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List of research projects available for prospective graduate students
Department of Anatomy
A/Prof George Yip
georgeyip@nus.edu.sg
Expression and Functional Analysis of Glycosaminoglycans and Proteoglycans in
Breast Cancer
Glycosaminoglycans are highly negatively charged molecules made up of repeating
disaccharide subunits consisting of an amino sugar and an uronic acid. They are
covalently linked to core protein backbones to form proteoglycans. Besides structural
roles, glycosaminoglycans and proteoglycans have important biological functions in
regulating cell behaviour through their interactions with growth factors and signalling
molecules. We aim to elucidate the effects of these molecules on cancer cell activities to
investigate if the molecules are clinically useful biomarkers and prognostic indicators,
and to develop novel therapeutic targets. A variety of cell and molecular biology
techniques will be employed in these studies, including cell culture,
immunohistochemistry, electron microscopy, ion exchange chromatography,
proteoglycan quantification assays, real-time PCR, in situ hybridisation, gene cloning,
and microarray analysis.
References:
1. Yip GW (2011) Breast cancer: Novel therapeutic targets. Recent Pat Anticancer Drug
Discov 6:164-165.
2. Koo CY, Sen YP, Bay BH, Yip GW (2008) Targeting heparan sulphate proteoglycans
in breast cancer treatment. Recent Pat Anticancer Drug Discov 3:151-158.
3. Yip GW, Smollich M, Götte M (2006) Therapeutic value of glycosaminoglycans in
cancer. Mol Cancer Ther 5:2139-2148.
4. Götte M, Yip GW (2006) Heparanase, Hyaluronan and CD44 in cancers: A breast
carcinoma perspective. Cancer Res 66:10233-10237.
Dr Hu Qidong
anthq@nus.edu.sg
Crosstalk between microRNAs and repeat-derived non-coding RNAs in neuronal
differentiation
For neurodegenerative diseases, the stem cell therapy represents a revolutionary strategy
to re-establish neural circuits. However, its wider application is hindered by the low
efficiency of differentiating pluripotent cells into desired neuronal cell types. Emerging
evidence has shown that miRNAs play a critical role in neuronal development. Our recent
study indicates that miRNA is regulated by a new type of small RNAs (riRNAs) derived
from the human Alu repeats in response to retinoic acid (RA) stimulation, a well-known
molecule to dictate neuronal differentiation. Hence, we aim to: 1) characterize the role of
riRNAs in regulating miRNAs; 2) investigate the mechanism underlying the crosstalk
between miRNAs and riRNAs in neurogenesis.
To identify those miRNAs regulated by riRNAs, we will carry out miRNA arrays under
various differentiating conditions and confirm the regulation by extensive biochemical
and molecular approaches. To explore the transcriptional regulation of miRNAs, we will
map the binding profiles of riRNAs by deep sequencing. The differentiation assay will
then be performed in human embryonic carcinoma cells and ES cells to establish the
functional crosstalk between riRNAs and miRNAs. To further explore the mechanism of
riRNA-regulated miRNA expression, we will carry out proteomics study to identify the
protein complexes functioning together with riRNAs. These proteins may serve as
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transcriptional activators and repressors. We will then map their genome-wide binding
profiles and correlate with riRNA binding patterns.
We expect to obtain a clear picture of riRNA-regulated miRNAs during neuronal
development and to be able to enhance the neuronal differentiation of stem cells. These
efforts will greatly benefit the patients suffering from neurodegenerative diseases.
Department of Biochemistry
Dr Chen Ee Sin
ee_sin_chen@nuhs.edu.sg
Anti-cancer drug resistance mechanism through the regulation of chromatin
structure
The genomic DNA of all eukaryotic cells are intricately organized around structural
proteins called histones into a higher order chromatin. The most fundamental subunits of
the chromatin is called nucleosome that contains 8 histone molecules organized around
about 146 base pairs of DNA. The nucleosome is the scaffold on which all DNAdependent processes including transcription, DNA repair and recombination occur and
the integrity of nucleosomal organization bears direct impact on important cell cycle
processes such as chromosome segregation and DNA replication. These two processes are
commonly targeted by many anti-cancer drugs to induce genomic instability in cancer
cells. However often, the effects of the drugs also affect normal cells resulting in profuse
side effects associated with chemotherapeutic treatments. This project is aim at finding
mechanisms that protect chromatin against the damage caused by chemotherapeutic
drugs. We will use fission yeast as a model organism to uncover universal mechanism in
order to facilitate targeted testing in human breast and gastric cancer cells.
Prof David M Virshup
gmsdmv@nus.edu.sg
Project 1:
Curing cancer with Wnt inhibition
david.virshup@dukenus.edu.sg
Novel drugs that block Wnt secretion show promise in stopping cancer growth. In this
project, we aim to learn more about the best way to use these drugs, inhibitors of the
enzyme PORCN. By understanding more about pathways affected by PORCN inhibitors,
the student will identify rational drug combinations, and strategies to minimize toxic side
effects in mouse models, and in patients. Techniques include molecular and genetic
analysis, and cell culture and mouse based models.
Project 2:
Defining Cancers Sensitive to Inhibition of the Wnt Secretion Pathway
Wnt signalling drives cancers and inflammatory diseases. We have developed novel,
specific and potent drugs that prevent Wnt secretion by inhibiting the O-acyltransferase
enzyme, PORCN. In this project, the student will develop and assess specific genetic
predictive biomarkers to select and characterize cancers and patients with drug-sensitive
disease. Techniques include molecular and genetic analysis, and cell culture and mouse
based models.
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List of research projects available for prospective graduate students
Dr Deng Lih Wen
lih_wen_deng@nuhs.edu.sg
Project 1:
Targeting MLL5β mediated transcriptional control of the E6/E7 oncogenes as a
basis for novel therapeutic strategies for HPV16/18 associated cancers.
Our lab has previously identified a new MLL5 isoform, MLL5β, which expression was
correlated to high-risk HPV16/18 cervical cancers. Our initial data suggest that MLL5β
is involved in the regulation and expression of key oncogenes E6 and E7 in cervical
cancer and targeted silencing of MLL5β could inhibit HPV16/18 cervical cancer
development. We have recently established that cisplatin, the current gold standard in
cervical cancer care modulates its anti-tumour effects through MLL5β. Our findings have
also suggested that targeted gene silencing of MLL5β has potential applications as a
mode of therapeutic intervention for HPV16/18-related cancers with less cytotoxic effects
compared to cisplatin. Currently we have identified a key post-translation modification
(PTM) on MLL5β, which is essential for the assembly of the transcriptional activation
complex required to initiate E6/E7 transcription. Based on this finding we have moved on
to identify potential small molecular inhibitors of this PTM and are investigating their
potential applications in cervical cancer therapy.
However, much remains to be done to better understand the full role of MLL5β in E6/E7
expression regulation such as what are other components of this MLL5β-associated
transcriptional complex as well as the exact role of MLL5β in the complex. A better
understanding of these molecular events will lead to the potential identification of better
therapeutic avenues for cervical cancer therapy. We are looking for an enthusiastic and
motivated candidate to focus on further understanding these molecular events. The
candidate will work on identifying the other components of the MLL5β transcription
activation complex as well as further understand the histone methyltransferase role of
MLL5β in the complex. The outcome of the project will be to harness this knowledge and
translate it to the development of novel molecular targeted therapeutic strategies for
cervical cancer therapy.
Project 2:
Targeting Cancer Metabolism In Inhibiting Tumor Progression
Metabolic switch is an important event during tumor progression. It is believed that
cancer cells reprogram their metabolic processes so as to exhibit high glycolysis to feed
their rapid proliferation, a phenomenon known as Warburg effect. The adaptation to
glucose metabolism in cancer cells produces high metabolic acid that in turn confers
tumor survival and invasion. The invasion of in situ tumor cells to distant sites, also
known as cancer metastasis, accounts for more than 80% of cancer-associated death.
Studies have also found that cancer cells which acquire high invasiveness are usually
highly resistant to chemotherapy. Conceptually, intertwining cancer metabolism,
particularly the glycolysis process would serve as an effective way to target cancer
progression. However, the inter-link between cancer metabolism and cancer invasiveness
is unclear at the moment.
Our preliminary data demonstrated that our compound of interest interfered with cancer
glycolysis and potentially affected cancer metastasis process. In this project, we are
interested in finding the underlying cellular metabolic changes in cancer cells across
different invasive stages and understand how these metabolic switches affect cancer
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hostile behavior. We also intend to screen for pharmacological compounds that exhibit
synergistic effects to achieve anti-cancer survival and anti-metastasis. Findings from this
project will provide further knowledge on cancer progression and shed light in developing
innovative cancer therapy strategy.
Adjunct Prof HUI Kam Man Project 1:
cmrhkm@nccs.com.sg
Validation of blood-based three-gene signature in improving the detection of early
hepatocellular carcinoma (HCC) from high-risk chronic hepatitis B virus carriers
(CHB)
Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers
worldwide. Early detection of HCC in high risk individuals would greatly improve the
clinical outcome of HCC. We profiled for transcriptome differences in the peripheral
blood mononuclear cells (PBMC) of CHB and HCC patients and identified a novel threegene signature which can accurately detect HCC from high-risk CHB patients. The
student candidate will be involved in the validation of this three-gene signature using
various techniques including RT-qPCR, QuantiGene assays, flow Cytometry and
fluorescence microscopy.
Project 2:
In vitro culture of hepatocellular carcinoma (HCC) tumor initiating cells (TICs)
Hepatocellular carcinoma is the fifth most common cancer and the third leading cause of
cancer related deaths worldwide. The extremely poor prognosis of HCC is partly due to
the fact that HCC shows high degree of heterogeneity not only between tumors but also
within each individual tumor. This poses great challenges in the proper research,
diagnosis and treatment of HCC.
Phenotypic and functional heterogeneity arises among cancer cells even within the same
tumor as a consequence of continual accumulation of genetic and epigenetic changes.
Yet, only a small proportion of cells in the tumor exist to convey tumorigenicity, the
tumor initiating cells (TIC). TICs have mainly be studied based on specific surface
markers and FACS sorting. However, such markers are usually hard to be reproduced in
solid tumors. In this project, we will attempt to adapt a feeder cells culture system to
culture HCC TICs to reduce a complex tumor to its crucial constituting clones. This will
form the basis for dissecting HCC tumor heterogeneity and to find suitable
targets/markers to improve the prognosis of HCC.
A/Prof Lee Guat Lay,
Caroline
caroline_lee@nuhs.edu.sg
Project 1:
Colorectal cancer (CRC) is the most common cancer in Singapore with around 2000 new
cases each year.
About a third of these cases are metastatic (stage IV) at diagnosis and around one third of
curatively resected cases (stages I-III) will relapse suggesting that a substantial proportion
of patients may need treatment for metastatic/relapsed CRC. The two commonest
combination drug regimes for this cancer in Singapore are CAPOX (capecitabine and
platinum-based oxaliplatin) or CAPIRI (capecitabine and topoisomerase-I inhibitor)
but the response rate to these regimes is only ~40-45%. Developing a reliable early
predictive biomarker of response to these drugs in metastatic CRC can lead to appropriate
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tailoring of treatment for individual patients and help move us closer to a truly
personalized care. It will not only be cost effective but will also reduce unnecessary sideeffects in patients who will not benefit from the drug treatment and novel treatments can
be explored for these patients. However, there are currently no reliable tests for early
prediction of response to chemotherapy in these patients. Genetic single-nucleotidepolymorphisms (SNPs) have strongly been implicated in the determination of differences
in drug response and hence can serve as a useful early predictive biomarker for response.
Here, we propose a novel pathway-based, potentially-functional SNP (pfSNP) approach
to identify SNPs that may serve as predictive biomarker of response to these drugs in 300
treatment-naïve CRC patients who receive either CAPOX or CAPIRI. Response that will
be assessed include tumor shrinkage according to standard RECIST criteria, toxicity
according to standard NCI-CTC criteria, progression-free survival and overall survival. A
simple, robust, cost-effective point-of-care genotyping assay will then be developed for
polymorphisms that can predict drug response. We will also automate this point-of-care
test so that it will be capable of translating genetic information to clinically relevant
information about drug response.
Project 2:
Hepatocellular carcinoma (HCC), amongst the top 5 cancers in Singapore is the third
leading global cause of cancer death with five-year survival of ~5%. Although molecular
advances have led to an increased understanding of the genetic changes that occur in
HCC, there is still inadequate knowledge about the full spectrum of molecular
mechanisms and epigenetic events in hepatocarcinogenesis. Current diagnosis of HCC
relies on routine screening of at-risk patients, including those with cirrhosis due to viral
hepatitis, by screening serum alpha-fetoprotein (sAFP) levels in conjunction with hepatic
ultrasonography but this combination has limitations. Although cost-effective,
ultrasonography has only 60% sensitivity and 97% specificity. sAFP is only 40-60%
sensitive as many tumors do not produce AFP or do so at very advanced stage. Hence,
there is an urgent need to identify better, more reliable non-invasive biomarkers with
higher sensitivity and specificity for early detection of HCC. Here, we propose to
interrogate miRNAs/small RNAs, by themselves or in combination with current available
AFP/Ultrasound biomarkers, as emerging novel biomarkers for predicting individuals
who are likely to develop cirrhosis or at the early stage of cirrhosis as well as those likely
to develop HCC or at early stages of HCC. Retrospective, as well as prospectively
collected samples with yearly follow-up and sample collection from 4 groups of
individuals, namely, Normal Non-cirrhotic (NNC), HBV chronic carriers (HBCC),
Cirrhotic and HCC will be examined. Next-Generation Deep Sequencing on prospective
samples at zero time point will be employed to identify novel miRNAs/small RNAs that
are that are associated with cirrhosis and/or HCC. Realtime RT-PCR will be used on the
retrospective samples to validate promising miRNAs while prospective samples collected
at 24 month time point will be used to confirm the robustness of these validated
biomarkers. These novel biomarkers either alone or in combination with current
biomarkers may be clinically useful.
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List of research projects available for prospective graduate students
Dr Lim Yoon Pin
bchlyp@nus.edu.sg
Function and mechanism of a novel WBP2 oncogene in breast cancer and metastasis
Through cutting-edge phosphoproteomics technology, we have identified a novel breast
cancer associated gene (1). Our lab is the first to demonstrate that WBP2 transcription
co-activator is a new breast oncogene that upon phosphorylation is able to transform
normal mammary cells into cancer cells and make mild cancer cells aggressive by
activating multiple other oncogenic pathways (2). We have also identified a new
interacting partner of WBP2 that modulates the expression of WBP2 and its Wntpromoting activities in breast cancer cells (3). The aim of the new project is to elucidate
how WBP2 turns on other oncogenes and contributes to breast oncogenesis and
metastasis using a wide array of in vitro and animal model assays associated with signal
transduction, transcription and cancer biology. The proposed study is likely to result in
the mapping of signaling cascades and transcriptional networks associated with the
WBP2 thus providing new insights into the molecular etiology of breast cancer. Our lab
also has a strong emphasis on translational research – for example in the exploitation of
WBP2 as a biomarker and drug target. A patent has been filed for the use of WBP2 in
cancer detection and therapy. Students will have a chance to participate in
commercialization of intellectual properties associated with WBP2.
1.
Chen, Y., et.al and Lim, Y. P. (2007) Differential Expression of Novel Tyrosine
Kinase Substrates during Breast Cancer Development. Mol Cell Proteomics 6, 2072-2087
(5 year impact factor: 9.4)
2.
Lim, S. K., et.al. and Lim, Y. P. (2011) Tyrosine phosphorylation of
transcriptional coactivator WW-domain binding protein 2 regulates estrogen receptor
alpha function in breast cancer via the Wnt pathway. FASEB J 25, 3004-3018 (5 year
impact factor: 7.2)
3.
Lim S.K., et.al. and Lim YP (2014) WIP negatively regulates WBP2 expression
and WBP2-mediated Wnt activation in breast cancer cells (manuscript in preparation).
Dr Long Yun Chau
bchlongy@nus.edu.sg
Project 1:
The role of energy sensing network in exercise-induced skeletal muscle adaptation
The beneficial effects of exercise on health are widely recognized. Exercise could
improve and prevent chronic metabolic disorders such as diabetes and obesity. However,
the cellular mechanism behind these effects is largely unknown. Optimization of exercise
regimen or pharmacological recapitulation of exercise effects requires close examination
of intramuscular events that triggers the exercise adaptations. Our previous and current
work has aimed to define the mechanism that drives exercise-induced metabolic
adaptation in skeletal muscle.
Exercise induces a multitude of cellular disturbances in muscle including decreased
intramuscular energy levels. Exercise-induced energy deficit is considered an important
stimulus for the beneficial metabolic outcomes. AMP-activated protein kinase (AMPK) is
an important signal transducer which is activated in response to energy stress during
exercise. Our previous work in mouse models provided evidence that activation of this
protein kinase is sufficient to increase skeletal muscle glycogen store, fatty acid
utilization and muscle endurance – benefits that are derived from exercise training.
Conversely, disruption of muscle AMPK signaling accelerated the progression of muscle
fatigue and impaired exercise-mediated metabolic gene expression. These results
provided important evidence that AMPK signaling mediates some of the exercise-
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mediated metabolic responses in skeletal muscle. In the current study, we hypothesize
that AMPK plays a role in autophagy and amino acids metabolism in skeletal muscle, and
this effect is critical for metabolic benefits of exercise and endurance.
Project 2
Role of insulin-induced glycolysis in the regulation of PGC1α-mediated myotube
lipid metabolism.
Our body is subjected to irregular nutrient supply, including the transition between fasting
and feeding. Therefore, the ability of our body to selectively store and utilize different
energy substrates is critical for energy balance. Given the substantial mass and energy
consumption, skeletal muscle plays a critical role in the regulation of energy balance, and
impaired skeletal muscle metabolism is closely associated with metabolic diseases such
as type 2 diabetes and obesity. Thus, investigation of skeletal muscle energy substrate
metabolism is critical for the understanding of pathophysiology of metabolic diseases.
Under postprandial conditions, insulin stimulates skeletal muscle glucose uptake and
utilization via Akt, a protein kinase that activates downstream effectors of insulin.
Conversely, skeletal muscle increases lipid oxidation under fasting conditions, which
coincident with the induction of PGC1α - a transcription coactivator that has been
implicated in the induction of lipid metabolic gene program. Nonetheless, the interaction
between the disparate insulin and PGC1α pathways which are activated under contrasting
nutritional states remains largely unknown. This research project aims to establish the
role of nutrient (glucose) and hormone (insulin) in the regulation of PGC1α cultured
myotubes, and the impact of this regulation on lipid metabolic gene program in myotubes.
Project 3
Role of insulin-like signaling in the regulation of SIRT1 deacetylase in cultured
myotube
The loss of skeletal muscle mass and function is a common feature of muscle pathologies
associated with inactivity (atrophy), aging (sarcopenia) and diseases (cachexia). The
cause can be attributed to elevated protein degradation and resistance to anabolic growth
hormones such as insulin and insulin-like growth factor 1 (IGF1). These anabolic
hormones are critical signals that stimulate skeletal muscle glycogen and protein
synthesis (anabolic metabolism), predominantly via the Akt kinase pathway. Conversely,
nutrient deprivation activates Silent Information Regulator T1 (SIRT1) deacetylase and
forkhead box protein O1 (FOXO1) proteins that induce catabolic metabolism in skeletal
muscle. The increase of catabolic over anabolic pathway may be a molecular basis for the
loss of muscle mass and function. Nonetheless, the mechanism by which such delicate
balance is regulated remains largely unknown. This proposal aims to evaluate the novel
role of insulin-like signals in the regulation of SIRT1, and the unknown function of
SIRT1 in the regulation of glucose and amino acid metabolism in cultured myotubes. The
specific aims are (1) to determine if insulin-like signals regulate SIRT1 via nutrient
metabolism, (2) to establish the role Akt in the regulation of SIRT1-induced FOXO1
pathways, and (3) to examine the impact of AKT-SIRT1 pathways on energy substrate
metabolism. Cultured myotubes will be used as a model. The research will provide
important insight into the pathophysiology of skeletal muscle metabolism, via the
identification of insulin-like hormones as regulators of SIRT1. It will also provide critical
evidence for SIRT1 and IGF1 as potential drug targets for muscle wasting diseases.
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List of research projects available for prospective graduate students
Dr Kenneth Ban
bchbhkk@nus.edu.sg
Project 1:
Development of Transposon-based Approach for Combinatorial Testing of Cancer
Genes in Human Cell Lines
Recent exome sequencing efforts have uncovered complex somatic mutations in human
cancer that underlie their heterogenous biologic behaviour and therapeutic response.
Understanding how these mutations cooperate and act to drive tumourigenesis is a major
challenge and current experimental approaches are focused on functional testing of one or
a few genes at a time.
The overall goal of this project is to develop parallelized approach in a human cell line to
determine what combinations of candidate cancer genes cooperate to drive
tumourigenesis. We hypothesize that the random integration and remobilization of
transposons carrying candidate genes can be used to identify tumourigenic combinations
when expressed combinatorially in a non-transformed human cell line. We propose to test
this hypothesis through the following aims:
1) To generate an inducible transposase-expressing non-transformed human cell line and
to determine the optimal conditions for induction of transposase activity.
2) To randomly integrate promoterless donor transposons containing candidate genes by
induced transposition in the cell line, and to identify which candidate genes are expressed
in tumour colonies.
Project 2:
Validation of Candidate Prostate and Breast Cancer Genes
Genome-wide surveys of human cancers have revealed complex and heterogenous
alterations, many of unknown significance, highlighting the critical need to distinguish
driver from non-contributory passenger alterations.
Forward genetic screens are a powerful unbiased approach for identifying genes/pathways
involved in a biological process. Our lab has conducted large-scale transposon-based
forward genetic screens in mice to uncover novel candidate
A/Prof Matthew Chang
Project 1:
matthew_chang@nuhs.edu.sg Engineering microbes for therapeutic applications
Synthetic biology aims to engineer genetically modified biological systems that perform
novel functions that do not exist in nature, with reusable, standard interchangeable
biological parts. The use of these standard biological parts enables the exploitation of
common engineering principles such as standardization, decoupling, and abstraction for
synthetic biology. With this engineering framework in place, synthetic biology has the
potential to make the construction of novel biological systems a predictable, reliable, and
systematic process. While the development of most biological systems remains largely ad
hoc, recent efforts to implement an engineering framework in synthetic biology have
provided long-awaited evidences that engineering principles can facilitate the
construction of novel biological systems. In our previous study, we engineered probiotic
microbes to effectively sense the presence of a human pathogen and kill the pathogen by
releasing antimicrobial peptide, as a proof-of-concept of a novel microbe-based
antimicrobial strategy. Our recent efforts are focused on developing therapeutic microbes
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with programmable and clinically relevant functionalities to fight against human
pathogen. To this end, we have implemented functionalities to include the ability of
probiotic microbes to swim toward a human pathogen upon sensing and disrupt biofilm,
and exert strong antimicrobial activity against the pathogen. This development suggests
the possibility that probiotics could potentially be engineered for prevention and
treatment of infectious diseases, which may provide an antimicrobial strategy that is
complementary to current antibiotic therapies. With this foundational therapeutic cell
engineering in place, our aim is focused on engineering microbes to be further endowed
with therapeutic functions toward other gut-related pathogens and conditions and other
human disease such as cancer and metabolic disorders.
