Displaying publications 1 - 20 of 27 in total

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  1. Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium
    Sim EU, Ang CH, Ng CC, Lee CW, Narayanan K
    J Hum Genet, 2010 Feb;55(2):118-20.
    PMID: 19927161 DOI: 10.1038/jhg.2009.124
    Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.
  2. Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters
    Lee CW, Ng AY, Bong CW, Narayanan K, Sim EU, Ng CC
    Water Res, 2011 Feb;45(4):1561-70.
    PMID: 21146847 DOI: 10.1016/j.watres.2010.11.025
    Using the size fractionation method, we measured the decay rates of Escherichia coli, Salmonella Typhi and Vibrio parahaemolyticus in the coastal waters of Peninsular Malaysia. The size fractions were total or unfiltered, <250 μm, <20 μm, <2 μm, <0.7 μm, <0.2 μm and <0.02 μm. We also carried out abiotic (inorganic nutrients) and biotic (bacterial abundance, production and protistan bacterivory) measurements at Port Dickson, Klang and Kuantan. Klang had highest nutrient concentrations whereas both bacterial production and protistan bacterivory rates were highest at Kuantan. We observed signs of protist-bacteria coupling via the following correlations: Protistan bacterivory-Bacterial Production: r = 0.773, df = 11, p < 0.01; Protist-Bacteria: r = 0.586, df = 12, p < 0.05. However none of the bacterial decay rates were correlated with the biotic variables measured. E. coli and Salmonella decay rates were generally higher in the larger fraction (>0.7 μm) than in the smaller fraction (<0.7 μm) suggesting the more important role played by protists. E. coli and Salmonella also decreased in the <0.02 μm fraction and suggested that these non-halophilic bacteria did not survive well in seawater. In contrast, Vibrio grew well in seawater. There was usually an increase in Vibrio after one day incubation. Our results confirmed that decay or loss rates of E. coli did not match that of Vibrio, and also did not correlate with Salmonella decay rates. However E. coli showed persistence where its decay rates were generally lower than Salmonella.
  3. Crude protein extraction protocol for phage N15 protelomerase in vitro enzymatic assays
    Chen Q, Narayanan K
    Anal Biochem, 2011 Jul 1;414(1):169-71.
    PMID: 21396906 DOI: 10.1016/j.ab.2011.03.006
    The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TelN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TelN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes.
  4. Escherichia coli bactofection using Lipofectamine
    Narayanan K, Lee CW, Radu A, Sim EU
    Anal Biochem, 2013 Aug 15;439(2):142-4.
    PMID: 23608053 DOI: 10.1016/j.ab.2013.04.010
    Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.
  5. Induction of protein expression within Escherichia coli vector for entry into mammalian cells
    Chen Q, Lee CW, Sim EU, Narayanan K
    Hum Gene Ther Methods, 2014 Feb;25(1):40-7.
    PMID: 24134118 DOI: 10.1089/hgtb.2012.188
    Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.
  6. Short homologies efficiently generate detectable homologous recombination events
    Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K
    Anal Biochem, 2014 Oct 1;462:26-8.
    PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030
    When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
  7. Recombineering linear BACs
    Chen Q, Narayanan K
    Methods Mol Biol, 2015;1227:27-54.
    PMID: 25239740 DOI: 10.1007/978-1-4939-1652-8_2
    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
  8. Control of Attachment of Pseudomonas aeruginosa and Burkholderia cepacia to Surfaces by Shear Force
    Hui YW, Narayanan K, Dykes GA
    Water Environ Res, 2016 Nov 01;88(11):2040-2046.
    PMID: 26704787 DOI: 10.2175/106143016X14504669767292
      The effect of physical shearing on the attachment of six Pseudomonas aeruginosa strains and six Burkholderia cepacia strains to glass, stainless steel, polystyrene and Teflon® was determined. A significant (p < 0.05) decrease in hydrophobicity was apparent for all P. aeruginosa strains (17-36%) and B. cepacia, MS 5 (20%) after shearing. A significant (p < 0.05) decrease in attachment of some P. aeruginosa (0.2-0.5 log CFU/cm2) and B. cepacia (0.2-0.4 log CFU/cm2) strains to some surface types was apparent after shearing. Significant (p < 0.05) correlation was observed for both numbers of flagellated cells and hydrophobicity against attachment to glass, stainless steel and polystyrene for P. aeruginosa while only hydrophobicity showed significant correlation against the same surfaces for B. cepacia. Scanning electron microscopy and protein analysis showed that shearing removed surface proteins from the cells and may have led to the observed changes in hydrophobicity and attachment to abiotic surfaces.
  9. Fulltext Human Ribosomal Proteins RPeL27, RPeL43, and RPeL41 Are Upregulated in Nasopharyngeal Carcinoma Cell Lines
    Sim EU, Chan SL, Ng KL, Lee CW, Narayanan K
    Dis Markers, 2016;2016:5179594.
