Theses (MSc. Molecular Medicine)

Background: Neural tube defects (NTDs) are congenital malformations of the brain, spine, or spinal cord resulting from genetics or nutritional deficiencies in folates and other micronutrients during early embryonic development. Inadequate folate increases homocysteine, which increases the risk of NTDs. Numerous discussions and studies on the efficacy of supplementation with activated 5-Methylhydrofolate (5-MTHF) versus synthetic folic acid in improving blood folate levels in women of childbearing age. This systematic review and meta-analysis aim to evaluate the available evidence and synthesise data on the comparative impact of these two forms of folate supplementation. Methods: A systematic review and meta-analysis were conducted by PRISMA guidelines. Relevant studies were identified through electronic searches of PubMed, Scopus, and ScienceDirect, covering randomised controlled trials published up to March 2024. Inclusion criteria focus on trials comparing 5-MTHF and folic acid supplementation across various populations, including pre-pregnancy, pregnancy, postpartum, lactation, and women with MTHFR gene polymorphisms. Risk of bias was assessed using the Cochrane Risk of Bias tool, which evaluated factors such as randomisation, blinding, and completeness of outcome data. Meta-analysis was performed using Comprehensive Meta-Analysis (CMA) and RevMan software to synthesize data on plasma folate, and red blood cell folate levels. Results: The study aims to review existing evidence from a total of ten studies involving 1,351 women across various stages of pre-pregnancy, pregnancy, post-partum, and lactation, as well as women with MTHFR gene polymorphism. The methodologies included randomised, controlled trials with different dosages of folic acid, 5-MTHF, or placebos. Key biomarkers measured included plasma folate, red blood cell (RBC) folate, homocysteine, and unmetabolised folic acid levels. Results indicate variability among studies where four studies showed higher blood or plasma folate levels with 5-MTHF compared to folic acid/placebo, while three others showed similar increases in folate levels but with potential additional benefits such as reduced unmetabolised folic acid or lower homocysteine levels in the 5-MTHF groups. The remaining three studies found comparable efficacy between the two forms of folate, one of which suggested a relationship between the MTHFR (methylenetetrahydrofolate reductase) genotype and high levels of plasma homocysteine, which can contribute to various health risks. There were ten studies assessed for risk of bias. Two studies were of some concerns due to lack of clarity in the reported outcome data and the remaining eight studies have a low risk of bias. Of the eight studies included in the analysis, two studies, short-term duration of 12 weeks or less weeks, reported significant positive differences in favour of 5-MTHF while six studies, long-term duration of more than 12 weeks, favoured folic acid over 5-MTHF. Discussion: Optimal blood folate levels are important in preventing NTDs. Many variations exist in these studies that warrant further standardised research to clarify optimal dosages and durations of supplementation with 5-MTHF versus folic acid. The benefits of reduced homocysteine and unmetabolised folic acid through 5-MTHF supplementation may bring additional benefits to high-risk groups. Conclusion: This review highlights the substantial heterogeneity among studies comparing 5-MTHF and folic acid supplementation. While short-term studies favour 5-MTHF, long-term studies generally favour folic acid. These findings highlight the need for further research to standardize methodologies and clarify supplementation guidelines.

