Theses (PHD)

There is an urgent need in search of an insulin-sensitising agent exhibiting good safety, efficacy and tolerability profile for the prevention and treatment of type-2 diabetes mellitus. Catalpol, an iridoid glucoside, is the main bioactive component of Rehmannia glutinosa radix. Catalpol is reported to possess anti-oxidant, neuro-protective and glucose-lowering effects. The work described in this study aimed to examine the effects of catalpol in improving glycaemic control as well as insulin sensitivity in both skeletal muscles and liver of type-2 diabetes mellitus mice. Male C57BL/6N mice aged 5-6 weeks were placed on a high fat diet (60% kcal) for two weeks prior to streptozotocin (50 mg/kg, i.p.) administration for three consecutive days. Group fed with normal chow diet served as the normal control. Four groups of diabetic animals were administered orally with saline, catalpol (100 mg/kg), catalpol (200 mg/kg) or metformin (200 mg/kg) once per day for four consecutive weeks. Treatment of catalpol (200 mg/kg) significantly lowered fasting plasma glucose compared to diabetic control (p < 0.01), along with attenuation of insulin resistance by reducing HOMA-IR index. Catalpol improved insulin sensitivity in skeletal muscles through activating AMPK/SIRT1/PGC-1a pathway. The downstream effect was observed in improved mitochondrial respiration. Administration of catalpol increased the insulin sensitivity in liver by up-regulating PPAR-g and suppressing the expression of gluconeogenic enzymes. Besides, catalpol treatment improved lipid metabolism by regulating plasma adiponectin, plasma triglycerides and liver triglycerides levels. Collectively, catalpol (200 mg/kg) exerted anti-diabetic effects by improving glycaemic control, insulin sensitivity and mitochondrial function in skeletal muscles. Besides, catalpol improved insulin sensitivity in liver and lipid metabolism.

α-Mangostin has been explored by many researchers, and it is effective against bacteria and other microbes such as fungi and mycobacteria. Enterococcus faecalis (E. faecalis), Staphylococcus epidermidis (S. epidermidis), and Candida albicans (C. albicans) are the most prevalent biofilms that induce endodontic flare-ups and secondary infections. This research aimed to formulate and characterize palm oil-in-water based α-Mangostin nanoemulsion (PO-AMNE) endodontic irrigant and test its antimicrobial effectiveness against E. faecalis, S. epidermidis, and C. albicans biofilms with smear layer removal capability. In addition, it was tested for cytotoxicity in Immortalized Oral Kerantinocytes-OKF-6 cells. The optimization of the 0.2% PO-AMNE irrigant was performed using the Box-Behnken statistical design. The optimized 0.2% PO-AMNE irrigant was found to have a particle size of 340.9 nm with 0.246 PDI of the dispersed droplets and zeta potential (mV) of -27.2±0.7 mV. The MIC/mL values showed that 0.2% PO-AMNE (1.22±0.02) was comparable to 2% CHX (1.33±0.01) and 3.25% NaOCl (2.2±0.09) had the least inhibition for E. faecalis. 3.25% NaOCL showed the maximum inhibition on S. epidermidis (0.26±0.05), whereas 0.2% PO-AMNE (1.25±0.0) was comparable to 2% CHX (1.86±0.07). For C. albicans, 2% CHX (8.12±0.12) showed the least inhibition as compared to 0.2% PO-AMNE (1.23±0.02) and 3.25% NaOCl (0.59±0.02). The 0.2% PO-AMNE irrigant was then evaluated for antimicrobial efficacy using colony-forming units (CFU) against E. faecalis, S. epidermidis, and C. albicans biofilms. The 0.2% PO-AMNE was comparable to 3.25% NaOCl and 2% CHX in inhibiting the growth of biofilms. The 0.2% PO-AMNE irrigant was investigated for smear layer removal, and 0.2% POAMNE and 17% EDTA demonstrated comparable efficacy in removing the smear layer with a mean score of p<0.001. Finally, in Immortalized Oral Keratinocytes-OKF-6 cells, 0.2% POAMNE was less cytotoxic compared to 17% EDTA, 3.25% NaOCl, and 2% CHX. The cell viability percentage of 0.2% PO-AMNE after 60 minutes was 32.51%, whereas there was a decrease in the cell viability percentages for 3.25% NaOCl, CHX, and 17% EDTA cell survival of 6.47%, 4.30%, and 4.15%, respectively. Overall, the formulated 0.2% PO-AMNE irrigant may potentially combat endodontic infection-related microbial biofilms. Keywords: α-Mangostin, palm-oil, nanoemulsion, root canal irrigants, antimicrobial, antifungal, smear layer, OKF-6 cells.

Background: A previous study on the effects of hot water extract of Morinda citrifolia (M. citrifolia) fruits on human blood coagulation suggested anticoagulant effects in vitro. This study was carried out to determine the effective extraction method of the fruit producing highest anticoagulant activity, along with the aim to identify its effective fraction exhibiting highest anticoagulant activity and its mechanism of action, and to chemically characterize this effective fraction. This study, which involved in vitro and in vivo methods, compared M. citrifolia extracts to antiplatelet/anticoagulant drugs, aspirin and warfarin (positive controls), and three known compounds of the M. citrifolia fruit (scopoletin, quercetin and rutin) with distilled water (vehicle) as the negative control. Methods: Phase I identified the best method of extraction to produce highest anticoagulant activity, while the phase II, determined the effective fractions of this identified extract, with highest anticoagulant activity using bioassay guided fractionation. Phase III of the study, revealed the mechanism of anticoagulant action via various antiplatelet and anti-coagulation cascade pathways. The effect on biochemisbtry, hematology and histology of treated animals as well as the interaction between warfarin and the M. citrifolia were also examined. Differences between groups were compared using the one-way analysis of variance (ANOVA), and students’t-test with SPSS. In Phase IV the effective anticoagulant fraction was chemically characterized. Result and discussion: The results suggests, crushed fruit extract (CE) as the most effective extract with dose dependent anticoagulant activity via coagulation profile in vitro. The water (McWF) and methanol (McMF) fractions of the M. citrifolia extract were identified as the most effective fractions, exhibiting highest anticoagulant activity, while phase III suggested significant inhibitory effects of theses fractions on platelets and the coagulation cascade. The interaction study of M. citrifolia with warfarin showed significant reduction of warfarin’s antiplatelet and anticoagulant activity. The biochemical studies saw increased liver enzymes while histological findings suggested liver degeneration with M. citrifolia. The LCMS profiling of the M. citrifolia effective fraction (McWF) showed the presence rutin which was quantified. Conclusion: M. citrifolia fruit extract exhibited anticoagulant and anti-platelet activity, in vitro and in vivo. The McWF was identified the most effective fraction, though the crude extract still remained more potent. M. citrifolia reduced the anticoagulant activity of warfarin. The prospects of using M. citrifolia as a possible anticoagulant requires further verification on its dose dependent, reversible and the safety index of its anticoagulant action and possible liver toxicity.

