EXPRESSION OF THE MICROFOLD CELLS IN THREE-DIMENSIONAL COCULTURE SYSTEM FOR IN VITRO CULTIVATION OF HUMAN NOROVIRUS

Introduction: Human Norovirus (HuNoV), a food-borne virus is the leading cause for acute gastroenteritis. However, its inability to propagate in vitropersists as major challenge in understanding HuNoV biology.Objective: This study aims to determine an effective culture system for HuNoV.Methods: The Caco-2 cells were cocultured with Raji B cells on alginate hydrogel beads. Scanning electron microscopy (SEM) was performed to confirmthe three-dimensional (3D) cells morphology. Western blot (WB) analysis was performed to detect protein markers expressed by Microfold (M) cells.Results: Optimization of Caco-2 cells monoculture in the alginate hydrogel beads showed optimum number of cells of 1 × 106 cells/ml, indicatedby the intact structure of the beads. Result of SEM showed clear structure of monoculture in the alginate hydrogel beads indicated by the presenceof smooth and regular apical surface while the coculture showed reduced apical surface of M cells. The result of WB showed downregulation ofUlex europaeus antibody expression.Conclusion: It is evident that the expression of M cells grown in 3D alginate hydrogel beads was successful, indicated by the structural morphologyseen under SEM as well as expression of protein marker by M cells. This established in vitro system is highly potential for cultivation of HuNoV

ABERRANT N-GLYCOSYLATION REGULATES INVASION OF MG-63 CELLS THROUGH EXTRACELLULAR MATRIX REMODELING

Objective: Despite advances in multimodal therapy, osteosarcoma (OS) still imposes big challenge due to its high rate of metastasis. The previousstudies reported that aberrant glycosylation in the cells mediates the invasion of several cancers including OS. However, its mechanism, particularlyN-glycosylation in OS progression, is still poorly understood. Thus, this study aims to investigate the effect of glycosylation inhibitions toward OS cellsinvasiveness.Materials and Methods: Both 1-deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (1-DMJ) were used to inhibit the activities ofalpha-glucosidase-I/II and alpha-1,2-mannosidase, respectively. Invasion assay and real-time polymerase chain reaction (PCR) (quantitative PCR[qPCR]) analysis of extracellular matrix-related genes were performed at post 24 h of treatment with the inhibitors, 0.5 mM 1-DNJ and 0.5 mM 1-DMJ,respectively, on the OS cell line, MG-63.Results: Results showed that the inhibition of N-glycosylation with 1-DNJ decreases the invasion rate of MG-63 cells while the inhibition ofN-glycosylation by 1-DMJ caused the invasion rate of MG-63 cells to increase. qPCR analysis showed downregulated expression of matrixmetalloproteinase (MMP2) gene in both types of treatments while the expression of its inhibitor, tissue inhibitor of metalloproteinase (TIMP2) wasupregulated in both types of treatments. In this study, MMP9 genes were not detected in both samples; however, the expression of its inhibitor, TIMP1was downregulated in MG-63 cells treated with 1-DNJ but upregulated in 1-DMJ treated cells.Conclusion: It is concluded that 1-DNJ reduced the invasion rate in MG-63 cells through downregulation of MMP2 gene which subsequently reduceddegradation of collagen type IV. However, the contrasting effect showed by 1-DMJ requires further investigation to elucidate its underlying mechanism