Project 2:
Engineering microbes to produce valuable biochemicals
Metabolic engineering of microbes has been applied to synthesize therapeutic chemicals,
such as analgesics, antibiotics, anti-malarials, anti-arrhythmics, parasympatholytics,
antioxidants and anti-cancer agents. Notably, synthetic biology strategies have shown
great potential in biosynthesis of value-added nutraceutical chemicals at low cost. In our
previous study, we have developed synthetic microorganisms that produce valuable
biochemicals, and show superior tolerance against toxic chemicals (products and byproducts). Additionally, we have also developed AND-gate dynamic controllers of gene
transcription and synthetic promoters for metabolite sensing and regulation, i.e. promoters
that combine inducible functionality with regulation of protein expression levels upon
detection of key intermediates towards the engineering and optimization of bio-catalytic
yeast cells. Our recent project with international collaboration on the synthetic yeast
genome project (Synthetic Yeast 2.0, http://syntheticyeast.org) would provide extensive
insights to explore the synthetic yeast for efficient production of valuable nutraceuticals.
By reconstructing various metabolic pathways, we aim to engineer microorganisms (e.g.
bacteria, yeast) to synthesize value-added nutraceuticals. Multiple approaches, such as
synthetic biology and omics (functional genomics, transcriptomics, proteomics and
metabolomics), will be integrated for pathway reconstruction and flux-balance, analyses
of targets (genes, transcripts, and metabolites), and tolerance improvement. Successful
delivery of this project could extensively expand our knowledge on value-added chemical
biosynthesis pathways in microorganism, and potentially lead to industrialization and
commercialization.
Project 3:
Developing synthetic genome and chromosomes in microbes
The International Synthetic Yeast Genome Project (Sc2.0) has been recently formed to
complete the design, synthesis and assembly of the genome of Saccharomyces cerevisiae,
which will accomplish the construction of the first ersatz eukaryotic genome. The
organism was chosen because its genome is relatively compact and well understood. One
underpinning hypothesis of this project is that the “synthetic yeast” approach offers a
customised platform that enables us to understand fundamental questions pertinent to
characteristics of genomes such as genome organisation, gene structure, gene regulations,
functions of RNA splicing and evolution. Yeast chromosome will be re-designed and
constructed to fully assemble synthetic chromosome. We envision that using synthetic
chromosomes and genome, we will be able to answer profound questions about the
properties of yeast metabolism and cellular machinery.
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A/Prof Mridula Sharma
bchmridu@nus.edu.sg
Epigenetic regulation of Sarcopenia
Sarcopenia is age related loss of muscle mass. Age related changes in the levels of
hormones and gene expression have been observed which affect the protein synthesis,
energy metabolism, inflammatory response and protein degradation in the muscles. The
epigenetic changes involved in the altered expression of an aging genome and
contribution of non-coding regulatory RNAs, such as microRNAs (miRNA) are not fully
known. Therefore, epigenetic changes underlying the altered expression of genes in
muscle during aging will be analysed. Because miRNAs appear to play a significant role
in gene regulation in the muscle under physiological and non-physiological conditions,
the role of miRNA during sarcopenia will be studied.
Perturbations in muscle metabolism during Sarcopenia
The aging muscle also shows perturbations in metabolic activity which may be due to
intrinsic intracellular changes or extracellular environment. Recently we have
demonstrated that one of the critical regulators of muscle mass, Myostatin induces
Reactive oxygen species in skeletal muscle and that inhibition of Myostatin leads to not
only reduced ROS levels but also, sarcopenia. Unpublished results from our lab also show
that absence of Myostatin helps to reduce insulin resistance while higher levels of glucose
induce Myostatin expression. These observations suggest a central role for Myostatin in
ROS mediated sarcopenia and insulin resistance and diabetes. The underlying
mechanisms involved in these processes will be analysed.
Dr Sudhakar Jha
bchsjha@nus.edu.sg
Chromatin remodeling and cancer prevention
Down-regulation of tumor suppressors or expression of oncogenes is a hallmark feature
of cancers. These alterations in the transcriptome arise as a consequence of a malfunction
in the chromatin organization. Chromatin remodeling complexes play an important role in
maintaining this organization as they create a histone code that is read by specific readers
resulting in an active or repressed chromatin. My laboratory is interested in studying the
regulation of chromatin remodeling complexes and their role in cancer prevention. Our
research focuses on understanding the function of TIP60 (a histone acetyltransferase) as a
tumor suppressor. Interestingly, TIP60 is down-regulated by E6, a viral oncogene
expressed by human papillomavirus (HPV). Using TIP60 as a candidate molecule, we
plan to investigate how deregulation of this chromatin remodeling complex can lead to
cancer.
Dr Takao Inoue
bchti@nus.edu.sg
Our lab is working on important biological problems using C. elegans genetics and
molecular biology:
Wnt signaling through receptor tyrosine kinases Ryk and Ror
Wnt is an important class of signaling proteins regulating, cancer, stem cell, neurons and
many aspects of development. Frizzled seven-transmembrane receptors are the bestunderstood class of Wnt receptors. However, receptor tyrosine kinases Ryk and Ror can
also function as alternative Wnt receptors, and their mechanism of function is not as well
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understood. Previous research identified p21-activated kinase (Pak) as a component of
Wnt signaling pathways (Goh et al., PNAS, 2012). We are continuing to elucidate the
mechanism of Ryk and Ror signaling through identification of novel signaling pathway
components and analysis of Ryk and Ror signaling in C. elegans.
Gene regulation by chromatin factors and lncRNAs
We are investigating the regulation of transcription by chromatin factors and lncRNAs
(long non-coding RNAs). We are currently studying the regulation of the bed-3 gene,
which likely involves the homolog of human Blimp-1/PRDM1 transcription factor and
lncRNAs.
Mitochondria and aging
We are interested in the relationship between mitochondrial organization (e.g.
fragmentation vs. fusion, subcellular localization) and aging.
K. Y. Goh, N. W. Ng, T. Hagen and T. Inoue, 2012. p21-activated kinase functions in Ror
receptor tyrosine kinase-mediated Wnt signaling. Proceedings of the National Academy
of Sciences of the United States of America. 109(39):15853-15858
A/Prof Tang Bor Luen
bchtbl@nus.edu.sg
Roles of Rab small GTPases in cellular physiology and pathology
Protein trafficking and membrane dynamics underly fundamental processes of cellular
physiology, and when these go wrong, result in pathologies ranging from cancer to
neurodegeneration. Work in our laboratory has identified two brain-enriched Rab
proteins, Rab23 and Rab31, which have fundamental roles in cells, and have been
associated with disease conditions. Rab23 is involve in developmental Shh signaling,
ciliary traffic and kidney diseases, while Rab31 functions in endocytosis and is associated
with cancer progression. The lab seeks to further define the pathophysiological roles of
these small GTPases, and to understand their mechanisms of action from both a cellular
and organismal perspective.
Dr Wu Qiang
bchwq@nus.edu.sg
Modeling metabolic changes in reprogramming and human disease with pluripotent
stem cells
Pluripotent stem cells which are capable of self-renewing hold great potential in
regenerative medicine. Interestingly, compared to differentiated cell types, pluripotent
stem cells have some striking metabolic features such as significantly elevated
dependence on glycolysis, less reactive oxygen species and unique crosstalks between
metabolic factors with genetic, epigenetic, signaling factors. While genetic and epigenetic
mechanisms that control stem cell self-renewal, differentiation, and reprogramming have
been intensely studied, stem cells have emerged as an important model to study
metabolism since cell fates can be changed by induced differentiation or reprogramming.
However, it largely remains unknown how stem cell metabolism is regulated during stem
cell differentiation and how it is reset during reprogramming. One of the specific
questions is how metabolic genes crosstalk with epigenetic factors in such cell fate
changes.
Dr Wu Qiang’s laboratory specializes in stem cell biology and the epigenetic regulation
of stem cell proliferation and differentiation. The laboratory seeks to direct pluripotent
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stem cells from undifferentiated state into multipotent adipose stem cells (intermediate
state) and finally into adipocytes. We are keen to investigate gene expression patterns of
these three cell types; in particular, we will focus on changes of key metabolic genes and
epigenetic factors (DNA methyltransferases, histone methytransferses and histone
deacetylases). In addition, we will examine the changes of DNA methylation content and
chromatin state at genomic regions of key glycolysis-regulating genes (such as
hexokinase and lactate dehydrogenase) and mitochondria biogenesis genes. Meanwhile,
we seek to reprogram mutilpotent adipose stem cells into induced pluripotent stem cells.
During this reprogramming process, the somatic oxidative phosphorylation will be
switched into glycolysis-dependence in iPS cells. We will thereafter explore how such
metabolic switch controls cell fate manipulation. This research is expected to provide new
insights into how metabolism and intermediate metabolites determine stem cell-centric
cell fate decision.
Adjunct Dr Xu Feng (SICS)
Xu_Feng@sics.a-star.edu.sg
Epigenetic Control of adipogenesis
Obesity and its related diseases such as diabetes increasingly are responsible for
significant economic and social burdens in both established and emerging countries. For
instance, diabetes alone, is affecting more than 285 million people worldwide and cost the
U.S. economy approximately $218 billion in 2007. Obesity develops when energy intake
exceeds energy expenditure, and the current treatments for obesity have been primarily
focused on reducing energy intake. Unfortunately, these measures were largely inefficient
in maintaining long-term weight loss. Therefore, new approaches to tackle this problem
are needed urgently.
Fat cells can be broadly divided into fat storage white adipocytes and fat burning brown
adipocytes. Previously brown adipose tissue (BAT) was thought to be present only in
small mammals and infants within the interscapular area. Recently these fat burning
brown adipocytes were also discovered in adult humans, providing an exciting new
therapeutic approach for the treatment or prevention of obesity by increasing energy
expenditure.
Epigenetic mechanisms play essential roles in modulating adipogenesis. Our current
research centers on the function of histone modifications and microRNAs in both white
and brown adipocyte differentiation from multipotent stem cells. To fully decipher the
epigenetic mechanisms controlling adipogenesis and the white/brown lineage
commitment, we utilize the advanced genomic and proteomic methodologies as well as
classic biochemistry and molecular biology techniques in our study. Through our
research, we aim to identify epigenetic modulators that drive brown but not white
adipocyte differentiation, thus increasing energy expenditure. Besides our basic research
into the molecular mechanism of adipogenesis, we are also interested in identifying
potential drug targets to treat obesity and metabolic diseases such as diabetes.
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Dr Yeong Foong May
bchyfm@nus.edu.sg
Understanding the Interplay between Cell Cycle control and Protein trafficking
We are interested in how cells regulate cytokinesis such that it occurs only after
chromosome segregation so as to produce two viable daughter cells. We plan to study the
links between cell cycle regulation and cytokinesis, with particular focus on how the cell
cycle machinery affects endocytosis at the cytokinesis site. Endocytosis is a key cellular
process that leads to the internalization of proteins and membranes from the plasma
membrane. During cytokinesis, endocytosis occurs at the cytokinesis site and
perturbations to endocytosis can cause a failure in cytokinesis.
Our preliminary data suggest that the key cell cycle, the cyclin-dependent kinase (Cdk1)
activity in mitosis has an apparent role in preventing endocytosis and cytokinesis. Indeed,
upon the destruction of the mitotic Cdk1 activity, the both cytokinesis and endocytosis at
the neck are triggered. This was evident from our initial data showing that key endocytic
components accumulate at the neck only during cytokinesis.
We plan to make use of budding yeast as our model for the proposed study, given that
both processes are conserved and that the budding yeast serves as a good system for
complex genetic manipulations, we plan to make use of budding yeast to understand the
regulatory relationship among Cdk1 activity and endocytosis during cytokinesis. Our lab
has the necessary tools and techniques to execute experiments including genetic and
physical screens. Moreover, we also conduct time-lapsed imaging experiments in addition
to physical interaction assays. We aim to provide insights into how cells normally prevent
cytokinesis until after nuclear division by studying how cells regulate endocytosis during
cytokinesis. The proper execution of cytokinesis is important, given that premature
cytokinesis can cause a loss of cell viability while failure to execute cytokinesis can lead
to tetraploidy in human cells, which is a precursor to tumourous growths.
Dr Yew Wen Shan
bchyws@nus.edu.sg
Our research interest focuses on protein engineering and biocatalysis, with emphasis on
using structural and mechanistic enzymological knowledge to develop therapeutics.
Current research projects in the lab center on using Synthetic Enzymology for biomedical
and bioremediation applications:
1.
Defining Synthetic Isoprenoid Enzymology as an enabling platform for the
Construction of Next-Generation Synthetic Biology solutions for nutrichemical
production.
2.
Using Synthetic Enzymology to discover and develop novel Polyketide-based
therapeutic biomolecules, in the areas of infectious diseases, cancer and aging.
3.
Developing anti-virulence Quorum-Based Technology for use in the
biomedical industry.
4.
Using Synthetic Lixiviant Enzymology for Biomining of Electronic Wastes
for Environmental Sustainability.
5.
Development of Lead Compounds Targeting Enzymes involved in Metabolic
Cancer.
6.
Using Synthetic Enzymology for the Construction of Therapeutic (Probiotic)
Cells for the Treatment of Metabolic Diseases and Infectious Diseases.
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Department of Medicine
Dr Chester Drum
mdccld@nus.edu.sg
Advanced pharmacokinetic models for drug effectiveness discovery
The way in which different ethnicities and individuals metabolize drugs can be
represented as an intricate web of biochemical changes to prescribed medicines. When
this variance in pharmacokinetic metabolism is cast against an electronic medical record
database that documents both adverse drug reactions and beneficial effects, the total data
set can be treated as a discovery mechanism for unexpected benefits and problems
relevant to commonly prescribed medicines. In this project you will interface with a
dedicated triple quadrapole tandem mass spectrometer to develop novel detection
methods for interesting drug metabolites and use this information to dissect exposure
dependent effects of the measured drugs. Using established, large market medicines in
new ways and understanding hidden risks specific to the Asian market will be critical to
healthcare in the upcoming decades. This project seeks to position the graduate candidate
to take advantage of this rapidly expanding scientific and regulatory field and develop
skills relevant to the implementation of large- market pharmaceutical safety and
effectiveness strategies. Because this project can be relevant to both cardiovascular and
cancer therapies, the ultimate experimental design will tightly incorporate the intellectual
goals of the student in addition to leveraging opportunities already established within the
lab.
Photon mediated therapeutic modulators
Electromagnetic radiation is an appealing choice for modulation of biological processes
within a living organism. From the historical treatment of cancers through X-ray therapy
to the recent advent of optogenetics, the ability of light to permeate living tissues without
creating off target effects is well proven. Converting incident radiation to physical work
or chemical activity, however, presents a major continuing challenge. Tissue and, in
particular blood, have wide absorption ranges and permit relatively narrow bandwidths of
electromagnetic radiation to penetrate beneath the skin surface. This project will use a
chemical biology approach to implement novel, highly photoisomerizable small molecule
sensitizers to convert relevant electromagnetic radiation into cell and protein-specific
biological actions. Our major work in this area is currently being prepared for publication
and will have many follow-on studies of equally high impact. Disease areas amenable to
this approach include cardiovascular, cancer and ophthalmic applications. Our lab has a
full time Ph.D. – level chemist, biochemist and spectroscopist to aid a motivated graduate
student in project design and completion.
If interested, please contact the PI for discussion as the ultimate experimental design will
tightly incorporate the intellectual goals of the student in addition to leveraging the
current results within the lab.
A Protein Dynamic Approach to Engineered Translational Therapeutics
Capitalizing on the combined M.D. and Ph.D. training of the professor, this project
leverages a structural biology approach (Drum et al. Nature. 2003) to create clinically
relevant engineered proteins for both diagnostic and therapeutic purposes. “The protein
folding problem” is one of the most interesting in biology --how does a linear peptide
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with more conformations than there are atoms in universe, know to fold into a single
functional conformation? Can biochemical engineering fundamentally effect this process
in a way to make it amenable to oncological or cardiovascular treatments? In this project
you will use a novel form of protein expression invented by our lab to study protein
folding dynamics and novel cellular uptake mechanisms for drug delivery. The project
will use novel nanoparticle materials for the delivery of active protein substrates into
cells. Our lab has a full time synthetic chemist, biochemist and materials engineer in
addition to full molecular biology and protein biosynthesis support which is an excellent
setting for a graduate student who wants an efficient approach to a high impact problem,
not bounded by a single technique. Although Prof. Drum was clinically trained as a
cardiologist (BWH, Harvard Med School) he is also an award winning structural biologist
and is currently focused on advanced materials as they apply to cutting edge biological
phenomena. Please enquire if interested. Ultimately the chosen project will reflect both
the intellectual input of the PI and the student, thus interested candidates should apply and
they will be welcome to have a discussion regarding their specific interests.
Prof Edward H KOO
mdckhme@nus.edu.sg
Molecular mechanisms of neuronal injury or neurodegeneration
The Laboratory for Molecular Neurodegeneration at YLL School of Medicine was
established by Dr. Ling Shuo-Chien and Prof. Edward Koo in late 2013 when we first
joined the NUS faculty. Our research focus is on the mechanisms of neurodegenerative
diseases, a group of brain disorders that is often age-associated and includes Alzheimer
and Parkinson diseases, amyotrophic lateral sclerosis, frontotemporal dementia, among
others. We take a cell and molecular biology approach to our studies with an emphasis
on animal models and translational research. My laboratory’s philosophy is to study both
the “normal and abnormal biology” of genes and proteins that are implicated in disease
pathogenesis. In many instances, mutations are found in genes with unknown function
and which require thorough investigations of the basic functions of these genes and
proteins before we can appreciate how their dysfunction contributes Alzheimer disease
where we have concentrated on both basic and translational studies as well as
experimental therapeutics. Ongoing and future projects include:
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Mechanisms of synaptic injury in Alzheimer disease
Role of caspase cleavage of the amyloid precursor protein (APP) in
neurodegeneration
Mechanisms of tau mediated synaptic dysfunction
Investigations into mechanisms of neuronal vulnerability in Alzheimer disease
Contribution of blood-brain barrier to neurodegeneration
Neurobiological basis of cognitive loss due to chemotherapy treatment in cancer
patients (“chemobrain”)
A/Prof Lee Chi-Hang, Ronald We invite students who are interested in clinical research to join our research team. We
mdclchr@nus.edu.sg
conduct a series of research projects on obstructive sleep apnea and cardiovascular
diseases that are highly relevant to patient case. Our projects are funded NMRC, MOE,
and medical industry. Students will be exposed to clinical research methodology and
medical health industry during the study. Publications in international, peer-reviewed
scientific journals at the end of the study are expected.
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Sleep Study Guided Multidisciplinary Therapy for Patients Presenting with Acute
Coronary Syndrome.
The aim of this randomized, open-label clinical trial is to determine the impact of Sleep
Study-Guided Multidisciplinary Therapy (SGMT, i.e. continuous positive airway pressure
and behavioral therapy) for obstructive sleep apnea (OSA) in the sub-acute phase of acute
coronary syndrome on cardiovascular outcomes. We hypothesize that SGMT will result
in a lower (1) plasma NT-pro BNP level, (2) 10-year risk of cardiovascular mortality
based on the European SCORE algorithm, and (3) cardiovascular event rate, when
compared with Standard Therapy.
OSA is an emerging cardiac risk factor and prognostic marker. We have reported that
OSA is a prevalent and independent predictor of adverse outcomes in patients with acute
coronary syndrome. In this clinical trial, a continuation of my research and publication
trajectory, 180 patients presenting with acute coronary syndrome will be randomly
assigned to SGMT (n=90) or Standard Therapy (n=90) groups. Both groups will receive
guideline-mandated treatment for acute coronary syndrome. Those assigned to SGMT
will undergo a sleep study. Those found to have OSA will attend the SGMT clinic run by
a multidisciplinary team. Advice on continuous positive airway pressure and behavioral
therapy (weight loss, exercise, positional therapy, abstinence of alcohol and sleeping
pills) will be given. The primary endpoint is plasma NT-pro BNP concentration at 6month follow-up. The secondary endpoint is 10-year risk of cardiovascular mortality
based on the European SCORE algorithm which includes age, sex, smoking status,
systolic blood pressure, and serum total cholesterol or total/HDL-cholesterol ratio.
Adverse cardiovascular events at 3-year follow-up will be determined.
In our aging population with an increasing prevalence of obesity, OSA will potentially
become an increasingly important contributor to cardiovascular disease. Leveraging the
collective expertise of a team of cardiologists and sleep physicians, our work will benefit
society by advancing our understanding of the cardiovascular benefits of screening for
and treating OSA.
A/Prof Ling Lieng Hsi
mdcllh@nus.edu.sg
Maternal vascular status and influence on fetal and child development
(For MSc project)
This research is part of the birth cohort study “Growing Up in Singapore Towards healthy
Outcome” (GUSTO) of the TCR flagship programme “Developmental Origins:
Singapore” (DevOS).
Prenatal and perinatal factors determine the subsequent growth of the child. This project
aims to determine if maternal vascular structure and function, which is influenced by
premature atherosclerosis and collagen/elastin degradation, has any influence on the
physical and metabolic characteristics of newborns and their subsequent development. A
preconception cohort will be studied with several established techniques to assess
structural atherosclerosis (carotid intima-medial thickness), arterial stiffness (carotid
artery pulsatility and carotid-femoral pulse wave velocity) and vascular reactivity
(brachial artery flow-mediated dilation). One or more of these measures will be repeated
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during pregnancy, at the height of cardiovascular stress.
The student will have opportunity to:
a) learn technical skills needed in conducting vascular testing
b) correlate these findings in preconception women with prevailing cardiometabolic
milieu
c) determine how the stress of pregnancy modifies these indexes
d) ascertain if abnormal vascular structure affects fetal and newborn outcomes and later
development.
Dr Mahmoud A. Pouladi
mdcmap@nus.edu.sg
Modeling neurodegenerative diseases in human neurons using genome engineering
and stem cell technologies: Towards the discovery of therapeutic targets and the
development of novel approaches for therapy
Neurodegenerative diseases pose a profound health, social, and economic burden and
remain an area of enormous unmet medical need. Therapies to prevent neurodegenerative
diseases remain lacking and existing treatments are largely palliative. Recent advances in
stem cell technology have enabled the generation of induced pluripotent stem cells from
readily accessible patient sources, such as fibroblasts, which can then be differentiated
into specific cell types that would otherwise be hard to access, such as those of the
neuronal lineage. Coupled with genome engineering technologies such as TALEN and
CRISPR-based approaches, we are now able to establish isogenic stem cell-based models
of neurodegenerative diseases in physiologically relevant cellular systems.
1. Delineation of disease-associated molecular pathways using isogenic stem cellbased models of Huntington disease. Huntington disease is an autosomal dominant form
of dementia. Using unbiased and large-scale genomic and biochemical approaches, we
aim to (a) identify disease-specific alterations in molecular pathways in stem cell-derived
Huntington disease neurons, and (b) validate their role in causing disease phenotypes.
Once validated, the identified molecular aberrations may serve as potential targets for
therapeutic intervention.
2. Identification of genetic modifiers of disease phenotype using stem cell-derived
models of neurodegenerative disease: Using forward genetic screening approaches and
Huntington disease as a model system, our research aim is to identify and characterize
genetic modulators of disease phenotypes in stem cell-derived neurons, with the goal of
uncovering candidate targets for therapeutic intervention.
Prof Nicholas Paton
nick_paton@nuhs.edu.sg
Development of Whole Blood Bactericidal Activity Assay for Tuberculosis
Therapeutics
Tuberculosis (TB) is one of the world's deadliest infectious diseases with an estimated 9
million new cases and 1.4 million deaths per year. It is caused by Mycobacterium
tuberculosis, which is becoming increasingly resistant to current drugs, thereby posing a
major public health problem in Asia and worldwide. Our group’s research is focused on
investigating new approaches for delivering improved, more efficient treatments for TB.