Epstein-Barr virus (EBV) is a class I carcinogen human herpes virus which has infected 90% of humanity and most prevalent among Asians, especially Chinese. After primary infection, EBV establishes the lifelong virus carrier state. EBV can be detected in two different tissues namely, B lymphocytes and epithelial cells. EBV is linked to the pathogenesis of a variety of human tumors and disorders, such as Burkitt’s lymphoma, and nasopharyngeal carcinoma. Algae are a potential source of antiviral compounds; however, there have been very few reports on the antiviral activity of microalgae extracts against EBV. The objective of this study was to investigate the antiviral activity of extracts from three microalgae, namely Ankistrodesmus convolutus UMACC 101, Synechococcus elongatus UMACC 105 and Spirulina platensis UMACC 161 against EBV in Burkitt’s lymphoma (BL) cell lines. Three EBV-positive BL cell lines, namely Akata, B95-8 and P3HR-1 were used as in vitro study model. A bioassay-guided fractionation approach was used for the screening of antiviral activity. The antiviral activity of the microalgae extracts was elucidated based on their inhibition efficacy in reducing number of cell-free viral particles being released by chemically induced lytic BL cells. This was assessed by quantifying the cell-free DNA using real-time PCR technique. In addition, the inhibition activity of microalgae extracts against the expression of the viral proteins LMP1, EBNA1 and ZEBRA in BL cells was assessed using immunocytochemistry technique. Two antiviral drugs namely acyclovir and foscarnet were chosen as positive controls. Methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity (IC50 >200 µg/mL) and reduced the cell-free EBV viral load most effectively (EC50 <0.01 µg/mL) and thus, displayed high therapeutic index (>28000). The extracts decreased the expression of EBNA1 (>45%), LMP1 (>38%) and ZEBRA (>67%) effectively in P3HR-1 cells. After column chromatography fractionation, the non-polar fraction of the extract from Synechococcus elongatus (SEF1) reduced the amount of cell-free EBV DNA most effectively (EC50= 2.9μg/mL; therapeutic index >69) with low cytotoxicity (IC50 >200 μg/mL). SEF1 inhibited the expression of EBNA1 and ZEBRA (>40%) effectively in P3HR-1 cells. When SEF1 was further fractionated using HPLC, the sub-fraction SEF1’a was most active in reducing the cell-free EBV DNA (EC50= 1.38µ/mL; therapeutic index >14.5). It inhibited the expression of LMP1 moderately (25%) in P3HR-1 cells. The microalgae extracts did not interact with the cytoskeleton components (actin and tubulin) of BL cells during the release of cell-free EBV particles as revealed by the immunofluorescence study. The active constituents in the microalgae extracts tested might consist of pigments such as chlorophylls, carotenoids, phaeophytins and phycobilins. In conclusion, methanol extracts from Ankistrodesmus convolutus, Synechococcus elongatus and Spirulina platensis showed antiviral activity by inhibiting the release of EBV from the BL cells and the expression of the viral proteins LMP1, EBNA1 and ZEBRA in the host cells. The potential of the microalgae as a source of antiviral drugs against EBV is worth exploring.