Antimicrobial effect of Tetraspanin CD9 Peptides on Pseudomonas aeruginosa

It is critical to find an alternative therapeutic approach to combat Pseudomonas aeruginosa (P. aeruginosa) that can simultaneously reduce the occurrence of bacterial resistance. The tetraspanin CD9, a highly expressed membrane protein in melanocytes was chosen for this study because it is highly expressed in keratinocytes and has been implicated in the pathogenesis of bacterial infections in a previous study. The antimicrobial activity of CD9 peptides against the standard strain P. aeruginosa (ATCC 27853) and a clinical multidrug-resistant P. aeruginosa (MDR- P. aeruginosa) was studied using the disc diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CD9 peptides were determined by broth microdilution assays with concentrations ranging from 1 mg/mL to 4.88×10-4 mg/mL. The antibiofilm activity of the CD9 peptides was also determined. CD9 peptides showed an 11.75 ± 2.36 mm inhibition zone against the standard P. aeruginosa strain but none against the MDR- P. aeruginosa. Both isolates had the same MIC value, 0.25 mg/mL. The MBC for the standard strain P. aeruginosa was 0.5 mg/mL, while for the MDR- P. aeruginosa strain, it was 1 mg/mL. CD9 peptides significantly inhibited up to 70% biofilm against both P. aeruginosa isolates. CD9 peptides showed a modest inhibitory effect against the standard strain P. aeruginosa but not against MDR- P. aeruginosa. Interestingly, CD9 peptides were found to be a good anti-biofilm treatment against both P. aeruginosa isolates. This study demonstrated that CD9 peptides have the potential to be an alternative antimicrobial treatment against P. aeruginosa

Siam-weeb based gelatin electrospun scaffolds

Siam weed (Chromolaena odorata) is a traditional herb used to soothe burns and scars and potentially improve dressing and tissue-engineered construct healing ability. The use of Siam weed is mainly in the form of extract. The state of liquid extract limits the use of Siam weed compared to the state of gels and membrane. However, the development of the hybrid of Siam weed in gel and membrane form is lacking. In this study, Siam weed was harvested and made into aqua extracts. The biocompatibility of the aqua extract with various concentrations was tested using Cell Proliferation Assay. The aqua extract was then incorporated into the gelatin solution and spun into nanofibers using an electrospinning technique. The cell proliferation study shows maximum cell proliferation at the 25 μg/ml concentration. The scanning electron microscope images of the electrospun scaffolds show fibrous networks’ microstructures without beads. The concentration of the hybrid solution was found to affect the morphology of the nanofibers by having diameters in the range of 160 ± 90 to 250 ± 150 nm. The Siam-weed-based gelatin electrospun scaffolds provide a native-like microenvironment and potentially improve wound healing ability for biomedical application

TOCOTRIENOL-RICH FRACTION MODULATE THE PHOSPHOINOSITIDE 3-KINASES/AKT SIGNALING PATHWAY GENES AND PREVENT OXIDATIVE STRESS IN NICOTINE-INDUCED PRE-IMPLANTATION EMBRYOS

Objective: This study aimed to determine the effects of the tocotrienol-rich fraction (TRF) on the regulations of phosphoinositide 3-kinases (PI3K)/Aktpathways related genes in preimplantation embryos induced by nicotine (Nic).Methods: Twenty-four female BALB/c mice were divided into four groups with Nic and TRF supplementation for 7 consecutive days. Animalswere superovulated before mating with fertile males. Plasma malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase weredetermined and analyzed accordingly. Embryos with two and eight blastomeres were assessed for gene expression analysis.Results: The levels of endogenous antioxidative enzymes for the group with TRF intervention and TRF only group showed no significant changes whencompared to the control group. The level of oxidative stress (OS) biomarkers was also significantly decreased when compared to the Nic-induced group.At 2-cell stage, Pten gene was significantly upregulated while Akt1, GSK3β, and Mapk1 were significantly downregulated almost similar to the baseline(control) in the Nic-induced mice. Intervention with TRF resulted in a significant downregulated of Pten gene followed by a significant upregulationof other genes. The same pattern was shown at the 8-cell stage.Conclusion: This showed that TRF evidently has OS protection capacity and it could be through modulating the PI3K/Akt signaling pathway

