeB toxin gene was up-regulated from day 2, following induction with 17-b-estradiol as compared with the expression level at day 1. The expression level was highest at day 3, after which the expression levels decreased at days 6 and 7 to levels lower than that of day 1. Thus, the bacterial YoeB toxin could potentially be developed as a specific cell ablation tool for plants. Profiling of Metabolites Contributing to Leaf and Mesocarp Development in Elaeis guineensis Bee Keat Neoh, Huey Fang Teh, Theresa Lee Mei Ng, Soon Huat Tiong, Asma Dazni Danial, Mohd. Amiron Ersad, Mohd. Zairey Azwan, Mohaimi Mohamed, Harikrishna Kulaveerasingam and David R. Appleton. Sime Darby Technology Centre Sdn Bhd, 1st Floor, Block B, UPM-MTDC Technology Centre III, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Email: [email protected] Oil palm (Elaeis guineensis Jacq.) is the most productive oil crop and can store up to 90% of oil in its fruit mesocarp. Oil palm leaves are the photosynthetic source and therefore the major contributor for vegetative and reproductive growth. Differential partitioning of resources produced by autotrophic leaves between heterotrophic spears and other sink organs (roots, trunk, bunches) ultimately affects bunch production and yield. Hence, the investigation of metabolic changes during the switch of leaves from heterotrophy to autotrophy could shed light on these physiological processes. A metabolomics approach was employed to study oil palm leaves and mesocarp at differentdevelopmental stages in order to better understand the progression of metabolite biosynthesis and partitioning. The rapid increase in leaf biomass requires a carbon allocation, presumably from both heterotrophic sources (trunk apex) and autotrophic leaves.Carbon and nitrogen flow within the oil palm depends on translocation of sugars and amino acids from the autotrophic to heterotrophic organs. Amino acids (building blocks for protein) were found to be more concentrated in younger leaves and mesocarp compared to the matured leaves. However, sugars (precursor for lipid biosynthesis) were found to be concentrated in the mesocarp before lipid biosynthesis and lower in leaf, suggesting that sugar participates in leaf growth during the fast elongation stage and are also accumulated in preparation for lipid biosynthesis in the mesocarp. These results provide useful information for oil palm photosynthetic and


translocation studies and lay the foundation for studying their involvement in lipid biosynthesis.

Session 4: OMICs The Genetics of Complex Diseases (with Specific Reference to Asthma and Allergic Diseases) Chew Fook Tim Allergy and Molecular Immunology Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore. Asthma and allergic rhinitis are major global health conditions affecting 10-25% of the worlds’ population. There are clear evidences that these allergic diseases are influenced by both genetic and environmental factors.A genetic background, specifically a family history of atopic disease, has been the strongest risk factor for the development of allergic diseases, irrespective of the varying prevalence and environmental risk factors present in different societies. The prevalence of these diseases, however, seems to have increased more rapidly over the last 2-3 decades than can be explained by genetics alone. Many environmental factors may thus influence and modify the genetic predisposition towards the development of these diseases. These include allergen exposures (increase indoor lifestyle), early infection/microbial load (the hygiene hypothesis), and influences of a modern lifestyle (including nutrition and use of medication). In this presentation, I will describe the large-scale epidemiological studies performed in Singapore and in the region, coupled with genome-wide association studies to assess the genetic components associated with asthma and allergic diseases in our populations.These diseases are complex multi-factorial conditions influenced by many gene-gene (epistatic) as well as gene-environment interactions, which would require large-scale international collaborations to evaluate. Does an RNA Computer Guide Construction of the Human Embryo? Laurence James Croft Chief Scientific Officer, Malaysian Genomics Resource Centre Berhad (MGRC) Recent advances in sequencing technology have unveiled an incredibly diverse transcriptome in eukaryotic cells. Over 80% of the human genome is transcribed, and published functional characterisation of new non-coding RNAs, such as long non-coding RNAs (lncRNAs), is expanding


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rapidly. lncRNA genes now outnumber protein-coding genes in the human genome and much discussion centers on their function in body plan development. In this talk I argue for the existence of an RNA-based computer in each mammalian cell. Such a parallel computer may guide, in a perturbation-resistant manner, the accurate microscopic construction of reproducible macroscopic structures, such as human twins. Proteomic Analysis of Callus Proliferation in Plant Tissue Culture Chin Chiew Foan School of Biosciences, Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia Callus formation represents the initial and vital stage of somatic embryogenesis in plant tissue culture. Many commercial tissue culture laboratories of crops, such as oil palm, utilize somatic embryogenesis technology to mass-produce clonal elite materials for the establishment of plantations. However, one of the main obstacles in somatic embryogenesis is the initiation and proliferation of callus and the subsequent conversion of the callus into embryos. In order to elucidate the fundamental mechanisms underlying callus initiation and proliferation at the cellular and molecular levels, proteomic approaches were used. This presentation demonstrates the use of 2D-PAGE, coupled with MALDI TOF-TOF mass spectrometry, to analyze callus proliferation in commercial crops, particularly oil palm and vanilla orchids. Differential proteins associated with callus proliferation will be discussed.

Session 5: Molecular Biology Cnidarians, Not So Simple After All Hwang Jung Shan Faculty of Applied Science, UCSI University, No.1, Jalan MenaraGading, UCSI Heights, 56000 Cheras, Kuala Lumpur, Malaysia The phylum Cnidaria includes a diverse group of animals with more than 10,000 species, including jellyfish, sea anemones, sea pens, hydroids, and corals. Cnidariansare considered simple animals, with a diploblastic body plan, a nerve net, a gastrovascular cavity and a few different cell types. To date, at least two cnidarian genomes (Nematostella and Hydra) have been fullysequenced and assembled. Both genomes containthe gene repertoire

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similar to those in vertebrates and are required for the formation of mesoderm, cell-junctions and nervous system. This indicates that a great deal of vertebrate genes might exist in cnidarians. Evolution also offers cnidarians a unique set of ‘novel’ genes that shares no sequence orthology with other bilaterians. In Hydra, many of these ‘novel’ genesare involved in structural novelties of a specialised organelle, called nematocyst, which is used in the capture of prey, for self-defense and in movement. Evidence has shownthat these nematocyst genes probably arose through the tandem duplication event. Agroup of these ‘novel’ genes encodes for proteins that appear to be low molecular weight, water-soluble and cytolytic. Cytolysins have so far been identified in sea anemones as well as in Hydra. Hydra cytolysinhas been cloned and its recombinant form was tested on human cells. The results suggested that Hydracytolysinis capable of binding to the sphingomyelinassociated membrane and form a cation-selective pore on human cells.Despite their morphological simplicity, cnidarians are ancient animals having levels of complexity and striking similarities with vertebrates,while carrying out specialised functions. Fungal Infectious Disease: Research on Post-Genomics and Proteomics Chong Pei Pei Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia,43400 Serdang, Selangor Fungal species, including Candida albicans, Aspergillusfumigatus and Cryptococcus neoformans, are medically important pathogens that cause opportunistic infections in immunocompromised hosts such as transplant patients, HIV-positive individuals, and cancer patients. The spectrum of infections that these fungi cause includes not only skin and mucosal infections, but also life-threatening sepsis and invasive infections. The advent of the genomic and proteomic era has resulted in a spike in research and publications that attempt to answer questions on the fundamental biological aspects of these infections through the use of genomic and proteomic platforms. In addition, the genomes of C. albicans and several other medically-relevant Candida species have also been completed. Most of the studies have focused on gene expression changes that occur in different in vitro models with conditions that mimic the actual physiology of the


infection process. Aspects of interest include morphogenesis, host-cell interaction, virulence factors and stress response to adverse conditions and antifungal drugs. Proteomics studies on the selected fungi have shed light on the cell wall biology, immune responses and pathogenicity of these fungal infections. Now, with the availability of new technology platforms, such as next-generation sequencing, whole-genome association studies, comparative genomics of related species and gene knockouts, we can anticipate with excitement the new information that will come forth. Hopefully, this will enhance our understanding on the factors that play a role in the success of these opportunistic fungi as human colonizers and pathogens as opposed to other harmless fungi. An insight onSubstrate Binding from 3D structure of Polygonum minusHuds NADP+-dependent nerol dehydrogenase Cheng Seng Tana,b , Sze Lei Panga, Maizom Hassan b, Zeti-Azura Mohamed-Hussein a,b, Ismanizan Ismaila,b, Amir Rabu b, Kok Lian Hoc,Chyan Leong Ngb*and Zamri Zainala,b* School of Biosciences and Biotechnology, Faculty of Science and Technology,Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia b Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, c Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia *Correspondence email: [email protected] and [email protected] a

The oxidation of nerolto neral is catalyzed by nerol dehydrogenase, an enzyme belonging to the medium-chain alcohol dehydrogenase/reductase (MDR) superfamily. To date, no three-dimensional structure of nerol dehydrogenaseis available.Recently, we have successfully crystallized the Escherichia coli-expressed Polygonum minus Huds recombinant nerol dehydrogenase (PmNeDH).All thecrystals werediffracted beyond 2.0 Å resolution. The crystal structure of the homodimerPmNeDHwas determined to be 1.54 Å. Our structural comparison analysis, sequence alignment and mutagenesis studies of PmNeDHwith other alcohol dehydrogenases has identified a fewaminoacid residues that play an important role in thisenzyme, hence providing further understanding of enzyme activity and substrate specificity of PmNeDH.



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Epigenetic Modulation of the miR-200 family is Associated with Transition to a Breast Cancer Stem Cell-like State

Production of Transgenic Tomato Plants Expressing Three Overlapping Regions (C1C2C3 Genes) Isolated from Tomato Yellow Leaf Curl Virus [Malaysia]

Lim Yat Yuen

Mohtaram Mahmodieh Koravieh1, Mohamad Roff, Mohd. Noor2, Jennifer Ann Harikrishna1.3and Rofina Yasmin Othman1.3

Institute of Biological Sciences, University of Malaya (UM)

Faculty

of

Science,

The miR-200 family is a key regulator of EMT; however, its role in controlling the transition between cancer stem cell-like and non-stem cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial cells (HMLE) to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem-like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200, indicating that similar molecular changes may occur during breast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200, as restoration of its expression decreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200 expression revealed that both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem-like phenotypes. In particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histone modifications, whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that miR-200 family plays a critical role in the transition between stem-like and non-stem-like phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therapy targeted against miR-200 family members and epigenetic modifications may therefore be applicable to breast cancer.

Institute of Biological Science, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, 2 Horticulture Research Centre, MARDI Headquarters, P.O.Box 12301, GPO, 50774 Kuala Lumpur, 3 Centre for Research in Biotechnology for Agriculture (CEBAR) and Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. Corresponding Author: [email protected] 1

Geminiviruses cause severe diseases on several crops throughout the world. Tomato yellow leaf curl virus (TYLCV) is a single-stranded DNA (ssDNA) plant virus in the genus Begomovirus of the family Geminiviridae. Yellow leaf curling of tomatoes is caused by TYLCV. The disease is one of the greatest constraints in tomato production, resulting in 20–100% yield loss of tomato plants, depending on the stage of plant growth at the time of infection.TYLCV originated in the Middle East-Mediterranean region and has been introduced into many other regions around the world, making it among the most virulent and damaging begomoviruses in tomato crops. The use of siRNAs, an intermediate in the gene-silencing pathway, has become a powerful tool for specifically down-regulating gene expression, as has been demonstrated successfully in a wide variety of cells and organisms. Constructs encoding self-complementary hpRNA have been shown to be capable of generating post-transcriptional silencing to result in undetectable levels of the targeted mRNA transcript. Here, a hairpin hpRNA construct (Phells-C1C2C3), containing viral sequences encompassing part of a replication initiation gene (Rep, C1), a transcription activator gene (TrAP, C2) and part of a replication enhancer (REn, C3), was produced. Transgenic tomato plants expressing this construct were produced using agrobacterium–mediated transformation

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and examined for the insertion of the viral genes by PCR, followed by sequencing. The transgenic lines were used for T0 seed production and gene silencing study in T1. Our results showed that construction of hp-RNAi and the production of transgenic tomato plants were successful. Identification of a S. typhi-specific Antigen of Diagnostic Potential Using Bioinformatics and Proteomic Approaches Goay Yuan Xin¹, Chin Kai Ling¹, Eugene Ong Boon Beng¹, Suresh V. Chinni4, Zaidah Abdul Rahman², Prabha Balaram3, Phua Kia Kien¹ Institute for Research in Molecular Medicine (INFORMM), USM Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia. 2 Department of Medical Microbiology and Parasitology, School of Medical Sciences, USM Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia. 3 Quest International University Perak, Ipoh, Perak, Malaysia. 4 AIMST University, Jalan Bedong, 08100, Bedong, Kedah Darul Aman, Malaysia. 1

Salmonella enterica subspecies enterica serovar typhi (S. typhi) causes a systemic infection called typhoid fever, which is restricted to humans. However, the conventional method for diagnosis of typhoidusing culture and serotyping methods is laborious, time-consuming and sometimes subjective in result interpretation. Antibody detection methods can provide a better strategy for faster and easier detection. As such, this work was carried out to identify antigenic S. Typhi-specific proteins that may serve as better diagnostic markers for typhoid fever. In silico and wet-lab PCR experiments were carried out in order to ascertain the specificity of 3 putative genes, which were tested for cross-reactivity with other enteric pathogens. The SCRATCH protein prediction software, a free online protein analysis tool, was used to predict the antigenicity value of the genes. The 3 genes, namely STYP3, STYP11 and STYP22, with high specificity and antigenicity prediction indiceswere studied. The genes were cloned into a vector, pET28a, and protein expression was carried out according to standard protocols. The affinity chromatography-purified proteins were tested using the Western blot technique for specific IgGs on 4 confirmed typhoid fever sera, with 4 normal sera as controls.This study identified a protein marker, STYP11, which is specific for typhoid fever. The preliminary result showed that an antigenic band at a molecular weight of 29kDa was


targeted by human IgG in all 4 typhoid sera, reaffirming that it has good diagnostic value. There were no cross-reactions in normal sera. In future, STYP11 will be further evaluated for its sensitivity and specificity with more serum samples for further confirmation of its value as a potential diagnostic marker. Pipeline for the de novo Assembly of Bacterial Genomes from Illumina Sequence Data Sabiha Shaik1, Ramani Baddam1, Narender Kumar1 and Niyaz Ahmed1,2 Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India 2 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

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The genome of an organism is a knowledge hub, and when properly mined can describe the organism in infinite detail. A wide range of questions about microbes, ranging from their origin to their lifestyle adaptations, can be answered by looking at their genetic makeup. Next Generation Sequencing (NGS) technology, with its ability to produce an enormous volume of data at low cost and in a short amount of time, has been attracting researchers all over the world. But short read lengths and huge amounts of data are still major concerns associated with NGS data. Though a variety of tools for assembly and alignment are available, they usually require some computational expertise. This necessitates the development of more user-friendly techniques and unified protocols for easy and rapid assembly of microbial sequence data. An optimal protocol is required for assembling and curating, so that meaningful information can be extracted from the sequence data. So far, no such guidelines have been put forward. We propose a complete pipeline for the de novo assembly of Illumina paired-end reads into a draft genome, specifically in the case of bacteria. This pipeline is a powerful combination of both publicly-available tools and in-house written scripts. To compliment this pipeline, a tool called Contig-Mapper was developed in-house, which reads and extracts information from a SAM (Sequence Alignment Map) file and then creates a map of all possible linkages between the assembled contigs. This pipeline will be of much help to beginners in the domain of bacterial genome sequencing. The validation of this proposed pipeline was performed using simulated data of the Sanger-sequenced genome of E.coli CFT073.


