Theses (MSc. Molecular Medicine)

Crude palm oil (CPO) contains several phytonutrients such as carotenoids, tocopherols (TP), tocotrienols (T3), sterols, phospholipids, squalene, and tripterpenic and aliphatic hydrocarbons. Tocopherols (α, β, γ, δ) along with tocotrienols (α, β, γ, δ) represent the two naturally-occurring sub-classes of vitamin E. Both sub-classes exhibit multiple antioxidant and immunity-enhancing properties. However, only tocotrienols have been shown to possess potent anticancer activities using in vivo and in vitro studies. Several studies have reported that the delta- (δ) and gamma- (γ) isoforms of T3 possess more potent anti-proliferative and anticancer effects compared to the other isoforms (α or β). The anti-cancer effects of T3 have been demonstrated in many types of solid cancers, including breast cancer (BC). Recently several studies showed that T3s exert their anti-cancer effects through targeting multiple cell signalling pathways. To date, the ability of γT3 inducing autophagy in breast cancer cells has not been fully explored. Therefore, in this study we investigated the ability of palm γT3 to differentially regulate genes involved in autophagy in the MDA-MB-231 human breast cancer cells using different culture conditions. Cell proliferation was quantified using the WST cell proliferation kit. The half maximal inhibitory concentration (IC50) of γT3 was calculated for two culture condition; (a) complete media with 10% FBS [IC50 at 24 hrs: 13.25 μg/mL; 48 hrs: 7.1μg/mL and 72 hrs: 6.5 μg/mL] and media supplemented with 1% FBS [IC50 at 24 hrs: 1.9 μg/mL; 48 hrs: 1.6 μg/mL and 72 hrs: 1.2 μg/mL]. For detection of autophagy genes, RNA was extracted post 48 hrs of exposure. The purified RNA was analysed using a commercial quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) array annotated with genes involved in human autophagy to identify differentially regulated genes associated with autophagy in the MDA-MB-231 cells. Results from the human autophagy qPCR array showed that a total of 54 genes were differentially regulated by γT3 in the MDA-MB-231 human breast cancer. Ten of these genes were significantly up-regulated. Most of the differentially regulated genes were from: (i) genes involved in autophagic vacuole formation [from genes categorised as belonging to the autophagic machinery components] (ii) co-regulators of autophagy and apoptosis [from genes categorised as belonging to the regulation of autophagy genes]. One of the major genes involved in the autophagy pathway inducers i.e. the BECN1 gene was found markedly up-regulated along with the EIF2AK3, GABARAP and GABARAP2 genes. The results from this study suggest that the potent cytotoxic effects observed when the MDA-MB-231 human breast cancer cells were treated with γT3 at 7.1 μg/mL for 48 hours could also be via regulation of several autophagy-inducing genes.

Propionibacterium acnes (P. acnes), plays a key role in acne pathogenesis by inducing the innate immune system cells to produce certain pro-inflammatory mediators which contribute to the formation of inflammatory acne lesion. The present study was conducted to evaluate the potential of Doani sidr honey and honey polysaccharide rich fraction and madecassoside for antimicrobial and anti-inflammatory activity against P. acnes. The broth dilution method was used to assess the antibacterial activity and the ultrastructural changes in cell morphology before and after exposure to tested samples were studied using transmission electron microscopy (TEM). The tested samples were further investigated for the suppression of pro-inflammatory cytokines tumour necrosis factor (TNF-α) and interleukin 8 (IL-8) by testing the supernatants in the co-culture of the human peripheral blood mononuclear cells (PBMCs) and heat killed P. acnes using enzyme immunoassay kits (ELISA). The statistical analysis was done using the Graph Pad Prism 5 program. The Doani sidr honey and its polysaccharide rich fraction were able to inhibit the growth of P. acnes with a noteworthy minimum inhibitory concentration (MIC) value of 18%(w/v) and 29 %(w/v) respectively and also the release of TNF-α and IL-8 pro-inflammatory cytokines by suppressing them by more than 90%. TEM micrographs revealed the lethal effects of Doani sidr honey against P. acnes .However, no significant inhibition of the P. acnes growth and the secretion of TNF-α and IL-8 was detected for madecassoside at its highest tested concentration. Our results suggested that Doani sidr honey possess both antimicrobial and anti-inflammatory effects against P. acnes and could possibly be used as therapeutic agents for acne. Furthermore, polysaccharide fraction derived from Doani sidr honey showed potent inhibitory effect toward P. acnes. Hence, we hypothesise the fraction prepared from Doani sidr honey might contribute to honey activity. Therefore, this fraction needs to be further explored and characterised to find the main contributing phytochemicals and the antibacterial and anti-inflammatory properties in this fraction.