Increasing human activities has led to anthropogenic contamination of heavy metals in the environment of Antarctica. Microalgae, the primary producers of the food chain, are the first target affected by heavy metal pollution. The primary aim of this study was to assess the toxic effects of copper (Cu) and lead (Pb) on microalgae isolated from Signy Island, Antarctica, with emphasis on their oxidative stress response and metabolomic profiles. In addition, the heavy metal contents and occurrence of microalgae in the soils from differently impacted sites of Signy Island were investigated. A total of 29 taxa of microalgae from Bacillariophyta, Chlorophyta, Cyanobacteria, and Tribophyta were recorded from cultured materials derived from the soil samples collected from Signy Island. The soil contents of 14 metals (Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, and Zn) from five sampling sites (Berntsen Point, Gourlay Peninsula, Jane Col, North Point, and Pumphouse Lake) were determined. Results showed that the Cu content was highest in Gourlay Peninsula (135.36 ± 71.99 μg/g) while the Pb content was highest in Berntsen Point (14.78 ± 23.91 μg/g). Twenty-two unialgal strains were isolated. Morphological analysis using light microscope showed that three of the unialgal isolates were from the genus Coccomyxa and 19 isolates were from the genus Chlorella and allies. Phylogenetic analysis showed that the isolates were clustered in separate clades consisting of Coccomyxa subellipsoidea, Micractinium inermum, Chlorella sp., and Chlorella vulgaris. Micractinium inermum BP-33 isolated from high Pb site (Cu=1.523 μg/g; Pb=127.643 μg/g) and C. vulgaris GP24 isolated from high Cu site (Cu=119.942 μg/g; Pb=0.893 μg/g) were chosen for 10-day Cu and Pb toxicity testing. Micractinium inermum BP-33 was more sensitive to both Cu and Pb while C. vulgaris GP24 was more resistant to both metals. Exposure of the microalgae to the metals resulted in decreased chlorophyll a, chlorophyll b, and carotenoid contents. The microalgae exposed to the metals for 4 and 10 days showed increased cell granularity, accumulation of large amounts of starch granules, abundant vacuoles with electron-dense polyphosphate bodies, and alteration in chloroplast structure. The reactive oxygen species (ROS) levels of M. inermum BP-33 exposed to Cu were similar to the control while superoxide dismutase (SOD) and catalase (CAT) activities increased significantly (P<0.05). The ROS levels in Micractinium inermum BP-33 exposed to Pb were significantly lower (P<0.05) than the control. In terms of antioxidant enzyme response, there was an increase in SOD activity but no significant change in CAT activity was observed. In C. vulgarisGP24, the ROS levels were significantly lower (P<0.05) than the control when exposed to Cu at EC50. A decrease in SOD activity and an increase in CAT activity were also observed. In comparison, the ROS levels in C. vulgarisGP24 exposed to Pb at EC10were significantly higher (P<0.05) than the control, with significant increase (P<0.05) in both SOD and CAT activities. The metabolic responses of microalgae after exposure to Cu and Pb for 4 and 10 days were analysed using 1H NMR spectroscopy. Subsequent PLS-DA scores plot showed that Cu and Pb-treated cells were clearly distinct from the control. The covariance plots calculated using permutation test revealed that the discrimination pattern in PLS-DA plots were mainly influenced by carbohydrates, some amino acids and organic osmolytes. The highest number of metabolite changes were detected in M. inermum BP-33 after exposure to Pb for 10 days (9 up-regulated; 22 down-regulated), while the least number of significant metabolites changes was detected in C. vulgaris GP24 after exposure to Cu for 10 days (2 up-regulated). Metabolites that confer potential oxidative protection such as glutathione, malate, myo-inositol, inosine, proline, and taurine were detected in the microalgae exposed to heavy metals. Glutathione and malate were up-regulated in both microalgae exposed to Pb while myo-inositol and inosine were up-regulated in M. inermum BP-33 after being exposed to Pb for 10 days. In addition, proline was up-regulated in M. inermum BP-33 on day 10 after exposure to Pb while in C. vulgaris GP24, this occurred on day 4. Down-regulation of taurine was observed in both microalgae after being exposed to Cu and Pb for 4 days. In conclusion, the findings of this study suggested that heavy metals could affect the occurrence of soil microalgae on Signy Island. Heavy metals could disrupt the growth, photosynthetic pigments, and cellular features of microalgae. Heavy metals could also disrupt oxidative balance of microalgae, trigger the increase in antioxidant enzymes activity, and alter the metabolites in the microalgae.

Pesticides are widely used in agricultural activities in Malaysia. Some of the common pesticides used include organophosphate compounds such as chlorpyrifos and diazinon, and organochlorine chemicals such as lindane, heptachlor, endosulfan, DDT, chlordane, aldrin, dieldrin and endrin. Pesticides can cause adverse effects on the ecosystems due to their long half-life, and ultimately may end up in the sea affecting the marine ecosystems. Humans are inevitably exposed to pesticides through environmental contamination or occupational use. The primary aim of this study was to assess the toxicity of five pesticides, namely chlorpyrifos, lindane, endosulfan, dichlorvos and malathion using an integrated approach based on microalgae and animal cells. In addition, the mutagenicity of the pesticides was assessed by Ames test using Salmonella typhimurium. Results showed that lindane was the only pesticide that could cause mutation after liver enzyme activation. The toxic effect of the pesticides on normal human liver (THLE-2) and mouse embryo fibroblast (3T3) cells was assessed based on 72 h MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide) assay. Of the pesticides tested, endosulfan was most toxic (EC50 = 20.4 μg/mL) against THLE-2 cells while chlorpyrifos was most toxic (EC50 = 2.5 μg/mL) against 3T3 cells. Toxicity testing of the pesticides on three marine microalgae, namely Pavlova gyrans, Isochrysis sp. (clone T-ISO), and Dunaliella tertiolecta was conducted based on their five-day growth response assessed by optical density at 620 nm (OD620). Of the five pesticides tested, endosulfan was most toxic against Pavlova gyrans (EC50 = 0.17 μg/mL) and Dunaliella tertiolecta (EC50 = 0.35 μg/mL). In comparison, Dunaliella tertiolecta was most resistant to all the pesticides tested (EC50 = 0.35 - 28.09 μg/mL). Random Amplified Polymorphic DNA (RAPD) technique was used to detect DNA damage caused by the pesticides in animal and algae cells. A total of 31 sets of primers were used and appearance/disappearance of bands in the pesticides-treated cells were compared with the control. The change in RAPD band patterns was more obvious in animal cells compared to microalgae. Most of the changes in PCR banding patterns were random, but the frequencies were higher when the DNA was amplified with primer OPB 5 series, OPB 7 series and OPB 8. Based on the RAPD analysis, Pavlova gyrans showed the least changes in terms of DNA band pattern compared to the other species of microalgae. The induction of apoptosis by the pesticides in animal and algae cells was assessed based on oxidative DNA damage, nucleus condensation, and caspase-3 activity. Endosulfan and dichlorvos caused oxidative DNA damage in both mouse embryo fibroblast and human liver cells, while chlorpyrifos only caused oxidative DNA damage in mouse embryo fibroblast cells. In nucleus and DNA condensation study by Hoechst stain, apoptosis was detected in malathion-treated mouse embryo fibroblast cells with obvious DNA condensation after exposure to the pesticides for 4 h. Results from the caspase-3 activation study showed that human liver cells were more susceptible to caspase-3 induction by pesticides than mouse embryo fibroblast cells. Endosulfan and lindane showed the ability to increase the caspase-3 activity in liver cells. The findings suggest that the pesticides tested could adversely affect the growth, and may cause DNA damage in both marine microalgae and animal cells. The pesticides may impact on the marine ecosystem as well as human health.