METABOLITES PROFILE OF COLORECTAL CANCER CELLS AT DIFFERENT STAGES

Objective: The aim of this study is to characterize the metabolite profiles of colorectal cancer (CRC) cells of different stages of the disease to understandthe pathophysiological changes that may help to identify prevention strategies as well as the sites for potential therapeutic drug actions.Methods: Six CRC cell lines of different stages (classified using the Dukes classification) were used, and they are SW 1116 (stage A), HT 29 and SW480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D). Metabolites were extracted using methanol and water, and metabolic profiling wasperformed using liquid chromatography-mass spectrometry. Mass profiler professional software was used for statistical analysis.Results: There were 111,096 compounds detected across the samples, and 24 metabolites were identified to be significantly different betweenthe CRC stages. Most notably, there were eight metabolites that were significantly upregulated in the more advanced stages (B, C, and D) comparedwith Stage A. These metabolites include flavin mononucleotide, l-methionine, muricatacin, amillaripin, 2-methylbutyroylcarnitine, lumichrome,hexadeconoic acid, and lysoPE (0:0/16:0).Conclusion: This study showed that the expressions of metabolites at different stages of CRC were different, which represent the metabolic changesoccurring as CRC advances. The knowledge may help identify biomarkers for the staging of CRC, which could improve its prognosis as well as providea basis for the development of therapeutic interventions

Metabolomic characterization of colorectal cancer cell lines highlighting stage-specific alterations during cancer progression

Introduction: Metabolomic studies on various colorectal cancer (CRC) cell lines have improved our understanding of the biochemical events underlying the disease. However, the metabolic profile dynamics associated with different stages of CRC progression is still lacking. Such information can provide further insights into the pathophysiology and progression of the disease that will prove useful in identifying specific targets for drug designing and therapeutics. Thus, our study aims to characterize the metabolite profiles in the established cell lines corresponding to different stages of CRC. Methods: Metabolite profiling of normal colon cell lines (CCD 841 CoN) and CRC cell lines corresponding to different stages, i.e., SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D), was carried out using liquid chromatography-mass spectrometry (LC-MS). Mass Profiler Professional and Metaboanalyst 4.0 software were used for statistical and pathway analysis. METLIN database was used for the identification of metabolites. Results: We identified 72 differential metabolites compared between CRC cell lines of all the stages and normal colon cells. Principle component analysis and partial least squares discriminant analysis score plot were used to segregate normal and CRC cells, as well as CRC cells in different stages of the disease. Variable importance in projection score identified unique differential metabolites in CRC cells of the different stages. We identified 7 differential metabolites unique to stage A, 3 in stage B, 5 in stage C, and 5 in stage D. Conclusion: This study highlights the differential metabolite profiling in CRC cell lines corresponding to different stages. The identification of the differential metabolites in CRC cells at individual stages will lead to a better understanding of the pathophysiology of CRC development and progression and, hence, its application in treatment strategies

Effects of bisphenol a on neonatal cardiomyocytes beating rate and morphology

Bisphenol A (BPA) has been utilised excessively at a global capacity of 2.9 billion kg/year. It is widely used in manufacturing polycarbonate polymers and epoxy resins. Hence, humans are potentially exposed to this chemical substance in their daily life. As a typical endocrine disruptor, BPA exhibits detectable hormone-like properties. Many studies have been linking BPA exposure in humans with the risk of developing cardiovascular disease, however the direct exposure of BPA on cardiomyocytes beating rates and morphology have not been entirely explored. Therefore, in this study, we aimed to investigate the effects of BPA on cells structure and function of neonatal rat cardiomyocytes culture. Cardiomyocytes were isolated from 0 to 2 days old newborn rats and treated with 0.001 to 100 µM concentration of BPA. All cardiomyocytes were subjected to immunostaining, beating frequency assessment assay, MTS assay and Scanning Electron microscopy (SEM). In immunostaining, cardiomyocytes showed positive staining for F-actin. This staining allows identification of the cells thus differentiate cardiomyocytes from other cell types. Significance effects of BPA on cardiomyocytes were observed in MTS assay (p<0.05) and beating rates (p<0.01). Significant reduction (48%-64%, ± 1.5280) was observed in beating rate of cardiomyocytes exposed to 0.1 to 100 µM of BPA. Meanwhile in MTS assay, significant reduction (54%, 0.067 ± 0.0026) in cell viability was observed in cells exposed to 0.1 µM of BPA only. Interestingly, under SEM, cardiomyocytes showed altered cell surface homogeneity after BPA exposure. Exposure of 0.1 to 100 µM BPA lead to flatten of cardiomyocytes cell surface and blurring of the cell borders. This study offers an in vitro evidence of BPA effects on cardiomyocytes morphology and beating rates, thus suggest the potential adverse effect of BPA exposure. However, further investigation would be required to understand how BPA effects normal cells morphology and beating rates of heart cells