The whole blood bactericidal activity assay (WBA) is an ex vivo model for measuring
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effects of administered drugs, host factors and strain factors on mycobacterial
sterilization. Mycobacteria added to human whole blood cultures undergo phagocytosis
and remain intracellular in 72-h cultures. During TB treatment regimens, drug levels in
the whole blood cultures mirror those in the circulation at the time of phlebotomy. If
performed in parallel with pharmacokinetics measurements, the method can be used to
evaluate the effect of drugs throughout the dosing cycle.
To date, the method has not been utilized to investigate the synergy between the immune
system and TB drugs on mycobacterial activity, nor has it been used to look at the effects
of drugs on different TB strains. Both of these are potentially very useful aspects of this
experimental paradigm which deserve to be explored. The aim of this research project
therefore is to establish WBA as a method in clinical trials investigating new drug
regimens against tuberculosis.
The PhD candidate in charge of this project will develop the method and extend it to
measuring host immune responses and effects on different mycobacterial strains. He/she
will have an opportunity to learn the principles of multinational clinical trials, will be
involved in clinical pharmacokinetics studies, will acquire specialized in-demand skills to
work in BSL-3 laboratory environment, and will learn to transform data into knowledge
for improving human lives.
Prof Nobuhiro Yuki
mdcyuki@nus.edu.sg
Autoimmune disease induced by novel mechanism associated with antecedent virus
infection
Background: We previously reported a patient with overlapping Guillain–Barré
syndrome and Bickerstaff’s brainstem encephalitis. The patient was characterized by
antecedent herpes simplex virus type 1 (HSV-1) infection and high IgG anti-GQ1b
antibody titer in the serum. The possibility has been reported to enhance the expression of
GM2 on the cell membrane after cytomegalovirus infection. A few studies have linked
viruses infection to modulating gangliosides expression: human T-lymphotropic virus to
GD2 and simian virus 40 to GM3.
Objective: To unveil the relationship between antecedent HSV-1 infection and IgG antiGQ1b antibody.
Materials and methods: Human cell lines, such as neuroblastoma and astrocytoma cell
lines, will be infected by HSV-1 and -2. To measure the change of gangliosides on cell
surface and related gene expression, comprehensive glycosphingolipids analysis,
immunocytochemistry and quantitative PCR will be performed.
Present status and vision: We have already obtained some data related to the change of
gene expression. Further studies to confirm the alteration of gangliosides expression on
the cell surface is currently in progress. We hope to demonstrate that antecedent virus
infection leads to an autoimmune disease and that our results will aid in the formulation
of new therapeutic strategies.
Identification of novel autoantibodies in patients with central and peripheral
demyelinating diseases
Background: Multiple sclerosis (MS) and neuromyelitis optica (NMO) are
demyelinating diseases of the central nervous system. Meanwhile, Guillain–Barré
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syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are
demyelinating diseases of the peripheral nervous system. GBS is classified into acute
inflammatory demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy
(AMAN).
Specific autoantibodies are identified in the patients with NMO and AMAN: astrocytic
water channel aquaporin-4 is a major target molecule for NMO patients and gangliosides,
such as GM1 and GD1a, for AMAN patients. In contrast, pathogenic autoantibodies in
patients with MS, AIDP or CIDP have yet to be identified, although some antigens have
been reported in the patients.
Objective: To identify the major pathogenic autoantibodies in the patients with MS,
AIDP and CIDP.
Materials and methods: We have obtained more than 100 patients’ sera and are
continuing to collect sera from the patients. Proteomic analysis will be used for the
identification of autoantigens. Immunological approaches such as cell-based ELISA and
immunocytochemistry will be performed in order to confirm the presence of
autoantibodies.
Present status and vision: Our preliminary results suggest the presence of novel
autoantibodies in the patients’ sera. As a next step, we will perform proteomic analysis to
identify the novel targets. This study will contribute to the understanding of the
mechanisms for the pathogenesis of demyelinating diseases and provide the formulation
of new therapeutic strategies.
Identification of novel autoantibodies specific for IgM paraproteinemic neuropathy
IgM paraproteinemic neuropathy is a kind of peripheral neuropathy associated with
monoclonal IgM gammopathy. It presents as a chronic, distal-predominant,
demyelinating, mainly sensory, ataxic neuropathy. Half of those with IgM
paraproteinemic neuropathies have IgM antibodies against myelin-associated
glycoprotein (MAG). However, target molecules for the other patients have yet to be
identified. Deposition of complement on myelin and improvement of the neuropathy with
reduction of IgM anti-MAG titers suggest the pathogenic role for these proteins.
Reactivity of the IgM paraproteins has been found with several neural antigens, including
gangliosides and sulfatides.
The aim of this study is to identify novel molecular targets related to the pathogenesis of
IgM paraproteinemic neuropathies. We currently have a number of IgM paraproteinemic
neuropathies patients’ sera. Additionally, we have already obtained brain protein samples.
Using these samples, we plan to identify the target molecule by use of
immunoprepicitation and mass spectrometry. These results will provide useful
information to find effective treatment for this disabling neuropathy.
IdeS: a streptococcal enzyme with therapeutic potential for Guillain-Barré
syndrome
Guillain–Barré syndrome (GBS) is an acute neuropathy with a monophasic course of the
illness. Pathogenic IgG autoantibodies bind to peripheral nerve gangliosides, and activate
complement in situ, resulting in the development of limb weakness. Plasma exchange
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which removes the pathogenic antibodies is used as treatment for GBS. IgG degradating
enzyme of Streptococcus pyogenes (IdeS) is an endopeptidase that specifically cleaves
IgG antibodies to generate Fc and F(ab’)2 fragments. A successful Phase I study has been
finished with healthy volunteers.
To determine whether IdeS blocks complement deposition mediated by IgG autoantibody
binding to gangliosides, we have obtained sera from patients with anti-ganglioside
autoantibody-mediated neuropathies and have measured the cleavage of Fc fragments
from autoimmune complexes and the inhibitory effect of the sequential complement
depositions by IdeS. We also plan to determine whether IdeS protects neuronal cells
expressing gangliosides against complement-dependent cytotoxicity. These data will
provide evidence that IdeS could be effective for the patients with GBS.
Non-radioactive serological diagnosis of myasthenia gravis
Myasthenia gravis (MG) is the most common autoimmune disease of the neuromuscular
junction with a prevalence of 200-300 per million populations. Most MG patients (~85%)
have autoantibodies against the muscle acetylcholine receptor (AChR), and about 6% of
MG patients have autoantibodies against the muscle specific kinase (MuSK) and lowdensity lipoprotein receptor-related protein 4 (LRP4). Currently, the most sensitive assays
for the detection of the autoantibodies in MG sera have been the
radioimmunoprecipitation assays (RIPA). However, the use of radioactivity limits
availability of these tests in many diagnostic laboratories. To improve non-radioactive
assays for detection of antibodies to the AChR, MuSK and Lrp4, we plan to perform cellbased ELISA assay using a cell line transfected with the target auto-antigens. This assay
has very high sensitivity and has been reported to detect auto-antibodies that cannot be
detected by RIPA. We hope that this study may possible to develop the assays as a
routine practice in future.
Identification of novel autoantibodies in ocular myasthenia gravis
Myasthenia gravis (MG) is an autoimmune disease majority mediated by antibodies to the
acetylcholine receptor (AChR) and other muscle antigens of skeletal muscle at the
neuromuscular junction (NMJ). Initial symptoms of MG usually manifest as ocular MG
(OMG) with characteristic extra ocular muscle weakness and ocular misalignment. Up to
50-80% of OMG patients develop generalized MG (GMG) within two years. GMG
causes a more severe phenotype compared with OMG. There are two AChR subtypes,
fetal (alpha, beta, gamma, delta) and adult subtypes (alpha, beta, epsilon, delta). The
gamma subunit is replaced by the epsilon subunit in adult subtypes. It has been reported
that anti-AChR antibody is related to an increased risk of GMG development. In addition,
the expression profile of extraocular muscle is unique in fetal AChR at the NMJ. We
hypothesize that OMG patients who have antibodies against the γ subunit of AChR will
not develop generalized MG. In ours study, we will use a cell-based assay to test the
AChR antibodies in OMG and GMG patients, with fetal and adult type AChRs as the
antigens. However, approximately 50% of OMG patients show detectable anti-AChR.
Therefore, It is possible that other auto-antibodies are also involved in OMG. Identifying
novel antibodies in OMG will be used as the biological marker of OMG patients who do
not develop GMG.
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Identification of true target of antibodies from Lambert-Eaton Myasthenic
Syndrome (LEMS)
LEMS is a rare autoimmune neuromuscular disorder in which patients manifest both
muscle weakness and autonomic nervous system disruption. It is believed to be due to
autoantibodies targeting presynaptic voltage-gated calcium channels (VGCCs)
extracellularly, specifically P/Q- and N- types VGCCs. However, the distribution of
VGCCs and the manifestation of symptoms suggested that VGCCs may not be the true
target.
We hypothesize that there could be an unidentified protein X that is closely associated
with VGCCs, resulting in the false identification of the true target. The aim of the study is
to prove that VGCCs is not the true target and identify this protein X as the true target in
which antibodies from LEMS patients bind to. We will use techniques of
immunocytochemistry, immunohistochemistry, immunoprecipitation for the study.
Identification of the pathogenic autoantibodies in Graves’ ophthalmopathy
Graves’ ophthalmopathy (GO) is a potentially debilitating and disfiguring orbital
inflammatory disease. Its association with autoimmune thyroid disorders is believed to be
caused by a putative cross-reactive antigen connecting the thyroid gland and the
extrathyroidal tissues, the nature of which remains contentious. A number of orbital autoantigens have been suggested: thyroglobulin, extraocular muscle antigens, thyroid
stimulating hormone receptor (TSHR), insulin-like growth factor 1 receptor (IGF-1R).
Although TSHR is considered as one of the major antigens in the orbit, TSHR antibodies
cannot explain the development of ophthalmopathy in patients in some patients with
Graves’ hyperthyroidism, Hashimoto’s thyroiditis or in newborns with neonatal
thyrotoxicosis. Ninety percent of patients with GO have Graves’ hyperthyroidism; the
remaining 10% have hypothyroidism or are euthyroid.
Thyroid stimulating antibodies recognize the extracellular domain of TSHR. We
hypothesize that patients with GO have autoantibodies against a protein with similar
structure to the TSHR receptor and we aim to identify these autoantibodies responsible
for the development of ophthalmopathy. These autoantibodies when bound to the TSHRlike receptor expressed by fibroblasts or adipocytes in eye muscle are able to induce
differentiation of adipocytes. Hence, fibroblasts when exposed to sera from patients with
GO would proliferate and develop GO.
We aim to identify the autoantibodies responsible for the immunopathologic mechanism
in GO. Using bioinformatics analysis of the epitope region of TSHR, we identified a
possible antigen with similar structure to the extracellular domain of TSHR. Now we aim
to target this TSHR mimic molecule and produce an overexpression plasmid of this
molecule. We plan to transfect with the candidate TSHR mimic molecule overexpression
plasmid and treat with normal serum or GO patient serum. This will be followed by
autoantibody recognition of TSHR and the TSHR mimic molecule evaluated with
immunocytochemistry.
Identification of autoantibodies in immune-mediated demyelinating neuropathies
Guillain–Barré syndrome (GBS) is an acute polyneuropathy often preceded by viral or
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bacterial infection and is a self-limiting autoimmune disease. It is the most common cause
of acute flaccid paralysis since the near elimination of poliomyelitis. Acute inflammatory
demyelinating polyneuropathy (AIDP) is a demyelinating variant form of GBS. In AIDP,
the target antigen remains to be elucidated as autoantibodies against myelin proteins and
paranodal proteins have only been detected in a relatively small number of patients.
Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic or relapsing
autoimmune disease with similar clinical manifestations as AIDP. Similarly to AIDP, the
etiology of CIDP remains unclear.
Charcot–Marie–Tooth disease (CMT) is the most common hereditary neuromuscular
disorder which is caused by mutations to a wide heterogenous group of genes. CMT
patients especially for the demyelinating subtype have hallmarks and pathological
changes that are similar to AIDP and CIDP such as destruction of myelin sheath, slowing
of nerve conduction and absence of tendon reflexes. Hence, in elucidating the target
antigens in AIDP and CIDP, a hypothesis has been made that CMT disease related
proteins could be the targets for autoantibodies in AIDP and CIDP.
The aim of this study is to determine the target antigens in AIDP and CIDP patients by
investigating the autoantibody reactivity of these patients towards CMT disease related
proteins. Cell-based ELISA which utilizes the over-expression of CMT disease related
proteins especially CMT proteins that are expressed on the cell surface, including GJB1,
MPZ and PMP22 in CHO-K1 cells will be employed. Reactivity of sera from AIDP and
CIDP patients’ towards these cell surface CMT disease proteins will be examined.
Reproduction of the disease by injection of target antigen in an animal model will further
confirm the result. Identification of potential targets for AIDP and CIDP will help extend
knowledge about these diseases in search of better treatments for the patients.
Identification of novel antigen targeted by
demyelinating disease using co-coculture system
antibody
from
autoimmune
In demyelinating diseases such as Guillain–Barré syndrome (GBS) and chronic
inflammatory demyelinating polyneuropathy (CIDP), autoantibodies are present in the
manifestation of demyelination of neurons. It was suggested that this immunoreactivity
could be directed to epitopes present in Schwann cell-axon interaction. Syndecans, being
proteoglycans enriched in nodes of Schwann cell-axon interaction, are believed to
contribute to node organization. We hypothesise that syndecans could be a potential
target of autoantibodies of these demyelinating diseases.
Coculture with the neural crest-derived pheochromocytoma cell line PC12 and the
immortalized adult rat Schwann cell line IFRS1 can develop myelination in vitro. The
stable PC12-IFRS1 coculture system is free of technical and ethical problems arising
from the primary culture and can be a valuable tool to study peripheral nerve
degeneration and regeneration. Using the system, we seek to identify potential targets of
the autoantibodies. We will employ techniques of cell culture, immunocytochemistry and
immunoadsorption to aid in the investigation.
Validation of the importance of contactin-1 in the pathogenesis of CIDP
Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy
(CIDP) are immune-mediated neuropathies of peripheral nerves. Several findings strongly
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suggest that the IgG autoantibodies play crucial roles in the pathogenesis.
Paranodal protein contactin-1 was reported as a target antigen in Guillain–Barré
syndrome and CIDP. Later, another group reported the importance of contactin1/contactin-associated protein 1 complex and mannose-rich contactin-1 for CIDP patients.
The aim of this study is to determine whether antibodies titers against contactin-1 are
specifically high in sera from patients with CIDP.
We have already established an ELISA system to detect the antibodies against contactin-1
in the patients’ sera. Our preliminary results with the ELISA have shown the presence of
autoantibodies to contactin-1. We will perform ELISA using contactin-1/contactinassociated protein 1 complex and mannose-rich contactin-1. These results will provide
useful information to explore the importance of contactin-1 or its related molecules such
as mannose-rich contactin-1.
Evaluation of anti-amyloid β antibodies titre in Singaporean patients with
Alzheimer’s disease
Several research groups have found that the serum level of anti-amyloid β (Aβ) antibodies
was lower in Alzheimer’s disease (AD) patients than in non-demented individuals.
However, there are other conflicting reports in which one reported that the titre of anti-Aβ
antibody was elevated in AD patients’ sera as compared to non-demented individuals and
another that found no difference in the levels of the antibody between both patients
groups.
One of the reasons there are conflicting reports is the existence of anti- Aβ antibodies to
free Aβ or Aβ complex in the patients’ sera. Some group detected antibodies to either free
Aβ or Aβ complex while the others detected both of them. The aim of this study is to
determine whether patients with AD have high titres of autoantibodies to Aβ especially in
Singaporean. We are trying to establish the method to detect anti- Aβ antibodies in the
patients’ sera to either free Aβ or to the Aβ complex by enzyme-linked immunosorbent
assay. The results will provide useful information about the pathogenesis of AD.
Identification of novel glycolipid X project
Fisher syndrome is characterized by ophthalmoplegia, ataxia, and areflexia. The IgG antiGQ1b antibodies often detected in patients with Fisher syndrome may have a role in the
pathophysiology of this ophthalmoplegia. The ganglioside GQ1b is expressed in the
paranodal portion of human ocular motor nerves and is the possible target molecule in
this disease.
Previously, Mori et al. reported a patient with acute ataxia and areflexia, but no
ophthalmoplegia, associated with IgG anti-GQ1b antibodies. Clinical findings,
electrophysiological studies, and postural body sway analysis suggested that this patient’s
ataxia was very similar to that seen in patients with Fisher syndrome. This patient did not
present with ophthalmoplegia. The clinical and laboratory findings suggest that this case
is a variant form of Fisher syndrome.
ELISA showed the presence of IgG antibodies to GQ1b and GT1a, a situation commonly
described in Fisher syndrome, Guillain-Barré syndrome (GBS) with ophthalmoplegia,
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GBS without ophthalmoplegia and acuteoropharyngeal palsy. TLC immunostaining using
the patient’s serum reacted with GT1a and more weakly with GQ1b and unidentified
glycolipid. We hypothesise that this unidentified glycolipid X is novel disease-related
antigen. Identification of glycolipid X using anti-ganglioside antibody assays and mass
spectroscopy is useful for elucidation of the pathophysiology.
A/Prof Roger Foo
mdcrfsy@nus.edu.sg
Dr Roshni R. Singaraja
mdcrrs@nus.edu.sg/
rsingaraja@tlgm.astar.edu.sg
Heart failure (HF), a syndrome resulting from different possible initiating causes, remains
a major problem of mortality and morbidity worldwide, and is set to worsen as the world
population rapidly ages. Novel therapeutic targets for the drug discovery pipeline are
urgently needed. Oxidative stress is a well-known component of heart failure. During
oxidative stress, reactive oxygen species (ROS) attack different components of the cell.
This grant proposal seeks to fill a gap in knowledge where it remains unclear whether and
how ROS-induced DNA damage lesions, predominantly 8-oxoG, predispose to HF onset
or progression. We have previously found evidence of extensive 8-oxoG damage in the
nuclei of failing human hearts and failing hearts of mouse models of disease. 8-oxoG
accumulation was in part due to the loss of base repair enzyme OGG1. Recombinant
OGG1 completely restored base excision repair activity in an ex vivo assay, implicating
its potential role as an important limiting factor. Here, we propose to generate 2 genetic
mouse models. One, a cardiac-specific Ogg1 knockout mouse, to investigate the
sufficiency of OGG1 loss in producing 8-oxoG damage, and consequent accelerated
cardiomyopathy, with or without treatment with surgical Transverse Aortic Constriction.
Next, we will generate an inducible cardiac-specific overexpression of Ogg1, so that we
may investigate the beneficial effect of restoring OGG1 following disease onset with
surgical TAC and Myocardial Infarction. If current experimental therapies treat only by
reducing oxidative stress or ROS, the lack of DNA repair in HF still means that critical
accumulation of 8-oxoG lesions will be left unattended. This set of experiments will
conclusively elucidate the role of 8-oxoG DNA lesions in the heart and potentially
contribute to an important novel area of HF therapeutics.
Novel genes in cardiometabolic diseases: Identification and validation
Cardiovascular and metabolic diseases are among the top ten causes of death world-wide,
and represent a major health burden predicted to continue well into the 21st century. Our
laboratory utilizes human genetics and experimental human clinical studies, as well as in
vitro cell culture and in vivo animal models to identify and validate novel targets
contributing to cardiometabolic disorders. Once validated, these targets represent novel
therapeutic avenues for the treatment of cardiometabolic diseases.
Characterization of the role of the novel cardiometabolic gene CYP8B1 in vitro and
in human experimental clinical studies
Using human genetic studies, we have identified a novel gene, CYP8B1, with a potential
role in lipid and glucose metabolism. We aim to further characterize the role of this gene
utilizing human experimental clinical studies, iPS-cell derived hepatocyte models, as well
as animal models, in order to validate this target, as well as derive mechanistic insight
into the impact of this gene in cardiometabolic diseases.
Characterization of the role of novel genes in lipid and glucose metabolism
Using a deep sequencing approach in a human cohort, we have identified genes
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associated with plasma lipid and lipoprotein levels. Genetic studies in humans suggest
roles for these genes in lipid/glucose metabolism. We aim to use in vivo CRISPR based
approaches to validate the role of these genes in mouse models, as well in ex vivo cell
culture models.
Select Publications:
 Kaur A, Patankar JV, de Haan W, Ruddle P, Wijesekara N, Groen AK, Verchere
CB, Singaraja RR, Hayden MR. Loss of Cyp8b1 improves glucose homeostasis
by increasing GLP-1. Diabetes. 2014 Oct 22.
 Singaraja RR et al. Identification of four novel genes contributing to familial
elevated plasma HDL cholesterol in humans. J Lipid Res. 2014;55(8):1693-1701.
 Singaraja RR et al. The impact of partial and complete loss-of-function
mutations in endothelial lipase on high-density lipoprotein levels and
functionality in humans. Circ Cardiovasc Genet. 2013;6(1):54-62.
Dr See Kay Choong
MICU data-mining
kay_choong_see@nuhs.edu.sg
Students will perform retrospective data collection using our Medical ICU database, and
will have the opportunity to perform data analysis under guidance from the PI. Previous
projects have dealt with fluid optimization, ventilator management, non-invasive
ventilation, and ICU discharge care. Students will also be highly encouraged and
supervised to produce an abstract for a local or regional conference. Project work can be
mixed and matched with clinical postings within the Division of Respiratory & Critical
Care Medicine. About 4 to 12 weeks will be required for the project work, depending on
the scope of research.
Pleural disease research
Students will perform retrospective data collection using our pleural disease database, and
will have the opportunity to perform data analysis under guidance from the PI. Students
will also be highly encouraged and supervised to produce an abstract for a local or
regional conference. Project work can be mixed and matched with clinical postings within
the Division of Respiratory & Critical Care Medicine. About 4 to 12 weeks will be
required for the project work, depending on the scope of research.
Smoking cessation research
Students will perform retrospective data collection using our smoking cessation database,
and will have the opportunity to perform data analysis under guidance from the PI.
Students will also be highly encouraged and supervised to produce an abstract for a local
or regional conference. Project work can be mixed and matched with clinical postings
within the Division of Respiratory & Critical Care Medicine. About 4 to 12 weeks will be
required for the project work, depending on the scope of research.
Dr Takaomi Sanda
csitakao@nus.edu.sg
mdcts@nus.edu.sg
Development of Novel Therapeutics Targeting Transcription Factor Abnormalities
in T-cell Acute Lymphoblastic Leukemia.
The primary focus of our laboratory is to understand transcription factor abnormalities as
therapeutic targets in cancer/leukemia, particularly T-cell acute lymphoblastic leukemia
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(T-ALL). Our laboratory will develop a novel means to target these oncogenic
mechanisms in T-ALL cells through combining cancer genomics with functional genetics
and bioinformatics.
The most frequent genetic abnormality in T-ALL is the dysregulation of transcription
factor genes. We recently identified the “core” transcriptional regulatory circuits
controlled by the oncogenic transcription factor TAL1 in T-ALL (Sanda et al, Cancer
Cell, 2012). This work establishes that T-ALL cells possess the same general motifs of
transcriptional circuitry that were identified earlier in embryonic stem cells. We also
identified potential therapeutic targets by loss-of-function RNA interference screens in TALL cells.
Our laboratory utilizes the zebrafish model for functional analysis in vivo. Zebrafish
offer many advantages as a human cancer/leukemia model: (i) easy monitoring of tumor
development in vivo due to its transparency and fluorescence-labeling of tumor cells and
vasculature; (ii) a high degree of genetic conservation with humans; (iii) simple
transgenic techniques; and (iv) the feasibility of conducting genetic and drug screens.
Zebrafish models of cancer are particularly suited to functional genetic investigations in
vivo (“forward genetics”).