Session 6: Microbes Pathogen Genomics, Evolution and Survival Mechanisms: Lessons from the Two Age-old Pestilences Niyaz Ahmed Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, India Correspondence email: [email protected] With the advances in massively parallel, Next-Generation Sequencing (NGS) technologies, the scientific community is confronted with the challenge of data handling, management and heralding sustainable and testable ideas out of the genomic information. The NGS platforms not only help in real-time decipherment of various pathways, but also hold potential for the maturation of the OMICS sciences as a whole. We have been involved in sequencing the genomes of pathogenic species and strains of Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis, Salmonella enterica, Vibrio parahaemolyticus, etc. for some time, and these genomes have been analyzed to dissect the various survival mechanisms based on new genes and functions. Over the years, our comparative genomic analyses have helped us identify several putative virulence-encoding genes in two major human pathogens: Mycobacterium tuberculosis and Helicobacter pylori. Some of these virulence factors possibly play crucial roles relevant in chronic persistence of these bacteria and provide them with survival advantages. We analyzed the signaling pathways pertaining to proapoptotic and/or proinflammatory behavior of certain virulence factors from H. pylori and pathogenic mycobacteria. In the former organism, many of such genes are encoded by the ‘plasticity region cluster’ of the genome and we looked at the functions of those proteins, from the cluster, which were predicted to be proinflammatory and/or proapoptotic. In M. tuberculosis, we studied novel genes/ proteins that have predicted ribosome binding and thereby regulatory function as well as innate immune functions. We envision that such unrelated, yet finely-orchestrated functional characteristics could be significant in controlling or optimizing the pathogens’ metabolic processes during infection, thereby ensuring long-term survival of bacteria in


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a dormant form. Better insights into the mechanistic aspects of dormancy survival and/or adaptive colonization would facilitate improved understanding of the blooms and boundaries of bacterial parasitism. RAPD-PCR and MLST of Candida albicans Clinical Isolates from Five Blood Cultures Gek Mui TAN1, Saranpal Singh CHHABRA1, Pei Pei CHONG2 and Crystale Siew Ying LIM1* Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras 56000, 2 Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, *Corresponding author: [email protected] 1

Candida albicans is the main causative pathogen of the potentially deadlyCandida bloodstream infections (BSIs) in immunocompromised and immunodeficient patients. However, differences in the genetic makeup of clinical isolates from C. albicans BSI and their epidemiologic associations are not well-studied in Malaysia. FiveC. Albicans BSI clinical isolates, obtained from Universiti Putra Malaysia, were investigated via randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with the arbitrary primer PST, and also multilocus sequence typing (MLST). RAPD-PCR found these five isolates to be genetically identical, but MLST based on the amplification and DNA sequencing of seven housekeeping genes according to standardized procedures at [http://calbicans.mlst.net], revealed them to be genetically dissimilar. This study showed that MLST analysis has a higher discriminatory power than RAPD-PCR, resulting in the identification of three new strain types (TGM_CA16, TGM_CA36 and TGM_CA37), with the assigned strain identification numbers of ST2089, ST2090 and ST2091, respectively. Two other isolates are hypothesized as new species or isolated cases of genetic exchange between C. albicans and C. orthopsilosis, warranting further investigation. This is the first study to report strain types from blood sources of infection from Malaysia, and contributes to the expansion of the C. albicans MLST.net global database.

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Partial Characterization of an Antimicrobial Peptide Produced by Lactobacillus Plantarum Lim Sue Wen, Koshy Philip* and Noni Ajam Division of Microbiology,Institute of Biological Sciences, Faculty of Science,Universiti Malaya *Corresponding author: [email protected] The aim of this study was to carry out partial characterization of an antimicrobial peptide produced by lactic acid bacteria K25 isolated from kimchi, a Korean fermented food. The identification of lactic acid bacteria K25 was done using API and 16s rRNA sequencing, and the bacterium was confirmed as Lactobacillus plantarum. The antimicrobial peptide was secreted into the supernatant of the L. plantarum K25 culture when it was cultured in MRS broth, and showed antimicrobial activity against the pathogenic bacteria Bacillus cereus and Pseudomonas aeruginosa. The antimicrobial peptide, which was produced by L. plantarum K25, was extracted from the MRS broth using ammonium sulphate precipitation, with further desalting using Gel Filtration G25. The antimicrobial compound showed stability at high temperature (90 °C for 30 min) and was also active at pH 3.0 – 6.0, with evidence of inhibitory activity on the test microbial strains mentioned. Sweet Food Hedonic Ratings, Intake Frequency and Salivary Counts of Lactobacilli and Mutans Streptococci in Association with Obesity and Menstruation Variables among Female University Students Yee-How Say*, Sheng-Pei Lim and Wey-Zheng Yeo Department of Biomedical Science, Faculty of Science, Universiti Tunku Abdul Rahman (UTAR) Perak Campus, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak. *Corresponding author: [email protected] The high prevalence of obesity in Malaysia could be due to high sugar preference (‘sweet tooth’). Lactobacillus sp. and Streptococcus mutans are bacteria commonly found in the human oral cavity and are significant contributors of tooth decay. This study investigated the association of: a) preference and intake frequency of a list of 20 sweet foods; b) sensory ratings of sweet solutions; and c) salivary S. mutans andlactobacilli counts; with obesity and menstrual variables among female UTAR students (101 Chinese, 19 Indians). Demographic and anthropometric data were taken. The preference and intake frequency of sweet foods were assessed using a 7-point hedonic scale, while the sweetness intensity and pleasantness ratings of increasing concentrations of sucrose solutions, regular and less sugar Vico® chocolate malt beverage were assessed using the Labeled Magnitude Scale and Labeled Affective


Magnitude Scale. This study showed that centrally obese subjects (by Waist-Hip Ratio) had significantly higher salivary counts of S. mutans andlactobacilli compared to lean subjects. Salivary lactobacilli counts were negatively correlated with pleasantness rating of less sugar Vico®, indicating that subjects with increased pleasantness rating of this beverage had lower salivary lactobacilli counts. Females who suffered from pre-menstrual fluid retention may crave for sweets during depression and those who craved chocolate during depression had significantly higher salivary S. mutans counts. However, salivary S. mutans andlactobacillicounts were not associated with the period and regularity of the menstrual cycle, pre-menstrual depression, pain and fluid retention, indicating that menstruation variables did not affect these two salivary bacterial counts. In conclusion, this study found that salivary S. mutans andlactobacilli counts of the female students were affected by central obesity, but not menstruation variables. Salivary S. mutans andlactobacilli counts are also a poor objective indicator of having a sweet tooth. Concurrent session 7A: Mammalian Molecular Biology & Biotechnology

Lack of Association between NFKBIA -826C>T and -881A>G Polymorphisms and Colorectal Cancer Risk Shing Cheng Tan, Mohd Suzairi Mohd Shafi’i, Siti Nurfatimah Mohd Shahpudin, Mohd Aminudin Mustapha, Ahmad Aizat Abdul Aziz, Ravindran Ankathil Human Genome Centre, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. The inhibitory protein IkBa plays an important role in regulating the activity of nuclear factor-kappa B (NF-kB), a transcription factor which has been implicated in the initiation and progression of cancers. Genetic variations, such as single nucleotide polymorphisms in the NFKBIA gene which encodes for IkBa, could potentially lead to functional consequences of the protein product which could, in turn, modulate the risk of various cancers, including colorectal cancer (CRC). In this study, we investigated the frequencies of NFKBIA -826C>T (rs2233406) and -881A>G (rs3138053) polymorphic genotypes in Malaysian CRC patients and in cancer-free controls as well as evaluating the associations of the polymorphisms with CRC risk.A case-control study comprising of 237 CRC patients and an equal number of cancer-free controls was carried out. The polymorphisms were genotyped via PCR-RFLP, followed by DNA sequencing, from the genomic DNA of the study subjects. The association between the



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polymorphic genotypes and CRC risk was evaluated using unconditional logistic regression analysis. Our results showed that the two polymorphisms were in complete linkage disequilibrium (LD). The frequencies of the homozygous wild type, heterozygote and homozygous variant genotypes among the cases were 71.3%, 25.3% and 3.4% respectively, while those among the controls were 68.8%, 29.1% and 2.1%. The distribution of the polymorphic genotypes did not deviate significantly from the Hardy-Weinberg equilibrium (Pcases = 0.36, Pcontrols = 0.46). The heterozygote genotype was associated with a slightly reduced risk of CRC (OR = 0.84, 95% CI = 0.56 – 1.26), but without statistical significance (P = 0.40). Similarly, no statistically significant association was observed between the homozygous variant genotype and CRC risk (OR = 1.54, 95% CI = 0.50 – 4.82, P = 0.45). In conclusion, the present study suggests a lack of association between NFKBIA -826C>T and -881A>G polymorphisms and CRC risk.

A total of 82 genes were found to be significantly different at the 8-cell stage; with 27 genes up-regulated and 55 genes down-regulated. At the blastocyst stage, 66 genes were significantly different; with 43 genes up-regulated and 23 genes down-regulated. Differentially expressed genes were annotated and visualized using the DAVID functional analysis software (http://david.abcc.ncifcrf.gov/tools.jsp).Through the KEGG database, 9 genes for the 8-cell embryo and 11 genes for the blastocyst were annotated into different categories of pathways, which mostly involved lysosomic functions, oxidative phosphorylation and fatty acid metabolism pathways. Elucidation of these changes has provided a greater understanding of molecular alterations that occur during post-biopsy recovery of preimplantation embryos.

Gene Expression and Functional Pathways of Biopsied Preimplantation Murine Embryos.

Jang Jih Aaron Chen1, Kek Heng Chua1, Elizabeth George2, Jin Ai Mary Anne Tan1

Norhazlin, J.M.Y., Hoh, B.P., Sheikh Abdul Kadir, S.H., Norita, S., Wan-Hafizah, W.J., Mohd-Fazirul, M., Razif, D., and Nor-Ashikin, M.N.K.

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Institute Molecular Medicine Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia Biopsy is a prerequisite procedure of Preimplantation Genetic Diagnosis (PGD) programmes. Removal of one or two intact blastomeres can be carried out without detriment to embryonic developmental. The most common technique for blastomere biopsy is direct aspiration of one blastomere through an opening in the zona pellucida, either by mechanical drilling or acid breaching. Thus far, the effect of mechanical piezo-assisted biopsy effect on gene expression in embryos remains to be elucidated. This study was conducted to investigate the effect of piezo-assisted biopsy, at the 8-cell stage, and upon development to the blastocyst stage. Following zona breaching by piezo-drilling (EppendorfPiezoXpert®), single blastomeres were removed from in vivo-derived 8-cell ICR mouse embryos which were pre-incubated in Ca2+/Mg2+-free M2 medium. Biopsied embryos were allowed to develop further in M16 medium supplemented with 5% BSA, and collected for RNA extraction after 1 h (8-cell) and 42 h (blastocyst). cDNA were converted, amplified, labelled and hybridized on the Affymetrix GeneChip®. Microarray data were analysed with GeneSpring GX 12 to generate significant differentially-expressed genes, comparing non-biopsied (controls) and post-biopsied embryos at the 8-cell and blastocyst stages, respectively, using unpaired t-test at pstem>bark,. These findings suggest that C. cauliflora has antioxidative potential. For biochemical studies, 42 rats will be taken and divided into 7 groups (n=6): group 1 (positive control - normal saline treated rats; group 2 (negative control - treated with steptozotocin); group 3 (treated with a high dose of plant extract - 300mg/kg body weight); group 4-6 (treated with plant extracts at 100mg/kg body weight to 300mg/kg body weight) and streptozotocin , and group 7 (treated with metformin). Further studies are in progress to evaluate the antidiabetic effects of C. cauliflora against streptozotocin in vivo.

H08: Evaluation of the Antioxidant Activity and Antidiabetic Property of Cynometra Cauliflora (Nam-Nam, Fabaceae)

Dengue, a mosquito-borne viral disease, is a serious public health concern in many countries around the world. In Malaysia, suspected cases of dengue infection increase dramatically every year, with 44,641 cases reported in 2010, of which, 134 cases were fatal. Dengue infections cause a variable spectrum of manifestations, ranging from undifferentiated fever and dengue fever (DF) to the more severe syndrome, called dengue haemorrhagic fever (DHF). Recent studies have suggested the potential role of Fc gamma receptors (FcGRs) in the pathogenesis of dengue. The aim of this study was to determine FCGR1 monoclonal antibody levels in a selected group of dengue patients. Serum was isolated and used to quantify the FCGR1 protein using enzyme-link immunosorbent assay (ELISA). The microtiter plate provided was pre-coated with a monoclonal antibody specific to FCGR1 and the colour change was measured spectrophotometrically. The concentration of FCGRI in each sample was then determined by comparing the OD of each sample to a standard curve.Samples from 105 consenting respondents (39 healthy control, 24 DF and 42 DHF) were used in this study. Analysis of FCGR1concentrations in two different groups of dengue patients compared to the control group

Azalina Farina Binti Abd Aziz*, Mohammad Iqbal Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, *Corresponding author: [email protected] Cynometra cauliflora, usually known as Nam-nam, is native to Malaysia, is grown mainly in the northern peninsula and possesses many medical properties. However, its antidiabetic and antioxidative effects have not been fully investigated. The aim of this study was to understand the phytochemical, antioxidative and pharmacological properties of C. cauliflora. Dry samples of young leaves, matured leaves, stem and bark of C.cauliflora were subjected to phytochemical screening for total phenolic acids flavonoids content and DPPH free radical scavenging activity. Phytochemical screening showed the presence of tannins, saponins and flavonoids in all parts of C. cauliflora. Terpenoids were present in all parts of the plant except the bark. The constituent of cardiac glycosides

H09: FCGR1 as a Potential Biomarker for Dengue Infections in Malaysia Zaiharina MZ, Abu Thalhah AZ, Umi SH, Zuraihan Z, BP Hoh Institute of Molecular Medical Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia

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was performed using ANOVA. FCGR1 was found to be significant different between healthy controls and DF (p=0.00), DHF (p=0.00) but not between DF and DHF (p=0.79). Therefore, there is an association between FCGR1 protein levels and dengue patients. The importance of FCGR in various autoimmune and inflammatory diseases has been well-documented. It is possible that the protein level of FCGR1 in dengue is much higher during the DF stage compared to DHF, which suggests this Fc receptor may serve as a predictive marker for the progression to more severe dengue. However, further studies on the gene expression are needed to confirm this finding. H10: Interaction of Cryptococccus neoformans Extracellular Proteins with Host Mammalian Cells Choo Khi Khi1, Law Lok Mun1, Chong Pei Pei2, Anthony Ho Siong Hock1 and Phelim Yong Voon Chen1 School of Biosciences, Taylor’s University, Subang, Selangor Department of Biomedical Science,Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 1 2

Cryptococcus neoformans is encapsulated pathogenic yeast that contributes to high morbidity and mortality among the immuno-compromised population of the world. Host-pathogen interaction at the cellular interface is a vital part of pathogenesis as it precedes colonization and subsequent invasion by the pathogen. Binding and invasion into the host cells are hypothesized to be mediated by protein-protein interactions between the host pattern recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs). The aim of this study was to extract and characterize the extracellular proteins that mediate host-pathogen interaction in both encapsulated and acapsular C. neoformans. We demonstrated that the encapsulated C. neoformans binds at a lower rate to mammalian alveolar epithelial and macrophage cells, compared to the acapsular strains. Extracted extracellular proteins from both strains exhibited similar protein profiles through SDS-PAGE. Latex beads conjugated with C. neoformans extracellular proteins from both encapsulated and acapsular yeasts mediate binding to mammalian alveolar epithelial and macrophage cells. The conjugated latex beads were also phagocytosed by alveolar macrophages in a process similar to viable acapsular C. neoformans. The results indicate that proteins mediating host cell adhesion in C. neoformans are present in both the encapsulated and acapsular C. neoformans, but are usually masked by a thick polysaccharide capsule in the encapsulated strains.


H11: The MC4R rs17782313 Variant is Associated with Obesity in Malaysian-IndianAdolescents Ja’afaru Sani Mohammed, Ng Zoe Yi, Muthu Kumar VeerapenandRenee Lim Lay Hong Department of Biotechnology, Faculty of Applied Sciences, UCSI University, No 1, Jalan Menara Gading, UCSI Heights, 56000 Cheras Kuala Lumpur, Malaysia The genetic variant rs17782313 (TC) on the melanocortin-4 receptor (MC4R) gene has been consistently associated with obesity risk in various East Asian and African populations. MC4R is a G-protein coupled receptor highly expressed in hypothalamic nuclei, mediating energy homeostasis. This study aimed to determine the association of rs17782313with obesity in a population of Malaysian adolescents, comprising of Malay, Chinese and Indian self-reported ethnic groups. A total of 564 adolescents were previously recruited for anthropometric measurements (BMI and BF%). Extracted genomic DNA from the subjects was used for genotyping using a Taqman-based qPCR assay. Subjects were categorised based on BMI and BF%, wherein 23.4% were overweight/obese and 11.7% were over-fat. Indians and Malays showed significantly higher mean BMI and BF% (p=0.0001) compared to Chinese subjects. The risk allele C has a MAF of 0.238, whereas the MAF of T, the major allele, is 0.762: both are in Hardy-Weinberg equilibrium. Correspondingly, the frequency of the CC genotype is significantly lower than the TC and TT genotypes, both for the study population and by ethnic groups. Subjects with a C allele (CC or TC genotypes) showed significantly higher mean BMI (21.64±4.71 kg/m2) and BF% (21.70±6.72%), compared to subjects with the TT genotype (p=0.005, 0.003 respectively). Interestingly, an increase in BMI and BF% was observed in females and not males. Analysis by ethnicity demonstrated a significant association of the C allele with increased BMI (p=0.001) and BF% (p =0.001) in Indian females. In addition, the TC and CC genotypes were significantly higher in overweight/obese (p= 0.003; OR 3.25, 95% CI: 1.48-7.16) or over-fat (p=0.005; OR 3.87, 95% CI: 1.47-10.18) Indian subjects, supporting previous reports on association with Asian Indians. This is the first study to reveal the association of the rs17782313 variant with obesity in a Malaysian-Indian adolescent population, indicating gender and ethnic disparity of the variant to obesity, which should be further explored.