INTRODUCTION: Quebrachitol, an optically active cyclitol derived from plants, has attracted interest as a possible natural product inspiration for pharmaceuticals due to its potential medicinal therapeutic qualities in diabetes, cancer, and drug production. However, the antimicrobial properties of quebrachitol were indefinite. This is the first systematic review to specifically determine, appraise, and consolidate the antimicrobial effects of quebrachitol. METHODS: A comprehensive database search from PubMed, Scopus, and Google Scholar from January 2000 to February 2024 was conducted. All English, full-text, original articles involving experimental, in vivo, or in vitro research were included. Data pertaining to quebrachitol’s effectiveness (nil, partial, or total inhibition) against microorganisms was extracted. In the presence of antimicrobial effects, the minimum effective dosage or duration and mechanism of action were determined. The quality assessments of the articles were performed with QUIN (in vitro) and SYRCLE RoB (in vivo). RESULTS: The research yielded 866 studies, but only 11 met the inclusion criteria, comprising 7 in vitro, 1 in vivo, 1 in ovo, and 2 papers, which combined both in vitro and in vivo trials. Quebrachitol was found to have an anti-infectious effect against Salmonella sp., Candida albicans, infectious bursal disease virus, Newcastle disease virus, and Plasmodium sp. Additionally, there was significant evidence of inhibitions against biofilm in Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA). QUIN tool revealed that 60% of in vitro studies had a low risk of bias, with overall scores ranging from 56.25 to 87.5%. SYRCLE, however, showed that none of the studies contained more than 50% of the studies exhibited low-risk bias. The allocation sequence demonstrated high-risk bias due to a lack of random sequence generation and reliance on non-random approaches like availability or predetermined rules. CONCLUSION: Quebrachitol had demonstrated significant and promising antimicrobial potential in the in vitro studies, showing efficacy against pathogens associated with typhoid, malaria, avian flu disease, and biofilm-related diseases. However, the in vivo findings were inconclusive due to high-risk bias. Thus, further well-designed in vivo research is required to establish the antimicrobial efficacy and safety of quebrachitol and to assess its potential role in developing alternative treatments to address current antimicrobial resistance and pharmacovigilance concerns. Keywords: Quebrachitol, antimicrobial, antibacterial, antifungal, antiviral, antiparasitic, antibiofilm

Breast cancer cells often respond poorly to treatment because of their different subtypes such as low expression of the oestrogen, progesterone and receptor tyrosine-protein kinase erbB-2. Hence, there is a need to identify new molecules for the treatment. Radix Bupleurix contains glucosides, a class of oleanane derivatives called saikosaponins that have been widely used in Traditional Chinese Medicine for over a millennium. There are two main active components in Radix Bupleurix, Saikosaponin A (SSA) and Saikosaponin D (SSD), known to have a wide range of pharmacological actions. This study identifies the anti-cancer effect of SSA and SSD on breast cancer cell lines, MDA-MB-468, MCF7 and T47D. Our findings suggested that SSA and SSD can inhibit the growth of the breast cancer cell line in a dose-response-dependent manner. The IC50 for SSA and SSD ranges between 4.48 μM to 6.97 μM. SSA and SSD can inhibit cell migration and colony formation. Scratch assay results showed a significant area of closure compared to control (p < 0.05) after 120 hours. Furthermore, a combination assay showed no synergistic effect between SSA and SSD. In conclusion, these results suggest that SSA and SSD could be potential molecules for the treatment of breast cancer but further study is required to understand the pathway involved in the anti-cancer effect.

Excessive or dysregulated inflammation can lead to Chronic inflammatory respiratory diseases (CIRDs), such as Chronic Obstructive Pulmonary Disease (COPD) and asthma, which are significant global health concerns characterized by persistent airway inflammation and tissue damage. While conventional treatments have been effective, they are often associated with severe side effects driving the search for safer, natural alternatives. Nobiletin, a flavonoid derived from citrus peels, has emerged as a promising candidate due to its potent anti-inflammatory. It’s clinical application is hindered by poor water solubility, low oral bioavailability, and extensive first-pass metabolism. To address these limitations, this study explores the use of Nanostructured Lipid Carriers (NLCs) as an innovative mechanism of delivery for nobiletin. This research focused on formulating and characterizing nobiletin-loaded NLCs (Nob-NLC) to optimize their physicochemical properties and evaluate their efficacy in targeting respiratory inflammation using mouse macrophage cell line (RAW 264.7). The optimized formulation exhibited desired particle characteristics and a sustained release demonstrating its potential for enhanced therapeutic efficacy. In vitro studies revealed that Nob-NLC when compared to free nobiletin, demonstrated a greater anti-inflammatory action. Nob-NLC in the incubation significantly decreased the production of Nitric Oxide to 26.5 ± 0.27 μM (41.3 % decrease compared to LPS alone, p < 0.0001) and Reactive Oxygen Species 76.5% (108.5 ± 0.32 μM, p < 0.0001 vs. LPS alone). Furthermore, it reduced the mRNA expression of pro-inflammatory cytokines : reducing TNF-α expression by 69.1% (0.34 ± 0.02, p < 0.0001 vs. LPS-only group) and IL-6 expression by 42.5% (0.62, p < 0.0001 vs. LPS-treated group. The difference between action of pure nobiletin and Nob-NLC were statistically significant (p < 0.0001) in all in vitro studies except the Nitric Oxide study (p > 0.05). The results underscore the potential of nobiletin-loaded NLCs as an effective treatment strategy for managing CIRDs.