The emergence of Methicillin Resistant Staphylococcus aureus (MRSA) which is resistant to many available antibacterial agents, especially the β-lactam antibiotics, has become a major threat to the health sector worldwide, giving impetus to the search for novel antibacterial agents efficacious against this bacterium. Four (4) Cinnamomum species i.e. C.iners, C.altissimum, C.impressicostatum and C. porrectum were investigated against a range of multidrug resistant bacteria, including Methicillin Resistant Staphylococcus aureus, our main subject of interest, for potential antibacterial activity and elucidation of their modes and mechanisms of action. Characterisations of their bioactive fractions were performed using bioassay guided isolation techniques. C.impressiscostatum stem-bark extract recorded the highest zone of inhibition and lowest minimum inhibitory concentration against MRSA. The presence of salt enhanced the killing efficiency of the plant extract against MRSA. Cellular materials leaked from MRSA after treatment with the plant extract and this was concomitant with marked cell wall damage, as observed using scanning electron microscopy (SEM). A total of 136 genes were found to be differentially regulated by C. impressicostatum stem-bark extract. Amongst these, 73 genes were upregulated and 63 genes were downregulated. The MRSA genes that fluctuated in expression when treated with the plant extract involved various pathways, such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, membrane transport, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, biosynthesis of secondary metabolites, energy metabolism, folding, sorting and degradation, glycan metabolism, pathways in pathogenesis, replication and repair, signal transduction, transcription, translation, translational ribosomal structure and biogenesis and xenobiotic degradation and metabolism. The possible mechanisms underlying the killing action of C.impressicostatum active fraction of water extract against MRSA are via inhibition of biofilm formation, inhibition of nucleotide metabolism as well as DNA replication and repair, downregulation of the mismatch repair mechanism, thereby enabling maintenance of deleterious and lethal mutations, ultimately causing cell death, induction of increased cellular vulnerability to the effects of reactive oxygen and nitrogen species, inhibition of essential lipid biosynthesis required for cellular structure and metabolism as well as the induction of apoptosis.

No abstract available

Colorectal cancer (CRC) is a major cause of morbidity and mortality worldwide. Given the limitations of current population based screening methods, chemoprevention approaches have been considerably gaining credibility and interest as effective additional measures for CRC prevention during the last few decades. The aim of this dissertation was to assess the benefits of such approaches in a population whose constituents had different risks of developing CRC. The dissertation outcomes can be used to make evidence based decisions about the administration of chemopreventive agents (CPAs) for optimised prevention of CRC in Malaysia. The study addressed three distinctive analyses; however, the three were related in significant respects. First, comparative efficacy and safety of CPAs for the prevention of CRC were evaluated using network meta analysis techniques based on a systematic review of randomised controlled trials that compared at least one CPA (aspirin at different doses, any nonsteroidal anti inflammatory drugs, any antioxidants, folic acid, vitamin B6, vitamin B12, calcium, vitamin D, alone or in combination) to placebo or other CPA. Second, the balance between benefits and harms of those CPAs with evidence of efficacy was assessed using either net clinical benefit or risk benefit integrated analysis. Third, both long term clinical and economic outcomes of using CPA in the Malaysian setting were evaluated by cost effectiveness analysis. The assessment of CPAs in average risk individuals for CRC (that is, no personal history of CRC) suggested that aspirin at the dose range of 75--325 mg/day could be a safe and effective primary prevention for long--term CRC with potential dose--dependent effects. None of the other interventions were found to be effective in this population. Net clinical benefit analysis combining mortality from CRC, cardiovascular (CV) disease and bleeding suggested that low--dose aspirin (>100--325 mg/day; ASA--LD) provided the highest net survival gain but data were too limited to make a definitive conclusion. The assessment of CPAs in increased risk individuals for CRC due to a personal history of colorectal adenomas suggested that celecoxib (400--800 mg/day) and very--low--dose aspirin (≤100 mg/day; ASA--VLD) were the most effective agents for the prevention of recurrent advanced colorectal adenomas. However, the risk--benefit profile potentially favours the use of ASAV--LD especially in those with a history of advanced colorectal adenomas. Therefore, a Markov model was developed to assess the cost--effectiveness of ASA--VLD for the secondary prevention of CRC in a population with a personal history of advanced colorectal adenomas in Malaysia. It suggested that combining ASA--VLD with routine 3--year surveillance colonoscopy offered the best value for money when compared to ASA--VLD alone or routine 3--year surveillance colonoscopy. It also suggested that for those individuals who were compliant to ASA--VLD and willing to take this treatment for a longer period, an extension of surveillance colonoscopy intervals to 5 years could be considered in those countries with limited resources. Overall, this dissertation concludes that the addition of ASA--VLD as recommended for the CV disease prevention in Malaysia with routine surveillance colonoscopy in individuals with a previous history of advanced colorectal adenomas is a simple and effective means of reducing morbidity and mortality, and results in overall cost--saving.