H12: Farnesol Affects Cell Proliferation and P53 Gene Expression in HeLa Cells YUSELI and Crystale Siew Ying LIM* Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras 56000, Kuala Lumpur, Malaysia. *Corresponding author: [email protected] A tumor suppressor protein, p53, modulates the transcription of genes that have roles in DNA repair, cell cycle arrest or cell death in response to stress. The p53 gene, known as the “guardian of the genome and policeman of the oncogenes,” has been shown to be associated with cancer development. Farnesol, an isoprenoid alcohol, had been shown to induce apoptosis and growth inhibition in cancer cells at a concentration of 30 μM. However, the influence of farnesol on p53 expression is not known. Thus, the effect of farnesol on p53 gene expression and cell proliferation was investigated in HeLa cells. HeLa cells were treated with 30 μM farnesol in a time-dependent manner, followed by MTT assays to determine cell proliferation. RNA was extracted from HeLa cells treated for 0, 4, 16 and 24 hours, and subjected to quantitative reverse transcription real-time PCR to investigate differential p53 gene expression over time. There was a slight increase in HeLa proliferation after 4 hours of treatment with 30 μM of farnesol compared to 0 hour, where p53 gene expression was significantly down-regulated (2.0-fold change). HeLa cell proliferation was reduced (to 84.5% and 86.7%) only after 16 and 24 hours of incubation, respectively, compared to 0 hours, where interestingly, there was no significant change in p53 gene expression. Thus, 30 μM of farnesol is able to derepresses the down-regulation of p53 in HeLa cells, thus causing reduction in cell proliferation. Further studies on the effect of farnesol on p53 protein levels are warranted to assess the magnitude of protein-level change in response to transcriptional change in p53 expression. H13: Raw Coconut Milk and Its Active Compound Lauric Acid Abrogates TNF-a Down-regulation of Scavenger Receptor Class B Type 1 (SRBI) Expression Wong Hong Kin, Cheah Hoay Yan and Chew Choy Hoong Department of Biomedical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, 31900 Perak Scavenger receptor class B type 1 (SRB1) is a type III transmembrane glycoprotein that acts as an important mediator of the reverse cholesterol transport pathway. This receptor governs cholesterol transfer from high density lipoprotein (HDL) into the liver, and is thus regarded as an important anti-atherosclerotic agent. Tumour necrosis factor alpha (TNF-a), on the other hand, is a


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pro-inflammatory cytokine that is involved directly in the induction and progression of atherosclerotic lesions. The main objective of this project was to first identify the antagonistic nature between TNF-a and SRB1, and subsequently to investigate the potential anti-inflammatory effects posed by coconut milk and its active compound, lauric acid, against TNF-a-regulated SRB1 expression. HepG2 cells were chosen for this study as SRB1 is proven to be abundantly expressed in the hepatic system. Real-time RT-PCR was performed to investigate the dose-response mRNA levels of HepG2 SRB1 following treatment with TNF-a alone or with TNF-a and coconut milk or lauric acid. The mRNA level of the target gene was normalised against beta-actin, and relative expression to the untreated control (1.00-fold expression) was quantified using the delta-delta CT method. Results obtained from this project showed that SRB1 mRNA levels were significantly repressed by TNF-a in a dose-dependent manner. However, this down-regulated response was diminished when coconut milk or lauric acid was administered following the cytokine treatment. Generally, SRB1 mRNA levels were elevated from 0.28-fold to 2.22-fold and 0.44-fold to 1.56-fold when HepG2 samples pre-treated with TNF-a were exposed to increasing concentration of coconut milk or lauric acid, respectively. In short, this study has shown that both coconut milk and lauric acid are able to alleviate the down-regulatory effect of TNF-a on SRB1 transcript levels in HepG2 cells. H14: Cloning and Sequencing Analysis Of Micro-Rna 17-92a from Hepg2 Cancer Cell Line Nur Serene Sofia binti Nor Azri and Mohd Shihabuddin bin Ahmad Noorden Faculty of Pharmacy, Universiti Teknologi MARA, Campus of Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Accumulating evidence suggests that micro-RNAs (miRNAs) may act as oncogenes or tumor suppressor genes. miRNAs are small non-coding, double-stranded RNA molecules that can mediate the expression of target genes with complementary sequences. miRNA-17, miRNA-18, miRNA-19a, miRNA-20a, miRNA-19b, and miRNA-92a are transcribed as one primary-miRNA transcript (pri-miRNA) on the upstream of the miRNA pathway and interestingly, these microRNAs act independently in promoting liver cancer. Therefore, as preliminary work, these oncogenic miRNAs were selected for cloning whereby they will be used in the functional study of non-coding RNAs. The full length of miRNA 17-92a gene was obtained from the NCBI database for primer design and amplifications were carried out on HepG2 genomic DNA using 3 sets of PCR primers with different annealing temperatures. Gel excisions

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were carried out to isolate and purify the PCR products. The PCR products were freshly inserted into appropriate TA-cloning vectors and the vectors were transformed to a JM109 competent cell where the insertions were screened using blue-white selection. Direct sequencing was performed on each vector from M13 forward priming site to validate the ligation of inserts. The sequence analyses by Nucleotide Blast (BLASTn) and BOXSHADEClustalW alignment showed a complete match between the inserts and the sequences from NCBI database. For further applications, the investigation of hepatocellular miRNA activities can be experimentally demonstrated in cell system by the ectopic expressions of these miRNAs or by the application of reporter vector.


individuals with the susceptibility of T2DM/CVD (P>0.05). CTLA-4 gene polymorphism did not confer an increased risk of susceptibility towards T2DM/CVD among investigated Malay subjects.However the gene can be considered as an important indicator to differentiate between T2DM/CVD and healthy individuals. H16: The effect of Lysyl Oxidase (LOX) and mechanical load, as the modulator of pre-metastatic niche and cancer metastasis on bone N. Ab Latif1, H.R. Evans2, N. Wang2, J.T. Erler3, A. Gartland2. Preclinical Department, Faculty of Medicine, Universiti Kuala Lumpur, Royal College of Medicine Perak, Ipoh, Perak, Malaysia 2 Human Metabolism, The University of Sheffield, Sheffield, UK 3 Section of Cell and Molecular Biology, The Institute of Cancer Research, London, UK 1

H15: Analysis of Ctla-4 Gene Polymorphism of Patients with Type 2 Diabetes Mellitus/Cardiovascular Disease in Malay Population Sumaiyah Binti Khalid1, Ali Etemad1, Ramachandran Vasudevan2, 3 and Patimah Ismail1 Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. 2 School of Science, Monash University Sunway Campus 3 Institute of Gerontology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. 1

This case control study utilized 120 samples from unrelated healthy individuals as control subjects compared to 150 samples from patients confirmed with T2DM/CVD as case subjects. The aim of this study is to analyse the rs5742909 and rs231775 polymorphism of the CTLA-4 gene in Malay subjects with T2DM/CVD and to determine the association of clinical characteristics of the subjects. Genomic DNA was extracted from buccal sample. Polymerase Chain Reaction (PCR) was done to amplify the CTLA-4 gene followed by Restriction Fragment Length Polymorphism (RFLP) using specific restriction enzymes. The 247bp and 162bp PCR product of c.-319C>T (rs231775) and c.49A>G (rs5742909) were digested with MseI and BbvI respectively at 37°C for 90 and 50 minutes respectively. Restricted fragments were subsequently separated using 3% agarose gel electrophoresis and visualized by Alpha Imager System. The c.-319 loci only produced a single banding pattern of 247bp (CC) while in c.49, A allele produced fragments of 162bp and G allele formed two fragments of 90/72bp creating 3 types of banding patterns which were AA, AG and GG. The genotypic frequency of the loci were 24.7%, 55.3% and 20.0% respectively for the control subjects and 30%, 55% and 15% respectively for the case subjects. The age, BMI, SBP, FPG, HbA1c, LDL, HDL, TG, Cholesterol and family history of diabetes were significantly different between the case and healthy control subjects. However, there is no significant association of polymorphism c.-319 and c.49 between healthy

Lysyl Oxidase (LOX) is a protein involved in collagen and elastin crosslinking to produce mature and stable extracellular matrix (ECM). However, the presence of tumor cell mass in the body causes elevated expression of this protein which may lead to severe neoplastic effect. Previous study found that LOX tends to accumulate at site of high mechanical load (such as high fluid stress at bifurcation of arteries and lung branches) and induces the adhesion of a cluster of immune cells at this pre-metastatic site prior to the invasion of tumor cells. In this study, we investigated the role of LOX secreted by highly metastatic murine mammary 4T1 tumor cell line, plus the incorporation of mechanical load in mediating osteolytic bone metastases. Mice injected with 4T1 cells at their mammary fat pad were sacrificed and their femur and tibia were scanned using microCT scanner. We found increased number and area of lytic lesion on the cortical bones in both femur and tibia. The trabecular bone volume, thickness and number also decreased significantly. We then repeated the same experiment on mice but using 4T1 conditioned medium (CM) containing high LOX expression and activity level. Similar effects were obtained, showing LOX alone is capable of introducing osteolytic lesion. To further nvestigate whether mechanical load has an impact on these changes, we injected mice with the same CM and also mechanically loaded the mice’s right tibia. In fact, mechanical load caused the cortical bones to have more lytic lesions. Interestingly, this osteolytic effect was suppressed when the mice were co-injected with LOX inhibitor. Thus this finding shows a role for LOX in mediating metastases to the bone from a primary tumor and mechanical load does increase osteolytic bone metastases through the role of LOX. Hence, both can be potential therapeutic targets in preventing cancer metastases.


H17: Identification of Novel Lung Cancer mRNA Transcripts by Alu-PCR Nurul Syahidah Mohd Yusof, Mohd Zaki Salleh, Teh Lay Kek, Rosmadi Mohd Yusoff Faculty of Pharmacy, Universiti Teknologi Mara, 42300 Bandar Puncak Alam, Selangor, Malaysia Lung cancer can be classified into Small Cell Lung Cancer (SLC) and Non Small Cell Lung Cancer (NSLC). It is the leading cause of cancer-related death worldwidewhere tobacco smoking is considered as the main cause of the disease. However, the carcinogenesis of lung cancer is yet to be fully elucidated. Alu profiling is one method that can be used to identify novel genomic alterations without the knowledge of the sequences. Alu elements are the most abundant repetitive elements, making up ~11% of human genome. They are 300 bp long and ancestrally derived from 7SL RNA gene. As the Alu elements are widely dispersed in human genome, they can be used as universal primer for the detection of insertion or deletions of sequences. The aim of this study is to clone novel expressed transcripts that are involved in lung cancer. Total RNA was extracted from A549 cell linesand was reverse transcribed. The cDNA was amplified by using Alu PCR where Alu sequence was used as universal primer. Multiple nonspecific bands were observed and cloned into TOP10 vector by using TA cloning method. We managed to clone 3 fragments of different sizes which are 260, 280 and 791 bp respectively. BLAST search revealed no significant match to Ref_Seq mRNA database, indicating they are novel transcripts. Alu profiling is a useful method in identifying novel genes that may cause genomic alteration in lung cancer A549 cell lines. Work is currently in progress to validate the expression of these sequences by using qPCRbetween lung cancer and normal lung cell lines.

AGRICULTURE A01: Standardization of an Effective Sterilization Protocol for the Micropropagation ofMusa coccinea (Musa spp)


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explant sterilization, three methods of aseptic techniques on Musa coccinea using male buds and sucker explants were carried out. A 69% survival rate was seen in sucker explants soaked in 100% Clorox for 6 minutes, then soaked and rinsed twice in 70% and 100% ethanol for three and five minutes, respectively, followed by rinsing with sterilized distilled water. However, a 99% survival of explants was observed after soaking and washing in 70% Clorox and ethanol for five and three minutes, respectively, followed by rinsing and washing with sterilized distilled water. A02: In Vitro Micropropagation of Stevia rebaudiana for Artificial Seed Production Irene Yoon and Rashid K. Department of Biology, Center for Foundation Studies in Science, University of Malaya, 50603 Kuala Lumpur. Stevia rebaudiana Bertoni (natural sweetener) is a medically important, zero calorie value, sweet tasting and anti-diabetic herb. Conventionally, it is cultivated from seeds or stem-cutting, but seed viability rate is poor. Thus, the in vitro micropropagation technique is considered the best alternative to increase the propagation rate of this species for commercial uses. An efficient protocol has been developed for in vitro regeneration using shoot tips, nodal segments and leaves as explants. Shoot tips, nodal segments and leaves of 1.5 to 2.0 cm long were surfacesterilized with 30% sodium hypochloride, NaOCl and 70% ethanol. Surface-sterilized nodal, leaf and shoot tip explants were then cultured on Murashige and Skoog (MS) medium supplemented with various combinations of the plant growth regulators 6-Benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) for shoot inductions. The best results were obtained from MS medium supplemented with BA+ NAA at concentrations of 1.0 mg/l. Numerous shoots were induced from young shoot tips, which showed direct embryogenesis. Callus was induced at the basal part of the shoot tips as the concentration of BAP was increased. 0.5 mg/l of NAA caused maximum root formation in the nodular stem sections of S. rebaudiana. Production of embryogenic callus from the shoot tips and leaves was also studied.

Reza Farzinebrahimi1, Kamaludin Rashid2, Rofina Yasmin Othman2 and Rosna Mat Taha1

A03: Comparison of Genes Involved in Anthocyanin Biosynthesis in Black and White Rice

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Centre for Foundation Studies of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

Foo Hou Tan, Christianto Putra, Gopal Ji Tiwari and Sadequr Rahman

In order to reduce contamination in tissue cultures of the Musa family and investigate different methods of

Anthocyanin is one of the common plant pigments. As in other plants, rice (Oryza sativa) has structural genes that

1

Monash University, Bandar Sunway, Selangor, Malaysia

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are responsible for anthocyanin biosynthesis including phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavanone-3b-hydroxylase (F3H), dihydroflavanol reductase (DFR), anthocyanin synthase (ANS) and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT). There are also two main regulatory loci that control anthocyanin biosynthesis in rice, which are the C locus and Pl locus. The expression profiles of these genes were compared in white rice and black rice. To differentiate the amplification products from gDNA and RNA, primers spanning introns were designed to amplify genes involved in anthocyanin biosynthesis. The primers were designed based on the rice genome database from NCBI and the Rice Genome Annotation Project. The genes that were amplified included PAL, CHS, F3H, DFR and the C locus. However, instead of obtaining a single band of expected size from PCR, multiple bands of unexpected sizes were observed. Similar results were obtained after repeating the PCR with modified parameters, suggesting that the multiple PCR products indicated splicing variants.

chloride assay, whereas to assay the total flavonoid content, quercetin was used as a standard.The DPPH assay showed that the stem and leaf samples were observed to have significantly high active antioxidative compounds. However, all of the plant samples had significantly lower anti-oxidant content compared to the control, which was ascorbic acid (p < 0.05). Phenolic content of the stem, leaf and root was 1.26 ± 0.12 mg/ml, 2.09 ± 0.18 mg/ml and 1.56 ± 0.21 mg/ml, respectively. Phenolic content of stem and root samples were significantly lower when compared to the leaf sample (p < 0.05). Flavonoid content of the stem, leaf and root was 0.99 ± 0.08 ug/ml, 1.08 ± 0.04 ug/ml and 0.79 ± 0.09 ug/ml, respectively. The root sample has significantly lower flavonoid content than that of the leaf sample. In conclusion, Croton caudatus aqueous extract demonstrated good antioxidant potential, which may be due to the presence of phenolic acids and flavonoids in them.