Lung cancer is the second most common malignancy globally and the cause of the highest number of cancer-related mortality worldwide. Non-small-cell lung cancer contribute to the most of the cases. Conventional treatment methods including radiation therapy, chemotherapy and immunotherapy do exist, however they are only partially effective due to their side effects and recurrence risk. In both in vitro and in vivo investigations, 18β-Glycyrrhetinic acid (18β- Gly), a naturally occurring substance extracted from glycyrrhizic acid/glycyrrhizin of licorice plant, has demonstrated anti-cancer potential by suppressing cancer cell growth, angiogenesis and metastasis. Although 18β-Gly possess potential therapeutic abilities, the application of free 18β-Gly into clinical practices hindered due to its poor physicochemical characteristics such as poor bioavailability and low water solubility. PLGA, a synthetic polymer is great tune-able options for nano-drug delivery system which can be used for various diseases including cancer therapy. Formulating 18β-Gly into PLGA will help to overcome challenges imposed by poor physicochemical characteristics of 18β-Gly. For this research, 18β-Gly-PLGA was tested for its anti-cancer ability in A549 cell lines found in lung cancer. This study has produced results showing 18β-Gly-PLGA has great physicochemical characteristics such as sustained in vitro drug release and good entrapment efficiency. 18β-Gly-PLGA also able to inhibit A549 cells proliferation and migration. The oncogenes such as KRT18, EGFR, BRAF and KRAS were transcriptionally down-regulated as part of the underlying mechanism of 18β-Gly-PLGA anti- cancer effects. 18β-Gly-PLGA also able to significantly down-regulate cancer proliferation and migration associated proteins such as ErbB2, Survivin, M-CSF and Mesothelin. The anti- cancer ability of 18β-Gly-PLGA compared to empty PLGA to demonstrate the true potential of 18β-Gly-PLGA as anti-cancer agent.

Zerumbone (ZER) is an 11-membered crystalline sesquiterpene from the rhizome of Zingiber zerumbet, which carries many beneficial properties, including anti-inflammatory and antioxidant properties. In chronic obstructive pulmonary disease (COPD), zerumbone targets several inflammatory and oxidative mediators released by broncho-epithelial cells and alveolar macrophages in various pathways resulting in the suppression of the disease progression. However, zerumbone has many several drawbacks such as low bioavailability, poor gastrointestinal (GI) absorption and non-specific targeting of tissues and organs, all of which limits the development of ZER as potential new drug formulation for the treatment of COPD. Liquid crystalline nanoparticles (LCN) are novel drug delivery carrier that have tuneable characteristics to enhance and ease the delivery of bioactive compounds. Loading ZER into LCNs can potentially overcome the drawbacks of free ZER on its own. In this study, ZERLCNs were investigated for their ability to inhibit cigarette smoke extract induced inflammation, oxidative stress and senescence (the main characteristics of COPD) through in vitro study in human healthy bronchoepithelial cell line (BCiNS1.1) and mice macrophage (RAW264.7). The study compared the biological activity of ZER-LCNs with pure ZER powder. The results from this study indicated that ZER-LCNs had advantageous physicochemical parameters including a sustained in vitro release as well as a high entrapment efficiency. Additionally, the in vitro cellular studies showed that the ZER-LCNs had potent anti-inflammatory and antioxidant properties in both the BCi-NS1.1 and RAW 264.7 by regulating the genes and cytokines produced during inflammation and oxidative stress. The broncho-epithelial cells treated with ZER-LCNs inhibited the senescence induced by cigarette smoke extract by regulating the gene and protein expression of p21 and p16 (a marker of senescence). The research found that ZER-LCNs have superior biological activity compared to free ZER against cigarette smoke extract induced oxidative stress, inflammation, and senescence. This suggests the unique tuneable characteristics of ZER-LCNs and promising potential development for widescale pharmaceutical use. Additional in-depth studies involving animal model or clinical study on the mechanistic effect of ZER-LCN for its anti-inflammatory, antioxidative and antisenescence properties is required to validate its potential to treat COPD. Keywords: Zerumbone, liquid crystalline nanoparticles, monoolein, P407, anti-inflammatory, antioxidant, entrapment efficiency, drug release.