Aims: Development of TaqMan Assay qPCR for multiplexing Giardia spp targeting GDH and Cryptosporidium spp targeting 18s rRNA. Multiplex SYBR-Green Assay qPCR as an alternative. Methods and Results: Specific primers and TaqMan probes were designed for Cryptosporidium 18S rRNA and Giardia spp GDH genes. The primers and probes for TaqMan qPCR were designed by using ‘Primer Express’ software commercially with help of value added services from the supplier. Probes were conjugated with FAM and HEX respectively; this combination was appropriate for multiplexing the real-time PCR reactions for two targets. Standard curve for each of the real-time qPCR assay was constructed with DNA concentrations ranging from 101 to 10-5 ng/μl. For each assay, the cycle threshold values (Ct) of dilution points were plotted as a function of the logarithm of the input DNA quantity. Analytical sensitivity or detection limit was calculated to be 0.0004 ng/ul for both TaqMan assays when standardized with standard positive DNA templates in contrast to 0.4 ng/ul as in case of conventional PCR. Diagnostic sensitivity and specificity of the real time PCR assays were calculated based on the results while testing against confirmed positive and negative control DNA templates. Both diagnostic sensitivity and specificity were 100%. Conclusions: The original objective of developing a multiplex real-time TaqMan assay to detect Cryptosporidium and Giardia DNA simultaneously is successfully achieved. In this study 18s rRNA gene of Cryptosporidium spp and GDH gene of Giardia spp were observed to be diagnostic as seen in conventional PCR, SYBR green qPCR and TaqMan qPCR assays in this study. The designing of the above two genes specific primers as well as probes, the methodologies choosen, and the appropriate control materials employed in the development and validation of the TaqMan qPCR assays for individual detection of either pathogens are justified to be appropriate. Extended studies on testing diagnostic efficiencies of these real time multiplex tests on clinical and/or environmental samples are recommended. Significance and Impact of the Study: The multiplex TaqMan assay developed in the present study is beneficial in terms of a single step detection of two most important protozoan pathogens known to cause diarrhoea in both human and animals. Hence it is advantageous over traditional detection techniques being in practice till date. Future development should be screening application of this multiplex assay in both hospitals as well as in epidemiological studies. Specific diagnostic genetic markers should be identified for other important diarrheogenic pathogens (including bacteria, viral, parasitic agents) for which if a panel of such targets are tested by multiplexing highthroughput detection systems, then a faster diagnosis of the accurate etiological agent as well as possible concurrent infections could be identified in one step. Success on multiplexing highly sensitive technique using specific TaqMan probes as experienced in the present study is certainly tempting to add other pathogenic targets to existing two pathogens Cryptosporidium and Giardia for their simultaneous detection.

General overview: Neuroinflammation plays an important role in Alzheimer's disease (AD) pathogenesis. Prolonged activation of microglia can contribute to excessive production of pro-inflammatory cytokines. Neuronal stem cells (NSCs), is a potential AD therapy due to its regenerative abilities. Therefore, this study aims to investigate the regulatory effect of NSCs in lipopolysaccharides (LPS)-stimulated BV2 microglia cells. Methods: The effect of NE4C neuronal stem cell on LPS-stimulated BV2 cells were examined using transwell co-culture system. Real time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were used to study the regulation of inflammasome receptors expression that regulates the pro-inflammatory protein productions. The tested samples were further investigated for the inhibition of caspase-1 and pro-inflammatory cytokine, interleukin 1β (IL-1β) by testing the supernatants in the co-culture using caspase-1 colorimetric assay and enzyme-linked immunosorbent assay (ELISA) respectively. Results: Transwell co-culture of LPS-stimulated BV2 cells with NE4C significantly downregulated (p<0.01) mRNA expression of inflammasome receptors. Further studies on inflammasome dependent caspase-1 expression and IL-1β secretion showed NE4C inhibited inflammasome-mediated inflammation by regulating pro-inflammatory cytokines involved in activation of inflammation. Conclusion: The NE4C inhibits LPS induced neuroinflammation in BV2 cells by suppressing caspase-1 expression and IL-1β secretion through regulation of inflammasome activation.

Anthracycline regimens are commonly used in the adjuvant setting for the treatment of breast cancer since it is considered that anthracycline-containing chemotherapy is beneficial for breast cancer patients. Nonetheless, anthracyclines carry serious side effects, including cardiotoxicity and bone marrow damage. Thus, predictive factors that identify benefit and sensitivity to these drugs are warranted in clinical practice. Topoisomerase II alpha (TOP2A) is proposed as a molecular target for anthracyclines and often amplified concurrently with human epidermal growth factor receptor 2 (HER-2) gene amplification. This study aimed to evaluate the association between TOP2A gene alterations (i.e. amplification or deletion) and oestrogen receptor (ER), progesterone receptor (PR), HER-2 and clinicopathological characteristics of breast cancer. Besides, we also investigated the relationship between TOP2A gene alterations and chromosome 17 polysomy. Two hundred twenty-seven formalin-fixed paraffin-embedded (FFPE) tissue samples collected from 2009 to 2015 were analysed. ER, PR and HER-2 protein expressions were evaluated using immunohistochemical staining. A total of 133 cases was further evaluated for their TOP2A gene alterations using dual hapten dual in-situ hybridisation (DDISH). Among these, 61.7% were HER-2 positive. TOP2A gene was co-amplified and deleted in 28% and 37.8% of the HER-2 positive tumours, respectively. TOP2A gene amplification was observed only in HER-2 positive tumours but not in HER-2 negative tumours. There were significant associations between TOP2A gene alterations and HER-2 gene, and TOP2A gene alterations and ER/PR expressions. TOP2A and HER-2 gene amplifications were frequently detected in patients with intermediate tumour size, lymph node metastases, high histological grade and invasive carcinoma, no special type (NST) phenotype. Tumours displaying chromosome 17 polysomy were frequently detected in TOP2A gene deletion compared to TOP2A amplified cases. There was significant association between TOP2A gene alterations and chromosome 17 polysomy. In conclusion, TOP2A gene alterations were associated with ER, PR, HER-2 and chromosome 17 polysomy.

