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Lui Y.L.E.,Center for Biomedical and Life science | Lui Y.L.E.,Queensland University of Technology | Tan E.L.,Center for Biomedical and Life science | Tan E.L.,National University Hospital Singapore
Journal of Virological Methods | Year: 2014

Hand, foot and mouth disease (HFMD) is a contagious viral disease that frequently affects infants and children and present with blisters and flu-like symptoms. This disease is caused by a group of enteroviruses such as enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However, unlike other HFMD causing enteroviruses, EV71 have also been shown to be associated with more severe clinical manifestation such as aseptic meningitis, brainstem and cerebellar encephalitis which may lead to cardiopulmonary failure and death. Clinically, HFMD caused by EV71 is indistinguishable from other HFMD causing enteroviruses such as CA16. Molecular diagnosis methods such as the use of real-time PCR has been used commonly for the identification of EV71. In this study, two platforms namely the real-time PCR and the droplet digital PCR were compared for the detection quantitation of known EV71 viral copy number. The results reveal accurate and consistent results between the two platforms. In summary, the droplet digital PCR was demonstrated to be a promising technology for the identification and quantitation of EV71 viral copy number. © 2014 Elsevier B.V.

Issac T.H.K.,National University of Singapore | Tan E.L.,National University Hospital Singapore | Tan E.L.,Center for Biomedical and Life science | Chu J.J.H.,National University of Singapore
Journal of Proteomics | Year: 2014

Chikungunya virus (CHIKV) is an arthropod-borne, positive-sense, single-stranded RNA virus belonging to genus Alphavirus and family Togaviridae. The clinical manifestations developed upon CHIKV-infection include fever, myositis, arthralgia and maculopapular rash. Thus, the re-emergence of CHIKV has posed serious health threats worldwide. Due to the fact that myositis is induced upon CHIKV-infection, we sought to understand the dynamic proteomic regulation in SJCRH30, a human rhabdomyosarcoma cell line, to gain insights on CHIKV pathogenesis. Two-dimensional gel electrophoresis (2DE) in combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to profile differential cellular proteins expression in CHIKV-infected SJCRH30 cells. 2DE analysis on CHIKV-infected cells has revealed 44 protein spots. These spots are found to be involved in various biological pathways such as biomolecules synthesis and metabolism, cell signaling and cellular reorganization. siRNA-mediated gene silencing on selected genes has elucidated the biological significance of these gene-translated host proteins involved in CHIKV-infection. More importantly, the interaction of vimentin with non-structural protein (nsP3) of CHIKV was shown, suggesting the role played by vimentin during CHIKV replication by forming an anchorage network with the CHIKV replication complexes (RCs). Biological significance: Chikungunya virus (CHIKV) is a re-emerging virus that has caused various disease outbreaks in Africa and Asia. The clinical symptoms of CHIKV-infection include fever, skin rash, recurrent joint paint, and myositis. Neuronal implications and death may be resulted from the severe viral infection. Up to date, there are no effective treatments and vaccines against CHIKV-infection. More importantly, little is known about the differential regulation of host proteins upon CHIKV infection, hence deciphering the viral-host cell interactions during viral infection provide critical information on our understanding on the mechanisms of virus infection and its dependency of host proteins for replication. In light of the muscle-related clinical manifestations of myositis resulting from CHIKV-infection, human rhabdomyosarcoma cells, SJCRH30 were utilized in this protein profiling study, in order to decipher the pathogenesis of CHIKV. This study has identified an arrays of host proteins that are differentially regulated upon CHIKV infection including that of the cytoskeletal protein, vimentin that plays significant role in aiding the replication of CHIKV within the host cells through 2DE assay. Immunofluorescence assay further shows that the novel interaction between cytoskeleton structure and CHIKV replication complex by forming an intercalating network around the replication complexes and facilitating various stages of the virus life cycle. This novel finding has inevitably led to a deeper understanding of CHIKV pathogenesis in revealing the importance of host proteins during CHIKV replication, as well as contributing to the development of specific antiviral strategies against this medically important viral pathogen. © 2014 Elsevier B.V.

Zhao F.,Center for Biomedical and Life science | Zhao F.,Advanced Materials Technology Center | Lee C.-L.K.,Center for Biomedical and Life science | Kwan W.L.,Advanced Materials Technology Center
Solar Energy Materials and Solar Cells | Year: 2011

Spray coating is a high throughput coating technique that is scalable and adaptable for organic photovoltaic manufacturing. To ensure uniform coating of the organic layers, the wettability, surface tension and boiling points of the solvents have to be optimized. Here, we used microscopic videos to understand the dynamics of the spray coating process. By optimizing the wettability and drying time of the PEDOT:PSS suspension on a hydrophobic surface, we attained a spray coated transparent anode without compromising on device performance. We further applied this vacuum-free process to a near infrared absorber to achieve a transparent organic solar cell with close to 60% transparency. © 2011 Elsevier B.V. All rights reserved.