Success in the projects will not only validate the therapeutic approach outlined above, but
should also generate a series of highly effective, first-in-class small-molecule inhibitors
for future clinical testing. The knowledge gleaned from this work could well extend to
other leukemia/cancer in which an oncogenic transcription factor controls the core
transcriptional machinery.
Dr Teo Boon Wee
mdctbw@nus.edu.sg /
Evaluating the role of dynamic renal function testing (GFR stress test) in predicting
kidney disease progression
A/Prof Evan Lee
mdcleejc@nus.edu.sg
Guidelines recommend using serum creatinine-based estimated glomerular filtration rate
(eGFR) calculated from equations for evaluating chronic kidney disease (CKD) by stages.
These point-estimates are imprecise, and achieve eGFR within 15% of measured GFR in
<60% of patients. Creatinine is affected by muscle mass and other factors, and a twobiomarker equation using cystatin C and creatinine improves eGFR accuracy. The poor
accuracy and precision arise from fundamental problems in the primary assessment of
kidney function (GFR). Intra-day physiologic variability and medications cause
fluctuations in GFR. Current clinical GFR measurement protocols do not standardize the
conditions for maximizing nephron recruitment and stimulation, and therefore, report
total GFR. Amino acid loading can maximally increase GFR. This added GFR is termed
the renal functional reserve (RFR). By maximally stimulating GFR, we believe it would
be possible to increase the precision of GFR measurements.
Stress GFR have not been routinely performed because of cost and inconvenience.
Moreover, methods of GFR measurement using inulin and radioisotopes have become
less reliable due to product supply problems. Recent technological advances in the
automation of separation techniques for the detection of exogenous markers have resulted
in the adoption of high-performance liquid chromatography (HPLC) for GFR
measurements. The proposed marker iohexol has been used safely and successfully for
this purpose. However, the development and standardization of a clinical method of
stress GFR using HPLC detection of iohexol has not been described.
The prediction of clinical outcomes (rate of GFR decline and end-stage renal disease-
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ESRD) of CKD patients is poor. Clinical prediction factors still rely only on crude
proteinuria and blood pressure. It has been shown that RFR declines with advancing
CKD stages. RFR has also been improved or restored in patients with hypertension and
CKD with medications. However, studies linking the magnitude of RFR, and the
restoration of RFR with treatment, to changes in the progression of CKD is unknown.
We hypothesize that:
1.
GFR measurements are more precise after maximal nephron recruitment and
stimulation (stress) with an amino acid load
2.
Combination endogenous biomarker (serum creatinine and cystatin C) stress GFR
are as accurate as an using an exogenous marker (iohexol)
3.
Dynamic renal testing is more predictive of kidney disease progression than
traditional predictors
We aim to:
1.
Develop a stress GFR measurement method using serum and urine iohexol
detection by high-performance liquid chromatography (HPLC)
2.
Measure stress GFR in a variety of CKD patients to establish a biospecimen
bank, and a longitudinal dataset assessing RFR and clinical outcomes
Is Retinal Vessel Diameter (IRED) a Potential Biomarker for Early Renal Disease?
Chronic kidney disease (CKD) is a major public health problem worldwide associated
with adverse renal and cardiovascular outcomes. The number of patients with earlier
stages of CKD is more than 50 times higher than that reaching end-stage renal disease
(ESRD). The risk of cardiovascular disease (CVD) in ESRD patients is 10 to 20 times
higher than the general population and increased risks are evident even in milder stages of
the disease. CKD has also been shown to increase the risk of death by more than two
times in general population samples. Accumulating evidence shows that early detection
and treatment of CKD could potentially delay or prevent these adverse outcomes.
However, CKD is often underdiagnosed and under-treated due to its asymptomatic
nature. Recent studies have shown that despite better trends in the prevalence of CVD
risk factors, the prevalence of CKD in is increasing. It is therefore essential to identify
novel biomarkers that help early detection of CKD, improves risk stratification of
progressive CKD, and, monitor and adjust therapy more accurately.
Retinal microvasculature reflects the systemic circulation in health and disease elsewhere
in the body. It allows for non-invasive visualization of the microcirculation and studying
its structural and pathological changes directly. Using advanced computer-based retinal
image analytical techniques, retinal vascular caliber and structural alterations variations in
retinal vascular caliber are associated with diabetes, hypertension, CKD and CVD
independent of known risk factors in large epidemiological studies. Studies have shown
that retinal arteriolar narrowing and retinal venular dilation represent different
pathophysiological processes in humans. We hypothesize that retinal vascular imaging
can predict progressive CKD.
In this cross-sectional study, we aim to correlate retinal microvascular findings to patients
with diabetic and non-diabetic CKD, and in a future study correlate these findings to
progression of CKD.
Derivation and validation of a post-prandial (protein) glomerular filtration rate
estimating equation using combination biomarkers (Protein Stress Equation Study)
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Guidelines recommend using serum creatinine-based estimated glomerular filtration rate
(eGFR) calculated from equations for evaluating chronic kidney disease (CKD) by stages.
These point-estimates are imprecise, and achieve eGFR within 15% of measured GFR in
<60% of patients. Creatinine is affected by muscle mass, and a two-biomarker equation
using cystatin C/beta-trace protein and creatinine improves eGFR accuracy. The poor
accuracy and precision arise from fundamental problems in the primary assessment of
kidney function (GFR). Intra-day physiologic variability and medications cause
fluctuations in GFR. Current clinical GFR measurement protocols do not standardize the
conditions for maximizing nephron recruitment and stimulation, and therefore, report
total GFR. Amino acid loading can maximally increase GFR. This added GFR is termed
the renal functional reserve (RFR).
We hypothesize that:
1.
GFR measurements are more precise after maximal nephron recruitment and
stimulation (stress) with a protein load
2.
Combination endogenous biomarkers (serum creatinine and cystatin C/beta-trace
protein) GFR estimation equation derived from stress GFR reduces imprecision
3.
Vitamin D repletion affects the magnitude of the RFR in diabetic nephropathy
We aim to:
1.
Develop GFR prediction equations using combinations of biomarkers after an
oral protein-load stress GFR measurement using serum iohexol detection by highperformance liquid chromatography (HPLC)
2.
Measure stress GFR in vitamin-D deficient patients with diabetic nephropathy
3.
Compare the performance of estimation equations derived from stress versus
fasting GFR measurements
Prof Yoshiaki Ito
csiitoy@nus.edu.sg
Study of regulation of mitosis: Targeting a novel aspect of mitosis
Mitosis is one of the most basic processes in living organisms. Precise transmission of
genetic information to progeny cells is necessary for normal growth. Moreover,
oncogenic stimuli often provoke changes to the transcription profile and chromatin
modification; mitosis represents the perfect stage for cells to reset their gene expression
and epigenetic profiles to their original differentiation state. RUNX proteins as master
regulators of cell lineage are well positioned during mitosis to prevent inappropriate
reprogramming and ensure stable lineage fidelity in daughter cells – unlike most
transcription factors, RUNX proteins are retained on condensed mitotic chromatin and
may serve as regulators of epigenetic memory. Indeed, Runx3 inactivation is associated
with retrodifferentiation of different cell types. Recently, we detected RUNX at key
mitotic structures such as centrosomes, the spindle and midbody. The interaction of
RUNX with mitotic kinases and the impaired ability of RUNX-deficient cells to undergo
nocodazole-induced G2 arrest further suggest a profound role for RUNX in mitosis.
Strikingly, we found that mutation of one of the Threonine residues within the
evolutionarily conserved Runt domain of RUNX3 resulted in cells that failed to progress
through mitosis. Instead, the cells underwent successive nuclear envelope disruption and
reassembly without chromatin condensation or formation of the mitotic spindle, finally
culminating in multi-lobular nuclei. That a single amino acid mutation in RUNX3 results
in such devastating effects points towards a direct as well as dominant role for RUNX3 in
licensing the early stages of mitosis. We propose that RUNX protects against abnormal
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transmission of genetic information, regulating mitotic progression as part of its antitumor repertoire. We will investigate the molecular mechanisms underlying the
regulatory role of RUNX3 in mitosis. Post-translational modification of RUNX3 is a
necessary aspect of RUNX3 mitotic activities. It is also potentially druggable. We will
conduct a thorough determination of the post-translational modification sites in RUNX3
and delineate their functional significance. Characterizing the interaction of RUNX3 with
chromatin modifiers forms the third stage in our strategy to solve the mitotic functions of
RUNX3.
Understanding uncontrolled proliferation and changes in differentiation are
complementary goals in cancer management. The clinical implications of our work can be
described in 2 broad themes: (1) innovation in targeting mitosis in cancer and (2)
pharmacological enhancement of RUNX3 anti-tumor activities. Although RUNX3
inactivation is highly prevalent in human cancers, knowledge of the vulnerabilities of
these cancers remains unknown. This study seeks to reactivate epigenetically silenced
RUNX3 and exploit the post-translational modification status of RUNX3 in mitosis.
Our results indicate a previously unappreciated role for RUNX in mitotic progression and
prompt a revisit of the fundamentals in mitosis. RUNX, previously described as a
transcription regulator, is an essential component of mitosis machinery. Moreover, our
study potentially re-defines the tumor suppressor role of RUNX3 – as guardian of the
epigenome. With the rapid emergence of epigenetic modifiers in tumorigenesis, this work
will provoke a paradigm shift in our perception of tumor suppressors and necessitate its
re-classification in the cancer field – a new category of genes that safeguard the
epigenome and cell identity.
Studies on molecular mechanism of gastric cancer development by using two types
of stomach corpus epithelial stem cells
Lack of molecular approach to activate or inactivate gene of interest in stomach epithelial
stem cells in corpus has been delayed the progress of understanding the biology of
development and carcinogenesis in stomach. A small Runx1 enhancer fragment that
induces expression of Runx1 specifically in hematopoietic stem cells (eR1) was found
also to drive expression of Runx1 in stomach corpus epithelium in two locations. One is
around Isthmus where stem cells are known to be present. We call this SCist. No other
groups found the way to molecularly mark this stem cell population to date. The other is
unusual in that a small subset of pepsinogen expressing chief cells which are fully
differentiated. We designated this SCb. Recently, Troy+ cells that also express
pepsinogen have been reported by others as reserve stem cells near the base of the corpus
epithelium. It is not clear at the moment whether Troy+ cells and SCb are identical.
Ability to mark stem cells of undifferentiated state (SCist) and those of fully
differentiated chief cells (SCb) provides us useful tool to study, among other things, cell
of origin: question of whether cancer cells are originated from undifferentiated stem cells
or fully differentiated cells that acquire stem cell property to become cancer cells.
Cells near the lumen are exposed to food, bacteria and other environmental materials and
secrete mucin. They are constantly tuning over in several days. Probably SCist is
primarily responsible for replenishing such cells. The cells near the base of gastric
epithelium, such as parietal cells that secrete acid and chief cells that express pepsinogen,
turn over every several months. We have to study whether these cells are replenished by
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SCb.
As the first step to examine the use of these two types of stem cells in step-wise
carcinogenesis in gastric epithelial cells, we expressed oncogenic K-rasG12D using eR1CreERT2. We observed dramatic changes in both stem cells. SCist differentiated rapidly
to form the structure similar to that gland that are present in the region of stomach close
to duodenum that do not have parietal cells and chief cells. During this process parietal
and chief cells are eliminated. This process is very similar to that observed in
Helicobacter pylori-induced chronic gastric inflammation observed in the early stage of
human gastric cancer. We are expressing or deleting genes of interest in these two types
of stem cells and studying the step-wise carcinogenesis in stomach.
Studies on the basic tumor suppressor pathway
Among multiple tumor suppressor genes, p53 is the most well known, since it is believed
to have central role to survey abnormal activation of oncogenic signal and, upon detecting
such signals, induce apoptosis, senescence and cell cycle arrest to protect cells from
becoming cancer. Therefore, inactivation of p53 would be dangerous to our body.
Multiple regulatory network is known to regulate the p53 activities. Of them, p14ARF (
referred to as ARF) is considered to be the most important activator of p53 when it
responds to activated oncogenes. ARF inhibits MDM2 whose function is to degrade p53.
So ARF-p53, has been considered the basic tumor suppressor axis in monitoring the
oncogenic signals. It was known that Ras oncogene activates ARF but molecular
mechanism of this activation was not known. During the study on lung cancer, we
recently found that RUNX3 gene transcriptionally activates ARF in response to the Ras
signal. Then, Ras activates RUNX3 which in turn activated ARF, which would stabilize
p53. There are two other RUNX genes and all three seem to have this activity. Then is
the RUNX-ARF-p53 the main tumor suppressor axis to survey abnormal oncogenic
activity in many different tissue ? We are investigating this possibility.
One of the experimental systems in which the relationship between ARF and p53 was
established was the use of mouse embryo fibroblast (MEF). Freshly prepared MEF from
mouse embryos do not express ARF nor p53. However, as MEF are passaged in culture,
these two genes are gradually expressed. If ARF-/- MEF are used, p53 is not stabilized.
From these experiments, it was established that in normal cells, both genes are not
expressed or expressed only at low levels. Upon serial cultivation, which is considered to
be stress to the cells, these genes are gradually activated. p53 expression depends on the
ARF expression. We are examining RUNX gene expression in MEF. The data suggest
that RUNX genes are also not expressed significantly in the primary MEF but they begin
to be expressed after serial passage. Preliminary results suggest that RUNX deficient
MEF blocks the expression of ARF and p53.
Another important discovery made in lung cancer study is that inactivation of RUNX3 is
sufficient to induce adenoma, premalignant tumor, whereas inactivation of p53 is known
not to do so. Furthermore, p53 is known to be activated during the progression stage of
carcinogenesis. Therefore, RUNX genes are gatekeeper of carcinogenesis and p53
functions in the later stage.
We will study the relationship among RUNX-ARF-p53
further and examine RUNX-ARF-p53 is the fundamental tumor suppressor pathway in
human cancer.
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Department of Microbiology
Dr Chu Jang Hann, Justin
miccjh@nus.edu.sg
1.
2.
3.
4.
5.
Dr Stephan Gasser
micsg@nus.edu.sg
Immuno-pathogenesis of mosquito-borne dengue virus and chikungunya virus
infection.
Clinical biomarker development for viral diseases.
Development of novel antiviral agents and strategies against emerging viral diseases.
Receptors and entry processes of human enteroviruses (Enterovirus 71), flaviviruses
(Dengue and West Nile virus) and alphavirus (Chikungunya virus).
Host factors contribution to severity of dengue virus infection in elderly patients.
The role of DNA sensor in Cancer
Humans are constantly threatened by the invasion of viruses and have evolved systems of
immune defense to eliminate viruses in the body. Viral infections are detected primarily
by immune recognition of viral nucleic acids in the cytosol. Many cytosolic DNA sensors
induce the expression of pro-inflammatory cytokines and type I interferons (IFN) through
the activation of the transcription factors IFN regulatory factor 3 (IRF3) and nuclear
factor kappa-light-chain-enhancer of activated B cells (NF-kB). The same defense
mechanisms have also been implicated in protecting organisms from cancer and the
understanding of the molecular basis of tumor recognition by the immune system is a
topic of intense research interest. Optimal immune responses generally only occur when
cells are first exposed to pathogen-associated molecular patterns including cytosolic
DNA, or are subjected to disease-related stress. We recently demonstrated that B cell
lymphomas and many tumor cell lines of various origin constitutively express cytosolic
DNA as a consequence of oncogene-induced DNA damage and repair (Lam et al., under
revision). The expression of cytosolic DNA correlated with the activation of IRF3 and
NF-kB, suggesting that cytosolic DNA is detected by DNA sensors in tumor cells. To
identify DNA sensors involved in the recognition of cytosolic DNA in tumor cells, we
analyzed a cancer microarray database. Our analysis showed that the expression of the
cytosolic DNA sensor Z-DNA binding protein 1 (ZBP1) is highly upregulated in human
B cell chronic lymphocytic leukemia, a type of B cell non-Hodgkin lymphoma (NHL).
NHL is one of the most common types of lymphoma in Singapore. Our hypothesis is that
the DNA sensor ZBP1 prevents replication stress-associated nuclear DNA damage, which
can act as anti-cancer barrier. In this proposal we suggest 1) to study the role of Zbp1 in
the tumorigenesis of B cell lymphomas, 2) to characterize the role of Zbp1 in DNA repair
and DNA damage 3) to identify and characterize proteins and DNA that interact with
ZBP1. We plan to address the role of ZBP1 in B cell lymphomas using Eµ-Myc mice and
various lymphoma cell lines. Different gain- and loss-of-function approaches will be
employed to elucidate the role of ZBP1 in tumorigenesis. The increased survival of Zbp1deficient Eµ-Myc mice and the deregulated expression of Zbp1 in certain human
lymphomas suggest that lymphoma is an appropriate type of cancer to study the role of
ZBP1 in tumorigenesis. In summary, our proposal aims to show that the DNA sensor
ZBP1, which was previously shown to limit viral infections, also plays a critical role in
tumorigenesis. Strikingly, our preliminary results suggest that ZBP1 plays a key role in
DNA repair and possibly in DNA transport.
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A/Prof Tan Yee Joo
mictyj@nus.edu.sg
Understanding viral-host interaction in hepatitis C virus infection
Hepatitis C virus (HCV), a positive-stranded RNA virus of the family Flaviviridae,
infects an estimated 3% of people worldwide and is one of the major causes of liver
diseases. The HCV genome encodes a precursor polyprotein of ~3,000 amino acids that is
processed cotranslationally and posttranslationally to give rise to at least three structural
(core, E1 and E2) and seven non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and
NS5B) viral proteins. Unlike the approximately 24,000 genes in the human genome, the
number of HCV proteins is very limited. Thus, HCV interacts extensively with the
cellular machineries in the human host and uses them to its advantage. The aim of this
project is to characterize the interactions between HCV and host proteins and determine if
their roles in the establishment of infection and disease progression.
Understanding cell death regulation in hepatitis C virus infection
In the fight for survival between virus and host, cell death regulation is an important
determinant and many viruses encode for proteins that can interfere with the cellular cell
death signalling, skewing it to their advantage. Apoptosis and other forms of cell death
have been observed in hepatitis C virus (HCV) infection in vitro and in vivo but the
detailed understanding of this intricate viral-host interplay is still lacking. Massive
apoptosis in the liver leads to acute liver failure, while the dysregulation of cell death
pathways is a major factor in the development of hepatocellular carcinoma (HCC).
Apoptosis of hepatocytes during HCV infection has been proposed to be a direct cause of
liver fibrosis, which can further lead to cirrhosis and liver failure. Hence, it is important to
understand the cellular mechanisms underlying HCV-mediated apoptosis as well as other
cell death pathways. Our hypothesis is that HCV proteins are engaged in a network of
interaction with a group of interconnected host cell death regulating proteins to yield a
tightly regulated system that ensures optimal HCV replication and survival in the host
cell.
Generating antiviral neutralizing antibodies and characterizing their mechanism of
inhibition
Seasonal influenza A virus causes significant morbidity and mortality yearly while newly
emerged strains continue to pose pandemic threats. Passive immunotherapy is now
increasingly being used to treat human infectious diseases and there is a demand for the
development of neutralizing mAbs for passive immunotherapy in the event of a highly
pathogenic flu pandemic as this could be particularly useful for protecting certain groups
of people, such as immuno-compromised patients or the elderly, who may not respond
well to vaccines. As the hemagglutinin (HA) protein mediates viral entry, it has been the
main target for the preclinical studies on antibody-based immunotherapy and these
studies suggest that it may be a viable option to administer neutralizing HA mAbs as a
form of passive immunotherapy for influenza A infection. However, viral escape mutants
were observed when anti-HA mAbs were used individually. Combination therapy, where
multiple steps in the virus life cycle are inhibited simultaneously, is highly recommended
to minimize the development of escape viruses. Hence, the aim of this study is to
determine if other viral proteins can stimulate neutralizing antibodies that prevent viral
infection and replication.
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Dr Volker PATZEL
micvp@nus.edu.sg
Genetic Reversal of Asian Hemoglobinopathies and Thalassaemias with a Novel and
Clinically Relevant Vector
Erythrocytic abnormalities (EAs) are the most prevalent inherited disorders worldwide. In
particular, Southeast (SE) Asia bears a considerable burden of heritable EAs (including
thalassemias, ovalocytosis, HbE and Glucose-6-phosphate dehydrogenase (G6PD)
deficiencies), which in most cases adversely affect normal human development, life span
and limit therapeutic choices for comorbidities. The only cure has been the allogenic
transplantation of bone-marrow cells (BMCs) whose success depends on the limited
availability of suitable donors. Therapies based on genetic correction of a patient’s own
BMCs using lentiviral gene delivery vectors have shown promise, however its clinical
application is limited by the alarming possibility of uncontrolled genomic vector
integration. Thus there is a pressing need to develop gene therapies that are both safe and
efficient. The aim of this proposal is to optimise and use safe, non-integrative DNA
minimal vectors that can trigger transient expression in human primary cells to deliver
RNA-guided genome editing in order to reverse the genotypes and phenotypes of two
important SE Asian EAs (E/0-thalassaemia-associated E-globin allele and the G6PDMahidol487A mutation). The technology to achieve this goal is the Cas9/CRISPR system, a
novel breakthrough tool for genome engineering. We further leverage on two important
resources; firstly ‘made to order’ and manufactured DNA minimal vectors. Secondly, a
library of fully phenotyped and genotyped Asian hematopoietic stem cells (HSCs),
normal and mutant, that have been banked in Singapore. These cells can be
reprogrammed ex vivo and could then be re-implanted into patients for future therapy.
The ex vivo genetic reversal of the two chosen EAs in HSCs should be considered a very
exciting and important therapeutic approach which might pave the way for future in vivo
demonstrations in rodent or non-human primate models.
Development of DNA minimal vectors for enhanced and sustained expression of
transgenes in human primary cells
The genetic manipulation of human primary cells holds great promises but is challenging.
While transgene expression from bacterial plasmids is silenced within 24 hours, viral
gene transfer harbours safety risks, and naked RNA triggers only short-term effects. Here
we aim to develop novel minimal genetic vectors for enhanced and sustained transgene
expression in human primary cells. We will focus on dumbbell-shaped DNA vectors
harboring expression cassettes for non-coding and/or coding RNA. We will investigate
the effects of additional regulatory genetic elements including introns, mirtrons,
posttranscriptional regulatory elements, enhancers, and nuclear import signals on the
levels and duration of transgene expression in human primary cells including T
lymphocytes. Due to the limitations involved with the use of state-of-the-art technologies,
alternative safe genetic vectors that escape transgene silencing in primary cells harbour
great potential for therapeutic applications including genetic therapy of inherited and
acquired genetic diseases, genetic vaccination, stem cell programming, somatic cell
reprogramming, and immunotherapy. As a therapeutically relevant transgene we selected
different human antiviral-specific T cell receptors (TCR) that have already been used to
redirect the specificity of T lymphocytes for adoptive T cell therapy. We will furnish the
vector also with microRNA genes known to further improve the CD8+ T cell response in
order to engineer virus-specific T cells with enhanced function that can be used in the
treatment of different chronic viral diseases (like HBV or HCMV). The development of
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safe and efficient genetic TCR vectors able to engineer anti-viral specific T cells can be
of high significance in the growing field of specific immune therapy.
Design and investigation of advanced RNA trans-splicing Vectors
Functional RNAs and RNA technologies are successfully used for manipulation of gene
expression in tissue culture cells and increasingly make it towards pre-clinical and clinical
investigations. Thereby, high on-target activity and specificity are imperative.