Introduction: According to the National Oral Health Survey of Preschool Children (NOHPS) in 2015, in Malaysia, caries affects 71.3% of preschool children, most of which remain untreated. Caries is preventable; thus, the caries lesion formation and progression can be controlled. There is a need to find a cost-effective caries prevention intervention for 5-6-year-olds that is attractive to governmental and private investment. This study investigates the effectiveness and costeffectiveness of delivering 6-monthly Dental Home Visits (DHVs) and Education Leaflets (ELs) compared to delivering only ELs in caries prevention of 5-6-year-olds in Malaysia. Objectives: This study aimed to evaluate the cost-effectiveness of 6- monthly DHVs and ELs in preventing new caries development in 5– 6-year-olds compared to those receiving only ELs over 24 months. Methodology: The study design is a community-based randomized controlled trial with two arms. A nested sample of 5–6-year-olds attending government-funded kindergartens in the state of Selangor within the 2015 NOHPS was randomized to receive 6-monthly DHVs and ELs for the intervention group and only ELs delivered to the control group over 24 months. This study analyzed the costeffectiveness of each arm in preventing new caries development, OHRQoL, and potential dental treatment cost avoidance in 24 months. This study adopted a government or health provider perspective. The cost data of this study was the estimated cost data, and a conservative approach was adopted to perform sensitivity analysis. Results: Fourteen out of 100 5-6-year-olds in the intervention group, and 60 out of 100 5-6-year-olds in the control group developed new caries at the 24-month follow-up. The estimated cost avoidance of dental treatment on the dental chair of 5-6-year-olds in the intervention group for 24 months was RM 1,500.25, and RM 4,800.00 was the estimated cost avoidance of 5-6-year-olds in the control group for 24 months. The estimated cost avoidance of comprehensive dental treatment under GA of 5-6-year-olds in the intervention group for 24 months was RM 21,945.25, and RM 90,007.70 was the estimated cost avoidance of 5-6-year-olds in the control group for 24 months. ICER for dental treatment on the dental chair was RM -72.50, and RM – 1,492.60 was ICER for comprehensive dental treatment under GA. The sensitivity analysis demonstrated consistent negative results, after calculating with different values of total estimated intervention costs and the total estimated control costs with the range of 15.0-30.0%. 6- monthly DHVs and ELs remained at quadrant II in the costeffectiveness plane. Conclusions: Delivering 6-monthly DHVs and ELs resulted in fewer number of 5-6-year-olds developing new caries at 24-month followup. It would reduce the number of 5-6-year-olds that need dental treatment and potentially avoid expensive dental treatment due to caries. 6-monthly DHVs and ELs could reduce the economic burden of caries in 5-6-year-olds in Malaysia

To address gaps in the understanding of host-parasite interactions, this study employed a systems biology approach to investigate system biological responses in Balb/c mice infected with Toxoplasma gondii and Trypanosoma brucei brucei. The study found distinct histopathological changes in Balb/e mouse organs post- infection. Spleen hyperplasia, chronic inflammation, and immune cell infiltrations were apparent in both infected arms. Cytokine profiling identified key players like tumour necrosis factor alpha, interferon-gamma, interleukin-2 in T. gondii group interleukin-6, and interleukin-IO in T. b. brucei group, providing insights into their roles in host defence or immunomodulation. Nuclear magnetic resonance metabolomics profiling was used to identify potential parasitic infection biomarkers, revealing alterations in metabolic profiles indicative of metabolic adaptations and gut microbiota dynamics during infection. Notably, short-chain fatty acids like acetate and butyrate, aromatic amino acid derivatives like phenylacetylglycine and tyrosine, and metabolites such as sueeinate and creatine were implicated in immune regulation and host-parasite interactions. This study also explores interactions between the gut microbiota and parasitic pathogens, highlighting their impact on disease outcomes. Specifically, Lactobacillus species were identified as key players in modulating the host's immune response and metabolic processes during parasitic infections. In conclusion, the systems biology approach enhances our understanding of host-pathogen interactions in Balb,/c mice, revealing potential therapeutic targets. While the findings are specific to the mouse model, they offer valuable insights that could inform strategies for managing parasitic infections in humans. This study thus lays a strong foundation for future research aimed at developing targeted interventions across various host systems, potentially bridging the gap from animal models to human disease management.

Gastrointestinal parasite (GIP) infections have a global distribution and a major impact on the socioeconomic and public health of the bottom billion of the world’s poorest people. In Malaysia, the Orang Asli community are prone to these infections due to environmental and personal hygiene practices. Aside from the socioeconomic development plans, mass drug administration (MDA) with albendazole, a benzimidazole (BZ) group of anthelminthic (AH) drug are carried out periodically by the government to control GIP infection in this community. Albendazole (ABZ) is commonly used to treat GIP, mainly due to soil transmitted helminths (STHs). However, emergence of drug resistant among GIP has been a recent concern, as BZ resistance in GIP have been reported due to single nucleotide polymorphism (SNP) in β-tubulin gene. In this study, we investigated the prevalence of GIP and potential risk factors for infection among the Orang Asli community at Kg. Serendah in Malaysia. We studied the efficacy of single dose albendazole and explored the presence of single nucleotide polymorphism frequencies (SNPs) β-tubulin gene which confers resistance towards benzimidazole group of anthelminthic drugs. Stool samples were collected from 110 villagers. The stool samples were concentrated using formalin ethyl acetate concentration technique (FEACT) and floatation technique. The stool samples were stained with trichrome stain and acid fast stain to identify the parasite species by microscopy. DNA extraction was carried out using Stool Kit (Qiagen) and followed by conventional PCR assay to detect the parasites. All the participants in the survey were treated as follows: 400mg of albendazole for above 2 years old and 200 mg of albendazole for ≤ 2 years old. After deworming exercise, the villagers were examined at 1 and 6 months post treatment. PCR amplification and sequencing of β-tubulin gene from parasites at pretreatment and post treatment (1 & 6 months) were analyzed. Demographic socioeconomic and behavioural characteristics were treated as categorical variables and presented as frequencies and percentages. Pearson’s Chi- square (X2) test was used to test the association of GIP prevalence with demographic socioeconomic and behavioral factors. Odds ratio (OR) and 95% CI were computed. To identify risk factors associated with GIP infection variables that showed association in the univariate analysis were used in multivariate analysis. Data analysis was performed using SPSS version 18. Stool samples were collected from 110 villagers. The total samples obtained from male were 50 and female were 60. The prevalence for GIP found in this study was 67% and the parasites detected were Trichuris trichiura (50%), Ascaris lumbricoides (39%), Blastocystis hominis (10%), Cryptosporidium parvum (7.2%), Taenia spp (2.7%) and Microsporidia (2.7%). The prevalence was 68% and 66% in females and males respectively. Higher GIP prevalence was observed among children below 6 years compared to the adults. Univariate analysis showed the avaibility of latrine, the source of drinking water, boiling water before drinking and place of defecation were potential risk factors for GIP infection. Multivariate analysis showed that not using the toilet and drinking unboiled water was predictive of GIP infection in this study area. The cure rate after 1 month post treatment for A. lumbricoides was 87% and for T. trichiura was 46% after a single dose of albendazole. The cure rate after 6 months post treatment declined to 33% for A. lumbricoides but remained the same at 46% for T. trichiura. The poor cure rate for STH was not due to drug resistant as presence of SNP was not detected in β-tubulin gene sequenced. The conventional PCR assay developed in this work was efficient to detect STH but not protozoa. The overall prevalence of GIP in this study was 67% and mainly due to STH infection. Females had slightly higher prevalence compared to males. Moreover, children below 6 years of age had the highest GIP infection followed by adults. The overall cure rate was higher for Ascaris lumbricoides infection compared to Trichuris trichiura with a single dose albendazole after 1 month treatment. No SNPs were detected among all the STH sequenced before and after treatment. Summing up the result shows, resistant markers do not contribute to the high prevalence of STH among Orang Asli at Kg. Serendah.