Comparison of non-alcoholic fatty liver disease (NAFLD) model using diet-induced NAFLD mice with genetically modified mice

Prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing steadily every year affecting all population both Western and Asian countries. The current treatments available for NAFLD are non-conclusive warranting newer effective pharmacological agents. Newly formulated agents require prior testing using animal models. However, in developing countries, these models are often costly. The possibility of using more affordable animal model in local settings should be investigated. In this study, ten Institute of Cancer Research (ICR) and seven B6.Cg-LepOb/J leptin-knockout (JAX) male mice were recruited. Five ICR and all JAX mice were subjected to high-fat diet (60% kcal fat) and remaining ICR mice were given standard diet (SD) for six weeks. Body weight and food intake were measured weekly while abdominal circumference, random blood glucose and liver span were measured at the end of the HFD study. Livers collected were subjected to histology assessment. Compared to ICR group, JAX group presented with significantly higher body weight (58 ± 0.72, p<0.05), larger body weight changes (16.57 ± 0.81, p<0.05), more HFD intake (197.14 ± 0.812, p<0.05) and larger abdominal circumference (11.79 ± 0.34: p<0.05). Liver from JAX group appeared with general steatosis and presentation of high-grade panacinar steatosis, low number of lobular inflammations and minimal fibrosis. Liver of ICR mice showed Zone 3 steatosis with high number of lobular inflammations without fibrosis. The NAFLD characteristics presented in JAX group suggested that B6.Cg-LepOb/J mice developed characteristics of NAFLD resembling human while ICR is suitable NAFLD model resembling human population resilient towards NAFLD

The impacts of intrauterine Bisphenol A exposure on pregnancy and expression of miRNAs related to heart development and diseases in animal model

This study aimed to examine the impact of BPA exposure on pregnancy and foetuses on cardiac tissues and the expression of cardiac microRNAs (miRNAs) related to heart development and diseases. Pregnancy is known to be the “critical windows” in determining the offspring physical and cells development in their life after birth. The increment of the risk of cardiovascular disease (CVD) in a later stage of life has been reported by few studies demonstrated from prenatal exposure of BPA. BPA has been shown to alter miRNAs expression profiles for organ development, regeneration and metabolic functions. These alterations have been associated with the risk of CVDs. However, the associations between pregnancy outcomes and miRNAs expression in cardiac of mother- and foetuses-exposed to BPA are still not entirely explored. In BPA-exposed pregnant rat groups, a significant weight gained was observed in comparison to control (p < 0.05). Interestingly, significant changes in systolic and diastolic blood pressure between the first and third trimester of BPA-exposed pregnant rats were also observed (p < 0.05). In BPA-exposed pregnant rats, miR-499-5p was significantly altered in the heart (p < 0.01). Meanwhile, altered miR-17-5p, -208-3p, and -210-3p expressions were observed in all heart of the foetuses from BPA-exposed pregnant rats (p < 0.05). In H&E staining, BPA-exposed foetal hearts showed a sign of fibrosis while BPA-exposed pregnant rats showed muscle remnant. Masson trichrome staining further confirmed the presence of fibrosis observed in BPA-exposed foetal heart and reduced expression of cardiac troponin I (cTnI) was also observed in BPA-exposed foetal heart. In summary, altered cardiac miRNAs with histological changes were observed in both mother- and foetus-exposed BPA These findings put forward the importance of future work to further understand how prenatal BPA exposure affect foetuses in their later stage of life