A004: Antioxidant, Total Phenolic Content and Total Flavonoid Content in Various Parts of Croton caudatus

Teo Chee How1,2*, Ma Lu2, Eszter Kapusi2, Götz Hensel2, Jochen Kumlehn2, Ingo Schubert2, Andreas Houben2 and Michael Florian Mette2

Mohamad Ikhtifar MR 1, Ebby Anuar B3, Afiqah S3, Kamalruazman D3, Razif D1, 2*, Farah Shafeera I1, 2 and Zulkhairi A1, 2 Natural Product & Phytomedicine Biotechnology Research Laboratory, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam, 42300 Selangor Darul Ehsan, Malaysia 2 Department of Basic Sciences, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam, 42300 Selangor Darul Ehsan, Malaysia 3 Department of Medical Laboratory Technology, Faculty of Health Sciences, Universiti Teknologi MARA, Puncak Alam, 42300 Selangor Darul Ehsan, Malaysia. 1

Croton caudatus, which belongs to the family Euphorbiaceae, is widely distributed around subtropical and tropical regions. Previous studies have reported that the plant have anti-malarial, anti-cancer and anti-diabetes properties. In Malaysia, the plant is used as a contraceptive agent for women. The aim of this study was to determine the antioxidative properties, phenolic and flavonoid contents of different parts of Croton caudatus. Stems, leaves and roots of Croton caudatus were separated and dried in an oven at 400C for one week, and ground into powder. Each sample was subjected to 10% aqueous extraction. The lyophilized extracts were used for the study. The free radical scavenging activity of the different parts of the plant was measured using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay. The total phenolic content was assessed using aluminium

A05: Telomere-Mediated Chromosomal Truncation of Arabidopsis thaliana

Agro-Biotechnology Institute Malaysia, Ministry of Science, Technology and Innovation, c/o MARDI Headquaters, 43400 Serdang, Selangor DE, Malaysia 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany *Corresponding author. E-mail: [email protected] 1

Minichromosomes offer an enormous opportunity to plant biotechnology development as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in Arabidopsis thaliana by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of A. thaliana telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into the natural tetraploid A. thaliana accession Wa-1, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by DNA gel blot analysis, polymerase chain reaction, and fluorescence in situ hybridisation. The addition of new telomere repeats in this process could start alternatively from within the T-DNA-derived telomere repeats or from adjacent sequences close to the right border of the T-DNA. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than the expected if according to Mendelian rules.


A06: Isolation and over-expression of endogenous chitinase in Musa cv. Berangan Mariam Binte Sulaiman1,2, Yusmin Mohd Yusuf1,3, Rofina Yasmin Othman1,2 and Norzulaani Abdul Khalid1,2 Institute of Biological Sciences, Faculty of Science, University Malaya, 50603, Kuala Lumpur 2 Centre for Research in Biotechnology for Agriculture (CEBAR), Dept. of Chemistry, Faculty of Science, University Malaya, 50603, Kuala Lumpur,Wilayah Persekutuan, Malaysia 3 Centre for Foundation Studies in Science, University of Malaya, 50603, Kuala Lumpur 1

Chitinases are enzymes that catalyze the hydrolysis of b-1,4-linkages in chitin, a polymer of N-acetylglucosamine (GlcNAc) residues. They are commonly induced in plants upon introduction of various stress stimuli, suggesting their involvement in the general stress response. However, numerous experiments have indicated that they may play a more active role in defense, especially against fungal pathogens. For instance, our previous microarray results showed an over-expression of chitinase in Musa acuminata cv. Mutiara upon infection with Fusarium oxyporum f. sp. cubense. In other studies, preparations of the enzyme were also found to be inhibitory to fungal growth in vitro. Hence, in an attempt to develop a banana cultivar resistant to fungal pathogens, we have isolated a chitinase gene from Musa cv. Berangan by polymerase chain reaction (PCR). Subsequently, the gene was characterized and cloned into a binary vector. Following that, it was re-introduced into Berangan cell suspension via Agrobacterium-mediated transformation. To test for positive transformants, several replicates of the cell suspension were randomly selected for GUS-staining. Preliminary results suggest that transformation was successful. However, further analysis is required to validate the stability of the transgene. A07: Isolation and Expression Analysis of putative Pathogenesis Related (PR) 10 from Musa acuminata cv. ‘Berangan’ in Response to Exogenous Salicylic Acid Supply Nadiya Akmal Baharum1,2,4, Wan Muhammad Farhan Syafiq Wan Mohd Nor1, Nazia Abdul Majid1,2, Yusmin Mohd. Yusuf2,3, Rofina Yasmin Othman1,2 and Norzulaani Khalid1,2 Institute of Biological Science, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysia 2 Centre for Research in Biotechnology for Agriculture (CEBAR), University Malaya, 50603 Kuala Lumpur, 3 Centre for Foundation Studies in Science, University Malaya, 50603 Kuala Lumpur, Malaysia 4 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra 1


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Malaysia, 43400 UPM Serdang, Selangor, Malaysia PR10 is a member of the huge pathogenesis related (PR) protein family. It has been characterized in different plant species, including monocots, dicots, eudicots and conifers. PR10 has been a subject of interest due to its ribonuclease-like, anti-fungal, anti-bacterial and anti-viral properties. Similar to other PR proteins, an elevated expression of PR10 has been demonstrated in many plants, such as hot pepper, rice, plum and white birch, in various stress conditions and treatments. However to date, the isolation and characterization of PR10 from Musa acuminata cv. ‘Berangan’ has yet to be reported. In this study, we isolated the coding sequence of the PR10 gene from M. acuminata cv. ‘Berangan’ and determined the effect of exogenous salicylic acid on gene expression. The PR10 gene encodes for a putative protein of 160 amino acids with a molecular mass of 17.4kDa and an isoelectric point of 5.64. Genomic DNA sequences of PR10 revealed that only one intron region was located in between 2 exons. The predicted PR10 protein has a functional domain known as Betv1-like with a glycine rich loop and hydrophobic ligand binding-sites. A prominent conserved glycine-rich motif, GXGXXG, found within the residues of the PR10 protein further confirmed its identity. Our results also indicated the possibility of PR10 to be up-regulated upon exogenous salicylic acid treatment by activating the defence-related signalling pathway. A08: WRKY transcription Factor Expression in Musa acuminata cv. Berangan Siti Nur Akmar Bt Mazlin1,2, Yusmin Mohd Yusuf2,3, Nadiya Akmal Bt Baharum1,2, Wan Muhammad Farhan Syafiq Bin Wan Mohd Nor1, Norzulaani Khalid1,2 and Rofina Yasmin Othman1,2 Institute of Biological Science, Faculty of Science, University Malaya, 50603, Kuala Lumpur, Malaysia 2 Centre for Research in Biotechnology for Agriculture (CEBAR), University Malaya, 50603, Kuala Lumpur, 3 Centre for Foundation Studies in Science, University Malaya, 50603, Kuala Lumpur, Malaysia 1

The WRKY transcription factor is one of the ten largest families of transcriptional regulators across the green lineage. It is an integral part of the signalling web that modulates many plant processes, such as embryogenesis, plant growth and metabolic pathways and plant immune responses against biotic and abiotic stimuli. In plant immune responses, WRKY participates in the control of defence-related genes as a positive or negative regulator, and plays roles in the repression and derepression of important plant processes. The up-regulation of WRKY genes are strongly affected by both internal and external stimuli. In Arabidopsis, 49 out of 72 WRKY genes respond

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to bacteria or the endogenous signal molecule salicylic acid. Our previous study had shown that the WRKY gene is one of the genes that is up-regulated in Musa acuminata cv. Mutiara upon Fusarium oxysporum f. sp. cubense infection. In the present study, we carried out a similar experiment using endogenous salicylic acid as a stress factor to study WRKY gene expression. In this study, leaf plantlets of Musa acuminata cv. Berangan were treated with endogenous salicylic acid, with water as a control, at different time intervals. RNA was isolated from banana samples and WRKY gene expression was screened by semi-quantitative PCR. Our preliminary results showed that the expression of the WRKY gene peaked at 6 hours in leaves under endogenous salicylic acid and water treatments, declining thereafter. This result suggests that in both treatments (endogenous salicylic acid and water), the expression of the WRKY gene is activated due to its function in plant processes and the endogenous salicylic acid-dependent defence signalling pathway. However, further work on WRKY expression studies in banana plants still needs to be done to measure the expression level in those treatments and to understand its regulation in different stress responses pathways. A09: Genomic Fingerprint Patterns of Enterobacter cloacae from Banana Bacterial Wilt, Papaya Dieback and Pitaya Diseased Plants Using Repetitive Extragenic Palindromic unit b1-primed pcr (repub1-pcr) Saiyidah Hazirah bt. Zurin Adnan1 and Noor Hana Hussain2 Faculty of Applied Sciences, Universiti Teknologi MARA, 404050 Shah Alam Selangor 2 Institute of Science, Universiti Teknologi MARA, 404050 Shah Alam Selangor. 1

Enterobacter cloacaewere reported to be frequently isolated from papaya dieback, banana bacterial wilt and pitaya diseased plants by several laboratories in Malaysia. Although it has yet to be proven the primary causative agent of papaya dieback and banana bacterial wilt, it has been shown to cause the latter. The genomic fingerprint patterns of 10 Enterobacter cloacae isolates from banana bacterial wilt, nine from papaya dieback and five from pitaya diseased plants were determined using repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) followed by agarose gel electrophoresis. Two different clusters were identified from the banana isolates with nine of the 10 isolates being identical to one another and one isolate having a different pattern. Nine banana isolates had five identical bands ranging in size from 310 to 1500 base pairs while the remaining one isolate had eight bands with the smallest size being 314 base pairs and the largest being 1750 base pairs. The nine papaya isolates were revealed to have two different clusters which only differed by two bands.


Eight papaya isolates had 10 similar bands between 175 and 1800 base pairs while the lone isolate of the second cluster had 12 bands. All five pitaya isolates had the same fingerprint pattern with nine identical bands between the sizes of 197 and 1800 base pairs. The results showed that there is variability within each plant group and between the three plant groups suggesting that the Enterobacter cloacae isolates may have originated from different sources. A10: Molecular Survey of Endosymbionts in Populations of Diaphorina citri from Huanglongbing Diseased Citrus Trees Nurul Fithriah Md Amin1 and Noor Hana Hussain2 Facultyof Applied Sciencesand 2Institute of Science, Universiti Teknologi MARA, 40000 Shah Alam, Selangor, Malaysia. 1

Huanglongbing (HLB) is a citrus disease which is prevalent in Asia, America, and Africa. The vector of this disease is Diaphorina citri, a psyllid that carries the causative agent, Candidatusliberibacter spp.,phloem-limited Gram negative bacteria. In this study, detection of Candidatusliberibacter and other endosymbionts namely, Syncytium symbiont, Mycetocyte symbiont, Wolbachia wsp., and Arsenophonus sp. were carried out in 61 Diaphorina citri from infected citrus plants; Citrus sinensis or locally known as Limau madu at Padang Ipoh, Kuala Terengganu by PCR using specific primers.A total of 90.2% of the psyllids carried Candidatusliberibacter. Syncytium and Mycetocyte which are known to be obligate endosymbionts in Diaphorina citri,were only present in 49.1% and 78.7% of the psyllids, respectively. The endosymbionts Arsenophonus, and Wolbachiawere present in 57.4 % and 55.7% of the psyllids, respectively. All the psyllids carried a minimum of one species of endosymbiont and a maximum of five species of endosymbionts. This survey showed that endosymbionts in Diaphorina citri are quite diverse. However, this does not represent the total species of endosymbionts present in Diaphorina citri. A11: Influence of Activated Charcoal and Peptone on Seed Germination of Brassavola nodosa, Cattleya intermedia, Gastrochilus patinatus and Dendrobium lasianthera. Nurul Athirah M.Y., Noor Anilizawatima S. Faculty of Pharmacy, University Technology Mara (UiTM) Puncak Alam, Selangor, Malaysia Activated charcoal and peptone have been used for micropropagation of plants in tissue culture to improve the seed development of plants. The aim of this study was to examine the influence of activated charcoal and peptone


on the seeds germination of orchids; Brassavola nodosa, Cattleya intermedia, Gastrochilus patinatus and Dendrobium lasianthera. The seeds were sown on half strength Murashige and Skoog (1/2 MS) medium supplemented with different concentration of activated charcoal (0.0, 0.5, 1.0, 2.0 g/l) and peptone (0.0, 0.5, 1.0, 2.0 g/l) by using Complete Randomized Design method. Three replicates of each media were utilized. The percentage of protocorm and number of shoots induced were observed after 16 weeks. The results showed that Brassavola nodosa showed the highest percentage of protocorm and number of shoots than the other three species. The most optimum growth of Brassavola nodosa was in the media that was supplemented with 2.0 g/l peptone and without charcoal while the least growth was observed in media that is supplemented with 2.0 g/l charcoal and without peptone. In Dendrobium lasianthera, the highest percentage of protocorm and shoots was found in 1.0 g/l peptone and 0.0 g/l charcoal while the media supplemented with 2.0 g/l charcoal and 0.0 g/l peptone showed the least growth. The media of 0.5 g/l peptone and 1.0 g/l charcoal showed a little growth of protocorm while the other media did not show any development at all in Cattleya intermedia. Gastrochilus patinatus did not show any development in all the media. The result showed that addition of activated charcoal and peptone could enhance the seed development of these orchid species except Gastrochilus patinatus.


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fascinator was observed in medium I (Half strength MS basal medium supplemented with 30g sucrose, 2g Gelrite agar, 1g/L charcoal and 150mg/L of Gaviota fertilizer). The optimum growth of Dendrobium cretaceum was observed in medium L (Half strength MS basal medium supplemented with 30g sucrose, 2g/L Gelrite agar , 1g/L charcoal and 300mg/L Gaviota). For media using B’GREEN Seaweed as fertilizer, it was observed that medium A (Half strength MS basal medium supplemented with 30g sucrose, 2g/L Gelrite agar , 1g/L charcoal and 1ml/L B’GREEN Seaweed fertilizer) showed positive influence on the growth of Bulbophyllum fascinator and Dendrobium cretaceum protocorms. Based on observations, the growth of these protocorms were retarded in medium F (Half strength MS basal medium supplemented with 30g sucrose, 2g Gelrite agar, 1g/L charcoal and 6ml/L B’GREEN Seaweed Fertilizer). From our findings, Gaviota can enhance the growth of both Bulbophyllum fascinator and Dendrobium cretaceum protocorms while a low concentration of B’GREEN Seaweed Fertilizer has been identified to have positive influence on the growth of the protocorms. A13: Induction of Protocorm-Like Bodies (PLBs) in Vanda Mimi Palmer and Phalaenopsis bellina Using 1-napthaleneacetic Acid and 6-benzylaminopurine Salamah ARB and Noor Anilizawatima S Universiti Teknologi MARA

A12: Effect of Local Fertilizers on Growth of Bulbophyllum fascinatorand Dendrobium cretaceum Protocorms Nur Fariha Y and Noor Anilizawatima S Universiti Teknologi MARA Currently, the orchid industry receives high demand globally. Plant tissue culture technique provides an alternative to propagate orchid protocorms in an effective and fast way compared to the conventional method. Thus far, researchers have manipulated basal medium by using different materials to enhance the growth of orchid protocorms. The aim of this study was to investigate the influence of two different local fertilizers which are Gaviota and B’GREEN Seaweed fertilizer on the growth of Bulbophyllum fascinator and Dendrobium cretaceum protocorms. The protocorms were cultured in half strength Murashige and Skoog (MS) basal medium supplemented with 30g sucrose, 2g Gelrite agar, 1g/L charcoal and various concentrations of Gaviota and B’GREEN Seaweed fertilizer. For media which used Gaviota as fertilizer, the optimum growth of Bulbophyllum

The objective of this study wasto investigates the possibility of inducing protocorm-like bodies (PLBs) from the explants of Vanda Mimi Palmer and Phalaenopsis bellina. The leaf explants were cultured in half strength Murashige and Skoog (1/2 MS) medium by using the Complete Randomized Experimental Design (CRED) method. Two types of plant regulator hormone which are 1-napthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) with different concentration of 0mg/ml, 0.5mg/ml, 1.0mg/ml and 3.0mg/ ml were used to study the effects of these plant growth regulators (PGRs) on PLBs induction for 16 weeks. After 16 weeks of observation, there were no PBLs induced in both type of orchid explants at all tested concentrations of plant growth regulator. There were only colour changes of the explants which was observed. For Phalaenopsis bellina, the colour of the explants started to change from green to brown at week 6 and turned - black at week 10 to 11. On the other hand, Vanda Mimi Palmer - remained green explants in sample I (1.0mg/l BAP, 0mg/l NAA), J (1.0mg/l BAP, 0.5mg/l NAA) and L (1.0mg/l BAP, 3.0mg/l NAA) until week 12, while other samples started to change colour from green to brown at week 10. In conclusion, the maximum concentration of 3.0 mg/l NAA and 3.0 mg/l BAP are unable to trigger the explants to induce PLBs in these two species.