The current study investigated the potential of Poly (e-caprolactone)/Polyethylene oxide (PCL/PEO) based intravaginal matrices for the controlled delivery of tinidazole in the therapy of bacterial vaginosis. PCL/PEO matrices loaded with the microbicide tinidazole (TD) were produced by rapidly cooling suspensions of drug powder in PCL/PEO solutions in acetone to −80ºC. The prepared matrices were investigated for their morphology, drug-polymer compatibility, thermal properties and in vitro drug release. The actual tinidazole loadings were 1.1% to 3.8% w/w related to a theoretical loading of 10% w/w, and the drug incorporation efficiency was up to 38%. In vitro release studies showed a ‘burst release’ of 35-74% tinidazole in the first 24 hours and a cumulative release of 50-100% tinidazole in seven days. The released tinidazole displays up to 79% antibacterial activity against Gardnerella vaginalis. The in vivo rabbit pharmacokinetic study demonstrated the controlled release of tinidazole from the surgically sutured IVR segment. The mean tinidazole concentration in vaginal secretions on day 1 and day 3 was found to be 2303.6 ± 1485.08 ng/mL and 1627.6 ± 981.71 ng/mL respectively. The mean tinidazole concentrations in proximal and distal vaginal tissues on day 3 were found to be 792±182.43 and 689.5±135.05 ng/g respectively. Serum levels below the lower limit of quantification (0.5 μg/mL), show the controlled release of TD from the matrices, which may limit drug absorption into the systemic circulation and lower resistance risk. The results of the histopathological analysis showed that one week of intravaginal implants with and without tinidazole was well tolerated, had no significant effect on the histological morphology of the rabbit vagina, and no macroscopic changes were observed. These results demonstrate that PCL/PEO based intravaginal ring (IVR) has all the potential for the controlled intravaginal delivery of TD in bacterial vaginosis and warrants further investigation.

Corneal neovascularization significantly contributes to global blindness statistics, affecting approximately 1.4 million individuals annually, with 12% experiencing total blindness. Conventional anti-angiogenic therapies often require repetitive doses and later develop drug resistance. Recent advancements in genetically engineered mesenchymal stem cells (MSCs) using viral vectors present a promising strategy to enhance the efficacy of anti-angiogenic therapies. Adeno-associated virus (AAV) has been utilized in clinical settings to deliver therapeutic genes to the cornea due to its characteristic of transient integration into the genome. This study aims to genetically engineer MSCs overexpressed sFlt1 (MSCs.hsFlt1) using AAV to treat corneal neovascularization. Small and large-scale MSCs.hsFtl1 were produced and validated with gene and protein expressions. The functional in vitro study, based on tube formation assay, showed significant growth inhibition in human umbilical vein endothelial cells (HUVECs) at a low dose of MSCs.hsFlt1 and demonstrated a comparable anti-angiogenesis effect to bevacizumab. The toxicity and efficacy in vivo study involved 24 New Zealand white rabbits with mechanical wound injuries inflicted on the cornea. All rabbits were randomly divided into 3 groups and were treated subconjunctivally with normal saline, MSCs and MSCs.hsFlt1. Neovessels were measured and graded clinically. On day 91 post-treatment, neovascular lengths in MSCs.hsFlt1 decreased significantly and exhibited better anti-angiogenic effects than control and MSCs. Subconjunctival injection of genetically engineered MSCs.hsFlt1 effectively inhibits corneal neovascularization in both in vitro and in vivo models. These findings demonstrated safety and efficacy in managing neovascular conditions, especially the cornea, paving the way for further advancements in cellular gene therapy in a clinical setting.

Antibacterial properties have been demonstrated by propolis and its products in the past. By encapsulating propolis in chitosan nanoparticles, the intrinsic property of propolis can be enhanced. The resulting chitosan-propolis nanoparticle formulation had ideal physicochemical properties viz. spherical shape, smooth surfaces, average particle size around 100 nm, good stability (zeta potential +40 mV) and encapsulation efficiency of 88.8%. Chitosan-propolis nanoparticles demonstrated a steady and sustained release over time. The improved effect of chitosan-propolis nanoparticles can be attributed to their penetration into the biofilms as tested on Enterococcus faecalis and Staphylococcus epidermidis, which are clinically significant microbes causing human ailments. This formulation inhibited both E. faecalis and S. epidermidis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 μg/mL concentration. When tested on pre-formed biofilms, the formulation reduced E. faecalis and S. epidermidis bacterial number in the biofilm by ~75% and ~90% at 300 μg/mL, respectively. The chitosan-propolis nanoparticles physically disrupted the biofilm architecture, as evident by imaging studies. Treatment of biofilms with chitosan-propolis nanoparticles altered the expression of cytolysin genes (cylB, cylLL, cylLS, cylR1, cylR2, cylM and cyl1) and virulence genes (gelE, ace, asa, fsrB, fsrC, ebpA, ebpB, ebpC, efa, gls24 and bopD) genes in E. faecalis. This formulation also altered the expression of matrix and adhesive genes (embp, rsbU, sarA, sepA, atlE, and icaABCD) in S. epidermidis. Therefore, chitosan-propolis nanoparticles can be a prospective therapeutic agent to prevent biofilm formation and/or improve clearance of chronic biofilm infections.