Preterm prelabour rupture of membranes (PPROM) is a common obstetric condition occurring in 3%-4.5% of pregnancies. The significance of PPROM is that it is the leading cause of preterm birth which is a major risk factor for neonatal mortality and morbidity. amniotic fluid infections caused by ascending microbes from the genital tract play a major role in leading to PPROM. these ascending infections are treated by antibiotics which also reduce the inflammatory process elicited by them. Antibiotics have shown to prolong pregnancy and reduce neonatal morbidity. The drug of choice in PPROM is erythromycin however other drugs including ampicillin, cotrimoxazole and co-amoxiclav are used. The rise of antibiotic resistance and adverse effects associated with antibiotics used in PPROM, has led to the need for new antimicrobial agents such as Malaysian propolis. In this study, we assessed the susceptibility of 42 clinical isolates from PPROM patients to Malaysian propolis including isolates that possessed antibiotic resistant plasmids. Propolis susceptibility was investigated at 100 μg/mL, 500 μg/mL,1000 μg/mL using agar dilution method. Bacteriostatic/Bactericidal effect was investigated in broth. Antibiotic susceptibility of antibiotics to PPROM isolates was assessed using gentamicin 10ug (CN), chloramphenicol 30ug (C), cotrimoxazole 25ug (SXT), ceftriaxone 30ug (CRO), ampicillin 10ug (AMP), erythromycin 15ug(E) and co-amoxiclav 30ug (AMC). Plasmid curing agents Ethidium bromide, Acridine orange and Sodium dodecyl sulfate at a concentration of 200 μg/mL, were used to identify presence of antibiotic resistant plasmids in the clinical isolates. Results from the study showed Malaysian propolis was effective for 62% of the isolates while 38% were resistant. Propolis was effective against Gram-positive isolates namely, Staphylococcus aureus, Enterococcus faecalis, Group B streptococcus, Clostridium spp, Corynebacterium spp, Bacillus spp, however, Gram-negative isolates, Escherichia coli and Klebsiella pneumoniae were resistant. Legionella pneumophila was the only Gramnegative isolate susceptible to propolis. Candida albicans isolates were susceptible to propolis. Overall propolis had a bacteriostatic effect on most susceptible clinical isolates. Compared to the antibiotics used in the study, propolis was more effective than erythromycin, ampicillin, cotrimoxazole and ceftriaxone which encountered high resistance against clinical isolates. Chloramphenicol and co-amoxiclav were more effective than propolis. Plasmids were present in all clinical isolates including those susceptible to propolis. Ethidium bromide was most effective at curing plasmids compared to acridine orange and sodium dodecyl sulfate. In closing, propolis was effective in majority of clinical isolates of PPROM, it was a more effective treatment than erythromycin, ampicillin and cotrimoxazole which are first line antibiotics used in PPROM. Furthermore, plasmids were present in the clinical isolates successfully treated with propolis.

Colorectal cancer is a global burden, today. Currently, the drop in the number of drugs approved by FDA and their limited success, enforce medicinal chemists and biotech industries to develop novel drugs to meet the demands of the patient population. In the present research study, we synthesized six novel compounds of methoxy substituted (Z)‒5‒Benzylidene‒1,3‒thiazolidine‒2,4‒dione via Knoevenagel condensation reaction. All the newly synthesized compounds were tested for their cytotoxic effect. Cytotoxic evaluation was carried out by using MTT reagent on viable human colorectal cancer cell lines: SW48, HT29 and Caco2 and normal colon cell line, CCD 841 CoN. The IC50 values were determined for tested compounds revealing cytotoxic activity. Among all the tested analogues, compound C#06 revealed highest potency on cell line SW48 with an IC50 value of 10.34 ± 2.25. Therefore, it may be implied that the novel compound C#06 appears to be a very promising agent for colorectal cancer treatment.