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.

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.

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.

Rheumatoid arthritis (RA) is a chronic inflammatory condition which primarily affecting the joints and is characterized by dysregulated peptidylarginine deiminase (PAD) activity. PAD enzymes play a crucial role in catalysing the citrullination process and are implicated in RA pathogenesis by modifying RA-associated autoantigens. Meanwhile, Porphyromonas gingivalis (P. gingivalis), a key bacterium responsible for periodontitis, produces P. gingivalis peptidylarginine deiminase (PPAD). PPAD serves as a critical link between periodontitis and RA by generating autoantigens that trigger immune responses in RA patients. Given its role in autoimmunity, inhibiting PPAD activity presents a promising approach for managing RA. This study aims to evaluate the in vitro inhibitory effects of five FDA-approved compounds on PPAD and to identify key structural features responsible for protease inhibition. In this study, PPAD gene was cloned into pColdI plasmid and transformed into DH5α competent cells of Escherichia coli, then PPAD were overexpressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). The overexpressed cells then were sonicated to extract the PPAD enzyme (protein). The cells then pelleted because PPAD was in soluble form, then only the supernatant subjected for the citrulline colorimetric assay. In colorimetric assay, five FDA-approved compounds which had subjected to prior structure-based virtual screening, were evaluated in this study to confirm their inhibitory activity against the PPAD. Then the compounds that have the highest ability to reduce the citrullination activity, were subjected to IC50 study in order to assess the inhibitory effects of this top compound. The result indicated that PPAD gene was successfully inserted into the pColdI plasmid and transformed into DH5α competent cells. The DH5α-pColdI-PPAD cells were successfully overexpressed and subjected to sonication for PPAD enzyme extraction. The supernatant obtained after high-speed centrifugation was used for the citrulline colorimetric assay. In this assay, five drugs (Amifostine, 4-amino-3-hydroxybutyric acid, Eflornithine, Fludarabine, and Levetiracetam) were tested at 50 μM concentration against the PPAD enzyme. Among them, only Eflornithine exhibited the highest reduction in PPAD citrullination activity, showing a 16.3% decrease. Then further IC50 analysis revealed that Eflornithine could inhibit the PPAD citrullination activity by 50%, with an IC50 value of 108.6 μM. Although Eflornithine has demonstrated potential in reducing the PPAD enzyme activity, further validation is required before Eflornithine can be considered as appropriate treatment for RA.

Chronic wounds represent a significant healthcare challenge, often leading to prolonged patient suffering, bacterial infections, and impaired tissue regeneration, causing untreated and long-lasting scars formation. This study explores the potential of curcumin and quercetin-loaded chitosan-pectin scaffolds as a novel solution for enhancing wound healing. Curcumin and quercetin, both are well-known for their antioxidant, anti-inflammatory, and antibacterial properties, were incorporated into chitosan-pectin scaffolds via a lyophilization method. Different ratios of curcumin and quercetin were being explored and tested for their biocompatibility, antioxidant capacity, antibacterial and antibiofilm capacities, drug release profiles as well as physicochemical tests to assess their suitability for wound healing applications. The surface morphology and pore sizes and distributions were assessed with scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Besides that, the antioxidant activity of the resulting scaffolds was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, while their anti-inflammatory effects were evaluated in vitro through a bovine serum albumin (BSA) denaturation assay. Moreover, the antibacterial efficacy as well as antibiofilm affinity of the scaffolds were tested against Staphylococcus aureus and Pseudomonas aeruginosa. Biocompatibility of the scaffolds was tested via cell viability MTT assay while in-vitro wound healing was assessed using scratch assay. All of the curcumin-quercetin loaded scaffolds demonstrated strong antioxidant activity, ranging from 83 – 90%, and exhibited comparable anti-inflammatory activity, with values between 83 – 91%. They also showed impressive wound healing potential in vitro, with rates ranging from 85 - 100% at the 48-hour. Additionally, the antibiofilm activity against Staphylococcus aureus was notably high, achieving a 75 – 85% biofilm reduction. In comparison, the reduction in biofilm formation for Pseudomonas aeruginosa was moderate, ranging from 45 – 59%. The curcumin-quercetin loaded scaffolds, particularly the scaffold containing curcumin and quercetin at ratio 1:3 demonstrated the most promising results. These findings highlight the potential application of curcumin and quercetin-loaded chitosan-pectin scaffolds in promoting wound healing, addressing bacterial biofilms and supporting tissue regeneration. The results contribute to advancing biomimetic scaffolds as innovative solutions for managing chronic wounds and improving patient outcomes.

