Edmund Ui-Hang Sim

Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium.

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.

Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters.

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.

Recombination between linear double-stranded DNA substrates in vivo

Recombineering technology in Escherichia coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors.

Escherichia coli bactofection using Lipofectamine

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.

The uS8, uS4, eS31, and uL14 Ribosomal Protein Genes Are Dysregulated in Nasopharyngeal Carcinoma Cell Lines

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.

Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis

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.

Bioinformatics Analysis of the Ribosomal Proteins, RPL27, RPL37a and RPL41: 3-D Protein Modeling and Protein-protein Interaction Prediction

Ribosomal proteins (RPs) are constituents of ribosome important for protein biosynthesis but likely to have extraribosomal functions. Many RPs are associated with various diseases and cancers. A previous study reported RPL27, RPL37a and RPL41 gene to be downregulated in nasopharyngeal carcinoma (NPC) derived cell lines compared to their normal counterpart. However, their actual physiological roles in organogenesis or tumorigenesis have not been properly defined. In this paper, we report on the findings of structural prediction of these three genes and infer their interactions with other proteins using structural neighbor prediction and molecular docking strategies. Our results revealed that RPL27 interact with SYNJ2 and UBC9. RPL27 is predicted to mediate RNA binding protein and deregulate sumolyation. RPL37a is suggested to interact with CTNNB1, SCMH1 and ATBF1. It is predicted to deregulate Wnt degradation pathway, inhibit β-catenin migration and regulate homeotic transcription. Our studies on RPL41 did not allow logical inference on possible interacting factors. Nevertheless, results on RPL27 and RPL37a provide rational data for the elucidation of their molecular activities.

Increased serum levels of interleukin-6 and von Willenbrand Factor in early phase of acute coronary syndrome in a young and multiethnic Malaysian population

Objective Interleukin-6 (IL6; proinflammatory marker), von Willebrand Factor (vWF; endothelial dysfunction marker) and P-selectin (platelet activation marker), may play important roles in defining the pathogenesis of vulnerable plaques in acute coronary syndrome (ACS). This study aims to investigate the expression and relationship of these markers in early phases of ACS in a young and multiethnic Malaysian population. Design Peripheral whole blood mRNA, and serum levels of IL6, vWF and P-selectin were measured in 22 patients with ACS, and in 28 controls with angiographically significant coronary artery disease without previous ACS events. Venous blood from ACS patients was obtained within 1 h of hospital admission. Results No significant differences of IL6, vWF and P-selectin mRNA levels between ACS and controls were seen. ACS patients had significantly higher serum levels of IL6 and vWF (p<0.001), compared with controls. P-selectin correlated with IL6 (r=0.697, p=0.003) and vWF (r=0.497, p=0.05) at mRNA levels, indicating a possible association between these three indices of ACS pathogenesis. Conclusions Increased serum levels of IL6 and vWF suggest that inflammation and endothelial dysfunction may play a prominent role in the pathogenesis of the disease during the early phase of ACS.

Patterns and Determinants of Attitudes towards Genetic Risk of Cancer: Case Study in a Malaysian Public University

Genetic risk to cancer is a knowledge largely confined to experts and the more educated sectors of the developed western countries. The perception of genetic susceptibility to cancer among the masses is fragmented, particularly in developing countries. As cancer diseases affect developing countries as much as developed nations, it is imperative to study perception and reception of genetic risk to cancer in Southeast Asia. Here, we report on a novel case study to gauge the awareness and attitudes towards genetic determination of cancer among the undergraduates of a Malaysian public university. A total of 272 university undergraduate students completed an online questionnaire. On causes of cancer, the respondents believed that cancer is caused by lifestyle and environmental factors, but those with science background were more likely to associate it with genetic factors. The results on awareness of genetic profiling of cancer risk showed that there are significant differences between those with science and nonscience background but there are no significant differences for gender and socioeconomic background. As for attitudes towards cancer risk, female respondents, those from middle socioeconomic status and science background, are more likely to believe in genetic determinism of cancer. The findings have implications on target population segmentation in strategic health communication on cancer.

The dynamics of attached and free-living bacterial population in tropical coastal waters

We investigated the dynamics of attached and free-living bacterial abundance over a period of 18 months in tropical coastal waters of Malaysia. We measured the abundance at both oligotrophic coastal water (Port Dickson) and eutrophic estuary (Klang), and hypothesised that attached bacteria are predominant in eutrophic waters. We found that bacterial abundance was higher at Klang than Port Dickson (Student’s t-test: t=4.87, d.f.=19, P<0.001). Attached bacteria also formed a large fraction of the total bacteria at Klang (75% ±13s.d.) relative to Port Dickson (56% ±22), and showed preference for chlorophyll-a-based particles rather than total suspended solids. The bacterial community structure was clearly different between the two stations but was similar between the attached and free-living bacterial population. Our results showed the importance of attached bacteria in eutrophic water where they could play a major role in carbon and nutrient cycling.