Berberine (BBR), an isoquinoline alkaloid naturally derived from Berberis vulgaris and many other plant families, constitutes various pharmacological effects including anticancer properties. Particularly in breast carcinoma, berberine has been reported to target multiple pathways ranging from cell cycle arrest to the suppression of angiogenesis to mitochondria-directed apoptosis. However, low bioavailability,hydrophobicity, and low plasma concentration render the compound poor pharmacokinetics which limits its current use to dietary supplements, consumed with other nutritional products. In this study, a biocompatible liquid crystalline colloidal system consisting of monoolein and Poloxamer 407was explored for improved delivery to cultured tumour cells via the enhanced permeation and retention (EPR) effect. BBR-LCN-THP, a formulation containing Transcutol® HP (THP), was negatively charged at -9.29 ± 0.68 mV, recorded mean particle size of 188.77 ± 0.80 nm, PDI 0.16 ± 0.01, and had demonstrated highest entrapment efficiency (88.14 ± 0.29%), and prolonged release (plateau ~80%) across 24 hours. Good colloidal stability was maintained during storage at 25°C and 37°C for about 10 days before degradation was detected. The IC50 of BBR-LCN was about 10-fold lower compared to plain berberine, while the IC50 of BBRLCN- THP and BBR-LCN-PEG were about 55-times significantly lower than free berberine. Enhanced in vitro cytotoxicity, enhanced cellular uptake in MCF7 human breast cancer cells as well as in Caco-2 human colonic cell line, and cell cycle arrest at G1/G0 phase to attenuate cell cycle progression were attractive advantages demonstrated by the fabricated liquid crystalline dispersion systems. Therefore, this study has unravelled the unique tunable characteristics of three formulated LCN carriers of berberine and the optimized LCNs may be potentially developed for oral administration, presenting opportunities as systemic treatment for heterogeneous and/or drug-resistant oestrogen-positive breast carcinoma. Further studies are required to validate nonlinear dose-response relationships and underlying molecular mechanisms of cellular interactions. Keywords: Berberine, liquid crystalline nanoparticles, colloid, monoolein, PEG, MCF7, Caco-2, anticancer, entrapment efficiency, drug release, cellular uptake, internalization

The high intake of fruits and vegetables contribute to sufficient physiological levels of flavonoids as flavonoids have numerous roles in improving general health. Ministry of Health (MOH) Malaysia reported that Malaysians did not meet the dietary guidelines for fruits and vegetables intake daily. Hence, this study was conducted to better understand the relation between flavonoids intake and the expression of histatin 5 in healthy adults. A total of 36 subjects were given Flavonoid Food Frequency Questionnaire (FFQ) and socio demographic questionnaire for assessment of their total flavonoid intake. The mean flavonoids intake amongst the subjects tested was 1721.88 mg/day. Saliva samples were collected and histatin 5 was extracted using zinc chloride in an alkaline condition (pH 9). The extracted protein was then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). When tested with RP-HPLC, all 35 successfully tested subjects showed the expression of histatin 5 in various concentrations. Subsequently, the extracts were analysed using SDS PAGE, using histatin 5 standards and a low protein marker. Amongst the 35 subjects that were successfully tested, 15 subjects showed the presence of bands which indicated the presence of histatin 5 and 20 subjects showed no presence of band, which indicated no histatin 5 indicated when tested using SDS PAGE. Numerical data were analysed using Pearson’s correlation and Student’s T-test. No significant correlation was established between total flavonoid intake and the expression of histatin 5 (p=0.544). However, a gradual modest increase of the salivary protein was seen with the increase of flavonoid intake among the subjects. The intake of fruits and vegetables remain an important source of flavonoids among Malaysian adults.

Background: Obesity is classified as low-grade chronic inflammatory disease and genetic predisposition may play a role in the susceptibility to obesity development. Previous studies have shown that pro-inflammatory cytokine, TNF-α promoter region polymorphism was associated with overweight/obesity in various population. Therefore, this study aims to investigate the association of three single nucleotide polymorphisms of TNF-α (G-308A, C-863A and G-238A) and their haplotypes with the risk of overweight/ obesity in Malaysian population. Methods: Classification of overweight/obesity followed the Malaysian Clinical Practice Guideline of Obesity (2004). Polymorphisms of TNF-α (G-308A, C-863A and G-238A) were evaluated on 105 overweight/obese subjects and 100 non-overweight/ non-obese subjects as controls through tetra-arms PCR. Chi square and logistic regression analysis were done using SPSS software while haplotype and linkage disequilibrium analysis were done using HaploView 4.2 software. Results: The rare A alleles of TNF-α G-308A, C-863A and G-238A did not occur more frequently in overweight/obese individual as compared to controls. Similar results were obtained in allele frequency comparison among Chinese and Korean population for G-308A, G-238A and C-863A polymorphism. Although weak linkage disequilibrium was found between the markers tested, there were six haplotypes occurred in more than 1% of the population, with 308G/-863C/-238G as the most frequent haplotypes. Conclusion: This study recorded no association of TNF-α G-308A, C-863A and G-238A with two overweight/obesity phenotypes, BMI and WHR. Further studies with larger cohort and other obesity-related phenotypes are required to investigate the possible association of TNF-a polymorphism with overweight/obesity in Malaysian population.