Spliceosome-mediated RNA trans-splicing is a mechanism that allows functional genetic
repair on the RNA level. Thereby, trans-splicing RNAs bind via antisense binding
domains to the target pre-mRNA and subsequently trigger splicing in trans between
acceptor or donor splice sites of the target and donor or acceptor splice sites of the transsplicing RNA in order to repair or label the endogenous message. The aim of this project
is to study the contribution of distinct molecular features such as the antisense binding
domains or the target splice sites to the efficiency and specificity of the overall transsplice reaction and to combine RNA trans-splicing with other RNA technologies. The
gained knowledge will then be used to design next generation trans-splicing RNAs for
mRNA repair or labelling towards therapeutic applications. Therefore the RNAs will be
expressed from dumbbell-shaped DNA minimal vectors which allow sustained expression
in human primary cells. Targets will be inherited and acquired genetic disorders including
cancer and virus infections. The methods cover computational (in silico) RNA structural
design, molecular biology in vitro techniques, and vector testing ex vivo (in human tissue
culture and primary cells) and possibly in vivo (mice). The outcome of this project is
expected to have significant impact for RNA trans-splicing-based genetic therapy.
Dr Zhang Yongliang
DUSP4 in intestinal inflammation and colorectal cancer
Yongliang_zhang@nuhs.edu.sg
Research in my laboratory focuses on understanding the roles of dual specificity
phosphotases (DUSPs), also known as MAPK phosphatases (MKPs), in health and
disease (Nature, 2004; EMBO J., 2009; JBC, 2009). One of the project is to understand
the role of DUSPs in colorectal cancer (CRC), which is the third most common cancer
globally and the commonest cancer in Singapore, accounting for roughly 1.2 million new
cases and 600,000 deaths annually. Genome wide association studies had been carried out
over the pass decades to identified genes that are related to the development of CRC.
Several (DUSPs) were identified to be associated with CRC in these studies. Of which,
polymorphisms and deregulation of DUSP4, DUSP6 and DUSP10 were most commonly
reported in CRC. However, the mechanisms and effect of these genes in CRC
pathogenesis have not been clearly defined. DUSPs are negative regulators of mitogenactivated protein kinases (MAPKs) which is one of the main drivers for tumour
formation. Understanding the role of DUSPs in CRC development will provide valuable
information on CRC aetiology and a possibility for novel therapeutic intervention for
CRC. We recently found that DUSP10 is critical in regulating intestinal epithelial cell
growth in response to damage and acts as a suppressor of colorectal cancer (Png et al.,
submitted). In this project, we will use DSS-induced colitis and DSS/AOM-mediated
colorectal cancer model performed on DUSP4 knockout and control mice, as well as gene
overexpression and knockdown of DUSP4 in human CRC cell lines to decipher the role
of DUSP4 in intestinal inflammation and tumorigenesis. Our group is also interested in
the regulation of innate immune response to microbial infection (Nature Immunology,
2007; PLOS Pathog., 2013; PNAS, 2013) and regulation of T helper cell generation and
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function (Nature, 2007; Immunity, 2010). Projects on these research areas are also
available.
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Zhang, Y., J. N. Blattman, N. J. Kennedy, J. Duong, T. Nguyen, Y. Wang, R. J.
Davis, P. D. Greenberg, R. A. Flavell, and C. Dong. 2004. Regulation of innate and
adaptive immune responses by MAP kinase phosphatase 5. Nature. 430:793-797.
Qian F., Deng J., Cheng N., Welch E. J.; Zhang Y., Malik A. B.,
Flavell R. A.,
Dong C., and Ye R.D. 2009. A non-redundant role for MKP5 in limiting ROS
production and preventing LPS-induced vascular injury. EMBO J. 28: 2896-2907.
Zhang Y., Reynolds J., Chang S. H., Martin-Orozco N., Chung Y., Nurieva R. and
Dong C. 2009. MAP kinase phosphatase 1 is necessary for T cell activation and
function. J. Biol. Chem. 284:
30815-30824.
Png C. W., M. Weerasooriya, M. Osato, R. A. Flavell, C. Dong and Y. Zhang.
DUSP10 regulates intestinal barrier function and tumorigenesis. (Submitted)
Hsu M.Y., Y. Zhang, Y. You, D. Wang, H. Li, O. Duramad, X. Qin, C. Dong, and X.
Lin. 2007. The adaptor protein CARD9 is required for innate immune responses to
intracellular pathogens. Nature Immunol. 8: 198-205.
Negishi H., K. Matsuki, N. Endo, H. Sarashina, S. Miki, A. Matsuda, K. Fukazawa,
N. Taguchi- Atarashi, H. Ikushima, H. Yanai, J. Nishio, K. Honda, Y. Fujioka, Y.
Ohba, T. Noda, S. Taniguchi, E. Nishida, Y. Zhang, H. Chi, R.A. Flavell, and T.
Taniguchi. 2013. Beneficial innate signaling interference for antibacterial responses
by a Toll-like receptor-mediated enhancement of the MKPIRF3 axis. PNAS. 111:
19884-19889.
Chung Y., T. Yamazaki, B. Kim, Y. Zhang, J. M. Reynolds, G.J. Martinez, S.H.
Chang, M.Birkenbach, and C Dong. 2013. Epsterin Barr virus-induced 3 (EBI3)
together with IL-12 negatively regulates T helper 17-mediated immunity to Listeria
monocytogenes infection. PloS Pathog. 9: e1003628
Nurieva R., Yang X., Martinez G., Zhang Y., Panopoulos A. D., Ma L., Schlun K.,
Tian Q.,Watowich S., Jetten A. M., and Dong C. 2007. Essential autocrine regulation
by IL-21 in the generation of inflammatory T cells. Nature. 448: 480-483.
Nurieva R., S. Zheng, W. Jin, Y. Chung, Y. Zhang, G. J. Martinez, J. Reynolds, S.
Wang, X. Lin. S. Sun, G. Lozano and C. Dong. 2010. The E3 ubiquitin ligase GRAIL
regulates T cell tolerance and regulatory T cell function by mediating T cell receptorCD3 degradation. Immunity. 32: 670-680.
Department of Paediatrics
A/Prof Heng Chew Kiat
paehck@nus.edu.sg
Prof Yechiel Friedlander
yechielf@ekmd.huji.ac.il
Updated in Apr 2015
Genetics of coronary artery disease
Coronary artery disease (CAD) is the leading cause of death and disability in many
developed countries and is the number two killer in Singapore. According to the World
Health Organization, 23.6 million deaths each year by 2030 are projected to be due to
cardiovascular diseases. CAD is a complex disease that is caused by multiple genetic and
environmental factors as well as their interactions. There are several ongoing projects
being conducted in our Cardiovascular Genetics Lab. The common objectives of these
projects are to determine the contributing genetic factors to CAD and to develop methods
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to better predict CAD risk.
We have 3 ongoing projects available for graduate students.
1. Genome-wide association studies (GWAS) of coronary artery disease and its
associated risk factors
This study aims to identify susceptibility genes for CAD and myocardial infarction in the
Singaporean population. Other risk factor phenotypes investigated include lipid profile,
blood pressures and inflammatory markers. We also participate in both regional and
international consortia such as Asian Genetic Epidemiology Network (AGEN) and
Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE). Students
wishing to work on this project are required to have a good grasp of biostatistics.
2. Investigation of a newly identified protein, androgen-dependent tissue factor
pathway inhibitor regulating protein (ADTRP), for its role in coronary artery
disease
Recently, a GWAS in the Chinese population has identified rs6903956 within the
C6orf105 gene (now known as androgen-dependent tissue factor pathway inhibitor
“TFPI” regulating protein or ADTRP) on chromosome 6p24.1 as a novel susceptibility
locus for CAD.
ADTRP is a protein encoded by C6orf105 that regulates TFPI expression. TFPI is the
major inhibitor of tissue factor-factor VIIa–dependent FXa generation. Hence, ADTRP
can be considered a protective factor of CAD. As it is a newly discovered protein, very
little is known about its characteristics. This provides ample scope for investigation.
The specific aims of this study are:
1. To determine which of the two known isoforms of ADTRP is the predominant
form through the use of Human Coronary Artery Endothelial Cell (HCAEC) line.
2. To determine the changes in ADTRP in response to androgen in HCAEC.
3. To select meaningful single nucleotide polymorphisms for genotyping to
determine if any are significantly associated with CAD in the Singaporean
population.
4. To conduct genetic variant screening in ADTRP gene
The methods employed in this study include cell culture, PCR, qRT-PCR, genotyping,
bioinformatics and statistical analyses.
3. Identification of rare variants with strong effects on early onset myocardial
infarction
There is an increasing trend of early onset of myocardial infarction (MI) in patients who
are below 40 years. A proportion of these do not have strong conventional risk factors
such as smoking, hypertension and diabetes. In fact some of the patients with early onset
of MI lead physically active lives and do not appear to have any conventional risk factors
except for being male. It is therefore of interest to investigate the genetic factors
underlying this early onset phenotype. Genome-wide association studies (GWAS) have
thus far identified genetic variants that may predispose individuals to MI. However, these
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are mostly variants that are common in the population and are hence unlikely to account
for the small subset of patients who had MI at a young age. We aim to carry out exome
sequencing using next generation sequencing (NGS) technology for MI patients <40
years old) who have been identified from the National University Heart Centre in NUH.
The number of variants identified from these patients is expected to be very large. We
will employ filtering strategies to obtain meaningful number of target genes that we can
conduct experiments to verify their functions. We believe be insightful information could
be gleaned from such an approach.
A/Prof Lai Poh San
paelaips@nus.edu.sg
Identifying cause of genetic and clinical heterogeneity in neuromuscular diseases
Neuromuscular disorders (NMDs) form a large group of clinically and genetically
heterogenous disorder which may affect the muscles, nerve-muscle junction, peripheral
nerves in limbs, and the motor-nerve cells in the spinal cord. More than 400 NMDs have
been identified. Patients within each disease group often have wide range of clinical
features, severity, age of onset, rate of progression, type of muscle groups affected, and
mode of inheritance. There is considerable clinical and genotype heterogeneity making
definite diagnosis difficult when phenotypes overlap. Some disorders can also be caused
by more than one gene while different types of mutations in a single gene can give arise
to different diverse disorders. Often, a wide range of clinical manifestations can be
expressed from a single disease. Recent exciting developments in technologies such as
massively parallel genome-wide sequencing and epigenomics are enabling the molecular
dissection of the human genomes such that we can unravel the underlying basis of the
genetic and clinical heterogeneity of these diseases. Such molecular studies will help us
discover new genes as well as identify new clinical sub-forms of diseases. The clinical
applications include improved diagnostics and therapy interventions. For example, in
Duchenne muscular dystrophy, precise identification of mutations will allow personalized
molecular treatment through anti-sense mediated splicing.
Prof Lee Bee Wah
paeleebw@nus.edu.sg
Upper airway nasal microbiota and its relationship with persistent rhinitis and
wheezing in early childhood
There is little data on the pattern of nasal microbiome colonization in early life, and its
influence on disease development. A Singapore birth cohort GUSTO) is followed up for
allergic disorders including rhinitis in early childhood. We have shown that the
prevalence of rhinitis for at least 4 weeks is common (18.8%) at the age of 18 months,
and is associated with wheeze and eczema. We aim to analyze the diversity and
abundance of upper respiratory microbiota from archived nasal swabs taken at regular
intervals in the first 18 months of life and correlate the microbiota data with the clinical
outcomes of early onset rhinitis, and subsequent development of allergic rhinitis and
wheeze/asthma at age of 5 years.
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A/Prof Lee Yung Seng
paeleeys@nus.edu.sg
1. Prenatal, perinatal, and postnatal determinants of adiposity and metabolic
phenotype in childhood
(For MSc or PhD project)
This research is part of the birth cohort study “Growing Up in Singapore Towards healthy
Outcome” (GUSTO) of the TCR flagship programme “Developmental Origins:
Singapore” (DevOS). Prenatal, perinatal, and possibly early postnatal factors determine
the subsequent growth of the child. This project aims to uncover the factors which
determine the size and body composition of the newborns, and predictors of the growth of
young children in the first few years of life, including maternal factors (diet and nutrition,
habitus, pregnancy complications), birth events, and the baby’s growth pattern in the first
few months’ of life. Specifically the candidate will determine the factors which predict
catch up growth in the group of children with lower birth weights, as well as those with
growth failure. The student will get opportunities to study epigenetic changes of
candidate genes which predict the subsequent growth pattern of the children, as well as
predictors of failed catch up growth.
2. Determining body composition of infants and young children
(For MSc or PhD project)
This research is part of the birth cohort study “Growing Up in Singapore Towards healthy
Outcome” (GUSTO) of the TCR flagship programme “Developmental Origins:
Singapore” (DevOS). The candidate will study the various modalities of body
composition assessment of infants and young children, including MRI of abdominal fat
compartments and whole body fat, bioelectrical impedance, and air displacement
plethysmography. There will also be opportunity to study correlation of the body
composition parameters with developmental factors and epigenetic biomarkers.
3. Diet during infancy (0 -3 years) and infant health outcomes
(For PhD project)
The early years of life are critical to the growth, development, and well-being of children.
Feeding practices and food consumption contribute to infants' nutrient adequacy or excess
and also establish dietary patterns and habits that can last a lifetime. Understanding food
consumption patterns and nutrient intake is important to identify areas for improvement in
establishing healthy eating patterns at an early age.
Compared to their Western counterparts, little is known about the food consumption
patterns and nutrient intakes of Asian infants. The existence of the three major ethnic
groups (Chinese, Malay and Indians) in the local Singapore population provides an ideal
backdrop for a study to investigate the dietary patterns and nutrient intakes of infants in
the Asian region.
This research will use data from the GUSTO (Growing Up in Singapore Towards healthy
Outcomes) mother-offspring cohort study in Singapore and the largest in Asia. It is a
longitudinal study designed to understand the developmental origins of health and disease
from an Asian perspective. A cohort of 1200 pregnant women were recruited during the
first trimester of pregnancy and followed up regularly throughout pregnancy and the
nutrition, health and development of the child tracked from birth to 8 years of age.
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Research aims: With this, research projects would be undertaken to examine the
following hypotheses:
1) What are the dietary patterns of Asian children during weaning (6-12 mths), and in the
early years of life (up to 36 mths)? Are there differences between ethnic groups?
2) What are the associations between nutrient intakes, dietary patterns of Asian children
and their growth and adiposity in the early years of life (0 -36 months)?
3) Does sleep duration in early life influences energy intakes and dietary patterns in Asian
children and modifies the effect on growth and adiposity?
4. Role of sleep in behavioral and metabolic outcomes
(For MSc project)
Good sleep is important for good health. Over the past decade, increasing evidence
suggests that short sleep negatively impacts cognition and contributes to increased risk of
weight gain and metabolic disorders. As part of the Growing Up in Singapore Towards
healthy Outcomes (GUSTO) birth cohort study in Singapore, we are investigating the
influence of sleep behavior on neurobehavioral and growth outcomes. We are examining
sleep behavior in children and their mothers using questionnaires and actigraphy. Our aim
is to characterize the role of sleep in mental and physical health, as well as factors that
influence sleep duration and sleep quality.
5. Effects of a BCAA (branched chain amino acids)-supplemented hypocaloric
diet versus a standard hypocaloric diet on weight loss, lean mass
preservation and improvement of insulin sensitivity, and its benefits over a
high-protein hypocaloric diet.
High-protein diets better preserve lean mass than conventional low-fat diets. However,
they are costly and have potential health risks. Preserving lean mass is important for
sustaining high resting energy expenditure, leading to greater initial weight loss, better
weight maintenance and improving blood sugar levels. Branched-chain amino acids
(BCAA) supplements are known to preserve lean mass but their effects during weight
loss have not been examined. We want to investigate if a BCAA-supplemented diet is
more effective than a standard hypocaloric diet in terms of the aforementioned benefits,
and yet has less detrimental effects than a high-protein diet for weight loss. Using a 16week weight loss and 8-week weight maintenance intervention, overweight and obese
men and women will be randomized to either a hypocaloric diet with BCAA or placebo
supplements or a high-protein diet with placebo supplements. Participants’ compliance to
the diet versus supplements will be compared. Body composition, resting and dietinduced energy expenditure, insulin sensitivity will be measured and blood samples taken
before and after weight loss. These findings will inform on the benefits of BCAAsupplementation during energy restriction and may offer an alternative cost-effective
strategy for weight loss and maintenance, without the adverse health effects of a highprotein load.
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A/Prof Lynette Shek
lynette_shek@nuhs.edu.sg
Maternal diet during pregnancy and risk of asthma and allergic outcomes in the
offspring
(For MSc project)
Background
Co-supervisor:
Dr Mary Chong
mary_chong@sics.a-star.edu.sg There is increasing evidence to suggest that the intra-uterine environment has a pivotal
impact on the developing immune function and health outcomes of the offspring.
Research has shown that low maternal consumption of leafy vegetables, malaceous fruits
(e.g. apple and pear), and chocolate were positively associated with the risk of wheeze in
children, while high maternal consumption of fruit and berry juices was positively
associated with the risk of allergic rhinitis. No associations were observed between
maternal food consumption and asthma (Erkkola M et al, 2012)
Maternal intake of vitamin D was found to be important, having an inverse association
with sensitization to food allergens (Nwaru BI et al, 2009) and significantly associated
with a decreased risk of islet autoimmunity in offspring during the first four years of life
(The Diabetes Autoimmunity Study in the Young (DAISY) cohort). On the other hand,
low vitamin D intake during pregnancy has been associated with an increased risk of
asthma, eczema or hay fever, while winter birth was associated with higher IgE levels and
lowers IL-10 than summer birth (AL Ponsonby, 2010).
No data looking at maternal dietary patterns and maternal vitamin D status during
pregnancy and the impact on asthma and allergic outcomes currently exists in Singapore.
Research aims: With these, research projects would be undertaken to examine the
following hypotheses:
1) Are there differences in dietary patterns of healthy (non-allergic) pregnant mothers
versus allergic pregnant mothers during pregnancy?
2) What are the associations of dietary patterns during pregnancy with asthma and
allergic outcomes in offspring?
3) What are the associations of maternal vitamin D status (using serum vitamin D as
nutrient biomarker) with asthma and allergic outcomes in the offspring?
Specifically, the outcomes of the different study fields will be:
1) Clinical phenotypes (wheezing, rhinitis, eczema, food allergy), and
2) Skin prick test results at the age of 18 months and 36 months.
A/Prof Samuel Chong
paecs@nus.edu.sg
Regulation and role of MAPKAPK3, an IRF6-regulated gene, in epithelial and skin
formation
Unlike all other members of the interferon regulatory factor (IRF) transcription factor
family, IRF6 plays no role in the regulation of host defense and oncogenesis. Instead,
mutations in IRF6 lead to congenital oral clefting syndromes in humans. In mice,
homozygous Irf6 knockout leads to late-stage fetal demise or severe neonatal
craniofacial and limb defects. Interestingly, dominant-negative disruption of Irf6
function in zebrafish and Xenopus results in early gastrulation-stage developmental
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arrest and embryo rupture. Together, these observations are highly supportive of a
critical requirement for Irf6 in both embryonic and fetal development.
We previously performed a whole transcriptome analysis of zebrafish early embryos
after Morpholino™ mediated irf6 knockdown, and observed a number of highly
dysregulated genes at the 40% epiboly stage, with some changes occurring as early as
the mid-blastula transition (MBT). Among the differentially regulated genes, mapkapk3
stood out, registering a >100-fold suppression after irf6 knockdown. MAPKAPK3, a
member of the mitogen-activated protein kinase (MAPK) family, is targeted by all 3
cascades of MAPK (ERK, p38, and JNK) and has been reported to be involved in
several important cellular processes.
A canonical Irf6 binding site is present in the promoter region of mapkapk3, and we
have demonstrated physical binding between the mapkapk3 sequence and Irf6 using an
electrophoretic mobility shift assay (EMSA). Knocking down mapkapk3 produced
kinking of the notochord and an aberrant skin epithelium reminiscent of the skin defect
in Irf6 knockout mice. Hypothesis: The skin defects observed in humans with
dominant IRF6 mutations, and the epithelial and skin defects in Irf6-null mice, are
mediated through MAPKAPK3. This project will focus on characterizing the role of
mapkapk3 in epithelial and skin formation.
Prof Yap Hui Kim
hui_kim_yap@nuhs.edu.sg
Role of angiomotin in the pathogenesis of membranous nephropathy
Idiopathic membranous nephropathy (IMN) is the commonest cause for nephrotic
syndrome in adults, and half of the patients progress to renal failure. Despite its
prevalence, the molecular mechanisms behind IMN are poorly understood. Current
treatment options are therefore empirical and often ineffective. IMN is a result of immune
complex deposition in the glomerular basement membrane. The triggers for immune
complex formation are unknown.
In our preliminary study, we have identified a local Chinese family with X-linked
recessive membranous nephropathy, Fanconi syndrome and anti-tubular basement
membrane (anti-TBM) antibodies. Using exome sequencing, we have found angiomotin
(AMOT) as a novel candidate gene, with the mutation p.S50G in the p130-AMOT
isoform which is critical in maintaining tight junctions between cells. In our preliminary
work, we have demonstrated that AMOT is expressed in human podocytes and renal
tubular cells, both of which depend highly on tight junctions for their cellular function.
Our hypothesis is that the mutation p.S50G in the AMOT gene is functional and the antiTBM antibodies present in the family are anti-AMOT antibodies.
Our specific aims are to:
1) Study the expression of angiomotin and p130-AMOT isoform in human podocytes and
renal tubular cell cultures, and in animal and human kidney tissues by immunostaining
2) Study the identity of anti-TBM antibodies present in the family with p.S50G AMOT
mutation.
3) Elucidate the function of the angiomotin mutation p.S50G by its transfection into
HEK293, human podocyte and renal tubular cell cultures, observing its effect on cell
morphology, angiomotin subcellular distribution and its interactions with known and
novel binding partners.
4) Introduce the p.S50G mutation in rats using Transcription activator-like effector
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nucleases (TALENs) and studying its phenotypic effects.
Our study will improve the understanding of the mechanisms of IMN, which can result in
more targeted therapy with more efficiency and less side effects.
Role of Soluble Urokinase Plasminogen Activator Receptor (suPAR) in
Pathogenicity of Focal Segmental Glomerulosclerosis (FSGS)
Immune-mediated focal segmental glomerulosclerosis (FSGS) is the most common cause
of acquired end-stage renal disease in children worldwide. Studies on the pathogenetic
mechanism suggested a plausible role for induction of urokinase plasminogen activator
receptor (uPAR) signaling with increase in circulating soluble uPAR. Several questions,
however, remained unanswered: i) High suPAR levels are also found in non-proteinuric
diseases (eg. sepsis); ii) Is the whole suPAR molecule or one of its subunits, the elusive
proteinuric-inducing circulating factor; iii) Are immune cells the only possible source of
suPAR in FSGS? Our preliminary studies demonstrated increased suPAR production in
monocyte supernatant and injured podocytes. We therefore hypothesized that:
1. Secreted suPAR from podocytes promotes podocyte migration, contributing to elevated
circulating suPAR levels in FSGS patients;
2. A specific isoform of suPAR may be responsible for podocye foot process effacement;
3. Filtration of suPAR into urine in the presence of podocyte damage may explain the
discrepant suPAR levels observed in FSGS patients, and the elevated suPAR levels in the
presence of decreased glomerular filtration rate.
Thus, the primary aims are to:
1. Demonstrate suPAR production by injured podocytes and its role in promoting
podocyte migration,
2. Identify the suPAR isoform responsible for foot process effacement,
3. Identify the upstream mediators leading to increased uPAR expression in podocytes
and monocytes/immune cells.
The secondary aim is to validate the above findings in both wild type and Plaur-/- (CD87/-) mice via introduction of these upstream mediators, to determine if uPAR is increased
on the mouse podocytes resulting in FSGS.
The tertiary aim is to assess the clinical utility of suPAR (plasma+urine) as an early
biomarker of podocyte injury.