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A14: Transcriptome Profiling Data of Boesenbergia rotundaCell Culture in Response to Exogenous Phenylalanine Treatment

A15: A Scheme for Biosafety Risk Assessment and Evaluation of Transgenic Banana for Confined Field Trials in Malaysia

Noor Diyana Md Mustafa1,2, Yusmin Mohd Yusuf1,3, Norzulaani Khalid1,2, Rofina Yasmin Othman 1,2*

Sivaganesh Loganathan1,2, Norihan Mohd Salleh1, Yusmin Yusuf2, Vilasini Pillai3 Norzulaani Khalid2, Rofina Yasmin Othman2

Centre for Research in Biotechnology for Agriculture (CEBAR), University Malaya, 50603 Kuala Lumpur 2 Institute of Biological Sciences, Faculty Science,Universiti Malaya,50603 Kuala Lumpur 3 Centre for Foundation Stidies, University Malaya, 50603 Kuala Lumpur 1

Boesenbergia rotunda is a medicinal plant from the Zingiberaceae family and is commonly used as food ingredient and traditional medicine in Southeast Asia and China. There have been several flavonoid compounds extracted from Boesenbergia rotunda that showed significant medicinal properties such as 4-hydroxypanduratin A, panduratin A, pinostrobin, pinocembrin, alpinetin and cardamonin. The increasing reports on health benefits of these flavonoids have resulted in increasing demands on their commercialization. However, some are naturally low in abundance and the limited knowledge of their biosynthetic pathway at the molecular level hinders its production in a large scale. In this study, transcriptome sequencing and differentially expressed gene (DEG) analysis of control and phenylalanine treated Boesenbergia rotunda cell suspension culture was carried out to elucidate the key genes that are involved in flavonoid pathway. Phenylalanine is an aromatic amino acid derived from the shikimic acid pathway and enters the phenylpropanoid pathway to produce most of the known flavonoids. Therefore for RNA-seq analysis, total RNA from untreated and 14 days post-phenylalanine treated cell suspension cultures were extracted and sequenced using next generation sequencing technology employing an Illumina-Solexa platform. Our transcriptome data generated 101,043 unigenes with 50,932 unigenes (50.41%) successfully annotated in the public protein databases; which included 49.93% (50,447) in the non-redundant (NR) database, followed by 34.63% (34,989) in Swiss-Prot, 24,07% (24,316) in Kyoto Encyclopedia of Genes and Genomes (KEGG) and 16.26% (16,426) in Clusters of Orthologous Groups (COG). Through DEGs analysis, we found 14,644 unigenes up-regulated and 14,379 unigenes down-regulated in response to exogenous phenylalanine treatment. In the flavonoid pathway, we found 2 up-regulated phenylalanine ammonia-lyase (PAL), 3 up-regulated 4-coumaroyl:coenzyme A ligase (4CL) and 1 up-regulated chalcone synthase (CHS). This will be the first report on Boesenbergia rotundade novo transcriptome data. Hence, it can serve as a reference for the Zingiberaceae family in the future.

Agro-Biotechnology Institute Malaysia (ABI), National Institutes of Biotechnology Malaysia 2 Institute of Biological Sciences and the Center for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Malaysia 3 The National Science and Research Council (NSRC), MOSTI, Malaysia e-mail: [email protected] 1

A wide range of living modified organisms (LMOs) have been successfully developed and used for commercial production worldwide. Modern biotechnology provides unprecedented avenues for exploring biological systems and with it comes biosafety concerns which require the appropriate science-based assessment and management. The Malaysian Biosafety Act 2007 and Biosafety (Approval and Notification) Regulations 2010 regulates matters related to LMOs in the country. At the University of Malaya, Musa acuminata cells have been transformed with genes related to fungal disease defense via Agrobacterium tumefaciens mediated transformation. The constitutive expression of the expressed proteins is expected to present a potential strategy for combating selected fungal pathogens afflicting banana in Malaysia. Contained and subsequently open field trials are mandatory steps towards the commercial release of these transgenic lines which will involve an evidence based risk assessment process. Risk assessment of transgenic crops not only involves collection of various data pertaining to the morphological and physiological traits of the LMOs, but also the effects of it or its products on succeeding trees and to the diversified microorganism population that lives within its ecosystem. The performance of the host organism in the natural habitat and the field for intended release has to be studied. Risk assessment of LMOs is principally performed on case-by-case evaluation. Environmental release of LMOs is possible only after the collected information is examined comprehensively in a scientific manner, and the target end-points are well identified. The collaborative effort of University Malaya and the Agro-Biotechnology Institute Malaysia strives to create a structured compliance pipeline that will ensure the transfer of safe transgenic crops into the field. The presentation outlines the strategy to be used to for the risk assessment of fungal-resistant transgenic banana in Malaysia. This work is funded by an HIR-MOHE grant H-21001-00-F00018


A16: In Vitro Flowering and Fruiting of Andrographis alata (Vahl.) Nees – A Valuable Medicinal Plant Mohammed Arifullah1, Vikram Paritala1, Kishore K. Chiruvella2, G. Rama Gopal2 Faculty of Agro Based Industry (FIAT), Universiti Malaysia Kelantan, Jeli Campus, Locked bag 100, Jeli 17600, Kelantan, Malaysia 2 Division of Plant Tissue Culture, Department of Botany, Sri Venkateswara University, Tirupati, India Corresponding author: [email protected] 1

An efficient and reproducible protocol for mass micropropagation, in vitro flowering and fruiting of Andrographis alata Nees (Acanthaceae), a valuable medicinal plant has been developed using nodal explants. Nodal explants were dissected and inoculated onto the MS medium containing varying concentrations and combinations of plant growth regulators (PGRs) for shoot initiation. The Murashige and Skoog’s medium (MS) fortified with 2.5 mg l-1 6-benzyladenine (BA) and 0.2 mg l-1 indole-3-butyric acid (IBA) was found optimum for production of high frequency of multiple shoots. In vitro flowering and fruiting was observed in the presence of 6-benzyladenine (2.5 mg l-1) and indole-3-acetic acid (1.5 mg l-1). The regenerated shoots rooted best on 1/2 MS medium devoid of plant growth regulators. This paper describes in vitro flowering system to overcome problems associated with flower growth and development as well as fruit and seed production in vitro.

BIOPROCESS TECHNOLOGY AND BIOPRODUCTS B01: The Investigation of Mass Transfer under High Cell Density Fermentation (HCDF) Conditions. Salehmin MNI and Annuar MSM Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Mass transfer under high cell density fermentation (HCDF) conditions was studied. A number of established correlations were successfully used to predict the kLa value at different agitation rates ranging from 200-1000 rpm for gas-liquid and liquid-liquid mass transfer under HCDF conditions. The investigation was simulated in xantham gum solution, which resembles the viscosity of HCDF at 1.68 mPa.s. A linear relationship was observed between agitation rate (rpm) and the kLa value for both mass transfers. However, the experimental as well as theoretical kLa values for gas-liquid mass transfer were


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unexpectedly reduced at 800 rpm. The reduction of the kLa values were attributed to the presence of a high number of tiny bubbles. The immobility of this type of bubbly surface with no internal gas circulation was believed to diminish gas diffusion into the bulk liquid. For the liquid-liquid system, the kLa values at the highest agitation rate (1000 rpm), determined via experimental and theoretical means, did not agree with each other. This showed that the correlations failed to predict kLa values at high Reynold’s number under HCDF. B02: Bioethanol Production fromMixed Lignocellulosic Substrates Using Cellulolytic Termite Gut Bacteriaand Yeast Mushafau Adebayo Oke1,2*, Koshy Philip1 and Noni Ajam1 Microbiology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, 2 Department of Microbiology, Faculty of Science, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria. *Corresponding author: [email protected] 1

Energy insecurity and global warming associated with the use of fossil fuels have led to intense efforts towards sustainable production of biofuels, such as bioethanol. Utilization of biomass for the production of biofuels has been fraught with challenges such as fluctuating feedstock supply, high cost of feedstock handling, and other logistic problems. Most studies on lignocellulosic ethanol production have been based on single biomass feedstocks. A mixed biomass feedstock approach to the utilization of lignocellulosic biomass for ethanol production has the potential to overcome the challenges of biomass utilization, positively improve the bioethanol production process, and generally boost the biorefinery concept. Oil palm biomass and wood wastes account for the greater portion of wastes generated annually in Malaysia, and their accumulation is a serious environmental problem. This study aims to investigate the potential of mixed lignocellulosic biomass (oil palm fronds and saw dust) as substrates for bioethanol production. Cellulolytic bacteria have been isolated from the gut of dry wood termites collected from infested wood. Screening of the isolates for cellulase production using cellulose hydrolytic capacity values on CMC agar plates and identification of the isolates are in progress. Different pre-treatment methods will be evaluated and optimized to determine the best conditions for high sugar yield and low levels of inhibitors from the mixed substrates as well as each single substrate. The composition of the substrates before and after pre-treatment will be analysed using HPLC in order to determine the yield of sugars and inhibitors generated. The best among the cellulolytic bacteria will be used for enzymatic hydrolysis of the substrates along with Saccharomyces cerevisiae in an SSF process for ethanol production. Ethanol yield from each of the substrates (single

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and mixed) will be compared. It is expected that the yield from the mixed biomass substrates will be greater than or comparable to that of the single substrates. B03: Optimising Xylanase Production by Aspergillus brasiliensis Using Agricultural Residuals in Solid State Fermentation (SsF) Stephanie Ak Sali and Hooi Ling, Ho* Faculty of Applied Science, UCSI University Kuala Lumpur Campus,No. 1, JalanMenaraGading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia. *Corresponding author email: [email protected] Aspergillus species are known to produce various types of enzymes and one of important the enzyme is xylanase. Over the years, xylanase has become valuable and attractive due of its vast applications in pulp and paper, food and beverage, detergent and textiles industries. Aspergillus brasiliensisATCC 16404 was investigated for the production of xylanase in solid state fermentation (SsF). SsF is the fermentation method of culturing microorganisms on the humid solid substrates without emerging in the culture broth. SsF has always been an attractive substitute of submerged fermentation for xylanase production due to its lower capital investment, energy demand and operation cost. Hence, different parameters for medium formulation were optimised to obtain the maximum activity of xylanase. In this study, 10g of wheat bran as the optimum agricultural residual was able to produce the xylanase activity of 6.7091±1.3102 U/mL. Subsequently, the maximum xylanase activity of 6.7115±1.3130 U/mL was detected using wheat bran as the carbon source at the moisture content ratio set to 1:1. Interestingly, when 2% of yeast extract was added into the wheat bran, further study reviewed the maximum xylanase activity increased to 8.6382±3.8197 U/mL. Thus, the optimum medium formulation for the production of xylanase by Aspergillus brasiliensisin SsF was achieved using 10g of wheat bran as the optimum agricultural residual as carbon source with addition of 2% of yeast extract at the moisture content ratio of 1:1 at 30oC for 120 h. B04: Screening of Significant Media Componentsfor Thermostable Alkaline Protease Production by Recombinant 50a in Submerged Fermentation Using Plackett- Burman Design Nor Hidayah Bohari*and Noor Azlina Ibrahim Bioindustrial Technology, Faculty of Agro Based Industry, Universiti Malaysia Kelantan, 17600 Jeli, Kelantan. *Corresponding author: [email protected]


Screening of medium components to improve production of intracellular thermostable alkaline protease by a newly cloned E.coliBL21(DE3) pLysS harbouring 50a protease gene was reported. Plackett-Burman two level factorial design was applied to evaluate the fermentation medium components. Effect of seven medium components was studied in 45 experimental trials under submerged fermentation. The screening among sorbitol (carbon source), tryptone (organic nitrogen source), ammonium nitrate (inorganic nitrogen source), potassium dihydrogen phosphate (phosphate source), mangan sulphate (metal ion), calcium chloride (metal ion) and sodium chloride (common salt), only five components showed significant effect and influence the protease production. However, among five significant components, only sorbitol, tryptone and calcium chloride were identified as the most critical components and have larger effect for enhanced protease production by this recombinant 50a strain. In addition, the adequacy of the design and model was supported by a coefficient determination (R2) of 0.9417. B05: Expression and Purification of Predicted Uncharacterized Protein from Bacillus lehensisGI Norhazlin Mohamad Khoiri, Siti Aishah Rashid and Rosli Md Illias* Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. *Corresponding author: [email protected] Bacillus lehensis GI; an alkaliphilic Bacillus was originally isolated from rubber plantation soil in Pekan Nenas, Johor, Malaysia. The genome was sequenced by 454 pyrosequencing and Solexa/Illumina technology, resulting of about 4Mbp genome in size. The project is still ongoing but gene annotation stage has been used to identify several genes encoding proteins with interesting biological functions for example; glycoside hydrolases, dehydrogenases, molecular chaperones and novel genes which is termed as ‘predicted, uncharacterized protein’. One predicted uncharacterized protein; designated as bleg1_3696 protein was chosen. This gene consisted of an open reading frame (ORF) of 540 nucleotides, and encoded a protein of 179 amino acids with a predicted molecular weight of 19.98 kDa. The deduced amino acid sequence displayed the highest identity (44%) to the Proteinase K enzyme from Serratia species. The gene was isolated using PCR techniques and was successfully cloned using Gateway system. IPTG induction and autoinduction was used to express the protein and purification procedure was carried out using IMAC system. SDS-PAGE analysis of the crude supernatants showed protein bands with


molecular mass of approximately 20 kDa. Homology modeling was used to build the protein structure since the protein identity exceeds 30% protein identity. Protein structure will reveal the biological function, enzyme mechanism, protein-ligand interactions and oligomerization(s) of the protein which then will bring to the discovery of a new pharmaceutical agent. OMICs


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search for diagnostic markers. Essentially, the whole genome information from this Malaysian S. Typhi strain would enhance the understanding of the pathogen in greater depth. O02: Phylogenetic Relationships of the Red Algal Parasite Congracilaria babae (Gracilariaceae, Rhodophyta) Poh-Kheng Ng1,2, Phaik-Eem Lim 1,2 , Siew-Moi Phang 1,2 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, 2 Institute of Ocean and Earth Sciences, Institute of Postgraduate Studies, University of Malaya, Kuala Lumpur, 50603, Malaysia 1

O01: Unveiling the Genome Dynamics of Sporadic-Associated Salmonella enteric serovar Typhi Strain in Malaysia Using Whole Genome Next Generation Sequencing Approach Yap Kien Pong,1,2Gan Han Ming,2 Cindy Teh Shuan Ju,1,2 Chai Lay Ching1,2 and Thong Kwai Lin1,2* Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, 2 Laboratory of Biomedical Science and Molecular Microbiology,UMBIO Research Cluster, University of Malaya, 50603, Kuala Lumpur, Malaysia. *Corresponding author: [email protected] 1

Typhoid fever is an infectious disease of global importance. The causative agent, Salmonella enteric serovarTyphi (S. Typhi) is responsible for 21.7 million cases and 217,000 deaths annually worldwide. In Malaysia, typhoid fever remains endemic and sporadic typhoid cases are occasionally detected. The disease can be effectively controlled if the pathogenicity and adaptation mechanisms of this bacterium are thoroughly understood. Unfortunately, comparison and genomic variation studies on S. Typhi remain a challenging task due to insufficient genomic data deposited in public databases. Hence, to better understand the pathogen and its virulence mechanism, we applied high-throughput whole genome next generation sequencing technology to elucidate the important genomic information of this pathogen. The unveiled information could provide a promising platform for the development of infection control strategies. Herein, we describe the genome dynamics of a S. Typhi strain ST0208 isolated from a sporadic case in Kuala Lumpur, Malaysia. The approximate size of the genome is 4,798,272 bp, with a GC percentage of 52. The genome reveals 4,890 CDS which account for 83.7% of the genome. As in the reference genomes, CT18 and Ty2, ten pathogenicity islands (SPI1 – SPI10) containing virulence gene clusters such as spaMNOPQRS and invABCEFGH, with its regulatory proteins HilA, HilC, HilD, were identified. Notably, there were many uncharacterized and hypothetical proteins genes which were not detected in the reference genomes. These unique genes might serve to improve current strain identification methods and

Red algal parasites provide a powerful model for investigating the evolution of parasitism from a photosynthetic ancestry, based on the unique relationships between these parasites and their hosts, as most of them have evolved directly from their host species and some radiated to exploit more distantly-related hosts. Prior to the establishment of any model system for further functional study, it is useful to first identify the potential model organism, with an understanding of the systematics and taxonomy of the red algal parasite with reference to its host species. In this study, we uncovered the phylogenetic relationships of the host-parasite association for the red algal parasite Congracilaria babae, which was found on G. salicornia and Hydropuntia sp., based on the DNA sequences of the plastid rbcL gene and the nuclear ITS region. Our data suggest that C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host, Hydropuntia sp. Further comparative developmental studies and functional genomics analyses of C. babae from G. salicornia and Hydropuntia sp. may shed light on the factors leading to the origin of red algal parasitism. O03: Computer Based Characterisation of Pteropusvampyrus endogenous betaretrovirus (pveb) from Bat Genome Project Database Razlan NFA and Kambol R Faculty of Applied Sciences, UiTM Shah Alam, Selangor, Malaysia Retroviruses, associated with numerous chronic diseases such as leukemia and immunodeficiencies, have been harbored by diverse host range of animals. Recently, one member of bat endogenous retrovirus has been reported as the basal of gammaretrovirus. However, in this study we showed that the bat endogenous retrovirus is also present in another family of retrovirus, which is the betaretrovirus

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through insilico screening. Here we report the discovery and characterization of bat endogenous betaretrovirus. The size of nucleotide sequence is 8.7kb which was obtained during screening and characterization of bat genome from large flying fox (Pteropus vampyrus) database genome project. It has a typical genomic organization of a retrovirus possessing all the major conserved motifs and LTR flanking the viral genes: 5’-LTR-gag-pol-env-LTR-3’plus the presence of dUTPase which is common in beta and delta retrovirus family.

the wide dispersal of the infection in Malaysia within four months duration in 2009.