Thermoplastic elastomers (TPE) is an elastomeric material suitable for use in medical applications due to its inertness, low leachables and biocompatibility. Plasticisers are added into TPE blends to improve its processability and flexibility. TPEs are usually plasticised with mineral oil (MO) which are associated with inflammatory effects. Its different hydrocarbon components can induce different mechanisms that produces and regulates autoantibodies. Palm oil-based plasticisers in other polymer blends showed favourable mechanical properties, thermostability and enhanced biodegradation rate. A TPE blend with palm oil based polyester polyol (PP) as plasticiser was prepared and its suitability for use in medical applications were investigated. The tensile properties of the TPE blend was optimised and tested on in vitro enzymatic degradation with lipase over 60 days. The water uptake, weight loss and changes in tensile properties were measured. The biocompatibility was assessed by cytotoxicity assay on human kidney embryonic (HEK 293) cells and hemolysis was tested by direct contact of samples on human blood. The TPE blends with PP plasticisers showed significant weight loss and formation of cracks after enzymatic degradation; but with sustained mechanical properties. It is also a non-cytotoxic and non-hemolytic material which can be considered for medical applications like tissue engineering.

Background: The histopathological hallmarks of Alzheimer’s disease (AD) are aggregation of senile plaques (SPs) and neurofibrillary tangles (NFTs) which resulting in progressive dysfunction of synaptic activity and eventually neurodegeneration. The aggregation of amyloid-β peptides and deposition of tau oligomers contributes to the pathological formation of SPs and NFTs respectively. Besides, neuroinflammation also contribute to pathogenesis and progression of AD. Neural stem cells (NSCs) have the ability to self-proliferate and protect the nervous system. Thus, this regenerative characteristic of NSCs provide great promise in regenerating neuronal cells in the brain for aging, neuroinflammation and neurodegeneration. This study aims to investigate the protective effect of NSCs (NE4C) against microglial-mediated neurotoxicity. Methods: Cell viability assay was used to study the protective effect of NE-4C on the viability of SH-SY5Y. Western blot was used to study the expression of tau and its signalling pathway: glycogen synthase kinase 3β (GSK3β) and p38α mitogen-activated protein kinase (p38α MAPK); also amyloid-β processing protein: amyloid-β precursor protein (APP) and beta-site APP cleaving enzyme 1 (BACE1). The expression of beta-amyloid (Aβ) was determined by enzyme-linked immunosorbent assay (ELISA). Results: The protective effect of NE4C on SH-SY5Y cells against microglia-mediated neurotoxicity was observed in cell viability studies. The expression of phosphorylated tau, Aβ, GSK3β and phosphorylated p38α MAPK in SH-SY5Y cells were downregulated when cultured in the conditioned media of co-culture of NE-4C and BV2 cells. However, LPS stimulation do not affect the expression of APP and BACE1 in SH-SY5Y cells. Co-cultured of NE-4C cells with BV2 cells resulted in decrease in APP and BACE1 expression regardless of microglial activation by LPS. Conclusion: The NE-4C cells confer protective effect on SH-SY5Y cells against microglia-mediated neurotoxicity by suppressing the expression of tau and its signalling pathway. The attenuation of Aβ by NE-4C cells when NE-4C cells co-cultured with BV2 cells were unlikely to be due to the attenuation of APP and BACE1 expression alone. Further studies to determine the mechanisms involved in the attenuation of Aβ by NE4C through regulation of BV2 cells is required. Collectively, these results suggested that the neuroprotective effect of NE4C against microglia-mediated toxicity involved the attenuation of tau phosphorylation and amyloidogenesis, adding to the inherent benefits of NSCs in AD treatment.

Geraniin, the natural polyphenolic compound extracted from the plant, Phyllanthus amarus sp, is used to study various diseases. It has been reported that geraniin possesses anti-tumour, anti-inflammatory, anti-microbial and anti-oxidative properties. In one of the previous investigation, it was noted that geraniin exerts the inhibitory effect on the proliferation of estrogen dependent human breast cancer cell line. In this study, the anti-tumour effects of geraniin on human breast cancer cell metabolism were investigated. This study assessed the anti-tumour effects of geraniin using various approaches such as cell viability assay, lactate assay and qPCR gene expression for the in vitro work. We have particularly investigated the effect of geraniin towards the two different breast cancer cell lines which are MCF-7 and MDA-MB-231. We would like to further investigate the treatment response between hormonal dependent (MCF-7) and non-hormonal dependent (MDA-MB-231) breast cancer cells. The results showed that geraniin exhibits the inhibitory effect against both MCF-7 and MDA-MB-231 as seen in the cytotoxicity assay. This was further strengthened by evidence of lactate assay experiment in which both MCF-7 and MDA-MB-231 revealed reduction in the lactate concentration. However, from the lactate result, MCF-7 showed significant lactate reduction as compared to MDA-MB-231. On the other hand, qPCR gene expression on the targeted genes of MCT 1, MCT 2 and MCT 4 were downregulated in MCF-7. As for the MDA-MB-231, the genes of MCT 1 and MCT 2 were also downregulated, however, it was upregulated in MCT 4. Overall, the result is promising as geraniin could have a beneficial effect in combating the breast cancer progression. In conclusion, this study shows that geraniin has powerful anti-tumour effects in estrogen dependent human breast cancer (MCF-7) than the non- estrogen dependent breast cancer (MDA-MB-231) as seen from the results. Hence, geraniin holds great potential as a chemotherapeutic agent in the treatment of breast cancer.