    PMID: 28018022 DOI: 10.1155/2016/5179594
    Apart from their canonical role in ribosome biogenesis, there is increasing evidence of ribosomal protein genes' involvement in various cancers. A previous study by us revealed significant differential expression of three ribosomal protein genes (RPeL27, RPeL41, and RPeL43) between cell lines derived from tumor and normal nasopharyngeal epithelium. However, the results therein were based on a semiquantitative assay, thus preliminary in nature. Herein, we provide findings of a deeper analysis of these three genes in the context to nasopharyngeal carcinoma (NPC) tumorigenesis. Their expression patterns were analyzed in a more quantitative manner at transcript level. Their protein expression levels were also investigated. We showed results that are contrary to previous report. Rather than downregulation, these genes were significantly overexpressed in NPC cell lines compared to normal control at both transcript and protein levels. Nevertheless, their association with NPC has been established. Immunoprecipitation pulldown assays indicate the plausible interaction of either RPeL27 or RPeL43 with POTEE/TUBA1A and ACTB/ACTBL2 complexes. In addition, RPeL43 is shown to bind with MRAS and EIF2S1 proteins in a NPC cell line (HK1). Our findings support RPeL27, RPeL41, and RPeL43 as potential markers of NPC and provide insights into the interaction targets of RPeL27 and RPeL43 proteins.
  10. Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis
    Osahor A, Deekonda K, Lee CW, Sim EU, Radu A, Narayanan K
    Anal Biochem, 2017 10 01;534:46-48.
    PMID: 28693990 DOI: 10.1016/j.ab.2017.07.008
    Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
  11. Fulltext The uS8, uS4, eS31, and uL14 Ribosomal Protein Genes Are Dysregulated in Nasopharyngeal Carcinoma Cell Lines
    Sim EU, Ng KL, Lee CW, Narayanan K
    Biomed Res Int, 2017;2017:4876954.
    PMID: 28791303 DOI: 10.1155/2017/4876954
    The association of ribosomal proteins with carcinogenesis of nasopharyngeal carcinoma (NPC) has been established in a limited subset of ribosomal protein genes. To date, three ribosomal protein genes, eL27 (L27), eL41 (L41), and eL43 (L37a), have been found to be differentially expressed in cell lines derived from NPC tumors. This raises the possibility of more ribosomal protein genes that could be associated with NPC. In this study, we investigated the expression profiles of eight ribosomal protein genes, uS8 (S8), uS4 (S9), eS31 (S27a), eL6 (L6), eL18 (L18), uL14 (L23), eL24 (L24), and eL30 (L30), in six NPC-derived cell lines (HONE-1, SUNE1, HK1, TW01, TW04, and C666-1). Their expression levels were compared with that of a nonmalignant nasopharyngeal epithelial cell line (NP69) using quantitative real-time PCR (RT-qPCR) assay. Of the eight genes studied, the expressions of four ribosomal protein genes uS8 (S8), uS4 (S9), eS31 (S27a), and uL14 (L23) were found to be significantly downregulated in NPC cell lines relative to NP69. Our findings provide novel empirical evidence of these four ribosomal protein genes as NPC-associated genetic factors and reinforce the relevance of ribosomal proteins in the carcinogenesis of nasopharyngeal cancer.
  12. Greener approaches to the measurement of polyaromatic hydrocarbons (PAHs) in unused and used crankcase motor oils from Malaysia
    Narayanan K, Miyagawa H, Kitano R, Nakagawa K, Hirooka M, Hashimoto S, et al.
    Environ Sci Pollut Res Int, 2018 03;25(8):7206-7211.
    PMID: 26387694 DOI: 10.1007/s11356-015-5403-9
  13. A polyol-stevia blended sugar replacer exhibits low glycemic response among human subjects
    Ng AWR, Loh KK, Gupta N, Narayanan K
    Clin Nutr ESPEN, 2019 10;33:39-41.
    PMID: 31451273 DOI: 10.1016/j.clnesp.2019.07.014
    BACKGROUND & AIMS: Consumption of sugars in food and beverages has increased at an alarming rate. While excessive daily sugar intake has been well-associated as the onset of medical complications, additional sugars are still used in manufactured food products just to satisfy the consumers' needs. Hence, there is a need to develop sugar replacers that have low glycemic response without compromising the organoleptic characteristics of food products. This study aimed to determine if SUITENA™, a novel sweetener containing erythritol, xylitol, and Stevia, has low glycemic response upon consumption by human subjects.

    METHODS: Six human subjects were randomly chosen and were healthy at the point of experimentation. Capillary blood was collected via finger-prick method to monitor the glycemic response of every individual for 90 min after ingestion of sugar solution.

    RESULTS: It was found that the mean area under the curve (AUC) of the dextrose standard was 11.8-fold higher (p 

  14. Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells
    Liew PS, Chen Q, Ng AWR, Chew YC, Ravin NV, Sim EUH, et al.
    Anal Biochem, 2019 10 15;583:113361.
    PMID: 31306622 DOI: 10.1016/j.ab.2019.113361
    Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
  15. Environmental control of Vibrio spp. abundance and community structure in tropical waters
    Wong YY, Lee CW, Bong CW, Lim JH, Narayanan K, Sim EUH
    FEMS Microbiol Ecol, 2019 11 01;95(11).