O04: Comparative Genomics Reveals the Improved Environmental Fitness and Increased infectivity of a Malaysian Vibrio cholerae O1 Altered El Tor Strain

Lai Kuan Tan1,2*, Peck Toung Ooi3, Kwai Lin Thong1,2

Cindy Shuan Ju Teh1,3* and Kwai Lin Thong2,3 Department of Medical microbiology, Faculty of Medicine Institute of Biological Sciences, Faculty of Science 3 Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya, 50603 Kuala Lumpur *Corresponding author: [email protected] 1 2

Vibrio choleraeO1 altered El Tor strains have been detected in Asian countries and responsible for major outbreaks in Thailand, Vietnam and Malaysia. In Malaysia, outbreaks that occurred in the state of Kelantan and Terengganu from September – December 2009 were caused by altered El Tor strains. In the present study, the genome content of an outbreak-associated V. choleraeO1 altered El Tor strain (VC1761/09) was investigated and comparative genomic analysis was carried out on this particular strain and eight O1 V. cholerae strains. The Malaysian O1 V. cholerae draft genome was 4,012,991 bp in size and carried 3701 genes. Among the eight compared genomes, 2834 homologous genes were identified. A phylogenetic tree based on the homologous genes revealed that VC1761/09 was more closely related to an O1 altered El Tor strain isolated during 2010 outbreak in Haiti with 2951 genes that were 100% similar. Compared to another altered ElTor strain, MJ1236, the numbers of 100% homologous genes were only 2923. In addition to the genes inheriting from 7th pandemic ElTor that promote environmental fitness,VC1761/09 also demonstrated greater infectivity due to the retention of virulence traits of the Classical O1. However, the Malaysian O1 V. cholerae strain could be distinguished from the El Tor and Classical strains based on a truncated V. cholerae seventh pandemic island II (VSP II), properties in CTX prophage and the presence of mobile genetic elements. Similar to those O1 altered ElTor variants, VC1761/09 containeda~98 kb ICE-like element that harboured antibiotic resistance clusters and belonging to the SXT/R391 family. The unique characteristics of the VC1761/09 genome provided the bacterium with a competitive ecological dominance with greater infectivity. Without further preamble, this explained

MOLECULAR BIOLOGY

MB01: PCR Detection of Virulence Genes inYersinia Enterocolitica from Foods and Pigs

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2 Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya 3 Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, University Putra Malaysia *Corresponding author: [email protected] 1

Yersinia enterocolitica is a food-borne pathogen that causes gastroenteritis in humans. Yersiniosis is often misdiagnosed as appendicitis, as similar symptoms such as diarrhea, fever and lower right abdominal pain are present in both diseases. The carriage of virulence genes in Y. enterocolitica plays an important role in infections. The aim of this study was to investigate the prevalence of virulence genes in Y. enterocolitica isolates from foods and pigs in Malaysia. Thirty-three Y. enterocolitica isolates were PCR-screened for 16 virulence genes (chromosomal- or plasmid-borne) that are involved in the production of invasin, adhesin, transcriptional activator, fimbriae, enterotoxin, enterochelin receptor protein, enterochelin ABC transporter, enterochelin esterase, insecticidal toxin complex-like protein, Yersinia modulator, subtilisin/kexin-like protease, streptograminacetyltransferase, or proteins for auto agglutination and serum resistance. The positive rates of virulence genes tested in 33 isolates were: hreP(100%), sat (100%), ymoA(100%), myfA(94%), inv(94%), ystA(94%), yadA(94%), virF(85%), rfbC(85%), ail (85%), tccC(85%), fepA(33%),fes(15%), fepD(15%), ystB(6%), and ystC(0%). There were four virulence genes carriage patterns found. Isolates of bioserotype 1A/O:5 (n=2) were hreF+,fes+, sat+,fepD+,ymoA+,ystB+ and fepA+; bioserotype 1B/O:8 (n=3) were hreF+,myfA+,fes+, sat+,fepD +,inv+,ymoA+,ystA+,yadA+ andfepA+; and bioserotype 3 variant/O:3 were either hreP+,virF+,rfbC+,myfA+, sat+,inv+, ail+,ymoA+,ystA+,tccC+,yadA+ and fepA+(n=6) or without the fepA gene (n=22). Interestingly, the bioserotype 1A/O:5, that was grouped as non-pathogenic, carried some of the virulence genes which were not traditionally tested. We also found the loss of the ail gene (involved in adhesion, invasion, and protection from bactericidal effects) in the pathogenic bioserotype 1B/O:8 isolates. In conclusion, PCR-dependent


detection of virulence genes is feasible in providing rapid genotypic characterization of Y. enterocolitica. MB02: High Resolution Melt Curve Analysis for Mutation Detection in Quinolone-resistant Determining Region of DNA Topoisomerase IV Genes in Salmonella SooTein Ngoi1,2and Kwai Lin Thong1,2 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, 2Laboratory of Biomedical Science and Molecular Microbiology, UMBIO Cluster, Institute of Graduate Studies, University of Malaya, 50603, Kuala Lumpur, Malaysia 1

The increasing prevalence of quinolone-resistant Salmonella serovars has raised public health concerns in Malaysia. The DNA topoisomerase IV enzyme plays an important role in bacterial DNA replication. Mutations in the quinolone-resistant determining region (QRDR) of the bacterial topoisomerase IV genes contribute to quinolone-resistance among the bacteria. However, mutations in the QRDR of these genes are not common among Salmonella. Hence, it is often too costly and ineffective to analyse large sample sizes via DNA sequencing. The objective of this study was to develop a high resolution melt curve (HRM) analysis for rapid mutation screening for topoisomerase IV genes. Two pairs of primers were designed to target the QRDRs of the parCand parEgenes, which encode the subunits of the DNA topoisomerase IV enzyme. Twelve quinolone-resistant Salmonella Typhimurium strains were used to develop the HRM assay. The target regions were subsequently sequenced to confirm the presence of base substitutions. The HRM primers amplified a 219 bp and 193 bp region in parC and parEQRDR, respectively. Sequence analysis showed that only one strain consisted of multiple base substitutions in the parCQRDR, but others were silent mutations. The majority (n=11) of the strains consisted of the wild-type sequence in parCQRDR. In parEQRDR, base substitutions were detected in two strains, but only one resulted in an amino acid change (Met438-Ile). HRM analysis was able to detect mutations in the QRDR of the genes of interest. Different plots showed that the melt curves of the mutants were distinctly separated from the wild-type. In conclusion, HRM analysis was able to detect mutations in the QRDR of the topoisomerase IV genes. Hence, this method provides a rapid and sensitive screening tool for mutation detection, thus enabling high-throughput analysis of large sample sizes. MB03: Development of TaqMan Genotyping Assays for Detection of Beta-Thalassaemia Mutations in Malaysia Kek Heng Chua, Siew Leng Kho, Elizabeth George*, Jin Ai Mary Anne Tan.


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Department of Biomedical Science, Faculty of Medicine, University of Malaya, Lembah Pantai, Kuala Lumpur Haematology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor * Thalassaemia is the most common autosomal recessive disorder in Malaysia, characterised by defects in the synthesis of globin chains. The unbalanced accumulation of globin subunits causes ineffective erythropoiesis and haemolytic anaemia. Beta-thalassaemia patients require monthly blood transfusions and iron chelation therapies for survival. Beta-thalassaemia is caused by point mutations, addition or deletions in the b-globin gene. More than 200 mutations cause b-thalassaemia and the mutations are specific in each ethnic group. Rapid characterisation of the mutations is essential for genetic counselling and prenatal diagnosis. This study aims to develop a rapid and effective molecular screening technique for the detection of b-thalassaemia mutations present among Malaysians. In-house TaqMan genotyping assays were developed to screen these mutations. Nine sets of primers and probes were designed and optimised to identify the common mutations in Malaysian Malays and Chinese: -28 (A-G), CD17 (A-T), HbE (G-A), IVS1-1 (G-T), IVS1-5 (G-C), CD 41/42 (-CTTT), CD71/72 (+A), IVS2-654 (C-T) and Poly A (AATAAA-AATAGA). Evaluations were carried out to determine the sensitivity and specificity of the developed genotyping assays. Established molecular techniques – amplification refractory mutation system and restriction fragment length polymorphism were used to confirm the results of the quantitative real-time PCR TaqMan genotyping assays. The in-house developed system identified b-thalassaemia mutations with 100% sensitivity and specificity. Molecular characterisation using quantitative real-time PCR allows rapid high-throughput screening and is applicable for prenatal diagnosis with good sensitivity and specificity. MB04: Sequence Analysis of the E/NS1 Gene Junction of Dengue Type 1 Viruses Isolated in Klang Valley from 2010-2012 Nur Liyana K1, 3, Ravindran T1, Zainah S1, Salbiah N2, Lau IS2, Ong YC2, Khadijah NR2, Sharifah Aishah WMA2, Shatrah O3, Fauziah MK1 Virology Unit, IDRC, Institute for Medical Research, Selayang Hospital, 3 Department of Molecular Medicine, Faculty of Medicine, University of Malaya 1 2

Dengue is a mosquito-borne viral disease caused by four serotypes of dengue virus, which has been affecting the human population for decades in many tropical and subtropical regions of the world. In Malaysia, usually all

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four dengue serotypes co-circulate during dengue season, though any one of the serotype can be predominate. In this study, serum samples were collected from dengue fever and severe dengue fever patients in the Klang Valley from 2010-2012 to determine the prominent dengue serotype. In addition, sequencing of the envelope/non-structural 1 gene junction of the virus isolated in C6/36 (Aedes albopictus) cells were performed to determine the presence of any “virulence factor” in the virus. The results showed that Dengue-1 (DEN-1) was the predominant circulating serotype. The envelope and non-structural protein 1 junction (E/NS1) of the isolates were amplified and sequenced for comparison and to trace the evolutionary history of the strains. All sequences of the isolates were compared with the DEN-1 prototype Hawaii strain and E/NS1 sequences from neighbouring regions as well as other parts of the world which were obtained from the GenBank database and used for phylogenetic tree analysis. Analyses showed that there was a 97% to 100% similarity among the ten isolates at the nucleotide level. Amino acid analogues, on the other hand, showed a 98% to 100% homology. However, all five non-severe dengue isolates showed variation at position 780, resulting in an amino acid change from Valine to Alanine, as compared to the severe dengue isolates. A rooted phylogenetic tree was performed using the neighbour-joining method with DEN-2 and DEN-3 as the outgroups. The results showed that all ten isolates were classified as genotype I. In addition, the five isolates from severe dengue patients were probably clustered together with JN697057 and JN697058, the Malaysian DEN-1 strains that were reported to show monoclonal antibody escape in 2005.

merization of the protease was also evaluated. NS2B/NS3pro was being expressed in Eoli XL1 Blue and subsequently purified via nickel affinity matrix treatment and was checked for purity via SDS-PAGE. The expressed protease was analyzed by Western blot analysis utilizing anti-his antibodies for recognition. The expressed protease was verified using Mass-Spectrometry. The protease was further purified by gel filtration chromatography using HiLoadSuperdex 16/600 75pg. DNA sequencing analysis of the NS2B/NS3 revealed it as NS2B/NS3pro from DENV2. NS2B/3pro was successfully expressed at 0.38975mg/ml/L after nickel purification with 95% purity. Western blot analysis confirmed the existence of the NS2B/3pro at 35kDa (monomeric form) and mass spectrometry of the protein confirmed it as a protease from DENV2. The purity was increased to 98% after gel filtration chromatography. According to the standard gel filtration marker, the protease was eluted with an approximate size of 200kDa and 160kDa at room temperature, and the last peak might be in a trimeric or tetrameric form, with a size of approximately 120kDa. In future studies, oilgomerization will be studied using native PAGE. It is concluded that NS2B/3pro has reached a high percentage of purity for the next experiments in kinetics and secondary structural analysis with the inhibitors from Boesenbergia rotunda.

MB05: Preliminary Findings of the Expression and Purification of Dengue Virus Type 2 NS2B/3 Protease

Universiti Tunku Abdul Rahman

RufaidahOthman,1,5Saiful Anuar Karsani,1,5 2,5 3,5 Aida Baharuddn, Rozana Othman, Shatrah Othman,2,5Noorsaadah Abdul Rahman,4,5Rohana Yusof2,5* Institute of Science Biology, Faculty of Science,University of Malaya, Malaysia 2 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Malaysia 3 Department of Pharmacy, Faculty of Medicine, University of Malaya, Malaysia 4Department of Chemistry, Faculty of Science University of Malaya, Malaysia 5 Drug Design and Development Research Group, University of Malaya, Malaysia *Corresponding author: [email protected] 1

Dengue outbreakshave occurred since the 17th century. During the 19th century, the dengue virus (DENV) two-component protease NS2B/NS3 was discovered to mediate virus replication by processing the viral polyprotein precursor. In this study, the NS2B/NS3 protease (NS2B/3pro) was expressed and purified. The oligo

MB06: Development of a Broad-Host Range Acetosyringone-inducible Heterologous Protein Expression System Po-Iian Eliza Loo, WaiKeat Toh,Pek Chin Loh,Hann Ling Wong

Gene expression systems are widely used in laboratories for research and industrial-scale production of goods. The most common used inducible gene expression system is based on the lac operon, which is induced by IPTG (isopropyl b-D-1-thiogalactopyranoside). However, the lac operon is not economical, besides being leaky; it gives highly intense responses and is potentially toxic. In response to these problems, we are developing a broad-host range acetosyringone-inducible heterologous protein expression system. This system will be able to overcome the disadvantages of the ¬IPTG-inducible system while presenting more benefits. The soil bacterium, Agrobacterium tumefaciens, utilizes phenolic exudates, such as acetosyringone, to activate its virulence (vir) genes as a cue to infect a plant. The virA and virG virulence proteins function as a two-component regulatory system to sense these phenolic compounds to activate the vir operons. Activated virG subsequently binds to the vir box sequences in the vir promoter and interacts with the RNA polymerase, hence stimulating the transcription of genes downstream. The acetosyringone-inducible system that we are constructing is more tightly regulated, cost-effective and gives a lower basal transcription level than that of


the IPTG-inducible system, in addition to its broad host range application. In this study, we replaced the transferred DNA (T-DNA) region of plasmidpGreenII0049 with the acetosyringone-inducible virB promoter to generate a new vector, pASE. In order to increase the flexibility of this plasmid, a Gateway® cassette was inserted downstream of the virB promoter. The broad host range ori of pSa in pASE allows it to be replicated in many other Gram-negative bacteria. For the acetosyringone-inducible system to work, the function of the virA and virG will be supplemented by pCAMBIA5105, which is co-transformed with pASE and pSoup into the bacterial host. For evaluating the inducibility of the gene expression system, aGFP reporter gene will be clonedinto the Gateway® cassette. Upon addition of acetosyringone, GFP fluorescence is expected to be observed.