Introduction: The current standard for storing human cells involves using DMSO and liquid nitrogen, but this method requires specialized equipment and has associated toxicity. This study aims to develop a DMSO-free preservation protocol potentially through lyophilization. Methods: Trehalose and sucrose at 20-800 mM, hydroxyethyl starch (HES) at 25-350 mg/mL, poly(vinyl alcohol) (PVA) at 1-34 mg/mL and glycerol at 1-10%v/v were evaluated for its toxicity on fibroblasts (MRC5), kidney (HEK293) or mesenchymal stem cells (MSCs). Next, tri-excipients formulations were compared with 10% v/v DMSO during freezing and dehydration. The effect of freezing rate, primary drying duration or choice of reconstitution medium on the cell viability were assessed as well. Cell viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay or trypan blue staining. The optimized protocol was evaluated on other human cells and storage at -20 oC and -80 oC for ≤30 days. Molecular dynamics (MD) simulation was used to elucidate the molecular mechanisms of the sugar and polymer combination under freezing conditions. Results and Discussion: The formulations failed to provide adequate protection during lyophilization. However, (i) 100 mM trehalose, 150 mg/mL HES and 2% v/v glycerol, (ii) 100 mM sucrose, 150 mg/mL HES and 2% v/v glycerol and (iii) 50 mM trehalose, 50 mM sucrose, 150 mg/mL HES and 2% v/v glycerol showed similar or better cryoprotective capabilities than 10% v/v DMSO during freezing and storage. MD simulation showed the sugar-polymers exert cryoprotective effect through hygroscopicity and water redirection. Conclusion: The optimized formulations demonstrated great potential in replacing DMSO as a cryoprotective agent. However, further investigation is warranted in lyophilization. We hypothesized that intracellular damage was the cause of cell death.

Carbon nanotubes (CNT) have been explored as a non-viral system for gene delivery due to their unique structure and properties. Various functionalised carbon nanotubes can act as a vehicle for the systemic delivery of siRNA-based therapeutics. Here, we investigated the functionalisation of commercially available high-purity HiPCo® SWNT by non-covalent adsorption of phospholipid-polyethylene glycol (PL-PEG) with terminal amine (PL-PEG-NH2; PA) or maleimide groups (PL-PEG-maleimide; MA). The stable aqueous suspensions of PL-PEG functionalised SWNT were then conjugated with siRNA as the delivery cargos for siRNA. The conjugates were found to successfully silenced genes in various cell lines (HeLa, HEK-293, H1299 and MCF-7) without causing much cytotoxicity. A dual targeting model was also established by investigating the possibility of the conjugates to silence two genes of interest simultaneously via conjugating two different siRNA to the SWNT. The mechanism of cellular uptake of SWNT-siRNA conjugates by the epithelial cells was studied and was found to be energy dependent. Over-expression of ABCB1 gene (multiple drug resistance gene) was found not to affect the siRNA delivery by the SWNT. Finally, we investigated the effect of SWNT-020PA-S-S-siLUC in luciferase gene knock-down mouse xenograft model. The conjugates successfully delivered the siRNA to the tumour site and carried out the RNAi mechanism without much acute toxicity being observed. This shows that functionalised SWNT-siRNA conjugates could induce sustainable RNAi mechanism upon the selection of a suitable siRNA which is the niche to the survival of the cancer cells for anti-cancer treatment.

Rheumatoid arthritis (RA) is a chronic, systemic disease characterised by persistent inflammatory synovitis that typically involves peripheral joints in a symmetric distribution. The synovial inflammation can cause cartilage destruction and bone erosions that are irreversible. Although various studies have been conducted on RA, currently there is no cure for this disease. The RA patients are clinically managed with steroidal and non-steroidal anti-inflammatory drugs (NSAIDs). These drugs pose various health risks and side-effects. Therefore, there is a need to find other therapeutic approaches that have anti-inflammatory effects but with lesser or no risk of side effect. Oils from ostrich and emu are well-known for their anti-inflammatory effects. To date, the anti-inflammatory effects of ostrich oil (OO) have not been evaluated as a potential therapeutic agent for RA. In this study, we evaluated the therapeutic efficacy of OO in an established rat model of RA. The aim of this study was to refine and enrich the OO with natural bioactive compounds and compare the therapeutic efficacy of the various formulations in a rat model of RA using various parameters (histopathology, biomarkers). While carrying out this study, we developed and optimised a new method to refine ostrich oil (patent filed). The refined oil is further enriched with extracts from various plants or pure natural compounds to improve its’ stability and therapeutic properties. The therapeutic efficacy of the refined and stabilised OO was tested using the well-established rat model of collagen-induced arthritis (CIA). Briefly, arthritis was induced in Dark Agouti (DA) rats by injecting the rats intradermally with a mixture of collagen type II emulsified in Complete Freund’s Adjuvant (CFA) on day 0. The arthritis rats were topically treated with 500 mg/kg of the various OO formulations prepared when they start showing signs of arthritis, usually from day 30 up to day 45, when the study was terminated. Control rats were not given any treatment. The therapeutic efficacy of this treatment approach was assessed by the ability of the formulated OO to reduce paw oedema as well as by evaluating histopathological changes, protein and gene expressions. The results show that refined OO stabilised with curcumin or grape seed extract showed the most significant (p<0.05) effect in reducing paw oedema compared to the other OO formulations. The paw oedema changes correlated partially with histopathological analysis where there was significant reversal of changes in groups treated with OO stabilised with curcumin or grape seed extract but the effects were not so remarkable with the other OO formulations.