Introduction: Saffold virus (SAFV), of the Picornaviridae family is currently associated with unknown pathogenicity although the virus had been detected in infants with neurological disorders, including meningitis and cerebellitis. Exposure to SAFV infection mouse models showed that the virus is able to induce apoptosis of neuronal and glial cells. Orientin and madecassoside are potential compounds having an effect against SAFV infection due to their anti-viral, anti-inflammatory, antioxidative and anti-apoptotic abilities. Therefore, the aim of this study is to investigate if orientin and madecassoside provide preventive, direct inhibitory or a rescue effect on BV2 microglia cells and NE-4C neural stem cells infected with SAFV. Methods: The effects of orientin and madecassoside on BV2 and NE-4C cells infected with SAFV were examined at three different time-points: prevention (cells were treated with the two compounds respectively first, followed by SAFV infection), direct inhibition (the two compounds respectively were administered onto SAFV first and then added to the two cell lines) and rescue (cells were first infected with SAFV followed by administration of the two compounds respectively). The IC50 dose of orientin and madecassoside, and the CCID50 of SAFV on BV2 and NE-4C cells were determined via MTT assays. Subsequently, the two compounds were administered on the BV2 and NE-4C cells infected with SAFV at the three aforementioned time-points. The cells’ viability was measured via the MTT assay while the virus titer was determined by real-time PCR. Effectiveness of the compounds was determined based on improvements in cell viability and reductions in virus titer compared to the negative control. Results: Results from the efficacy experiments indicated that orientin was more effective than madecassoside in inhibiting SAFV as shown in the direct inhibition and rescue modes. However, both compounds were ineffective when administered in a preventive manner. Direct inhibition was selected as the optimum method as it provided more consistent results. Orientin significantly improved cell viability in BV2 cells and NE-4C cells by 94% and 90% respectively and had significantly lower virus titer when administered via direct inhibitory mode at a dose of 1μg. Whereas, madecassoside was only effective in NE-4C cells during direct inhibition with a significant improvement in cell viability of 87% and significantly lower virus titer compared to the negative control at a dose of 1μg. Conclusion: This study enabled the development of a cell-based assay with suitable methodology and criteria for analysis of results for drug testing against SAFV. Positive results were obtained for both rescue and direct inhibition modes but were more consistent for latter mode of action. Hence, direct inhibition of SAFV was the preferred mode of action and results indicated that orientin was superior to madecassoside in improving cell viability and reducing SAFV virus titer.

Diabetes mellitus (DM) is a chronic metabolic disorder, characterised by constant elevated levels of glucose in the blood. Currently, type 2 diabetes mellitus (T2DM) emerged as global burden disease. Despite the significant progress in treating diabetes by hypoglycaemic drugs, search for new drugs continues as the present synthetic drugs have several limitations. The herbal drugs with antidiabetic activity are attracting scientists' attention. Commonly, the aims of using herbs are to enhance insulin sensitivity and secretion. Madecassoside (MAD) and catalpol (CAT) are antioxidant herbal compounds. This study aimed to investigate the effect of MAD and CAT on insulin sensitivity and release in INS-1E cells. INS-1E cells were cultured in high glucose medium for 48 h. Subsequently, the medium was removed then MAD and CAT were added for 24 h. Then glucose stimulated insulin secretion (GSIS) and insulin signalling proteins were investigated. Results showed that 30 μM MAD and 40 μM CAT significantly increased the insulin concentration 26.4 ± 0.1 (P<0.01) and 26.1 ± 0.2 μg/L (P <0.05), respectively. Furthermore, these compounds improved insulin resistance through a significant enhancement of Phospho- (Tyr608) insulin receptor substrate-1 (IRS1), Akt, phospho-Ser473 Akt and GLUT2 expressions. In conclusion, 30 μM MAD and 40 μM CAT markedly increased the sensitivity of the cells to glucose and insulin.