Understanding the role of suPAR and its isoforms in podocyte injury will facilitate the
design of novel targeted therapies for this disease, as well as provide a potential
biomarker.
Department of Pharmacology
A/Prof Bian Jinsong
phcbjs@nus.edu.sg
Project 1: Activation of Na+/K+ ATPase (NKA) is a new strategy to treat ischemic
heart diseases
Na+/K+ ATPase (NKA) is responsible for maintaining the electrochemical gradient, and
hence the membrane potential, of the cell membrane. The -subunit of NKA is important
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in maintaining the function of α-subunit. In oxidative stress, glutathionylation of 1subunit impairs this interaction and decreases NKA activity. In chronic heart injury,
NKA loss contributes to the development of CHF. Our preliminary data showed
disruption of this interaction may induce heart dysfunction and injury. In this project, we
will express the soluble extracellular β ectodomains, which lack glutathionylation site
but are still able to interact with and activate α1-NKA, and study the cardioprotective
effects of these ectodomains.
Project 2: Inhibition of abnormal protein aggregation by hydrogen sulfide: a
potential approach to treat neurodegenerative diseases
Neurodegenerative disorders represent a major cause of disability and death, with an
unmet need for therapies that alter disease progression. Hydrogen sulfide (H 2S) has
recently been hypothesized to be an important neuromodulator in the brain. Abnormal
generation and metabolism of H2S may be actively involved in the pathogenesis of
central nervous system (CNS) diseases. We recently reported that both endogenous and
exogenous application of H2S produces therapeutic effects on Parkinson’s disease and
Alzheimer’s disease. However, the molecular mechanisms are still unclear. We
hypothesize that H2S may have the potentials to retard pathological process by
prevention of abnormal protein aggregation. In this project, we will examine the effects
of H2S on Tau phosphorylation, α-synuclein nitration and amyloid β aggregation. H2Sinduced S-sulfhydration, autophagy preservation, anti-oxidative stress and mitochondrial
protection will be studied.
A/Prof Christopher Chen
phccclh@nus.edu.sg
1) Dementia Neuroimaging
We propose to use multi-modal magnetic resonance imaging (MRI) and retinal imaging
techniques to systematically study brain functional and anatomical abnormalities and rate
of annual changes as well as vascular deficits associated with prodromal dementia
(Vascular Cognitive Impairment No Dementia, VCIND), mild VaD, AD.
We hypothesise that multi-modality MRI methods and novel retinal imaging will provide
novel insights in evaluating brain atrophy and lesions, cerebral artery stenosis, as well as
functional deficits associated with different types of dementia and thus identifying
dementia-subtype specific imaging markers.
Recent publications
S Hilal, YT Ong, C Cheung, CS Tan, N Venketasubramanian, W Niessen, H Vrooman,
AR Anuar, M Chew, C Chen, TY Wong, MK Ikram. Microvascular Network
Alterations in Retina of Subjects with Cerebral Small Vessel Disease. Neuroscience
Letters
S Hilal, M Saini , CS Tan, JA Catindig, ,YH Dong , RL Hollandez, WJ Niessen, HA
Vrooman, E Ting, TY Wong, C Chen, N Venketasubramanian , MK Ikram. Intracranial
stenosis, cerebrovascular diseases and cognitive impairment in Chinese Alzheimer
Disease & Associated Disorders (accepted)
JYT Thong, J Du, N Ratnarajah, YH Dong, HW Soon, M Saini, MZ Tan, AT Ta, C
Chen, A Qiu Abnormalities of Cortical Thickness, Subcortical Shapes, and White Matter
Integrity in Subcortical Vascular Cognitive Impairment Human Brain Mapping 35
(2014) 2320-2332
CY Cheung,,YT Ong, MK Ikram,, SY Ong, X Li, S Hilal, JA Catindig, N
Updated in Apr 2015
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List of research projects available for prospective graduate students
Venketasubramanian, P Yap,D Seow, CP Chen, TY Wong Microvascular network
alterations in the retina of patients with Alzheimer’s Disease Alzheimer's and Dementia
10 (2014) 135-42
2) Clinical Trials in Dementia and Stroke
Our current focus is on developing a regional network for innovative trials into
conditions prevalent and pertinent to Asia. We also plan to enhance Singapore's
capability for conducting increasingly sophisticated early phase studies utilising
biomarkers such as neuroimaging.
Recent publications include
CLH Chen, N Venketasubramanian, CF Lee, KSL Wong, MG Bousser for the CHIMES
Investigators. Effects of MLC601 on Early Vascular Events in Post-stroke Patients – The
CHIMES Study Stroke 44 (2013) 3580-3583
CLH Chen, SHY Young, HH Gan, R Singh, AY Lao, AC Baroque II, HM Chang, JHB
Hiyadan, CL Chua, JM Advincula, S Muengtaweepongsa, BPL Chan, HA de Silva, S
Towanabut, NC Suwanwela, N Poungvarin, S Chankrachang, KSL Wong, GB Eow, JC
Navarro, N Venketasubramanian, CF Lee, M-G Bousser, for the CHIMES Study
Investigators Chinese Medicine Neuroaid Efficacy on Stroke Recovery A Double-Blind,
Placebo-Controlled, Randomized Study Stroke 44 (2013) 2093-2100
Wong KS, Chen C, Fu J, Chang HM, Sulawela N, Huang Y, Han Z, Tan KS,
Ratanakorn D, Chollate P, Zhao Y, Koh A, Hao Q, Markus HS for the CLAIR Study
Investigators. Clopidogrel plus aspirin is more effective than aspirin alone in reducing
embolisation in patients with acute symptomatic intracranial stenosis - the CLAIR study
Lancet Neurology 9 (2010) 489-97.
3) Biomarker Discovery in Stroke and Dementia
Investigating the pathological basis and biomarkers for stroke and vascular cognitive
impairment using tissue samples from well characterized clinical cohorts
Recent publications
K Narasimhalu, L Ma, DA De Silva, MC Wong, HM Chang, C Chen Elevated plateletderived growth factor-AB/BB is associated with a lower risk of recurrent vascular events
in stroke patients. Journal of Stroke (accepted)
Datta A, Chen CP, Sze SK Discovery of Prognostic Biomarker Candidates of Lacunar
Infarction by Quantitative Proteomics of Exosome Enriched Plasma PLOS-1 (accepted)
Lam SM, Wang Y, Duan X, Wenk MR, Kalaria RN, Chen CP, Lai MK, Shui GH. The
brain lipidomes of subcortical ischemic vascular dementia and mixed dementia
Neurobiology of Aging (accepted)
Datta A, Qian J, Chong R, Kalaria RN, Francis PT, Lai MK, Chen CP, Sze SK. Novel
Pathophysiological Markers are Revealed by iTRAQ-based Quantitative Clinical
Proteomics Approach in Vascular Dementia. Journal of Proteomics 99C (2014) 54-67
4) Animal Models and Mechanisms of Dementia
Investigating novel shared mechanisms between Alzheimer’s Dementia and Diabetes by
use of cell cultures and animal models of hyperglycemia and AD. Investigating novel
treatments for dementia.
Recent publications
YA Lim, LA Murray, MKP Lai, C Chen NeuroAiD™ (MLC601) and Amyloid
Updated in Apr 2015
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List of research projects available for prospective graduate students
Precursor Protein Processing Cerebrovascular Diseases 35 (2013) 30-37
Götz J, Lim YA, Eckert A. Lessons from two prevalent amyloidoses-what amylin and
Aβ have in common. Front Aging Neurosci. 2013
Lim YA, Rhein V, Baysang G, Meier F, Poljak A, Raftery MJ, Guilhaus M, Ittner LM,
Eckert A, Götz J. Abeta and human amylin share a common toxicity pathway via
mitochondrial dysfunction. Proteomics. 2010 10(8):1621-33
5) Neurochemistry and Molecular Pathology of Dementia
Investigating the neurochemistry molecular pathology and their clinical correlates in
dementia using tissue samples from well characterized clinical cohorts.
Recent publications
NE Mohamed, JH Lee, PT Francis, D Aarsland, CG Ballard, CP Chen, MKP Lai. Loss
of NMDA NR1 subunit immunoreactivity correlates with dementia severity in Lewy
Body dementias J Neural Transm 121 (2014) 71-8
MG Tan, CL Lee, JH Lee, PT Francis , R Williams, C Chen, PTH Wong, MJ Ramírez,
MKP Lai. Decreased rabphilin 3A immunoreactivity in Alzheimer’ Disease is associated
with Aβ burden Neurochemistry International 64 (2014) 29-36
Y Sheng, JH Lee, AD. Medhurst, GK. Wilcock, M Esiri, PTH. Wong , CP Chen, MKP.
Lai Cortical histamine H3 receptors in ischemic vascular and mixed dementias J of
Neurological Sciences 315 (2012) 110-4
6) The Neuropsychology of Dementia and Cognitive Impairment
Improving the diagnosis of Cognitive Impairment by prospective longitudinal clinical
studies, the multi-ethnic Epidemiology of Dementia in Singapore study and studies in
primary health care.
Recent publications
Dong Y, Slavin MJ, Chan BP, Venketasubramanian N, Sharma VK, Collinson SL,
Sachdev P, Chen CL. Improving screening for Vascular Cognitive Impairment at 3-6
months after Mild Ischemic Stroke and Transient Ischemic Attack. International
Psychogeriatrics 26 (2014) 787-93
Dong Y, Lee WY, Hilal S, Saini M, Wong TY, Chen C, Venketasubramanian N, Ikram,
MK. Comparison of Montreal Cognitive Assessment and the Mini-Mental State
Examination in detecting multi-domain Mild Cognitive Impairment in communitydwelling Chinese elderly. International Psychogeriatrics 25 (2013) 1831-8
YH Dong, DZQ Gan, SY Tay, WI Koay, SL Collinson, S Hilal, N Venketasubramanian,
Chen C. Patterns of neuropsychological impairment in Azheimer’s Disease and Mixed
Dementia. J of Neurological Sciences 333 (2013) 5-8
YH Dong, WS Pang, LBS Lim, YH Yang, JC Morris, S Hilal, N Venketasubramanian,
CLH Chen The informant AD8 is superior to participant AD8 in detecting cognitive
impairment in a memory clinic setting J Alzheimer's Disease 35 (2013) 159-68
Dr Deron Raymond Herr
phcdrh@nus.edu.sg
Investigating a novel molecular regulator of cochlear integrity and hearing loss
Our previous studies have identified a cell surface receptor that is essential to maintain
the organ of Corti in the inner ear in mice. Without this receptor, the organ of Corti
degenerates and the mice lose all sense of hearing and balance. We reason that activation
of this receptor can prevent the degeneration of the organ of Corti that normally occurs in
Updated in Apr 2015
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humans with aging, or exposure to noise or toxins. This work will use a series of
experiments in mice and in cell culture to understand the molecular pathways of cochlear
degeneration, and to test a novel drug compound for its ability to prevent hearing loss.
Dr Gautam Sethi
phcgs@nus.edu.sg
NF-κB and STAT3 are ubiquitously expressed transcription factors involved in cell
survival, adhesion, inflammation, and differentiation.
NF-κB and STAT3 are constitutively active in most cancers and responsible for the
transcription of various genes involved in initiation, promotion, and perpetuation of
cancer. Further, these proteins can be activated by most cancer treatment modalities and
mediates treatment resistance. Hence, the drugs that can suppress NF-κB/STAT3
activation have a great potential as effective therapeutics for treatment of various solid
cancers and hematological malignancies. Our group is therefore interested in
investigating the following major aims:
1. Molecular mechanism(s) of activation of transcription factors NF-κB/STAT3 by
carcinogens, tumor promoters, infectious and inflammatory agents.
2. How tumor cells express constitutive active NF-κB/STAT3, evade apoptosis and
develop resistance to chemotherapeutic agents and radiation?
3. Identification of novel pharmacological compounds that can suppress dysregulated
NF-κB/STAT3 activation in tumor cells/animal models and patient samples.
4. Potential use of these novel compounds to overcome chemoresistance and
radioresistance in xenograft and transgenic cancer models.
Adjunct A/Prof Goh Boon
Cher
phcgbc@nus.edu.sg
Biofluid-based Exosomal Molecules (microRNA/protein) as Biomarkers for Use in
Personalized Medicine
Exosomes are small (30–120 nm) membrane-bound vesicles containing nucleic acid and
protein cargo involved in immune response, antigen presentation and intercellular
communication, either supporting or perturbing (in the case of cancer) different
physiological processes. Exosomal microRNAs and proteins are emerging as promising
biomarkers in the detection of several cancers. However much of their roles and functions
are not well elucidated. Our group is therefore interested in the discovery of potential
exosomal microRNAs and proteins that are suitable to be used as biomarkers for
diagnosis and chemotherapy treatment prediction so as to improve detection of cancer at
early stage and increase survival rate of cancer patients.
The graduate project will aim to identify and validate i) potential diagnostic biomarker
candidates by characterizing the differential profiles of exosomal microRNAs and
proteins from cancer patients and healthy volunteers; ii) potential predictive biomarker
candidates by examining the exosomal microRNAs and proteins expression profiles of
cancer patients before and after receiving chemotherapy. In addition, we will investigate
the cellular functions regulated by these biomarker candidates and determine the
underlying molecular mechanisms leading to the changes in cellular behavior.
Prof Peter Lobie
csipel@nus.edu.sg
phcpel@nus.edu.sg
Updated in Apr 2015
Role of secreted oncogenic proteins in breast cancer and development of
therapeutics to such
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List of research projects available for prospective graduate students
The aim of our projects is to determine the biology, mechanisms of action and potential
use as therapeutic targets in breast cancer of a number of secreted oncogenic proteins.
The expression of these proteins are highly associated with patient survival, play specific
roles in tumours of the female reproductive system and are of potential translational
importance. A range of techniques are utilized to address the biology of these proteins. I
would be happy to discuss further with interested students.
Prof Philip K Moore
dprmpk@nus.edu.sg
Research in this laboratory centres upon an evaluation of the physiological and
pathophysiological roles of naturally occurring gases most notably hydrogen sulfide.
Current areas of interest are the vasculoprotective effect of H2S and related sulfur
compounds with particular reference to their role in inflammation, atherosclerosis and as
vasodilator agents with additional effects on blood cell/endothelial cell interactions. This
laboratory also has a keen interest in the role of this intriguing gas in both ageing and
cancer. An additional aim is the discovery and further development of novel
therapeutically relevant drugs based on the delivery of biologically active sulfur species.
Dr Pieter Eichhorn
csipjae@nus.edu.sg
Identification and roles of Dubiquitinating enzymes in the TGFB pathway
TGF-β is essential for embryogenesis and tissue homoeostasis in multicellular organisms.
Furthermore, in advanced cancers TGF-β can act as an oncogenic factor and according to
growing clinical evidence, TGF-β can be considered a therapeutic target in cancer.
Ubiquitin modification of the TGF-β signaling pathway is emerging as a key mechanism
of TGF-β pathway control. However, the role of deubiquitinating enzymes (DUBs),
which mediate the removal and processing of ubiquitin, is less well understood. I am
interested in studying and elucidating the roles of DUBs in the TGF-β pathway.
A/Prof WS Fred WONG
phcwongf@nus.edu.sg
NUS-Hebrew University at Jerusalem Joint PhD program
Role of MITF and TFE3 in Allergy and Asthma
and
Prof Ehud RAZIN
ehudr@ekmd.huji.ac.il
PhD project focuses on the regulation of gene transcription in mast cells due to their
interaction with their growth factors and due to their trigger by immunological stimulus.
Prof RAZIN mainly investigates the role played by the transcription factors MITF and
TFE3 in immune related diseases such as asthma, allergy and others. Prof WONG’s
research interests are in drug discovery for the treatment of asthma and COPD. Their
research has been published in top peer-reviewed journals such as Immunity, J Clin
Invest, Blood, Mol Cell, Am J Respir Crit Care Med, J Allergy Clin Immunol, and others.
Recently, the large gaps that existed in our understanding of mast cell signaling have
started to be closed. This is due to the various mouse models which are extremely useful
in our understanding of these signaling pathways in human mast cells. The main aim of
the PhD project is to discover the definitive role played by mouse mast cells in asthma
and allergic disease models. We propose here a multi-disciplinary research approach for
exploring the function of mast cells in asthma. The great advantage of Prof RAZIN’s
participation is that he will be able to produce most of the tools for studying these
networks of proteins using both cell biology and molecular biology approaches.
Updated in Apr 2015
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List of research projects available for prospective graduate students
Department of Physiology
A/Prof Christopher Ang
Beng Ti
gmsabt@duke-nus.edu.sg
beng_ti_ang@nni.com.sg
Neuro-Oncology Research Proposal
Sialylation and Tumor Cell Invasiveness
Brain tumors are among the most devastating of cancers, with a mean survival
period of just 15 months upon diagnosis (Louis et al, Acta Neuropathol, 2007).
Currently, clinicians rely on mainly histology to guide treatment decisions. In line with
international efforts, we recently showed that resistance to current chemotherapeutic
drugs and tumor cell invasiveness can be attributed to a self-renewing, cellular fraction
within the tumor mass (Koh et al, Antioxid Redox Signal, 2013; Ng et al, Clin Cancer
Res, 2012). This tumor heterogeneity is the reason that brain tumors are notoriously
difficult to treat. We further showed that patient genetic profiles can be mapped to such
gene activation profiles enriched for stem-like properties. Our proposal here will focus on
one such important gene activation program, regulated by a sialylation enzyme, ST3Gal1.
We recently demonstrated that ST3Gal1 expression marks a persistent cellular fraction,
and is highly enriched in mesenchymal molecular features, typical of aggressive and
recurrent tumors. Our orthotopic animal model established from patient cells further
demonstrated that ST3Gal1 knockdown abrogated tumor-initiating and -sustaining
capability (under review).
Moving forward, we will focus on signaling mechanisms that mediate ST3Gal1
activity. Our lab has expertise to interrogate patient clinical databases, and we identified
several methylation hallmarks that potentially regulate ST3Gal1. This is a significant
endeavor as DNA methylation accounts for up to 70% of tumor profiles (Noushmehr et
al, Cancer Cell, 2010). We will test such methylation hallmarks in vitro and in animal
models. Our lab deals with several techniques such as primary cell culture, biochemical
and molecular biology approaches and flow cytometry. Histology forms a significant
thrust of our analyses, along with mouse models that recapitulate the patient
heterogeneous spectrum. Our study is unique as we rely on patient genomic content to
guide our bench experiments. The successful candidate will be exposed to crossdisciplinary approaches to address clinically important questions in glioma biology.
References
Updated in Apr 2015
1.
Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer
BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central
nervous system. Acta Neuropathol 114: 97-109
2.
Koh LW, Koh GR, Ng FS, Toh TB, Sandanaraj E, Chong YK, Phong M, TuckerKellogg G, Kon OL, Ng WH, Ng IH, Clement MV, Pervaiz S, Ang BT, Tang CS
(2013) A distinct reactive oxygen species profile confers chemoresistance in gliomapropagating cells and associates with patient survival outcome. Antioxid Redox
Signal 19: 2261-2279
3.
Ng FS, Toh TB, Ting EH, Koh GR, Sandanaraj E, Phong M, Wong SS, Leong SH,
Kon OL, Tucker-Kellogg G, Ng WH, Ng I, Tang C, Ang BT (2012) Progenitor-like
Traits Contribute to Patient Survival and Prognosis in Oligodendroglial Tumors.
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List of research projects available for prospective graduate students
Clin Cancer Res 18: 4122-4135
4.
Prof Carlos Ibanez
phscfi@nus.edu.sg
Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Berman BP, Pan
F, Pelloski CE, Sulman EP, Bhat KP, Verhaak RG, Hoadley KA, Hayes DN, Perou
CM, Schmidt HK, Ding L, Wilson RK, Van Den Berg D, Shen H, Bengtsson H,
Neuvial P, Cope LM, Buckley J, Herman JG, Baylin SB, Laird PW, Aldape K
(2010) Identification of a CpG island methylator phenotype that defines a distinct
subgroup of glioma. Cancer Cell 17: 510-522
Growth Factor Receptor Signaling and Biology in Nervous System Development and
Metabolic Regulation
With a focus on growth factor receptor signalling, the aim of this research is to discover
novel, general biological principles and mechanisms important for nervous system
development and metabolic regulation.
We plan to concentrate the main thrust of our future work on two programmes that
complement and expand current areas of expertise. The first one focuses on the
mechanisms of action and biological activities of the death receptor p75 NTR (p75
neurotrophin receptor), and reflects our continued interest in neuronal growth factors,
their signalling mechanisms and their potential for the development of novel therapies for
neurodegenerative diseases and neurotrauma. This is a program that involves signal
transduction studies, mouse genetics approaches, and a drug discovery effort to identify
novel p75NTR inhibitors.
The second program focuses on the role of a novel regulatory network, formed by
members of the transforming growth factor-beta (TGFreceptors, in the control of glucose homeostasis, fat accumulation and energy balance. It
is based on our original discovery of the TGFrecent studies on the functions of activin signalling through ALK7 and ALK4 in
pancreatic islets, adipose tissue, and hypothalamus. This program offers a new inroad to
the study of metabolic regulation and involves complementary studies in cell culture as
well as conditional and knock-in mouse models.
A/Prof Herbert Schwarz
phssh@nus.edu.sg
How does the cytokine receptor CD137 contribute to the pathogenesis of Hodgkin
lymphoma?
Hodgkin’s lymphoma (HL) is a cancer of the lymphatic system, and one of the most
common cancers among the young adults. No effective therapies exist for HL. HL is
characterized by an extensive tumor stroma which is essential for the persistence and
pathogenesis of HL. This tumor stroma is induced by the malignant cells in HL, the
Hodgkin Reed-Sternberg (HRS) cells, which are a small minority among the cells of the
tumor stroma that consists mainly of infiltrating leukocytes. It is largely unknown by
which mechanisms HRS cells cause HL.
Recently we found that the tumor necrosis factor receptor family member CD137 is
ectopically expressed by HRS cells, but not by corresponding healthy cells (Cancer
Research. 73(2):652-61, 2013). We have identified several changes induced by CD137 in
HRS cells that may cause or enhance the malignancy of HL. These chances involve the
Updated in Apr 2015
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List of research projects available for prospective graduate students
recruitment of leukocytes that form the tumor stroma, the secretion of growth factors for
HRS cells, and escape from immune surveillance.
The project will characterize the contribution of CD137 to the pathogenesis of HL, and
aim at identifying novel immunotherpeutic approaches for therapy.
Prof Edward H. KOO
phskhme@nus.edu.sg
Molecular mechanisms of neuronal injury or neurodegeneration
The Laboratory for Molecular Neurodegeneration at YLL School of Medicine was
established by Dr. Ling Shuo-Chien and Prof. Edward Koo in late 2013 when we
first joined the NUS faculty. Our research focus is on the mechanisms of
neurodegenerative diseases, a group of brain disorders that is often age-associated
and includes Alzheimer and Parkinson diseases, amyotrophic lateral sclerosis,
frontotemporal dementia, among others. We take a cell and molecular biology
approach to our studies with an emphasis on animal models and translational
research. My laboratory’s philosophy is to study both the “normal and abnormal
biology” of genes and proteins that are implicated in disease pathogenesis. In
many instances, mutations are found in genes with unknown function and which
require thorough investigations of the basic functions of these genes and proteins
before we can appreciate how their dysfunction contributes Alzheimer disease
where we have concentrated on both basic and translational studies as well as
experimental therapeutics. Ongoing and future projects include:
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Adjunct A/Prof Paula Lam
Yeng Po
cmrlyp@nccs.com.sg
paula_lam@nuhs.edu.sg
Updated in Apr 2015
Mechanisms of synaptic injury in Alzheimer disease
Role of caspase cleavage of the amyloid precursor protein (APP) in
neurodegeneration
Mechanisms of tau mediated synaptic dysfunction
Investigations into mechanisms of neuronal vulnerability in Alzheimer
disease
Contribution of blood-brain barrier to neurodegeneration
Neurobiological basis of cognitive loss due to chemotherapy treatment in
cancer patients (“chemobrain”)
Elucidating the mechanism of action of interleukin-13 receptor alpha 2 in normal
and cancer cells
Interleukin 13-receptor alpha 2 (ILregarded as a decoy receptor in retarded signaling via interleukin 13-receptor alpha 1.
