Yoke Kqueen Cheah

A synthetic curcuminoid derivative inhibits nitric oxide and proinflammatory cytokine synthesis

Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and inflammatory related diseases. However curcumin has one major important limitation in which it has poor bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of inflammation. BHMC showed a significant dose-response inhibitory action upon the synthesis of NO and we have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS activity with no effect at all upon the secretion of PGE(2) but has a strong inhibitory effect upon MCP-1 and IL-10 secretion and gene expression. Secretion and gene expression of TNF-alpha and IL-6 were moderately inhibited whereas IL-8 and IL-1beta were not altered. We conclude that BHMC selectively inhibits the synthesis of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and further pharmacological studies.

Molecular characterization of Antarctic actinobacteria and screening for antimicrobial metabolite production

The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231T, Staphylococcus aurues ATCC 51650T, methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44T and Pseudomonas aeruginosa ATCC 10145T, respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus—polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.

High Genetic Diversity of Enterococcus faecium and Enterococcus faecalis Clinical Isolates by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing from a Hospital in Malaysia

Little is known on the genetic relatedness and potential dissemination of particular enterococcal clones in Malaysia. We studied the antibiotic susceptibility profiles of Enterococcus faecium and Enterococcus faecalis and subjected them to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). E. faecium and E. faecalis displayed 27 and 30 pulsotypes, respectively, and 10 representative E. faecium and E. faecalis isolates (five each) yielded few different sequence types (STs): ST17 (2 isolates), ST78, ST203, and ST601 for E. faecium, and ST6, ST16, ST28, ST179, and ST399 for E. faecalis. Resistance to tazobactam-piperacillin and ampicillin amongst E. faecium isolates was highly observed as compared to E. faecalis isolates. All of the isolates were sensitive to vancomycin and teicoplanin. The presence of epidemic and nosocomial strains of selected E. faecium STs: 17, 78, and 203 and E. faecalis ST6 as well as high rates of resistance to multiple antibiotics amongst E. faecium isolates is of a particular concern.

Effect of organic-phase solvents on physicochemical properties and cellular uptake of astaxanthin nanodispersions.

A simplex centroid mixture design was used to study the interactions between two chosen solvents, dichloromethane (DCM) and acetone (ACT), as organic-phase components in the formation and physicochemical characterization and cellular uptake of astaxanthin nanodispersions produced using precipitation and condensation processes. Full cubic or quadratic regression models with acceptable determination coefficients were obtained for all of the studied responses. Multiple-response optimization predicted that the organic phase with 38% (w/w) DCM and 62% (w/w) ACT yielded astaxanthin nanodispersions with the minimum particle size (106 nm), polydispersity index (0.191), and total astaxanthin loss (12.7%, w/w) and the maximum cellular uptake (2981 fmol/cell). Astaxanthin cellular uptake from the produced nanodispersions also showed a good correlation with their particle size distributions and astaxanthin trans/cis isomerization ratios. The absence of significant (p > 0.05) differences between the experimental and predicted values of the response variables confirmed the adequacy of the fitted models.

Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 exhibit strong antifungal effects against vulvovaginal candidiasis-causing Candida glabrata isolates

This study investigates the antagonistic effects of the probiotic strains Lactobacillus rhamnosus GR‐1 and Lactobacillus reuteri RC‐14 against vulvovaginal candidiasis (VVC)‐causing Candida glabrata.

The effects of a synthetic curcuminoid analogue, 2,6-bis-(4-hydroxyl-3- methoxybenzylidine)cyclohexanone on proinflammatory signaling pathways and CLP-induced lethal sepsis in mice

We previously showed that 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), suppressed the synthesis of various proinflammatory mediators. In this study we explain the mechanism of action of BHMC in lipopolysaccharide (LPS)-induced U937 monocytes and further show that BHMC prevents lethality of CLP-induced sepsis. BHMC showed dose-dependent inhibitory effects on p38, JNK and ERK 1/2 activity as determined by inhibition of phosphorylation of downstream transcription factors ATF-2, c-Jun and Elk-1 respectively. Inhibition of these transcription factors subsequently caused total abolishment of AP-1-DNA binding. BHMC inhibited p65 NF-κB nuclear translocation and DNA binding of p65 NF-κB only at the highest concentration used (12.5μM) but failed to alter phosphorylation of JNK, ERK1/2 and STAT-1. Since the inhibition of p38 activity was more pronounced we evaluated the possibility that BHMC may bind to p38. Molecular docking experiments confirmed that BHMC fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We also show that BHMC was able to improve survival from lethal sepsis in a murine caecal-ligation and puncture (CLP) model.