MB07: Development ofMicrosatellite Markers for Genetic Analysis of Kappaphycus Doty and Eucheuma J. Agardh (Rhodophyta)from Various Localities Sze-Looi Song1,2, Phaik-Eem Lim1,2, Weng-Wah Lee3, Ji Tan1,2, Siew-Moi Phang1,2 Institute of Ocean and Earth Sciences (IOES), University of Malaya, 50603 Kuala Lumpur, 2 Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia 3 ACGT Laboratories, Lot L3-I-1, Enterprise 4, Technology Park Malaysia, Bukit Jalil, 57000 Kuala Lumpur, 1

Kappaphycus and Eucheuma are important sources of carrageenan production which are widely used in the food industry due to their gelling, thickening and stabilizing properties. However, the high morphological plasticity of seaweed often leads to misidentification of these economically important species. Microsatellite markers were developed from the EST database of Kappaphycusalvarezii which was later tested on species of Kappaphycus and Eucheuma from various localities. Seven EST-SSRs were obtained in which two primer-pairs showed polymorphismsin on the specimens examined. Primer-pair K1 indicated that there were two possible genotypes from Kappaphycus striatum, while primer-pair K7 distinguished Kappaphycus from Eucheuma which were collected from different locations. These preliminary results showed the potential of the microsatellite markers that we developed in the genetic analysis of these species. More samples with larger collection areas are needed for a better understanding of these commercially important Rhodophytes.


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MB08: Tracing the Natural History of House Crows Corvussplendens) Using Microsatellite and Mitochondrial DNA Markers. UrszulaKrzeminska, Robyn Wilson,DillonSarim, Chris Austin and Sadequr Rahman School of Science, Monash University, Bandar Sunway, Malaysia The common house crow (Corvussplendens), is amongst the most wide-spread species of birds. It has attained a high population density and pest status in many locations with breeding colonies in over than 20 countries outside its native range. House crows live in close proximity to humans with negative consequences for their health, economic activity and the regions ecology. Thus, it is important to study the population genetics of this invasive species in order to understand its movements. However, it can be difficult to obtain DNA samples from live birds. In this study, DNA was extracted from different parts of its feathers. A set of genetic markers was designed to analyse migration patterns and genetic differentiation between populations. The markers included autosomal microsatellites described in previous studies of crows and markers based on chicken DNA sequences. In addition, the mtDNA cytochrome oxidase subunit I (DNA barcode region) was amplified for the purpose of species identification. In the future we propose to use the mtDNA control region to identify the origin of crows in Malaysia and to compare maternal lineages from various locations. A broad set of markers can be used to answer a number of questions about the natural history of crows.

MB09: A TILLING Resource for Functional Genomics in Arabidopsis thaliana Accession C24 Kok Song Lai1*,Pulla Kaothien Nakayama2 and Seiji Takayama2 Agro-Biotechnology Institute Malaysia, Ministry of Science, Technology and Innovation, c/o MARDI Headquaters, 43400 Serdang, Selangor DE, Malaysia 2 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan *Corresponding author: [email protected] 1

TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that can be employed to generate an allelic series of induced mutations in targeted genes for functional analyses. To date, TILLING resources in

36


Arabidopsis thaliana are only available in Columbia and Landsberg erecta accessions. Here, we extended the Arabidopsis TILLING resources by developing a new population of ethyl methanesulfonate (EMS)-induced mutant lines in the commonly used A. thaliana accession C24.A permanent collection of 3,509 independent EMS mutagenized M2 lines was developed in A. thaliana accession C24 and designated as C24TILL. Using the TILLING method to search C24TILL for mutations in four selected genes identified a total of 73 mutations, comprising 69.6% missense, 29.0% sense and 1.4% nonsense mutations. Consistent with the propensity of EMS to induce guanine alkylation, 98.4% of the observed mutations were G/C to A/T transitions. Based on the mutations identified in the four target genes, the overall mutation density in the C24TILL collection was estimated to be 1/348 kb. TILLING the DUO POLLEN 1 (DUO1) gene from the C24TILL collection identified a truncation mutation leading to a deficiency in sperm cell differentiation.Taken together,a new TILLING resource, the C24TILL collection, was generated for A. thaliana accession C24. The C24TILL collection provides an allelic series of induced point mutations that will serve as a useful alternative reverse genetic resource for functional genetic studies in A. thaliana. MB10: Transformation of the pezT Toxin Gene from the Bacterial pezAT Toxin-antitoxin System into the Model Plant Arabidopsis thaliana Hasrif Nurhana Yasin1, Fauziah Abu Bakar1, Chew Chieng Yeo2 and Jennifer Ann Harikrishna1* Centre for Research in Biotechnology for Agriculture (CEBAR) and Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur 2 Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Kampus Gong Badak, 21300 Kuala Terengganu *Corresponding author: [email protected] 1

Toxin antitoxin (TA) systems are widely found in bacteria and archaea, and function in the regulation of cell growth and cell death. Prokaryotic TA systems have been found to be functional in eukaryotic cells, such as the yeast Saccharomyces cerevisiae, Xenopuslaevis and also in human cell lines. However, no such studies have been carried out in plants. The pezAT toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae consists of the PezA antitoxin and the PezT toxin. Expression of the pezT toxin gene has been found to be lethal in Escherichia coli. Here, we report the transformation of the pezT toxin gene into Arabidopsis thaliana as a model plant. The pezT toxin gene was fused with a green fluorescent protein gene, GFP, and cloned into the inducible plant expression vector pMDC221, where its expression will be dependent on the XVE transcriptional activator that is activated by 17b-estradiol. In parallel, the constitutive CaMV


35S promoter was cloned into the pMDC150 activator vector to enable the constitutive expression of the XVE activator. Both pMDC221-pezT: GFP and pMDC15035S recombinant vectors were transformed into Arabidopsis thaliana via Agrobacterium tumefaciens using the floral dip protocol. PCR which was carried out on DNA extracted from the T1 generation of transgenic Arabidopsis showed that the pezT toxin gene had successfully integrated into plant genome. MB11: Elucidation of Anoxybacillus Amylase Function Using Protein Engineering Approach Ranjani Velayudhan and Goh Kian Mau* Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia *Corresponding author: [email protected] a-Amylases are important industrial enzyme in hydrolyzing starch. This work aimed to elucidate the role of distinct amino acids in the conserved regions of a novel a-amylase in Anoxybacillus sp. (ASKA) (accession number: I1VWH9) by site directed mutagenesis. Based on the multiple sequence alignment of the conserved region II, Tyrosine 187 and Leucine 189 are the two invariant residues and were replaced with Phenylalanine and Leucine, respectively by the mega-primer mutagenesis approach. Analysis of the purified native ASKA and the mutant amylases revealed that mutagenesis has no direct relationship with the optimum temperature and pH. In comparison to the native amylase, the specific activity of the mutant L189I showed a decrease of 0.8 fold while no significancechanges were observed for Y187F mutant. Substitution of Y187F and L189I resulted in a dramatic reduction in thermostability, the half-life was reduced by 6% and 14%. There was a 25% and 17% reduction in total sugar production for each mutant respectively, compared to the native enzyme. In conclusion, for the first time, this study suggests that residues 187 and 189 at conserved region II are important for both thermostability and catalytic efficiency. MB12: Homology modelling of alpha-amylase from Anoxybacillus sp, SK3-4: Rational design that may alleviate calcium-dependence Chai Kian Piaw1, Štefan Janeček2, Mohd Shahir Shamsir Omar1, Mohamed Abu Naser1 and Goh Kian Mau1* Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia 2 Slovak Academy of Sciences, Institute of Molecular Biology, Bratislava, Slovakia *Corresponding author: [email protected] 1

A high maltose forming alpha-amylase derived from


Anoxybacillus sp. SK3-4 was recently purified and characterized. The alpha-amylase (denoted as ASKA herein) has temperature and pH optima of 60°C and 8, respectively. The activity and thermostability of ASKA is calcium-dependent, but calcium-dependence is not a desired trait for an industrial enzyme, as calcium oxalate which is formed may block process pipes and heat exchangers. This research was aimed at reducing calcium-dependence of ASKA via rational design. Homology model of ASKA was constructed using I-TASSER. The homology model was further improved by molecular dynamics simulation using Gromacs package. After the simulation, the structure contains 91.2 % of residues in favored region, 7.3 % in allowed region and 1.5 % in outlier region. The improved structure of ASKA was used as the model for rational design. Variants of ASKA (L154E and A161E) were proposed with the concept of salt bridge introduction at the calcium binding groove. From bioinformatics analyses, salt bridges E154-R226 and E161-K137 could potentially stabilize the calcium binding groove and thus alleviate calcium-dependence. MB13: Development of a DNA Barcoding Database for the Commercially Important Marine Fish Species in Malaysia Darlina Md Naim1, Abdul Rahman Abdul Majid2, Masazurah A Rahim2,3, Tun Nurul Aimi4,5, 5 Mark de Bruyn5,Gary Carvalho and Siti Azizah Mohd Nor1,3 School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia 2 Fisheries Research Institute, Jalan Batu Maung,11960, Batu Maung, Penang, Malaysia 3 Centre for Marine & Coastal Studies, Universiti Sains Malaysia, 11800 Penang, Malaysia 4 Faculty of Fisheries and Aqua Industry, Universiti Malaysia Terengganu, Terengganu, Malaysia 5 Molecular Ecology and Fisheries Genetics Laboratory, Environment Centre Wales, Bangor University, Bangor, Gwynedd, United Kingdom 1

We report here our on-going efforts to molecularly identify all the major commercially important marine fish species in the surrounding seas of Malaysia, namely in the Straits of Malacca, South China Sea, Sulu Sea and Sulawesi Sea. To date we have analysed more than 100 of the approximately 150 presumed fish species commonly obtained at fish landing sites throughout Malaysia based on the DNA barcoding cytochrome oxidase subunit 1 (COI) gene sequence. These are composed of 17 families: Ariidae, Balistidae, Caesionidae, Carangidae, Chirocentridae,


37

Clupeidae, Engraulidae, Haemulidae, Leiognathidae, Lutjanidae, Mullidae, Nemipteridae, Stromateidae, Sciaenidae, Scombridae, Serranidae and Triacanthidae from more than 1000 specimens. Detailed analyses of the Carangidae and Ariidae have revealed that there are potentially many more undocumented or cryptic species. We discuss this data in the light of fisheries management. This project is registered in the BOLDSYSTEMS under the Barcoding fish (FishBOL) campaign - Barcoding of Commercially Important Marine Species of Malaysia. This project is funded under the Delivering Excellence Grant (APEX - USM) and in collaboration with the Department of Fisheries Malaysia and Bangor University (United Kingdom). MB14: DNA barcode reveals taxonomic ambiguities in the Malaysian Ariidae Mohd Lutfi Abdullah1, Masazurah A Rahim1,2, Abdul Rahman Abdul Majid2, Mansor Mat Isa3 and SitiAzizah Mohd Nor1,3 Centre for Marine & Coastal Studies, Universiti Sains Malaysia, 11800 Penang, Malaysia 2 Fisheries Research Institute, Jalan Batu Maung, 11960 Batu Maung, Penang, Malaysia 3 School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia 1

A 621 bp of mitochondrial cytochrome oxidase subunit I (COI) or DNA barcode gene was utilized to resolve phylogenetic relationships and molecular taxonomy of eight presumed Malaysian Ariid species: Netumathalassina, Plicofilisargyropleuron, Plicofilispolystaphylodon, Nemapteryxcaelata, Hexanematichthyssagor, Arius maculatus, Osteogenosismilitaris and Arius venosus. The monophyly of most species was well established with a mean Kimura-2-parameter (K2P) interspecies distance of 9.6%. However, a few discrepancies were observed involving i) Nemapteryxcaelata and A. venosus. ii) A. maculatus and GeneBank A. arius. Possible explanations for such discrepancies could arise as a result of misidentifications in this study or errors in GenBank database input, hybridisation or incomplete lineage sorting. Our aim is to conduct a more detailed study of this group for closer morphological inspection. This study is part of an on-going project on a national DNA barcode initiative of commercially important marine fish species. To date more than 100 species have been barcoded from more than 1000 specimens obtained from Peninsular Malaysia, Sabah and Sarawak. The on-going project is funded under the Delivering Excellence Grant (APEX - USM) and in collaboration with the Department of Fisheries Malaysia.

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MB15: A Specific Single-chain Fv Antibody AgainstToxoplasma gondii Tachyzoites Generated Through Single Round Cell Biopanning Approach

MB16: Application of Molecular Approaches to Study Antibacterial Effect of Red Algae against Methicillin-Resistant Staphylococcus aureus

Sherene Lim Swee Yin1*, Go Pei See, Nazrin bin Abd Aziz, Fatin Iffah Rasyiqah binti Mohamad Zoolkefli, Fong Mun Yik, Chua Kek-Heng2 and Rofina Yasmin Othman1

Nagi. A. AL-Haj1*, Nurmas .I. Mashan2, and Mariana. N.Shamsudin2, 3

Genetics and Molecular Biology Division, Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, *Corresponding author: [email protected] 1

The Toxoplasma gondii parasite remains a challenging infection to eradicate with no vaccines or immunotherapy available, and drug treatmentassociated toxicity. The development of specific antibodies againstnative parasite antigens is therefore an approach that could be useful for multifunctional studies of immunity against this disease as native protein structures may confer functional significance. In this study , the phage-display system was utilized to develop a single-round subtractive biopanning procedure to isolate single-chain variable fragment (scFv) antibodies probed against tachyzoites. Through this optimized approach using an immunized scFv antibody library (complexity of 1.62 X 104 independent transformants), phage-scFv output clones from the biopanning showed an average of at least 5.6-fold higher binding titer to T. gondii relative to unpanned clones, and an average of 5-fold higher binding titer relative to the negative control human cell line WRL68. Statistical assessment of the specificity of the isolated monoclonal scFv - TG130 showed significant antibody binding selectivity for T. gondii compared to negative control cell line (Mann-Whitney test, P = 0.0303, n = 12), which demonstrated the utility of this approach in generating cell-specific scFvs. Immunoassay procedures showed that antigen binding activity of developed scFv was retained in its expressed soluble form. Through this approach, our findings have important implications for recombinant antibody development intoxoplasmosis disease.

Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana’a University, Yemen 2 Department of Microbiology and Parasitology, Faculty of Medicine and Health Sciences,University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 3 Department Marine Science and Aquaculture, Institute of Bioscience,University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 1

Methicillin-resistant Staphylococcus aureus(MRSA) accounts for a large proportion of hospital acquired infections and is constitute a tremendous burden on hospitals worldwide in terms of morbidity and considered a serious problem because of its multi-drug resistant properties. Therefore, this study was designed to explore an alternative antibacterial product of bioactive compound from Gracilaria changii through study of DNA and RNA encoding genes of interest in MRSA and non-MRSA. Several genes of MRSA were chosen to study the effect of both seaweed extract on treated and untreated genes that are potentially involved in the antibacterial activities by Reverse Transcriptase Polymerase Chain Reaction analysis. The extract of G. changii showed a significant antibacterial activity against S. aureus. The minimum inhibitory and bacterial concentrations (MICs and MBCs) were determined in the setting of clinical MRSA isolates. However, the in vitro effects of seaweed extract on treated and untreated gene was not fully cleared in this study. The overall results can be used as potential of the extract was found to exhibit distinctive antibacterial activity.