Parkinson’s disease is a common neurological disease characterised by motor and non-motor symptoms. Parkinson’s disease is associated with the loss of dopamine in the substantia nigra pars compacta neurons. Current treatment for Parkinson’s disease is ingestion of levodopa (dopamine precursor) complemented with the administration of dopamine agonists and inhibitors of dopamine degrading enzymes. However as the syndrome advances, constant use of dopaminergic medications becomes less effective and can lead to motor complications called dyskinesia. Hence, cell therapy is a promising alternative therapeutic strategy in Parkinson’s disease. It restores the damaged neurons in the nigrostriatal pathway. Transplanted neuronal stem cells are multipotent and are capable of differentiating to several types of cells in the central nervous system, including neurons and other brain cells. However, salsolinol, a neurotoxin exerts substantial toxicity on the transplanted neuronal stem cells. Salsolinol has diverse neuropharmacological effects including inducing oxidative stress besides being able to inhibit the mitochondrial energy supply. Salsolinal also inhibits some key enzymes involved in the production of dopamine such monoamine oxidase, tyrosine hydroxylase and catechol-O-methyltransferase. Additionally, salsolinol also prevents the uptake of catecholamines. This study aims to evaluate neurotoxicity and apoptogenic effect of salsolinol on NE-4C neuronal stem cells through mammalian target of rapamycin signalling pathway. Salsolinol was added to NE-4C cells. Cell viability and apoptosis were evaluated by using trypan blue exclusion method and mitochondrial membrane potential staining assay, respectively. Additionally, some key regulatory proteins that could mediate cell apoptosis through mTOR signalling pathway was analysed. The relative expressions of mTOR, phospho-mTOR, raptor and GßL proteins were determined using enzyme- linked immunosorbent assay. Results revealed that when NE-4C cells were treated with salsolinol (0-100μM) for 24, 48 and 72 hours, salsolinol induced death in NE-4C cells mainly by alteration of mitochondrial membrane potential. Additionally, the change of the level of phospho-mTOR indicates the contribution of this protein in the death of the NE-4C cells. In conclusion, salsolinol induced neuronal cell death via mitochondrial membrane potential alteration and down-regulation of phospho-mTOR protein.

Intracanal medicament is an important step in reducing the microorganisms in root canals. Drug synergism occurs when two or more drugs work together to produce an effect more potent than if each drug were applied individually. E. faecalis and C. albicans are the most common microorganisms responsible for root canal treatment failure. The current study aimed to determine the antimicrobial efficacy of AgNps, PrNps, and their combination against E. faecalis and C. albicans, to determine the effect of their combination on the adherence and hydrophobicity of E. faecalis and C. albicans on the tooth surface and to study the effect of their combination on proliferation and expression of vascular endothelial growth factor (VEGF) from PDLSCs. The antimicrobial efficacy was studied by colony forming unit method and adherence assay was assessed by fluorescence microscopy. To measure the cell proliferation, MTT assay was performed and VEGF concentration was measured by ELISA. The synergisms (combination) of AgNps and PrNps have similar antimicrobial efficacy compared to AgNps and PrNps alone. The synergisms had least hydrophobicity and adherence values compared to AgNps and PrNps alone. PDLSCs viability treated with combination of AgNPs and PrNPs showed better cell viability compared to AgNPs, PrNps alone and CHX. VEGF plays a pivotal role in improving the angiogenesis and osteogenesis for regeneration. Synergisms (combination) of the of AgNps and PrNps improve the VEGF secretion compared to the AgNPs, PrNps alone and CHX. The combination of AgNps and PrNps can be used as potent intracanal medicament.

Dengue fever is a mosquito-borne viral infection that imposes significant public health concern in Malaysia. Evidence has shown that Monocyte plays an essential role in dengue pathogenesis. This study evaluates the value of monocyte chemoattractant protein-2 (MCP-2) as an early severity biomarker in dengue. Total of 150 laboratories confirmed adult dengue subjects in the febrile phase arm with “Mild Dengue (MD)” and “Dengue with warning Sign/Severe Dengue (WS/SD)” were chosen for this study. Subjects in the WS/SD arm were recruited from Tuanku Ampuan Rahimah (TAR) Hospital, Klang while those in MD arm were recruited from several primary care clinics around Shah Alam, Selangor. Initially, plasma from the subject’s blood was collected for the MCP-2 protein level measurement using ELISA while statistical analysis was done using SPSS. Subject were follow-up to obtain the clinical outcome of infection. Results showed no significant difference in the mean MCP-2 protein between the MD and the WS/SD arm (p=0.841). However, age (p value=0.04) and day of illness (p value=0.049) were significantly associated with the MCP-2 ELISA concentration. In conclusion, our results suggested that MCP-2 protein is not a suitable biomarker to predict the severity of dengue in the early phase of dengue fever. Nonetheless, subject age and day of illness are independent confounding factors for MCP-2 protein concentration. MCP-2 concentration was highest on day 1 and day 2 of illness and subsequently reduce after day 2 of illness. Separately, MCP-2 concentration increases with increasing age group, with the highest concentration at more than 60 year old age group.