Recently, IL-13 has been shown to signal though the IL-1dependent manner to trans-activate the TGFβ-1 promoter in macrophages and monocytes.
The enhanced TGF-β1 levels can lead to fibrosis in some situations. The ILsignaling is also associated with tumor growth by preventing the engagement of CD8positive T cells in tumor immune surveillance. In this project, we aim to study the
crosstalk activities between IL-
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proliferation and metabolism. Furthermore, we are also keen to explore targeting ILntion of inflammation-associated fibrosis and cancer progression.
We will use cell and gene therapy in relevant animal models to evaluate the efficacy of
targeted therapy, standard biochemical and molecular approaches will be used to study
the various functions of this receptor.
Dr Lee Chi Wai
phsleec@nus.edu.sg
Cellular and molecular
neuromuscular diseases
mechanisms
underlying
neurodevelopment
and
Synapses are specialized cell membrane domains that facilitate neuronal communication
in the intricate nervous system. These synaptic specializations develop in response to
molecular interactions between pre- and postsynaptic cells. A major goal of current
research in developmental neuroscience is to elucidate the mechanisms underlying how
synapses are assembled. The nerve-muscle synapse, neuromuscular junction (NMJ),
which controls all muscle movements, has been considered as the best model for the
study of synaptogenesis due to its large size, simplicity and accessibility. When neurons
and muscle cells are cultured together, functional NMJs are formed spontaneously. The
structure and physiology of mature vertebrate NMJs are well understood. Currently, our
laboratory specifically focuses on the signal transduction and cytoskeletal mechanisms
underlying synapse development, disease, and regeneration. Three major areas are being
pursued in our lab: (1) postsynaptic receptor trafficking in the pathogenesis of muscular
dystrophy, (2) cytoskeletal dynamics in neuronal growth cones during axonal outgrowth
and pathfinding, and (3) axonal trafficking of mitochondria in synaptic formation,
function, and elimination. Using the simple and elegant Xenopus primary culture system,
a variety of techniques, including live-cell time-lapse fluorescence microscopy, superresolution microscopy, molecular biology, immunocytochemistry, and Western blotting
will be applied to these experimental systems to gain understanding to the cellular and
molecular mechanism of synaptic development. Our goal is to not only gain a mechanistic
understanding of the molecular and cellular aspects of neuronal structure and function,
but also provide insights into the cellular basis for neurological disorders.
A/Prof Lina Lim
phslhkl@nus.edu.sg
Control of PPARγ and NFκB by Annexin-A1 in inflammatory macrophages and
cancer cells
Inflammation occurs through dynamically varying levels of pro- and anti- inflammatory
cytokines competing for an upper hand either by activation of signalling cascades or
inhibition of downstream signals. NFKB is a master transcription factor involved in the
transcription of a number of pro-inflammatory as well as anti-inflammatory genes which
could control inflammation and resolution of inflammation. Natural NF-κB inhibitors
exist that would assist to regulate the production of cytokines during inflammation. One
such natural inhibitor of NF-κB is PPAR-γ, which has been shown to be antiinflammatory in a number of models of inflammation. In the following investigation, we
will define the regulation of PPAR-γ by another anti-inflammatory molecule we have
been working on for more than 10 years, Annexin-1. Preliminary studies in our lab that
has linked ANXA1 as a potential PPAR-γ regulator in cancer cells. As PPAR-γ is a
known natural NF-κB inhibitor acting through NFkB p65/RelA subunit, and since
Updated in Apr 2015
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List of research projects available for prospective graduate students
ANXA1 was shown to interact tightly and regulate upstream elements of NF-κB signaling
pathway, this project will focus on the regulation of PPAR-γ by ANXA1 in the context of
inflammatory macrophage function and cancer and will answer important questions
related to PPAR-γ agonist use clinically in the treatment of inflammatory diseases.
Annexin-A1: a host factor modulating influenza virus replication
The influenza virus infects millions of people each year and can result in severe or even
fatal complications. Understanding host responses to influenza infection will enable the
development of more effective anti-viral therapies. The host immune system recognizes
viral RNA via specific receptors and intracellular sensors that directly activate anti-viral
immune responses. Previous research has revealed diverse yet important roles for
Annexin family proteins in modulating the course of influenza infection. However, the
role of Annexin-A1 (ANXA1) in influenza virus infection has not been addressed.
ANXA1 is increased in nasal swab samples obtained from influenza infected patients. In
addition, presence of ANXA1 increases virus replication and results in more weight loss
after virus infection. We therefore hypothesize that ANXA1 may play a critical role in
host anti-viral responses. To test this hypothesis, we propose to assess how ANXA1
expression and activation in different cell types can influence the immune response to
influenza in vivo and to determine the molecular role played by ANXA1 in the innate
immune response to influenza. These data will enhance our understanding of disease
pathogenesis and may lead to the identification of novel targets for anti-viral
immunotherapies.
Dr Ling Shuo-Chien
phsling@nus.edu.sg
Mechanisms of Neuronal
Neurodegeneration
and
Synaptic
Dysfunctions
in
Aging
and
Aging poses both a fascinatingly biological question and a growing medical problem as
aging is the leading risk factor for age-associated diseases, many of which are late adultonset neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s
disease (PD), Frontotemporal dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS,
also known as motor neuron disease). The current goal of my lab is to define and
understand the processes underlying normal and pathological brain aging by elucidating
the underlying molecular and cellular mechanisms and how perturbation of the at-risk
systems cause premature failing that leads to neurodegeneration.
The overall strategy is to use disease-related genes as molecular handles to probe the
gene-phenotype relationship systematically in mice that are genetic mimics of human
diseases during aging and neurodegeneration with top-down and bottom-up approaches.
Specifically, we focus on two RNA-binding proteins, TDP-43 (TAR DNA-binding
protein 43 KDa) and FUS/TLS (fused in sarcoma/translocated in liposarcoma), whose
mutations are causal for ALS and FTD and inclusions of both proteins are the defining
pathological hallmarks in the majority of ALS and FTD patients. On the top-down
approach, we will interrogate molecular and synaptic changes in selective brain regions
and in neuronal populations as well as the contribution of non-neuronal neighboring cells
by combining mouse genetics with genome-wide quantitative methodologies. On the
bottom-up approach, we will reconstruct an in vitro neural network by building a
mechanically- and chemically-defined microfluidic system to analyze at a single cell
resolution as well as to manipulate with temporal and spatial precision. By integrating
neuroscience with leading edge technology, we expect that we will identify quantitative
Updated in Apr 2015
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List of research projects available for prospective graduate students
changes in gene expression that are critical for initiating and/or accelerating agedependent neurodegeneration.
The current projects in my laboratory will continue to use an integrated approach to
address the following critical issues: (1) how does disease-causing mutations provoke
neurodegeneration; (2) why specific neuron types are more susceptible to these mutations,
and (3) how do different cell types, such as astrocytes and oligodendrocytes, contribute to
disease progression. The students will learn various techniques, ranging from molecular,
cellular, biochemical, genetic and physiological levels as well as making use of many
cutting-edge techniques, such as high-throughput sequencing, microfluidic platform,
super-resolution imaging and single cell analysis. There will be opportunities to integrate
with bioinformatic-computational as well as nanotechnological and engineering scientists.
The long-term goal of our laboratory is to revert the molecular and cellular alternations to
rescue neuronal damages and ultimately to slow down aging and subsequent
neurodegeneration.
Prof Shazib Pervaiz
phssp@nus.edu.sg
Redox Regulation of protein phosphatase PP2A in carcinogenesis
Over the years, our work has highlighted the critical role of an altered redox metabolism
on cell survival and death signaling in cancer cells. Using a variety of model systems such
as drug-induced apoptosis, receptor mediated death signaling, and oncogene-induced cell
survival, we demonstrated that the intracellular ratio between the two main reactive
oxygen species (ROS), superoxide and hydrogen peroxide (O2 - :H2O2), determines
cancer cell response to death signals; a tilt in favor of superoxide promotes cell survival
whereas an increase in hydrogen peroxide favors death execution via activation of the
death promoting protein Bax. Of note, we have highlighted a novel biological activity of
Bcl-2 by providing experimental evidence linking Bcl-2-induced increase in
mitochondrial superoxide levels to the anti-apoptotic activity of Bcl-2. Interestingly, an
elevated O2 - :H2O2 ratio induced by either pharmacological inhibition (DDC) or gene
knockdown of Cu/Zn SOD resulted in an increase in phosphorylation of Bcl-2,
specifically at Ser70 (S70), and this site specific phosphorylation of Bcl-2 enhanced the
anti-apoptotic activity of Bcl-2, thereby rendering cancer cells resistant to chemotherapyinduced apoptosis. To that end, we have uncovered a novel mechanism in which an
increase in intracellular O2 - endows cancer cells with a survival advantage via tyrosine
nitration-mediated detachment of B56δ from PP2A catalytic core, and the eventual
accumulation of S70 phosphorylated Bcl-2 with potent anti-apoptotic activity. The
mechanism of redox modulation of PP2A in the context of carcinogenesis is under
investigation.
Specific targeting of mutant K-Ras expressing cancers via Aktdependent ROS
production
This project involves investigating the molecualr mechanism(s) underlying the activity of
a novel small molecule compound against mutant Kras expressing human cancers.
Identification of a novel protein TMTC2 in death receptor sensitization of human
cancers
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Our preliminary studies indicate that ligation of the death receptors DR4 and DR5
(TRAIL R1 and TRAIL R2) upregulates the expression of a novel transmembrane protein
TMTC2. Upregulation of TMTC2 increases death receptor sensitivity while its gene
knockdown inhibited TRAIL-mediated cell death in human nasopharyngeal carcinoma
cell lines. The sub-cellular localization of TMTC2 appears to place it at the ER, however
this has not been validated in more than one cell lines. Notably, TMTC2 induction
appears to be under the influence of intracellular reactive nitrogen species (RNS). The
mechanism of induction, protein structure and localization as well as the death sensitizing
activity of TMTC2 is under study.
Identification of surrogate death signaling pathways in cisplatinresistant human
cancer cells
Platinum-based compounds are the main line of treatment for a number of clinical
cancers. However, development of drug resistance remains a therapeutic challenge. We
have generated cisplatin-resistant clones from human lung and ovarian carcinoma cell
lines. These cells become resistant to most chemotherapeutic drugs, thus suggesting a
MDR phenotype. Although, the mechanism of this drug resistance is not clearly
understood, we have made a remarkable observation during the course of these studies, i.e
that cells that become resistant to cisplatin become highly sensitive to death receptormediated apoptosis. The mechanism of activation of this surrogate death signaling in the
context of cisplatin resistance is under investigation.
Mechanism of statin-induced apoptosis in cancer cells
We recently demonstrated that the cholesterol lowering drug, simvastatin, induces
apoptosis in human colorectal and breast carcinoma cells. We identified a critical role for
intracellular ROS and downstream JNK activation in statin-induced apoptosis.
Interestingly, we showed a massive induction and activation of Rho family of proteins,
Rac1, Rho and cdc42 in statin treated cells, upstream of ROS production. Of note, the
Rho family of proteins is prenylated despite their localization in the cytoplasm as opposed
to the plasma membrane. The mechanism of this non-membrane dependent prenylation as
well as ROS production in this model is under investigation.
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Dr Wong Boon Seng
Role of prion protein in breast cancer cell metabolism?
boon_seng_wong@nuhs.edu.sg
There is growing interest exploring the differential susceptibility of malignant versus
normal cells to energy metabolism to identify new cancer drug targets. Cancer cells gain
growth advantages by shifting cellular metabolism to aerobic glycolysis, a process known
as the Warburg effect. Accumulating evidence has linked mutations and altered
expression of several metabolic enzymes to this metabolic transformation. However, it is
unclear how these aberrant metabolic enzymes regulate this metabolic remodelling
process.
Prion protein (PrP) is well studied for its pathogenic role in prion diseases. Interestingly,
increasing PrP expression was shown to promote the process of invasiveness and
metastasis in several cancer types. But, little is known about the mechanisms underlining
this association.
While PrP pathogenic role in human prion diseases is known to be modulated by the
polymorphism at codon 129, this is not a risk factor in colorectal cancer. So, what is the
effect of other known mutations on human PrP? How does PrP modulate cancer growth?
One possibility is activating the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and other
associated signalling pathways that lead to increased glucose uptake. This suggests a
functional role for PrP in regulating in insulin signalling and possibly triggers the
metabolic transformation (Warburg effect).
In this project, we will examine the role of PrP in this metabolic remodeling process. We
will examine how PrP expression regulates the pathogenic cross-talking between tumour
energy metabolism and insulin signalling at the cellular and tissue levels. We will also
determine the functional effect of known human PrP mutations on cancer cell growth and
metabolism. Lastly, we will explore the effect of two different metabolic targeting anticancer drugs, metformin and dichloroacetate, on PrP function in regulating cancer growth
and metabolism in vitro and in vivo.
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A/Prof Celestial T. Yap
phsyapc@nus.edu.sg
To investigate the roles of cytoskeletal proteins in oncogenic signaling and cell
survival
Gelsolin (GSN) is an actin-associated cytoskeletal protein involved in binding and
severing actin filaments, thus controlling cytoskeletal turnover in response to cellular
signals (eg. migration, apoptosis). Gelsolin has also emerged as a new player in pathways
regulating signaling and gene transcription. We previously uncovered a novel role of
gelsolin in promoting invasion of colorectal tumour cells by enhancing extracellular
matrix breakdown, through the upregulation of urokinase plasminogen activator (uPa).
We aim to ascertain if specific oncogenic signaling pathways interact with gelsolin to
influence cancer cell behaviour. Our preliminary analysis of microarray data from human
breast cancer tissues and in vitro work on cell lines showed a correlation between gelsolin
expression with various oncogenic signaling pathway modulators, suggesting possible
involvement of gelsolin in regulating oncogenic signaling. The specific signaling
pathways identified to have potential interactions with gelsolin are known to induce
aggressive tumour cell behavior, including invasion and epithelial-mesenchymal
transformation. Gelsolin knockdown by siRNA decreases the expression of target genes
controlled by oncogenic signaling, while gelsolin overexpression increases signaling
activity. In addition, we found that induction of specific oncogenic signaling in breast
cancer cells also increased the expression of gelsolin, suggesting that the activities of
gelsolin may be recruited by certain oncogenic signaling pathways in a feedback loop to
enhance their target effects. Collectively, these data suggest novel regulatory roles of
gelsolin in signaling pathways that promote cancer progression. We will confirm the
interactions between gelsolin and the specific signaling pathways identified by our
laboratory, as well as examine the significance of these interactions on cancer cell
behavior and clinical outcomes.
Department of Psychological Medicine
Prof Kua Ee-Heok
pcmkeh@nus.edu.sg
Dr Feng Lei
pcmfl@nus.edu.sg
Diet and Healthy Aging in Asian Elderly
The diet and healthy aging study aims to identify dietary factors that are associated with
physical, cognitive and mental health among community-living Singaporean elderly.
Dietary measures include a locally validated Food Frequency Questionnaire (FFQ) and 3day food records. Health outcomes are self-rated health, chronic medical conditions,
physical performance (hand grip and walking speed), cognitive performance, subjective
cognitive impairment, sleep quality, depressive symptom and anxiety symptoms. We
have recruited seven hundred senior Singaporeans aged 60 and above from a
geographically defined area in Jurong district of Singapore. We plan to conduct a followup assessment to quantify cognitive decline among individuals who are identified as
normal cognitive aging or major and mild neurocognitive disorders at baseline. This study
is the first to systematically examine the relationship between local diet and mental health
in old age.
Choral singing for the prevention of dementia: a randomized controlled trial
Aims: To evaluate the efficacy and mechanisms of choral singing in the prevention of
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dementia and examine the underlying neural mechanisms of the expected efficacy using
Magnetic Resonance Imaging (MRI) technique.
Hypotheses: Choral singing could prevent cognitive decline among community-dwelling
elderly who are at high risk of dementia. The underlying neural mechanisms involve the
changes in brain structure and function that can be quantified using MRI technique.
Study design: randomized controlled trial. Sample size: 200 in total
Treatment Arms: (1) Intervention arm: weekly choral singing for 2 years; (2) Control
arm: weekly general health education and group activities for 2 years
Primary outcome measures: Cognitive decline measured by the Mini-Mental State
Examination (MMSE) and the Montreal Cognitive Assessment (MoCA); Test scores from
a standard neuropsychological test battery: Rey Auditory Verbal Learning Test, Digit
Span, Color Trails Test, Block Design, Verbal Fluency, Symbol Digit Modality Test.
Fasting venous blood samples will be collected, processed and stored for future analysis.
A/Prof Ng Tze Pin
pcmngtp@nus.edu.sg
Vascular factors are strongly implicated to play aetiological roles in the development of
neurocognitive disorders (NCD) due to Alzheimer’s disease (AD) or vascular disease
(VD). Traditional vascular-related factors such as hypertension, obesity, diabetes,
smoking and physical activity are well studied biomarkers of the risk of NCD due to AD
and VD. Novel vascular biomarkers based on measurement of arterial thickness and
stiffness (such as intima-media thickness, carotid-femoral pulse wave velocity, central
aortic waveform and pressure), endothelial function, and circulating molecular
biomarkers including NTproBNP, ST-2, GDF15, procollagen-III, urocortin 1, urotensin
II, troponin, signal peptides are potential candidate biomarkers for assessing risk, severity
and prognosis of NCD due to AD and VD. This research project will involve the
assessment of neurocognitive disorders among participants in the Singapore Longitudinal
Ageing Studies (SLAS) and clinical vascular measurements to explore and validate the
use of novel vascular biomarkers of cognitive function in NCD.
Dr Roger Ho Chun Man
pcmrhcm@nus.edu.sg
Neuropathic pain is a chronic pain condition resulting from nerves injury costing 40
billion annually in USA. One of the common clinical causes is spinal nerve root
compression due to intervertebral disc extrusion. Nerve root compression cause severe
back and leg pain affecting patient’s quality of life. Based on Yeo SN et al. epidemiology
study, 9% of Singapore population suffers from chronic pain leading to burden the
society. Even though MRI can show radiological changes in multiple prolapsed discs, it
fails to localize pain generator. Selective diagnostic nerve root block is common clinical
practice to sort out pain generators. However it is far from satisfactory due to diffusion
problem, which may lead to misdiagnosis and wrong level surgery. Pain persists
postoperatively even following “successful” surgical decompression. Unresolved case can
also be caused by changes in central nervous system and underlying psychological
factors. Up to now, pain is subjective; it cannot be visualized by existing diagnostic tool.
From our previous rodent neuropathic pain study, a few matrix metalloproteinase are
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significant up-regulated in affected dorsal root ganglion. We will develop a probe
targeting matrix metalloproteinase to visualize pain generator.
Our group will provide training for neuropathic pain model surgery, behavior study and
probe characterization technique.
Department of Obstetrics & Gynaecology
Prof Ariff Bongso
obgbongs@nus.edu.sg
1) Evaluation of the uses of umbilical cord Wharton’s jelly stem cells and its
extracts for cell based therapies, cord blood banking, anti-tumorigenesis and wound
healing.
2) Development of bioengineered tissue constructs using Wharton’s jelly stem cells
and nanofibrous scaffolds for tissue repair.
Dr B Srilatha
obgsb@nus.edu.sg
1) Molecular and functional characterization of hydrogen sulphide effects in the
area of sexual medicine (as a main PI)
Hydrogen sulphide (H2S) as a potential treatment for male and female sexual dysfunction
has been indicated. This project is aimed to understand the modus operandi of H2S with
the known neuromodulators and second messenger systems at the cellular level in sexual
system.
2) Clinical prevalence and associated factors of erectile dysfunction in uremia (as a
co-PI)
Erectile dysfunction is a common feature of uremia and it can have a strong negative
effect on life quality. In view of its high prevalence in uremia, this project is aimed to
estimate the magnitude of this problem in the adult uremic male patients in Singapore and
elucidate its genesis in this population.
A/Prof Chong Yap Seng
obgcys@nus.edu.sg
1) The role of epigenetic pathways present in umbilical cord derived mesenchymal
stem cells in fetal programming of metabolic disease
Co-supervisor:
Dr Walter Stunkel
walter_stunkel@sics.astar.edu.sg
It has been shown that an adverse intrauterine environment can largely influence the risk
for non-communicable diseases later in life. Maternal gestational diabetes (GDM) and
intrauterine growth restriction are correlated with increased adolescent type 2 diabetes,
obesity and cardiovascular disease in the offspring. As birth weight per se does not fully
explain obesity in the offspring of diabetic mothers, we speculate that epigenetic
transgenerational programming is a leading cause for the increased metabolic disease risk
susceptibility in these children.
The project involves work with established primary cell lines from umbilical cord
(Mesenchymal Stem Cells, MSCs and Human Umbilical Vein Endothelial Cells,
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HUVECs). The cells were taken from growth restricted neonates, as well as those with
maternal GDM background and matched with controls.
The student will apply state of the art laboratory technologies for assessing the
transcriptome, as well as the epigenetic state of MSCs by analyzing genome wide DNAmethylation and histone modifications. MSC lines will be differentiated into various cell
types such as adipocytes, myocytes and beta-like cells. The goal of the project will be to
functionally characterize candidate genes which may play an important role in the
development of metabolic diseases. These efforts may lead to novel biomarker
hypotheses for patient stratification, as well as potentially supporting the identification
and validation of new therapeutic targets.
A/Prof Chong Yap Seng
obgcys@nus.edu.sg
2)
Maternal diet during pregnancy and infant neurodevelopment
Omega 3 fatty acids such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are
important to foetal and infant growth and development. Deficiencies and imbalances of
Co-supervisors:
long chain polyunsaturated fatty acids (LCPUFAS including omega 3 and omega 6 fatty
Dr Anne Rifkin
anne_rifkin@sics.a-star.edu.sg acids) are associated with impairments in cognitive and behavioural performance.
While some RCTs during pregnancy suggest that prenatal DHA status might have subtle
Dr Mary Chong
mary_chong@sics.a-star.edu.sg positive effects on neurodevelopmental and behavior outcomes (Jensen CL, 2005;
Cheatham CL, 2011, Drover JR, 2011), these beneficial effects have not been consistently
replicated in other good quality studies (Dunstan JA, 2008; Judge MP, 2007; Makrides M,
2010). This is said to be in part due to the methodological limitations of the study and
also the genetic heterogeneity in fatty acid metabolism.
In addition, compared to their Western counterparts, little is also known about these
associations in Asian populations. Research on the associations between maternal dietary
patterns during pregnancy and the effects on infant neurodevelopment is also sparse.
This research will use data from the GUSTO (Growing Up in Singapore Towards healthy
Outcomes) mother-offspring cohort study in Singapore and the largest in Asia. It is a
longitudinal study designed to understand the developmental origins of health and disease
from an Asian perspective. A sub-cohort of 500 pregnant women were recruited during
the first trimester of pregnancy and followed up regularly throughout pregnancy and the
nutrition and neurodevelopment of the child tracked from birth to 8 years of age. The
existence of the three major ethnic groups (Chinese, Malay and Indians) in the local
Singapore population provides an ideal backdrop for a study to investigate the
associations of maternal omega 3 fatty acids on infant neurodevelopment and how this
may be modified by differential polymorphisms across ethnic groups.