Toll-like receptor 4 (TLR-4) is recognised for its role in host innate immunity. Studies on TLR-4 over-activation had linked it to cancer survival and progression. Nevertheless, there is no TLR-4 inhibitor available for cancer treatment. Therefore, the aim of this project is to identify small molecules as TLR-4 inhibitors for cancer treatment. Computational screening methods were used to identify potential TLR-4 ligands (hits). In silico model was found to be in agreement with the results from the in vitro TLR-4 signalling assay. Lead compounds were then designed. Molecular docking reported that a potent TLR-4 inhibitor forms strong binding with both TLR-4 and myeloid differentiation 2 (MD-2). NF-κB reporter assay and Griess assay studies further confirmed these lead compounds induced potent and selective TLR-4 inhibitory effects. Immunoblotting on macrophages and HEK-BlueTM hTLR4 cells further confirmed that the most potent lead compounds, BZD3 and EP13, inhibited lipopolysaccharide (LPSEc) induced MyD88 dependent pathway, but not MyD88 independent pathway in macrophages and HEK-BlueTM hTLR-4 cells. These two lead compounds also induced potent cancer specific anti-proliferative effects and III apoptotic cell death in cancer cells. TLR-4 and MD-2 ELISA also confirmed that BZD3 and EP13 exhibited TLR-4 inhibitory effects on cancer cells by lowering the expression of TLR-4 and MD-2 levels in the cancer cells. Immunoblotting experiments supported that BZD3 and EP13 inhibited the phosphorylation of downstream MyD88 dependent and MyD88 independent pathways in cancer cells. In conclusion, this study has identified a panel of TLR-4 inhibitors that may play a role in cancer survival and progression.

For the past decades, anthropogenic impacts on natural systems are of growing concern as the human population expands and global biological diversity declines. Agricultural practices often involve the use of fertilisers and pesticides to improve the yield of crops. Agricultural runoff with excess nitrogen (N) and phosphorus (P) could stimulate the excessive growth of algae in the aquatic ecosystems, including lakes and coastal regions, resulting in eutrophication. Besides fertilisers, pesticides are also used in large amounts for pest controls. Microalgae have been frequently used as bio-assay organisms to assess the toxicity of pesticides. However, most of the reports were on the effects of single or mixed pesticides without considering the possible influence of nutrients such as N and P on its toxicity. Therefore, our study aims to assess the changes of various biomarkers in microalgae after exposure to pesticides under nutrient-limited and nutrient-enriched conditions. In this study, two commonly used pesticides, atrazine and endosulfan were chosen. Atrazine, a herbicide of the triazine family, is one of the most frequently used herbicides worldwide while endosulfan is an organochlorine insecticide that causes acute neurotoxic to insects and mammals. A total of 10 isolates from Tasik Jaya and Cameron Highlands were selected to determine their sensitivities to atrazine and endosulfan. The EC50 for atrazine ranged from 43.07 µg/L to 1313.90 µ/L, while the EC50 for endosulfan ranged from 1.51 mg/L to > 50 mg/L after 96 h of exposure. Two of the microalgae, namely Scenedesmus arcuatus (highly sensitive) and Chlorella sp. (highly resistant) were selected for further studies, and compared with the model species, Pseudokirchneriella subcapitata. These three microalgae were also cultured in mediums with different levels of N (in the form of sodium nitrate, ammonium chloride and urea) and P (in the form of potassium phosphate) to determine the levels of N and P that limit/enhance the growth of the microalgae. The N-limited and N-excess levels were determined as 0.0117 mM and 3mM respectively while the P-limited and P-excess levels were 0.0002 mM and 1.7210mM respectively. To determine the effects of different nutrient conditions to the toxicity of atrazine and endosulfan, the three chosen microalgae were exposed to EC10 and EC50 of atrazine and endosulfan, being determined earlier in the dose-response assay in four different culture media: N- and P-limited (LNLP); N-limited, P-excess (LNHP); N-excess, P-limited (HNLP); as well as N- and P-excess (HNHP). In general,all three microalgae showed better tolerance to atrazine and endosulfan when they were cultured in nutrient-limited conditions as compared to nutrient-enriched conditions based on their growth response. In addition to growth response, the effects of different nutrient conditions to the toxicity of atrazine and endosulfan were also assessed using different biomarkers such as oxidative stress biomarkers, photosynthetic biomarkers and morphological biomarkers. The oxidative stress biomarkers of microalgae showed different changes when exposed to atrazine and endosulfan at different nutrient conditions. Reactive oxygen species (ROS) production and lipid peroxidation were induced significantly when microalgae were exposed to atrazine and endosulfan in nutrient-excess conditions, while superoxide dismutase (SOD) levels were generally higher in microalgae grown in nutrient-limited conditions. At N-limited conditions, exposure to atrazine and endosulfan has caused inhibition of SOD in P. subcapitata. The catalase (CAT) enzyme did not show many changes after exposure to atrazine and the response varied in different microalgae in different nutrient conditions after exposure to endosulfan. The photosynthetic biomarkers also showed different changes when microalgae were exposed to atrazine and endosulfan at different nutrient conditions. The maximum potential quantum efficiency of photosystem II (Fv/Fm) was significantly reduced with decreasing nutrient levels, while exposure to atrazine and endosulfan has stimulated the Fv/Fm regardless of the nutrient conditions. Similar trends were observed for the photosynthetic pigments. However, alpha and rETRmax were generally being reduced after exposure to atrazine and endosulfan in various nutrient conditions, except for Chlorella sp. 5 that showed significant elevation in alpha and rETRmax after exposure to endosulfan in nutrient-limited conditions. The presence of atrazine and endosulfan in nutrient-limited conditions has caused more severe damage to the algal cellular structure compared to those in nutrient-excess conditions, such as cell wall disruption and chloroplast damage. On the other hand, increased number of lipid bodies and starch granules were observed in nutrient-limited conditions, together with more severe damage to the cell wall and chloroplast of the microalgae compared to nutrient excess conditions. In conclusion, both atrazine and endosulfan caused significantly different adverse effects to different algal strains, based on their effects on various biomarkers assessed. In addition, nutrient conditions do affect the toxicity of pesticides.