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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.
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
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.
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.
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.
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.
Distribution of faecal indicator bacteria in tropical waters of Peninsular Malaysia and their decay rates in tropical seawater

Wong YY, Lee CW, Chai SCY, Lim JH, Bong CW, Sim EUH, et al.

Mar Pollut Bull, 2022 Dec;185(Pt A):114297.
PMID: 36327936 DOI: 10.1016/j.marpolbul.2022.114297

We investigated the appropriateness of faecal indicator bacteria in tropical waters. We compared total coliform (undetectable to 7.2 × 105 cfu 100 mL-1), faecal coliform (undetectable to 6.1 × 105 cfu 100 mL-1) and enterococci (undetectable to 3.1 × 104 cfu 100 mL-1) distribution in Peninsular Malaysia. Faecal indicator bacteria was highest in freshwater, and lowest in seawater (q > 4.18, p
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.
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.
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
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.
Identification of dual active sites in Caenorhabditis elegans GANA-1 protein: an ortholog of the human α-GAL a and α-NAGA enzymes

Cheong CSY, Khan SU, Ahmed N, Narayanan K

J Biomol Struct Dyn, 2023 Jul;41(11):5261-5276.
PMID: 35694994 DOI: 10.1080/07391102.2022.2084162

Fabry disease (FD) is caused by a defective α-galactosidase A (α-GAL A) enzyme responsible for breaking down globotriaosylceramide (Gb3). To develop affordable therapeutics, more effort is needed to obtain insights into the underlying mechanism of FD and understanding human α-GAL A structure and function in related animal models. We adopted C. elegans as a model to elucidate the sequence and 3D structure of its GANA-1 enzyme and compared it to human α-GAL A. We constructed GANA-1 3D structure by homology modelling and validated the quality of the predicted GANA-1 structure, followed by computational docking of human ligands. The GANA-1 protein shared sequence similarities up to 42.1% with the human α-GAL A in silico and had dual active sites. GANA-1 homology modelling showed that 11 out of 13 amino acids in the first active site of GANA-1 protein overlapped with the human α-GAL A active site, indicating the prospect for substrate cross-reaction. Computational molecular docking using human ligands like Gb3 (first pocket), 4-nitrophenyl-α-D-galactopyranoside (second pocket), α-galactose (second pocket), and N-acetyl-D-galactosamine (second pocket) showed negative binding energy. This revealed that the ligands were able to bind within both GANA-1 active sites, mimicking the human α-GAL A and α-NAGA enzymes. We identified human compounds with adequate docking scores, predicting robust interactions with the GANA-1 active site. Our data suggested that the C. elegans GANA-1 enzyme may possess structural and functional similarities to human α-GAL A, including an intrinsic capability to metabolize Gb3 deposits.Communicated by Ramaswamy H. Sarma.
Improved DNA Delivery Efficiency of Bacterial Vectors by Co-Delivery with Exogenous Lipid and Antimicrobial Reagents

Osahor AN, Narayanan K

Methods Mol Biol, 2021;2211:15-27.
PMID: 33336267 DOI: 10.1007/978-1-0716-0943-9_2

Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.
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.
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.
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.
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.
Large BACs transfect more efficiently in circular topology

Wong YC, Osahor A, Al-Ajli FOM, Narayanan K

Anal Biochem, 2021 10 01;630:114324.
PMID: 34363787 DOI: 10.1016/j.ab.2021.114324

The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.
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.
Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.

ACS Synth Biol, 2023 Apr 21;12(4):909-921.
PMID: 37026178 DOI: 10.1021/acssynbio.2c00580

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

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