Background Immunosuppressive microenvironment of pancreatic cancer pancreatic ductal adenocarcinoma cell (PDAC) and pancreatic stellate cell (PSC) comprise myeloid derived suppressor cells (MDSC) which greatly contribute in impaired antitumour response, as well as progression of tumour. It was previously (unpublished) reported that physical contact of PDAC and PSC with peripheral blood mononuclear cells (PBMC) can convert PBMC into MDSC, causing suppression of T cells proliferation and activity. Little is known on the role of PDAC or combined with PSC in MDSC differentiation. In order to better mimic the tumour microenvironment, the effects of PDAC and PSC on PBMC was investigated via incubation with conditioned media prepared from supernatant of PDAC and PSC co-culture. Methods PBMCs were incubated with conditioned media prepared from supernatant of PDAC and PSC co-culture. The differentiation of PBMC to MDSC and their subtypes, polymorphonuclear-MDSC (p-MDSC) and mononuclear-MDSC (m-MDSC) were analysed using flow cytometer. Results This study had shown that secreted molecules from PDAC and PSC alone can induce the differentiation of MDSC and thus increase the percentage of MDSC in the total cell population. Secreted molecules from PDAC and PSC combination can also increase the ratio of p-MDSC to m-MDSC. Conclusion The secreted molecules in conditioned media alone is able to increase the ratio of p-MDSC : m-MDSC, even though there is no physical contact between PDAC, PSC and PBMC.

Moringa oleifera L (Family: Moringacea) is a type of perennial angiosperm tree that consists of a few plant species. Moringa is a common vegetable used by inhabitants of tropical and sub-tropical countries. At present, M. oleifera is noted to possess a wide range of biological roles including anti-cancer, neuroprotective, anti-inflammatory and hepato-protective activities. In addition, there are numerous studies that have reported many other therapeutic effects of M. oleifera, which include anti-rheumatoid arthritis, anti-infertility, diuretic, thyroid regulation, anti-depression, anti-diabetes, pain relief and atherosclerosis. Over the last decade, the bioactivity of M. oleifera has achieved vast attention due to these bio-activities. The anti-cancer activities could be attributed to the presence of several bioactive compounds such as β-sitosterol-3-O-β-D-glucopyranoside, 4-(α-L-rhamnosyloxy) benzyl isothiocyanate and niazimicin in the Moringa oleifera leaves extracts. It has been noted that Moringa oleifera leaf extracts have anticancer effects, which is reported to take place through induction of apoptosis. To date, the ability of extracts from M. oleifera leaf to induce apoptosis in breast cancer cells has not been fully elucidated. This study involves evaluating the ability of extracts of Moringa oleifera, from various solvents, to modulate apoptosis. Cell based assays using two types of human breast cancer cell lines (MCF-7 and MDA MB-231) as well as a normal breast epithelial cell line (MCF-10A) were used in the experiments. Expression of several genes (human Caspase9, Bcl2, Ccnd1 and Brca2 genes) in the cells when treated with the extracts were also investigated. A commercial source of dried Moringa oleifera leaf powder was subjected to sequential extraction using n-hexane, followed by ethyl acetate, methanol and water-based extraction. The effect of these extracts (0, 10, 20, 40, 60, 80, 100 μg/mL) on cell proliferation was evaluated using the MTT cell proliferation assay. Data analysis was carried out on the MTT assay results. The half maximal inhibitory concentration (IC50) of M. oleifera extracts following 72 hours of exposure was found to be: (a) MCF-7 [IC50 for ethyl acetate (hex-EA): 95 g/mL; water: 100 g/mL and quercetin: 90 g/mL] and MCF-10A [IC50 for ethyl acetate (hex-EA): 52 g/mL]. The IC50 values for MDA MB-231 cells could not be attained in the studied concentrations range. Difficulty in obtaining the IC50 values for MDA MB-231 cells might be due to as this cell line is a triple-negative breast cancer [lack ER, PR and human epidermal growth factor receptor 2 (HER2) amplification)], which means it is an aggressive kind of breast cancer with limited therapeutic approach. The RNA from MCF-7 and MCF-10A cells treated with various extracts for 72 hrs of exposure was extracted for gene expression studies. The genes studied were [human Caspase9, Bcl2, Ccnd1 and Brca2 genes]; all of which are reported to be involved in the regulation of apoptosis. Following this, the purified RNA was analysed for gene expression using the real-time PCR approach. Gene expression studies showed that Caspase9, Bcl2, Ccnd1 and Brca2 genes were upregulated in MCF-7 cells treated with water extract of Moringa oleifera leaf powder. In the MCF-10A cells, the expression of the Bcl2 gene was downregulated in cells treated with hexane extract whilst the expression of the Ccnd1 gene was downregulated in cells treated with ethyl acetate (hex-EA) and water extracts. The findings from this study suggest that Moringa oleifera extracts can differentially regulate genes related to apoptosis in MCF-7 cells. More studies need to be conducted to elucidate the molecular signalling pathways.