Research aims: With this, research projects would be undertaken to examine the
following hypotheses:
1) What are the associations between maternal plasma LCPUFAs and infant
neurodevelopmental outcomes in early life (0 -2 yrs)?
2) Do FADS gene variants among individuals and possibly across ethnic groups modify
the effects of maternal plasma LCPUFAs and infant neurodevelopment?
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3) What are the associations between maternal dietary patterns and other nutrient intakes
on infant neurodevelopmental outcomes in early life (0 -2 yrs)?
Dr Citra Nurfarah bte Zaini
Mattar
obgcnzm@nus.edu.sg
Investigating efficacy and safety of the dual approaches of intrauterine gene transfer
and haematopoietic stem cell transplant for fetal treatment of thalassaemia major in
a knock-out murine model.
A/Prof Fong Chui Yee
obgfong@nus.edu.sg
1) Evaluation of the uses of umbilical cord Wharton’s jelly stem cells and its
extracts for cell based therapies, cord blood banking, anti-tumorigenesis and wound
healing.
2) Development of bioengineered tissue constructs using Wharton’s jelly stem cells
and nanofibrous scaffolds for tissue repair.
3) Applications of Wharton’s jelly stem cells in regenerative medicine.
Dr Gong Yinhan
obggy@nus.edu.sg
1) Chromatography tandem mass spectrometry research program: Development
and application of safe, fast and ultra-sensitive chromatography tandem mass
spectrometry technologies, including LC-MS/MS and GC-MS/MS, to determine
trace level of bioactive compounds in complicated biological samples.
2) Chiral drug separation research program: Preparation and application of novel
chiral stationary phases with high enantio-selectivities for high-performance liquid
chromatography and capillary electrochromatography to separate and purify
enantiomers of a wide range of chiral drug compounds.
A/Prof Mahesh Choolani
obgmac@nus.edu.sg
1) Non-invasive prenatal diagnosis using fetal erythroblasts derived from first
trimester maternal blood.
2) Enchancing enrichment of fetal primitive erythroblasts from maternal blood by
identification of unique fetal surface antigens and the development of a novel MEMS
device to deplete adult anucleate erythroyctes.
3)
Human fetal messenchymal stem cells for intrauterine treatment of
mucopolysaccharidoses.
4) Rapid diagnosis of common chromosomal aneuploides (chromosomes 13, 18, 21,
X and Y) using quantitiative fluorescent polymerase chain reaction.
5) Ultra-rapid prenatal diagnosis using uncultured amniocytes.
6) Characterisation of low molecular weight tumor specific markers for ovarian
cancer from serum of ovarian cancer patients.
7) Protein pattern based early diagnosis of epithelial ovarian cancers.
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8) Development of diagnostic kits for early detection of ovarian cancers.
Prof P Ganesan Adaikan
obgadaik@nus.edu.sg
Endothelial progenitor cells as a novel therapeutic option in vasculogenic ED
Erectile dysfunction (ED) is an increasingly common disease, afflicting both young and
old men. The problem is exacerbated by aging population, as well as changes in diet and
lifestyle. Metabolic diseases, a major risk factor in ED, are also on the rise.
Pathophysiological studies demonstrate that ED in these patients stem from compromised
vascular function leading to ischaemic cavernosa, and resultant loss of erectile function.
Consequently, existing palliative drugs, such as sildenafil, that are unable to restore
vascular function are also ineffective for reversing or curing ischaemic ED found in
patients with metabolic disease.
Based on our interesting pilot study, it was hypothesised that the injection of endothelial
progenitor cells (EPC) are expected to facilitate the formation of de novo vasculature in
the ischaemic penile tissue in animal models of ED and improve vascular networks and
restore erectile function through increased perfusion, as well as re-innervation.
The project involves validation of penile ischemia and vasculogenic erectile dysfunction
(ED) in rabbit and rodent models of metabolic diseases: (1) characterization of subpopulations of EPC to stimulate angiogenesis and support nervous networks. (2)
evaluation of EPC therapy in reversing endothelial and erectile dysfunction in animal
models of vasculogenic ED. Results arising from this study is expected to be useful for
cardiovascular diseases.
Prof Yong Eu Leong
obgyel@nus.edu.sg
Icaritin for the treatment of Androgen-Deprivation Therapy induced osteoporosis in
prostate cancer animal model
Androgen-deprivation therapy (ADT) by gonadotropin-releasing hormone (GnRH)
agonists is the mainstay of treatment for progressive prostate cancer. However, ADT is
associated with bone loss and an increased risk of fracture. Prostate cancer is a disease of
the elderly and thus, older men are at great risk when receiving androgen suppression
therapy. Denosumab, a newly FDA-approved monoclonal antibody against NF-kB, was
found to enhance bone mineral density and reduce the fracture probability in men
receiving androgen-deprivation therapy for prostate cancer. However, osteonecrosis of
the jaw occurred in 2.3% of the Denosumab treated group. The prenylflavone icaritin, a
phytoestrogen consumed in East Asian societies, has been widely shown to remarkably
improve the bone density and thus effective in treating osteoporosis. In addition, icaritin
was found in our previous study to suppress androgen receptor (AR) positive prostate
cancer by AR proteolysis through ubiquitin-proteasomal pathway. In consideration of the
unique dual effect of icaritin on the prostate cancer and bone health, it has an intrinsic
advantage over Denosumab on osteoporosis induced by ADT.
In this project, we hypothesize the combination of GnRH agonist and icaritin may relieve
the severe osteoporosis in prostate cancer animal model receiving androgen-deprivation
therapy and synergistically improve the androgen-deprivation therapy by masking AR.
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Department of Orthopaedic Surgery
Dr Angelo All
dosaha@nus.edu.sg
Induced Human Oligodendrocyte Precursor Cell Transplants for Improved
Functionality after Spinal Cord Injury
We propose to directly convert adult human fibroblasts into "induced
human oligodendrocyte precursor” cells and to study the remyelination capability of these
cells both in vitro and in vivo. We will generate patient-specific cells in a shorter time
frame than iPS cells as direct conversion does not require reprogramming to base cells.
These newly generated cells would also have significantly reduced risk of immune
rejection even when compared to properly differentiated OPs from currently available
human iPS cell lines. The goal of this project is to study whether these
cells remyelinate demyelinated axons in the hostile post-injury spinal cord microenvironment in rats. We will also test the cells’ myelin production capabilities in a microchamber in vitro.
Hypothermia after Spinal Cord Injury: Early Markers of Recovery
Spinal cord injury (SCI) is a devastating condition that can lead to paralysis of the limbs
below the injury level. Beyond the initial trauma of injury, the secondary phase of injury
comprises inflammation, demyelination and apoptosis, which are all major pathological
factors that exacerbates the progression of injury. Since mitigating this secondary phase
of injury greatly improves patient outcomes, it has motivated the search for an
early neuroprotective therapy. In animal models, primary focus has been on the effects of
post-SCI hypothermia on motor behavioral and histological outcomes with only a few
studies of the electrophysiological function of descending spinal cord pathways during
cooling. Thus, there is a critical need for establishing the full benefits of
hypothermic neuroprotection after SCI. Unlike other studies, our unique focus is on the
hypothermia-induced enhancements of the afferent sensory conduction, a vital function of
the sensory-motor system, as assessed by multi-limb somatosensory evoked potentials.
The cooling will be followed by a single recording session to acquire local field potentials
(LFPs) and multi-unit activity (MUA) from the dorsal pathways in the vicinity of the
injury, followed later by somatosensory evoked potentials (SSEPs), which are cortical
response waveforms drawn out by peripheral stimulation. The acute microelectrode
recordings will be used to develop an early statistical marker to identify animals with
maximal long-term recovery of SSEPs. The standard motor behavioral scoring (BBB) and
histopathology will be used to provide complementary measures of recovery. The goal of
this project is to use the contusive rat model of SCI, followed by an early, local cooling of
the injured region, to optimize a hypothermia treatment process that can adequately
mitigate the pathological factors in the second phase of injury.
Transplantation of Conjugated Nanoparticles to Limit Spinal Cord Injury
We propose to synthesize and administer conjugated nanoparticles and study their
capability to limit spinal cord injury (SCI) in a rat model. This treatment approach will
enable us to spare more healthy axons and neuropathways within the spinal cord
parenchyma and slow down progress of injury after trauma. This is important as SCI is a
time-sensitive pathology with a very short window of treatment. The goal of this proposal
is to generate a new generation of nanoparticles, which will be safe and effective in
preventing progress of trauma in our in vivo model of spinal cord injury.
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Crosstalk among Neural Pathways after Incomplete Spinal Cord Injury
Spinal Cord Injury (SCI) in the pediatric population is fairly rare but bears huge
socioeconomic consequences. Patients with SCI before their adolescence have different
mechanisms of injury and have a better neurological recovery potential for incomplete
and mild to severe injuries when compared to adults. Although the SCI is usually
diagnosed with the use of MRI, often pediatric SCI are without radiological anomaly.
There is also no evidence for the use of neuroprotective approaches for the treatment of
SCI in children. There is the need for a better determination of the mechanism of SCI at
the epicenter of injury. An understanding of the mechanisms that help promote the
improved neurological recovery observed in pediatric patients would also enhance future
therapies for all SCI patients. This project deals with incomplete SCI and the study of
post-contusion electrophysiological changes in the vicinity of the injury for pediatric
demographics. The goal of this proposal is to characterize functional reorganization
of neuropathways on the neuronal reconnection formation at the epicenter of contusion
induced SCI.
Behavioral Assessment after Spinal Cord Injury
Spinal Cord Injury (SCI) is a serious condition that severely impairs mobility and quality
of life with no treatment options. After injury, some axons are anatomically continued but
not functional. These neurons are electrically excitable cells and they are the focus of our
treatment in the acute phase. One approach to treat SCI is through limiting and preventing
the destruction of healthy neurons in the spinal cord parenchyma, thus aiding functional
recovery. A set of motor behavioral assessments, such as BBB open field locomotion test,
thermal sensation, rotorod, tactile allodynia and balance beam will be proposed to
thoroughly investigate the onset of injury as well as long-term progress of injury. We will
study phenotypic outcomes and the possible limitations during the progress of injury.
These assessments will be statistically analyzed and compared with the histological
examination as well as our previous electrophysiological assessments.
Prof James Hui
doshuij@nus.edu.sg
Best in class: Enhanced self renewal and therapeutic potential of adult human
mesenchymal stem cells.
Adult stem cell based therapies are an evolving pillar of clinical medicine for treating
conditions where body tissue needs to be replaced, repaired or regenerated. However, the
clinical use of adult stem cells relies on them being isolated from a patient’s bone marrow
and then grown in hospital laboratories until there are enough cells for re-plantation. This
process is costly and requires lengthy cell culture strategies that adversely affect the
quality of the patient’s stem cells. Our project is based on molecules that improve the
microenvironment of cultured stem cells to that we can produce ‘best in class’ stem cells
for clinical use.
Cartilage tissue engineering with mesenchymal stem cells
Tissue engineering (musculoskeletal) strategy requires a combination of three crucial
components: appropriate cell source, scaffolding/carrier matrix, and the temporal and
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spatial delivery of bioactive agents. With the lack of tissue derived mature cell, stem
cells, especially mesenchymal stem cell (MSC), due to its ease of use, proliferative
competence and the capability to differentiate to a variety of musculoskeletal lineages,
has emerged as the prospective cell source in tissue engineering. Our research proposal is
aimed to investigate the interplay of the biochemical and structural and biomechanical
aspects of the microenvironment that influences the initiation of the stem cell
differentiation and its subtle functional outcome specifically to the zonal and phenotypic
of the derived tissue. Combinatory effects of selected positive micro environmental cues
in a complex structural environment, and develop novel pre-implantation differentiation
protocols (2 approaches will be tested initially). In situ delivery of the bioactive factors in
a spatial and temporal control would be assessed. Further more in vitro exploration of an
appropriate controlled delivery method in combination with a suitable scaffold vehicle
would be tested. In vivo validation of the engineered tissue constructs in animal (rabbit or
porcine) cartilage defect models would be assessed.
Prof Lee Eng Hin
dosleeeh@nus.edu.sg
Effect of substrate patterning on the chondrogenic differentiation of mesenchymal
stem cells.
The main focus of our lab is the optimization of chondrogenesis towards a normal
cartilage phenotype for cartilage tissue engineering. Mesenchymal stem cell (MSC)
differentiation is influenced by its microenvironment. Manipulation of extracellular
biophysical and/or biochemical microenvironment of stem cell can improve the efficiency
of tissue engineering approaches using MSCs. We have identified specific biochemical
and topographical cues for the directed differentiation of MSCs to specific cartilage
phenotype. However, the mechanisms induced by these microenvironmental cues have
not yet been fully understood. The project will investigate the effect of incorporating
specific biochemical and/or physical cues in the substrate to achieve the desired lineage
and phenotype specification of the differentiated cells. The study will aim to elucidate the
mechanotransduction mechanisms that affect specific functional outcomes, including the
optimisation of the manipulation conditions, and validation of the approach in in vivo
animal model. Primary stem cell culture techniques and molecular biology analysis will
be employed in these studies, including immunohistochemistry, confocal microscopy,
scanning electron microscopy, real-time PCR and Western Blot.
References:
 Wu Y, Law JBK, He AY, Low HY, Hui JHP, Lim CT, Yang Z, Lee EH. Substrate
topography determines the fate of chondrogenesis from human mesenchymal stem cells
resulting in specific cartilage phenotype formation. Nanomedicine: NBM 2014 (in
press).
 Raghothaman D, Leong MF, Lim TC, Toh JKC, Wan ACA, Yang Z, Lee EH.
Engineering cell matrix interactions in assembled polyelectrolyte fiber hydrogels for
mesenchymal stem cell chondrogenesis. Biomaterials 2014 Mar;35(9):2607-16.
A/Prof Wilson Wang
doswang@nus.edu.sg
Updated in Apr 2015
Characterization and In Vivo Assessment of a Novel Patented Process to
Functionalize Antibiotic Bone Cements with Nanostructured Materials for
Enhanced Antimicrobial Activity
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The aim of this project is to synthesize and characterize novel bone cement using a
patented bioengineering approach to enhance antimicrobial activity in bone cement, using
conjugation of MSN and antibiotics to bone cement, with the aim of achieving prolonged
antimicrobial activity against orthopaedically-important bacteria. We will be assessed
antibacterial activity in vitro/in vivo using small (rabbit)/large animal model, toxicity
against mammalian cells and changes to mechanical properties.
Evaluation of Magnetic Resonance Image (MRI)-Based Novel 3-Dimensional (3-D)
Printed Scaffold Meniscus to Replicate and Replace Native Meniscus in an Ovine
Model: In Vivo Studies of Morphology, Function, Viability and Response
The knee menisci are fibro-cartilaginous crescent-shaped wedges that are essential to
knee joints in load bearing, shock absorption and joint stability. Their morphology and
internal structure are crucial to their normal functioning. Meniscal injuries are common in
both younger individuals (sports injuries) as well as older patients (degenerative tears),
and increase the risk of post-traumatic knee osteoarthritis. Current treatment modalities
(meniscectomy, repair) are often suboptimal due to poor healing potential and disruption
of normal structure, while allograft transplantation is limited by mismatch and availability
issues.
This clinical need has prompted research in new scaffold technologies for meniscal
replacement, but current techniques do not recreate the internal microstructure crucial to
meniscal function. Our group has developed a novel patented 3-D printing process using
drop-on-demand micro-dispensing technology that can create multi-material meniscal
scaffolds with morphology and microstructural patterns similar to native menisci. We
believe this specific advantage in being able to replicate internal microstructural
orientations is a breakthrough in bioscaffold technology that will enhance the chances of
long-term bio-implant success.
Our 3-D printed cell-laden polymer/collagen meniscal scaffolds have been characterized
in vitro for material and biomechanical properties and biological responses, with
promising results. The proposed present investigation aims to develop this technology
further, using an in vivo ovine model; we aim to validate the customized 3-D replication
of meniscal morphology by MRI-based scaffold printing in a large animal model. The
meniscal scaffolds (without and with autogenous cell seeding) will be surgically
implanted in the sheep knees, and assays performed for knee function and complications,
biocompatibility and inflammatory response (with sham surgery and meniscectomy
controls). After euthanasia and knee harvesting at time intervals, further evaluations will
be made for biomechanical function and histological, cellular and molecular changes.
This project aims to support the downstream development of customized meniscal
implants in clinical applications.
Prof Wong Hee Kit
doswhk@nus.edu.sg
Bone regeneration using NELL-1, a non-BMP2 osteoinductive growth factor,
delivered by novel carriers in a lumbar spinal fusion model
Bone regeneration in spine is a common technique used to treat trauma, low back pain
and deformities. With the aging population, the number of spinal fusion surgeries
performed annually is increasing significantly, which adds to the health care burden
globally. The use of autologous bone graft, gold standard for spinal fusion, was limited by
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associated complications such as donor site pain and unsatisfactory fusion rate, with
reported failure of fusion of up to 45%
Osteobiologics were introduced as substitutes for autologous bone graft for bone
regeneration. Bone Morphogenetic Protein-2 (BMP-2) is one of most potent bone growth
factor approved by FDA for human spinal fusion procedure. However, safety concerns
from complications associated with BMP-2 usage were raised recently. There is still an
unmet need to develop a safe and efficient bone regeneration device.
Due to the adverse effects reported recently that are associated with the BMP-2 usage in
clinical situation, there is a need to substitute BMP-2 with new and more specific bone
regeneration growth factors such as NELL-1. NELL-1, one of the new osteobiologics
under investigation, is known to be more specific in bone regeneration than BMP-29. It
has been shown that it could induce bone formation in vivo models from rodents to large
animals without significant complications.
We hypothesize that localization and controlled release of a non-BMP-2 based growth
factor, NELL-1 in one or both of our two growth factor carriers, PEC and BNPP, will
improve the efficiency and efficacy of bone regeneration in spinal fusion. This effect will
be evaluated in small and large animal lumbar interbody fusion models. If successful, this
study will introduce a new generation of non-BMP based bone graft substitute consisting
of NELL-1 matched with an optimal carrier that will significantly improve the clinical
outcome of spinal fusion procedure.
Department of Otolaryngology
A/Prof Wang De Yun
entwdy@nus.edu.sg
Quantitative assessment of the virulence of rhinoviruses, influenza viruses and
respiratory syncytial viruses and host defense mechanisms of human nasal
epithelium in vitro
The viral upper respiratory infections (URIs), including common cold, influenza and
acute rhinosinusitis, are very common disease, affecting millions of people annually.
They can be caused by any of the more than 200 different strains of viruses, such as
rhinovirus, coronavirus, respiratory syncytial viruses (RSV), influenza, parainfluenza and
adenoviruses. With the SARS pandemic of 2003 and that of H1N1 in 2009, global
concerns was raised, stemming from virulent new strains of viruses such as corona virus
(or SARS virus), H1N1, H5N1, which appear to be more promiscuous and circulate in
several species and are endemic in humans, birds and pigs. In addition to continuous
efforts in surveillance of naturally occurring viruses in humans and domestic animal
hosts, experimental studies elucidating susceptibilities to respiratory viruses and response
to infection by human nasal epithelium, which is the primary target site for common cold
and influenza viruses, are required. In this study, we aim to develop an in vitro model for
screening and quantitative assessment of the virulence of common cold and influenza
viruses, and the host defense mechanisms of human nasal epithelium using our newly
developed in vitro experimental models of human nasal epithelial stem/progenitor cells
(hNESPCs) and differentiated epithelial cells derived from hNESPCs in an air-liquid
interface (ALI) culture. In addition, we will investigate the host defense functions
including viral fusion and uncoating, transport of viral ribonucleoprotein (RNP)
complexes from nucleus, replication, transcription and translation of the viral genome,
export of viral RNP complexes from the nucleus, viral assembly and budding, and
potential drug targets, which will provide us with a useful reference for public health risk
assessment, interventions, disease control and prognosis for viral upper respiratory
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infections. This information is particularly useful in preparation for eventual pandemics
or outbreak of viral infection.
Department of Surgery
1. Euroscore I and II comparison
Dr Ooi Oon Cheong
oon_cheong_ooi@nuhs.edu.sg
A comparison of the accuracy of predicting adverse events in cardiac surgery
between Euroscore I and Euroscore II
2. Reno score
A statistical analysis to identify negative prognostic indicators for renal failure after
cardiac surgery in present-day Asian population
3. Stress in cardiac surgery
A questionnaire-based project to estimate the prevalence and severity of emotional
stress among present-day cardiac surgery patients
4. Quality of life in adult cardiac patients
A questionnaire-based project to assess the quality of life among present-day cardiac
surgery patients before and after the operation
A/Prof Stephen Chang Kin
Yong
cfscky@nus.edu.sg
CARE – Cord-stem cell Application For Regeneration Enhancement, Pre-Clinical
Large Scale Porcine Study
Specific Aims& Hypothesis
We would further validate the biological efficacy of hepatic-like cloned Cord Lining
Epithelial Cells (CLEC) in promoting liver regeneration through a well powered large
scale large animal study and to acquire scientific evidence from this pre-clinical trial.
The hypothesis is Hepatic-like cloned CLEC can promote liver regeneration and abate
liver failure in pigs who have undergone extensive hepatectomy.
Background & Clinical Significance
There are approximately 750,000 new cases of liver cancer diagnosed globally in 2008 or
5.9% of all cancers (excluding non-melanoma skin cancer)16 Liver cancer is the third
most common cause of death from cancer worldwide, estimated to be responsible for
nearly 700,000 deaths in 2008.17
To date, liver resection remains the main treatment modality for effective eradication and
cure for liver cancers localized to the liver. However, the disease burden at presentation
is often extensive and to fully resect all the cancer in the liver, there may not be adequate
residual liver volume. In addition, such cancers are often found in livers with a
background of liver cirrhosis and insufficiency which further limit the amount of
resection that can be performed safely. Aggressive liver cancer eradication via liver
resection must thus be substantiated by measures that promote liver regeneration early
and effectively.
Our preliminary studies on the porcine model using hepatic like CLEC has shown
promising results but more experiments would have to be conducted to gather substantial
scientific evidence that this treatment modality is safe and is an effective means of
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enhancing liver regeneration.
This would potentially enable early medical intervention opportunities for patients who
have gone through extensive liver resection and in the verge of liver insufficiency and
possibly for patients dying from liver failure from other causes.
Detection of RNA editing/mutation in peripheral blood of hepatocellular carcinoma
patients for early diagnosis
Aim: This study aims to to identify the aberrant RNA editing events in peripheral blood
of HCC patients for early diagnosis and correlate RNA editing patterns to clinical and
pathological features of HCC patients for prognostic and therapeutic relevance.
Methods: We will extract total RNAs from buffy coat blood samples of normal
individuals and HCC patients, followed by the reverse transcription and PCR. The
purified PCR products will be subjected to Sanger sequencing or pyrosequencing for
measuring the RNA editing frequency of specific sites. The correlation between the RNA
editing frequencies of 5 known editing targets (AZIN1, COPA, COG3, TTPA and FLNB)
and clinico-pathological features will be analyzed in HCC patients.
Importance: Until now, AFP has been used for many years as a serum/plasma marker for
HCC diagnosis and screening. However, it has been recognized that AFP levels often
increase even in the absence of HCC (chronic hepatitis or cirrhosis). Therefore, there is an
urgent need for identifying new biomarkers for diagnosing HCC. So far, there is no study
focusing on RNA editing/mutation in the blood of HCC patients.
Potential benefits & risks: For early diagnosis, there are many obvious advantages to a
peripheral marker in terms of risks, cost, and ability to monitor individuals over time.
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