In comparison to traditional organic synthesis, catalyst-free synthetic techniques provide several advantages and have been proved to be environmentally friendly. Here, a facile catalyst-free procedure for the synthesis of derivatives of diarylsulfonylureas was developed. The simple process involves reacting p-toluenesulfonyl isocyanate with various substituted primary amines in the presence of dichloromethane as the solvent at room temperature to produce the desired diarylsulfonylureas derivatives. Nine compounds with up to 94% yield have been synthesized successfully through this protocol. These products' structures were characterised using a variety of techniques, including fourier-transform infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy, physical analysis, and melting point. Overall, this method has many benefits, including convenient room temperature synthesis, a straightforward workup procedure, rapid reaction times, and quantifiable reaction yields. Keywords: Diarylsulfonylureas, Green Chemistry, Catalyst-Free, Room Temperature synthesis

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.

Pancreatic ductal adenocarcinoma (PDA) has a complex tumour microenvironment (TME) that are made up of pancreatic ductal adenocarcinoma cells (PDACs) and pancreatic stellate cells (PSCs). PDACs and PSCs’ interaction allows pancreatic cancer cells to evade the immune system and enhance tumour progression. Hence, identifying and comparing the proteomic profile of PDACs, PSCs and their co-culture will determine which genes are involved in the progression of PDA. PANC10.05 cells (primary PDAC cell line), PSC (hPSC21-S/T) cells and PANC10.05/PSCs co-culture were cultured in 6-well plate for 3 days. Cells collected were lysed to extract the proteins within the cells. The extracted protein lysate then undergoes insolution digestion of proteins. The protein in the samples were analysed and quantified through LCMS/MS before proceeding to data analysis via DEBrowser. The results showed that VIM, KRT8 and KRT18 were differentially expressed proteins (DEPs) detected when compared between PANC10.05, PSCs and their co-culture. In this study, monoculture of PANC10.05 and PSCs were able to express proteins without any interaction between PANC10.05 and PSCs. Hence, PANC10.05-PSCs co-culture is not required for the expression of proteins. The expression of DEPs in PANC10.05 and PSCs are associated in the progression, invasion and metastasis of PDA.

Pancreatic ductal adenocarcinoma (PDAC) is an almost always deadly illness, and early identification is a crucial factor in enhancing survival. The tumour microenvironment (TME), a possible source of biomarkers in PDAC, has a significant impact in cell proliferation, metastasis, and treatment resistance, all of which contribute to a poorer clinical outcome. Metastatic PDAC (SW1990 cell line) and pancreatic stellate cells (PSCs), one of the major cell types that comprise the tumour microenvironment (TME) of pancreatic cancer, foster an immunosuppressive environment around the tumour. The purpose of this investigation was to examine the proteome profiles of conditioned media (CM) from PSCs, SW1990, and PSC+SW1990 co-culture. To establish the probable proteins responsible for the different immunosuppressive efficacy of PSCs, SW1990, and PSC+SW1990 co-culture, the CM containing the secreted proteins from all 3 cell line groups were analysed by Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) and then subjected to bioinformatics analysis, including identification of differentially expressed proteins (DEPs), Gene Ontology (GO) terms analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway. TGM2, KRT8, KRT18, KRT19, MYH9, NME1, NME1-NME2, and LDHA were the eight DEPs that were significantly elevated in all three cell line groups comparisons with a false discovery rate (FDR)  0.05 and fold change (FC) ≥ 2. Multiple signalling pathways, including oestrogen signalling, actin cytoskeleton, protein processing in endoplasmic reticulum, vascular smooth muscle contraction, the hypoxia-inducible factor 1 (HIF-1) and the central carbon metabolism, were enriched in the comparison study. The DEPs detected, their GO terns, and the signalling pathways were integrated and matched with literature and proteomic data from prior research in order to better comprehend their association with PSCs and metastatic PDAC. In conclusion, this comparative proteomic research extends 3 the evidence of protein alterations linked with PSCs and metastatic PDAC that should be studied further as possible biomarkers or therapeutic targets