Synthesis of a DNA-targeting nickel (II) complex with testosterone thiosemicarbazone which exhibits selective cytotoxicity towards human prostate cancer cells (LNCaP).

Testosterone thiosemicarbazone, L and its nickel (II) complex 1 were synthesized and characterized by using FTIR, CHN, (1)H NMR, and X-ray crystallography. X-ray diffraction study confirmed the formation of L from condensation of testosterone and thiosemicarbazide. Mononuclear complex 1 is coordinated to two Schiff base ligands via two imine nitrogens and two tautomeric thiol sulfurs. The cytotoxicity of both compounds was investigated via MTT assay with cisplatin as positive reference standard. L is more potent towards androgen-dependent LNCaP (prostate) and HCT 116 (colon). On the other hand, complex 1, which is in a distorted square planar environment with L acting as a bidentate NS-donor ligand, is capable of inhibiting the growth of all the cancer cell lines tested, including PC-3 (prostate). It is noteworthy that both compounds are less toxic towards human colon cell CCD-18Co. The intrinsic DNA binding constant (Kb) of both compounds were evaluated via UV-Vis spectrophotometry. Both compounds showed Kb values which are comparable to the reported Kb value of typical classical intercalator such as ethidium bromide. The binding constant of the complex is almost double compared with ligand L. Both compounds were unable to inhibit the action topoisomerase I, which is the common target in cancer treatment (especially colon cancer). This suggest a topoisomerase I independent-cell death mechanism.

Multiplex PCR for the concurrent detection and differentiation of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium

Salmonellosis outbreaks involving typhoid fever and human gastroenteritis are important diseases in tropical countries where hygienic conditions are often not maintained. A rapid and sensitive method to detect Salmonella spp., Salmonella Typhi and Salmonella Typhimurium is needed to improve control and surveillance of typhoid fever and Salmonella gastroenteritis. Our objective was the concurrent detection and differentiation of these food-borne pathogens using a multiplex PCR. We therefore designed and optimized a multiplex PCR using three specific PCR primer pairs for the simultaneous detection of these pathogens. The concentration of each of the primer pairs, magnesium chloride concentration, and primer annealing temperature were optimized before verification of the specificity of the primer pairs. The target genes produced amplicons at 429 bp, 300 bp and 620 bp which were shown to be 100% specific to each target bacterium, Salmonella spp., Salmonella Typhi and Salmonella Typhimurium, respectively.

Formation of biofilm by Listeria monocytogenes ATCC 19112 at different incubation temperatures and concentrations of sodium chloride

Biofilm formation can lead to various consequences in the food processing line such as contamination and equipment breakdowns. Since formation of biofilm can occur in various conditions; this study was carried out using L. monocytogenes ATCC 19112 and its biofilm formation ability tested under various concentrations of sodium chloride and temperatures. Cultures of L. monocytogenes ATCC 19112 were placed in 96-well microtitre plate containing concentration of sodium chloride from 1–10% (w/v) and incubated at different temperature of 4 °C, 30 °C and 45 °C for up to 60 h. Absorbance reading of crystal violet staining showed the density of biofilm formed in the 96-well microtitre plates was significantly higher when incubated in 4 °C. The formation of biofilm also occurs at a faster rate at 4 °C and higher optical density (OD 570 nm) was observed at 45 °C. This shows that storage under formation of biofilm that may lead to a higher contamination along the processing line in the food industry. Formation of biofilm was found to be more dependent on temperature compared to sodium chloride stress.