    PMID: 31688899 DOI: 10.1093/femsec/fiz176
    We measured Vibrio spp. distribution and community profile in the tropical estuary of Port Klang and coastal water of Port Dickson, Malaysia. Vibrio spp. abundance ranged from 15 to 2395 colony forming units mL-1, and was driven by salinity and chlorophyll a (Chl a) concentration. However, the effect of salinity was pronounced only when salinity was <20 ppt. A total of 27 Vibrio spp. were identified, and theVibrio spp. community at Port Dickson was more diverse (H' = 1.94 ± 0.21). However species composition between Port Dickson and Port Klang were similar. Two frequently occurring Vibrio spp. were V. owensii and V. rotiferianus, which exhibited relatively higher growth rates (ANCOVA: F > 4.338, P < 0.05). Co-culture experiments between fast- and slow-growing Vibrio spp. revealed that fast-growing Vibrio spp. (r-strategists) were overwhelmed by slower-growing Vibrio spp. (K-strategists) when nutrient conditions were set towards oligotrophy. In response to resource availability, the intrinsic growth strategy of each Vibrio spp. determined its occurrence and the development of Vibrio spp. community composition.
  16. A Self-Replicating Linear DNA
    Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K
    ACS Synth Biol, 2020 04 17;9(4):804-813.
    PMID: 32196315 DOI: 10.1021/acssynbio.9b00478
    TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.
  17. Influence of elevated river flow on hypoxia occurrence, nutrient concentration and microbial dynamics in a tropical estuary
    Lee CW, Lim JH, Heng PL, Marican NF, Narayanan K, Sim EUH, et al.
    Environ Monit Assess, 2020 Sep 25;192(10):660.
    PMID: 32975666 DOI: 10.1007/s10661-020-08625-3
    We sampled the Klang estuary during the inter-monsoon and northeast monsoon period (July-Nov 2011, Oct-Nov 2012), which coincided with higher rainfall and elevated Klang River flow. The increased freshwater inflow into the estuary resulted in water column stratification that was observed during both sampling periods. Dissolved oxygen (DO) dropped below 63 μM, and hypoxia was observed. Elevated river flow also transported dissolved inorganic nutrients, chlorophyll a and bacteria to the estuary. However, bacterial production did not correlate with DO concentration in this study. As hypoxia was probably not due to in situ heterotrophic processes, deoxygenated waters were probably from upstream. We surmised this as DO correlated with salinity (R2 = 0.664, df = 86, p  6.7 h), hypoxia could occur at the Klang estuary. Here, we presented a model that related riverine flow rate to the post-heavy rainfall hypoxia that explicated the episodic hypoxia at Klang estuary. As Klang estuary supports aquaculture and cockle culture, our results could help protect the aquaculture and cockle culture industry here.
  18. Inhibition of lysosomal vacuolar proton pump down-regulates cellular acidification and enhances E. coli bactofection efficiency
    Akinsola RO, Lee CW, Sim EUH, Narayanan K
    Anal Biochem, 2021 03 01;616:114088.
    PMID: 33358938 DOI: 10.1016/j.ab.2020.114088
    Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
  19. Fulltext The roles of ribosomal proteins in nasopharyngeal cancer: culprits, sentinels or both
    Sim EU, Lee CW, Narayanan K
    Biomark Res, 2021 Jun 30;9(1):51.
    PMID: 34193301 DOI: 10.1186/s40364-021-00311-x
    Ribosomal protein genes encode products that are essential for cellular protein biosynthesis and are major components of ribosomes. Canonically, they are involved in the complex system of ribosome biogenesis pivotal to the catalysis of protein translation. Amid this tightly organised process, some ribosomal proteins have unique spatial and temporal physiological activity giving rise to their extra-ribosomal functions. Many of these extra-ribosomal roles pertain to cellular growth and differentiation, thus implicating the involvement of some ribosomal proteins in organogenesis. Consequently, dysregulated functions of these ribosomal proteins could be linked to oncogenesis or neoplastic transformation of human cells. Their suspected roles in carcinogenesis have been reported but not specifically explained for malignancy of the nasopharynx. This is despite the fact that literature since one and half decade ago have documented the association of ribosomal proteins to nasopharyngeal cancer. In this review, we explain the association and contribution of dysregulated expression among a subset of ribosomal proteins to nasopharyngeal oncogenesis. The relationship of these ribosomal proteins with the cancer are explained. We provide information to indicate that the dysfunctional extra-ribosomal activities of specific ribosomal proteins are tightly involved with the molecular pathogenesis of nasopharyngeal cancer albeit mechanisms yet to be precisely defined. The complete knowledge of this will impact future applications in the effective management of nasopharyngeal cancer.
  20. An antibody binding-based fluorescent assay for the rapid quantification of globotriaosylceramide levels in human Fabry cells
    Ng AWR, Narayanan K
    Anal Biochem, 2021 09 01;628:114287.
    PMID: 34119486 DOI: 10.1016/j.ab.2021.114287
    Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb3). Here, we describe a microplate Gb3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb3 deposits from human biological samples, for example, from urine and blood.
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