Tan L.-M.,Center for Biomedical and Life science | Sem Z.-Y.,Center for Biomedical and Life science | Chong W.-Y.,Center for Biomedical and Life science | Hendra,National Engineering School of Mechanical and Aeronautical Engineering | Kwan W.L.,Singapore University of Technology and Design
Organic Letters | Year: 2013

We report the development of a heterogeneous catalyst system on continuous flow chemistry. A palladium (Pd) coated tubular reactor was placed in line with copper (Cu) tubing using a continuous flow platform, and a Sonogashira C-C coupling reaction was used to evaluate the performance. The reactions were favorably carried out in the Cu reactor, catalyzed by the traces of leached Pd from the Pd reactor. The leached Pd and Cu were trapped with a metal scavaging resin at the back-end of the continuous flow system, affording a genuine approach toward green chemistry. © 2012 American Chemical Society.

Lee J.J.,National University Hospital Singapore | Chow V.T.K.,National University of Singapore | Poh C.L.,University of Malaya | Tan E.L.,National University Hospital Singapore | Tan E.L.,Center for Biomedical and Life science
Journal of Proteomics | Year: 2011

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16. © 2011 Elsevier B.V.

Chen G.,Center for Biomedical and Life science | Zhu W.,CAS Shanghai Institute of Materia Medica | Zuo Z.,Curtin University Australia
Bioorganic and Medicinal Chemistry Letters | Year: 2010

Nowadays identification of novel non-peptide β-secretase (BACE-1, hereinafter) inhibitors with low cytotoxicity and good blood-brain barrier (BBB) property holds common interest of drug discovery for Alzheimer's disease. Twenty SPECS compounds were tested in BACE-1 FRET assays and methylthiazoletetrazolium (MTT) cytotoxicity experiment. Two compounds: 2 and 15 demonstrated IC50 values of 0.53 and 9.4 μM. In addition, 2 showed least toxic effect to the neuroblastoma cells. The results from both in silico and in vitro studies provided new pharmacophoric entities for chemical synthesis and optimization on the current discovered BACE-1 small molecule inhibitors. © 2010 Elsevier Ltd. All rights reserved.

Chen G.,Center for Biomedical and Life science | Zhu W.,CAS Shanghai Institute of Materia Medica | Zuo Z.,Curtin University Australia
Bioorganic and Medicinal Chemistry Letters | Year: 2010

A similarity search on the structural analogs of an inhibitor of BACE-1 with IC50 2.8 μM, which contained a P1 benzothiazole group together with a triazine ring linked by a secondary amine group, was described in this Letter and some more potent inhibitors against BACE-1 were identified. The most potent compound 5 (IC50 = 0.12 μM) increases the inhibitory potency by 24 folds. Our results suggest that a pyrrolidinyl side group at the P3′ and P4′ of the inhibitors are favored for strong inhibition and a small aromatic group at the P4 position is also essential to the potency. © 2010 Elsevier Ltd. All rights reserved.

Tan C.W.,University of Malaya | Chan Y.F.,University of Malaya | Sim K.M.,University Tunku Abdul Rahman | Tan E.L.,Center for Biomedical and Life science | Poh C.L.,Sunway University
PLoS ONE | Year: 2012

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6-9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71. © 2012 Tan et al.

Tan H.Y.,Monash University | Ng T.W.,Monash University | Neild A.,Monash University | Liew O.W.,Center for Biomedical and Life science
Analytical Biochemistry | Year: 2010

A major advantage of the image-based fluorescent microplate detection approach is the capability of using lower magnification to maximize the field of view so that color or intensity can provide a first step in isolating samples of interest in a high-throughput fashion. Lenses with low magnification typically have low numerical aperture, and consequently the effect of the point spread function and the liquid meniscus effect of small liquid volumes on standard microplates conspire to affect the intensity readings. We demonstrate this and show that the capillary well microplate design overcomes this limitation. © 2009 Elsevier Inc. All rights reserved.

Tan H.Y.,Monash University | Ng T.W.,Monash University | Neild A.,Monash University | Liew O.W.,Center for Biomedical and Life science
Journal of Biomolecular Screening | Year: 2010

The presence of bubbles in liquid samples residing in microplate wells causes inaccuracies in fluorescence measurements. In addition, pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results, particularly in the context of high-throughput screening. In this work, the authors describe an adapted design to the capillary wells microplate approach that permits side viewing. They demonstrate a prototype that detects bubbles and pipetting errors during actual assay runs to ensure accuracy in screening. © 2010 Society for Laboratory Automation and Screening.

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