MICROBES

M01: Rare Shigella sonnei Mannitol-Negative Phenotype Caused by Insertion Sequence IS4 Koh Xiu Pei1,2 , Noni Ajam1, Thong Kwai Lin 1,2 Institute of Biological Sciences, Faculty of Science;2Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya, 50603 Kuala Lumpur, Malaysia. Email:[email protected] 1

Bacteria of the genus Shigella causes shigellosis, an acute intestinal infection which is endemic in developing countries.Shigella comprises of four species, differentiated by antigenic properties and biochemical characteristics. Fermentation of D-mannitol has been used as a key characteristic in Shigella classification. D-mannitol is fermented by S. sonnei, S. flexneri, and S. boydii, and is not fermented by S. dysenteriae. Shigella sonnei is thepredominant etiologic agent of shigellosis in Malaysia. In this study, rare mannitol-negative S. sonnei were isolatedfrom four sporadic cases in Malaysia. The isolates were characterized based on carbohydrate fermentation profiles using API 50CH strip and API 50CHE medium (bioMérieux, SA, France). The molecular basis of the mannitol-negative phenotype was then investigated. Primers were designed to detect the mtl genes in these isolates and amplicons were sequenced and analyzed. The carbohydrate fermentation profiles of these isolates were typical of S. sonnei,except for mannitol. More than 70% of the 49 tested carbohydrates were not fermented. All three structural genes of the S. sonnei mannitol operon (in the order: mtlA, mtlD, and mtlR) were intact in these mannitol-negative phenotypes but the amplicons for mtlA were larger than expected. An insertion sequence, IS4, was inserted 12 bases before the start codon of the mtlA gene in each isolate. The insertion appeared to disrupt the regulation of the mannitol operon. Such an insertion was not detected in the mannitol-fermenting S. sonnei. Even though mannitol-negative S. sonnei are rarely reported, a negative result for mannitol fermentation should not ex-


39

clude the possibility of an isolate being S. sonnei. It remains to be seen if more S. sonnei mannitol-negative phenotypes will arise and whether the loss of mannitol fermentation is adaptive or due to reduction in purifying selection. M02: Antimicrobial Properties of Pandanus Extracts against Staphylococcus aureus Ahmad, M. S., Armayni, U. A., Md. Yusof, N., Abdul Wahab, I., Sultan, S. & Mohsin, H. F. Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Puncak AlamSelangor Darul Ehsan. E-mail: [email protected] Intensive studies on the biological properties of Pandanus pygmaeus (locally known as yellow pandan from the Pandanaceae plant family) have not been done. Hence, the aim of this study was to assess the antimicrobial activity of hexane, dichloromethane and methanol Pandanus extracts and their chromatographic fractions, as well as to determine their bioactive constituents. The antimicrobial assay was performed using the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] method. The results showed that the hexane and methanolic extracts of P. pygmaeus (0.125 to 1 mg/ml) possessed a moderate inhibitory activity against Staphylococcus aureus ATTC 12228, with 40 to 60% inhibition by hexane and 50 to 67% inhibition by methanolic extracts. The inhibitory activity of the dichloromethane extract ranged from 0.015 to 1 mg/ml, and had a relatively weaker inhibitory activity of 33 to 47% against S. aureus. Vancomycin was used as the positive control in the assay. Furthermore, one of the hexane fractions (labelled as H07) also showed moderate inhibitory activity (50%) against S. aureus at a concentration of 0.125 mg/ml. Phytochemical studies on H07 resulted in the purification of a triterpenoid which had slight resemblance to the structure of an antitubercular triterpene, previously isolated from Pandanus tectorius Soland. var. laevis. It is believed that the isolated triterpenoid could demonstrate bactericidal properties against S. aureus, warranting further antimicrobial assays for other Pandanus compounds in future.

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M03: Pathotyping of Swine Escherichia coli Strains Wing Sze Ho1,2*, Lai Kuan Tan1,2, Peck Toung Ooi3, Chew Chieng Yeo4 and Kwai Lin Thong 1,2 Institute of Biological Sciences, Faculty of Science,University of Malaya 2 Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya 3 Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, University Putra Malaysia. 4 Faculty of Agriculture and Biotechnology, Universiti Sultan Zainal Abidin, Kuala Terengganu, Malaysia. Email: [email protected] 1

Escherichia coli can exist as harmless commensals in the intestinal tracts of animals while others can be pathogenic. Pathogenic forms of E. coli can cause a variety of diarrhoeal diseases in hosts due to the presence of specific colonisation factors, virulence factors and pathogenicityassociated genes. In this study, a total of 527 presumptive E. coli were obtained from swine farms located in two states in Malaysia. Pathotyping targeting of the VT, LT1, LT2, ST and eaeA genes, which are associated with three pathotypes (ETEC, EPEC and EHEC), was performed and the prevalence of serogroup O157 E. coli was investigated. Out of 527 isolates, 357 (67.7%) were confirmed to be E. coli by PCR and ten isolates were verocytotoxin (VT) -positive but negative for eae (attaching and effacing) gene. All VTEC (verocytotoxin-producing E. coli) isolates were non O157. All VTEC were isolated in farms located in Penang and were found in three types of swab samples (nasal, rectal and tongue). Five of them were isolated from healthy pigs while another five were from unhealthy pigs. No significant correlation was found between the health conditions of pigs and the presence of the VT gene (p > 0.05). This report indicates that eae-negative VTEC was the most common E. coli pathotype isolated from local swine samples. eae negative- VTEC were thought to be less virulent than classical EHEC, which possesses the eae gene. Although the VTEC isolates did not possess eae gene which is needed for the expression of full virulence, its presence should not be overlooked, as eae-negative VTEC has been reported to cause diseases worldwide.


M04: Blood, Faecal and Water-Borne Salmonella Typhi Exhibit High Clonality but Vary in Motility and Biofilm Forming Ability Kalaivani Kalai Chelvam Kwai Lin Thong 1,2

,

1,2

Lay Ching Chai

1,2

and

Institute of Biological Sciences, Faculty of Science, University of Malaya 2 Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya E-mail: [email protected] 1

Salmonella Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It is incapable of infecting other living organisms, yet able to cause both acute and chronic infections displaying various disease manifestations, and is able to transform the human host into an asymptomatic carriage with periodical dissemination via urine and faeces. The principal factors underlying the unique lifestyle of motility and the biofilm forming ability of S. Typhi remain largely unknown and are therefore the main objective of this study. To do so, swim and swarm motility was performed with 0.25% and 0.5% agar concentration respectively; while biofilm formation was determined by growing the bacterial strains for 48 hours in 96-well microtitre plates. Out of 60 S. Typhi strains, all showed swarming ability with small, smooth, flat colonies on the agar surface that remained at the inoculation point. Only two strains were non-motile in the swim plate assay. Three swimming patterns of Salmonella Typhi were observed for swim assay: featureless (96.7%), bull’s eye (1.8%) and vortex (1.8%). The majority of strains (21; 38.2%) were found to be weak biofilm producers while 20 (33.3%) strains did not form any biofilm. Twelve and seven strains were moderate and strong biofilm producers, respectively. A high degree of variation was observed in swimming and swarming motility. No distinct correlation between motility and biofilm forming capability was observed. In conclusion, the diversity observed among this set of S. Typhi strains suggests the possible involvement of surface motility and biofilm formation that might be related to virulence and pathogenesis. Motility is significant for long-term survival and persistence of S. Typhi biofilms, yet motility and biofilm formation might involve similar components at certain stages and specific conditions. These findings serve as caveats for future studies to understand the characteristics and the transmission mechanism of this pathogen.


M05: Genetic Characterization and Diversity of Listeria monocytogenes Isolated from Ready-to-Eat Foods Hossein Jamali* and Kwai Lin Thong Email: [email protected] The objectives of this study were to determine the prevalence and antibiotic resistance as well as to characterize Listeria monocytogenes isolated from ready-to-eat (RTE) foods. From November 2010 to January 2012, a total of 250 RTE food samples were purchased from street-side hawker stalls and hypermarkets in Klang Valley, Malaysia. The presumptive isolates were biochemically characterized and further conﬁrmed by Polymerase Chain Reaction (PCR). Out of 250 samples, Listeria spp. was detected in 52 (20.8%) samples, out of which 32 (12.8%) were positive for L. monocytogenes. Out of the 32 L. monocytogenes isolates, 21 (40.4%) were grouped into serogroup “1/2a, 3a”; 7 (13.5%) belonged to serogroup “1/2c, 3c”; and 4 (7.7%) belonged to the serogroups “4b, 4d, 4e”. All the L. monocytogenes harbored the inlA, inlB, inlC and inlJ virulence genes. L. monocytogenes showed highest resistance to Penicillin G (53%), followed by tetracycline (15.6%), amoxicillin-clavulanic acid (12.5%), vancomycin (9.4%) erythromycin (6.3%) and clindamycin / streptomycin / kanamycin / chloramphenicol (3.1%). All isolates were susceptible to gentamicin, rifampicin, clindamycin, kanamycin and vancomycin. REP-PCR and PFGE generated 30 (D = 0.996), and 27 (D = 0.988) patterns, respectively. These results indicated that L. monocytogenes isolates from RTE food were diverse. Since different subtyping methods often give different discriminatory powers, the usage of more than one subtyping approach is necessary in providing a more accurate picture of the clonality of L. monocytogenes. Furthermore, recovery of potentially pathogenic L. monocytogenes from RTE foods is a matter of public health concern. Therefore continued surveillance of the prevalence of L. monocytogenes and its emerging antibiotic resistance is needed for the recognition of foods that may pose a risk as well as to warrant the effective treatment of listeriosis. M06: The Application of invA, lamB and toxR Genes in Screening Bacterial Contaminants in Anadara granosa Adila Nadiah Ahmad, Roslina Sulaiman, Azlin Sham Rambely, Emida Mohamed and *Siti Nazrina Camalxaman Department of Medical Laboratory Technology, Faculty of Health Sciences,Universiti Teknologi MARA, 42300 Bandar Puncak Alam, Selangor, Malaysia. Email: [email protected] The consumption of Anadara granosa,or “bloody clams” is popular worldwide. This filter-feeding shellfish can accumulate enteric pathogens from polluted aquatic


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environments rendering them as a source of infection for food-borne diseases. Limited data are currently available in Asia, as most cases are often unreported, go unnoticed or uninvestigated. Therefore, the objective of this study was to screen for microorganisms native to estuarine waters in Selangor, for future surveillance and risk assessment efforts. A total of 450 raw Anadara granosa samples were screened for E. coli, Salmonella and Vibrio spp. from 15 different wet markets using microbiological, serological and molecular-based detection methods. Microbiological assessment and serological screening revealed that from the 450 samples, 27% (120/450), 30% (135/450) and 47% (210/450) of the isolates were of Salmonella, E. coli and Vibrio origin, respectively. PCR amplifications of the invA, lamB and toxR genes yielded single bands at 284 bp, 309 bp and 368 bp, respectively for the microbes mentioned above. Our preliminary results denote the incidence of bacterial contaminants in Anadara granosa as well as the potential use of the invA, lamB and toxR genesas diagnostic tools to aid their detection. Since gastroenteritis confers a substantial burden to society, a greater awareness and enhanced public health efforts should be underlined to minimize the risk of outbreaks. Further analytical studies are warranted to substantiate our findings. M07: Cloning, Expression and Immune Studies of a Specific DNA Marker for Salmonella Typhi Noradilin Abdullah1,2, Chua Kek Heng3, Kwai Lin Thong1 Institute of Biological Science, Faculty of Science, University of Malaya. 2 Faculty of Biosciences and Medical Engineering, UTM, 3 Department of Molecular Medicine, Faculty of Medicine, UM. Email:[email protected] 1

ST332, a 332 bp gene marker is one of the components incorporated in the Salmonella EZplex Kit for simultaneous detection of the foodborne pathogens Salmonella Typhi (S. Typhi), S. Paratyphi A and Salmonella spp. It has been identified as a hypothetical protein of S. Typhi strain CT18 (STY4528). In a previous study, the gene marker showed specificity in DNA-based detection of S. Typhi via polymerase chain reaction (PCR). Therefore, in this study, its specificity in an immunoassay was examined. For this purpose, the gene was amplified by PCR and cloned into a pGEX-4T-1 expression vector. Successful recombinants were produced by transformation of the cloned vector into E. coli BL21 grown on selection media (LB with 100 µg/ml carbanecillin). Analysis of the clones by PCR screening and sequencing proved that the gene insert was cloned at the right open reading frame and the BLAST identity of the cloned gene insert also matched that of the previous study, STY 4528. Therefore, the fusion gene was expressed by IPTG and the total protein was purified by NP-40 extraction buffer. The extracted pro-

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tein was hybridized against pooled typhoid patients’ sera that had antibodies specific to S. Typhi antigens by dot-blot immunoassay. The results revealed that the expressed protein has a weak affinity for the immunoassay. M08: Actinomycetes as Potential Producers of Quorum Sensing Inhibitors Kavimalar Devaraj, Geok Yuan Annie Tan, Kok Gan Chan. Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur. Email: [email protected]; [email protected] Actinomycetes are now targeted for their ability to inhibit quorum sensing in pathogenic strains of bacteria due to their capability to synthesise and produce a wide variety of secondary metabolites. In this study, a total of 147 actinomycete isolates from the Microbial Resources Laboratory Culture Collection were screened for their ability to inhibit violacein production by Chromobacterium violaceum CV026, with exogenously supplied C6-HSL. Nine isolates were observed to have potential anti quorum sensing activity. Phylogenetic characterization indicated that the isolates belong to the genera Streptomyces and Micromonospora. This study exhibited the prospective ability of actinomycetes in quenching quorum sensing signals accountable for bacterial virulence. The discovery of potential quorum sensing inhibitors is essential in attenuating the emergence of antibiotic-resistant bacteria. M09: Effect of Bacterial N-Acyl-L-Homoserine-Lactone on Efficiency of Agrobacterium-Mediated Transformation in Tobacco Plants Chin-Fong Chen1, Norzulaani Khalid1, Chan1

2

and Kok-Gan

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, 2 UMBIO, University of Malaya, 50603 Kuala Lumpur. Email: [email protected];[email protected] 1

Many bacteria use quorum sensing (QS) molecules, such as N-acyl-L-homoserine-lactones (AHLs), as an intercellular signal molecule to regulate its cell population density and certain gene expression. However, only N-3-oxo-octanyl-L-homoserine-lactone (3OC8HSL) has been found to control the conjugation process in Agrobacterium. To date, Agrobacterium-mediated genetic transformation remains as the most commonly used method to produce transgenic plants. However, low transformation frequency of this technique continues to be a significant limitation. Only a limited plant species has been


transformed, probably due to experimental, host plant and bacterial factors. Therefore, the aim of the present study was to investigate the effect of 3OC8HSL in the cell-to-cell communication system during Agrobacterium-mediated transformation. Our results revealed that 3OC8HSL significantly influenced Agrobacterium-mediated transformation in tobacco plants. The highest percentage of transformation (92.2 %) was recorded on the inoculated leaf explants that were cultured on MS medium containing 3% (w/v) sucrose, 0.2% (w/v) GelriteTM, 1.0 mg/L BAP, 0.1 mg/L NAA and supplemented with 100 µM 3OC8HSL. This study will be extended to explore the potential of other bacterial AHLs on Agrobacterium-mediated plant transformation. M10: Mutational Analysis of Drug Resistance-Associated Genes in Plasmodium vivax Isolates from Sabah Nor Afizah Nuin1, Tan Lii Lian1, Timothy William2, Praba Dhanaraj3 and Lau Tiek Ying1* Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 KotaKinabalu, Sabah, 2 Infectious Diseases Department, Queen Elizabeth Hospital,Kota Kinabalu, 88560, Sabah, 3 Kudat District Hospital, Peti Surat No. 22, 89057, Kudat, Sabah, Malaysia. Email: [email protected] 1

Plasmodium vivax, which is known to be the most geographically-widespread malaria parasite, is found throughout South and Central America, Asia, the Middle East and parts of Africa. High-level resistance to antifolate antimalarial drugs is known to be associated with mutations in the dihydrofolate reductase (dhfr) gene. Meanwhile, P. vivax pvcrt-o (pvcg10) is an ortholog of pfcrt and was identified to be associated with chloroquine resistance. The aim of this study was to identify single nucleotide polymorphisms in the full-length dhfr (1.8 kb) and crt (4.2 kb) genes of P. vivax isolated from Kudat, Keningau and Kota Kinabalu, Sabah. Nested-PCR was conducted to amplify the pvdhfr and pvcrt-o genes on a single confirmed P. vivax infection, based on species-specific nested-PCR. A total of 6 complete P. vivax dhfr and crt sequences were amplified. Four point mutations in the pvdhfr gene associated with S-P drug resistance were identified in this study, namely F57 (83%), S58 (100%), T61 (67%), and S117 (100%). Interestingly, a mutation at residue 57 (F->L) in our study resulted in a novel codon, CTC, instead of TTA and TTG that were described previously. As in the pvcrt-o gene, 4 synonymous mutations, 8 nonsynonymous mutations, 1 nucleotide deletion and 1 insertion of amino acid lysine (K) at amino acid position 10 (16.7%) were observed. However, all the nonsynonymous singleton mutations in pvcrt-o found in this study, namely N74S, V115A, T148I, V184I, Q205R, S226P, M342T, and


D410G (16.67% each), were randomly found in the studied P. vivax isolates and were novel. This study suggests that P. vivax in Sabah isolates may not be susceptible to S-P. As for the pvcrt-o gene, no resistance-conferring mutations were discovered. Therefore, mutational analysis of drug resistance-associated genes from this study may provide guidelines for appropriate treatment in P.vivax malaria.


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