Pancreatic ductal adenocarcinoma (PDAC) is a carcinoma with high malignancy and fatality. It is characterized by its surrounding stroma which occupy about 70% of the total tumour volume. The tumour microenvironment is made up of several tissue component, for example, fibroblasts, pancreatic stellate cells (PSC), immune cells and extracellular matrix (ECM) proteins. The crosstalk between PDAC and stroma promotes the desmoplastic reaction around the tumour while enhancing the proliferation of PDAC. Treatment targeting the PDAC stroma tissues have yielded heterogenous result. The increase of use of omics technology in studying diseases have led to the comparison of proteomic and transcriptomic profiles of PDAC and stroma cells in this study. We have collected and applied data integration steps on a number of transcriptomic and proteomic studies which contain human PDAC and stroma samples. RStudio and DEBrowser analysis tool are utilized to explore the expression profile of the samples, as well as GO term and KEGG pathway analysis for the enriched genes. These steps were also applied to study the gene expression profile of PDAC and its metastases to identify the differentially expressed genes responsible for the proliferation of the carcinoma. Six differentially expressed genes between PDAC and stroma have been found to be commonly existing in both Transcriptomic datasets and Proteomic datasets; which are FLNB, CTSB, C1S, EFEMP2, ABI3BP, and FSTL1 genes. FGA and FGB are the two genes found to upregulated in the liver metastases of PDAC. The functional enrichment and KEGG pathway analysis of these genes were also discussed in this study.

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.

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.

Potential cardiotoxicity remained the crucial factor limiting the clinical use of doxorubicin-based chemotherapy, despite their effectiveness in various malignancies. Combination therapy involving doxorubicin has therefore emerged as a potential therapeutic option, in an effort to enhance anticancer efficiency at the same time minimise cardiovascular side effects. Epidermal growth factor receptor (EGFR) inhibitors are a group of tyrosine kinase small molecules inhibitor recently approved for clinical use in cancer therapy. The combination of doxorubicin and EGFR inhibitors has shown great promise by demonstrating enhanced anticancer effects and ability to reverse chemoresistance associated with doxorubicin. Although EGFR inhibitor alone displayed minimal cardiotoxicity, it has raised concern that concurrent use would potentiate doxorubicin-induced cardiotoxicity. This study therefore aimed to investigate possible synergistic cardiotoxic effect of doxorubicin and EGFR inhibition. By utilising human AC16 cardiomyocytes, we first assessed the EGFR inhibitors including erlotinib, gefitinib, afatinib and lapatinib, with concentration ranging from 1-100 μM for erlotinib, gefitinib and lapatinib whereas 1-10 μM for afatinib, which revealed that all EGFR inhibitors tested elicited cytotoxic responses towards cardiomyocytes at different potency levels. When combined with doxorubicin, only erlotinib was found to synergise doxorubicin-induced cardiotoxicity. Further cell apoptotic assay confirmed this synergism, suggesting that erlotinib may enhance apoptosis induced by doxorubicin. Intriguingly, this synergism was not observed in gefitinib, afatinib and lapatinib, which proposed that synergistic cardiotoxicity may be specific to each inhibitor instead of a drug-class effect. In summary, this study suggested that the clinical use of concurrent erlotinib and doxorubicin should be approached with caution due to the possibility of increased cardiotoxicity, and this warrants further mechanistic studies for understanding the precise underlying mechanisms and developing cardioprotective agents.