IPRC - 2018
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/19163
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Item Protective effect of coconut oil meal phenolic antioxidants on mitochondrial DNA damage in HEp-2 human epithelial cells(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Nisansala, A.; Hapugaswatte, H.; Seneviratne, K.N.; Jayathilaka, N.Coconut oil meal is a rich source of phenolic antioxidants. In most of the reported research, antioxidant activities of phenolic extracts have been tested using chemical systems. However, similar studies conducted in biological systems is relatively less common. In this study, glutathione oxidation and mitochondrial DNA (mt-DNA) damage in HEp-2 cells were used as biological reactions to assess protective effect of coconut oil meal phenolic antioxidants (CMPA). CMPA were extracted with ethanol water solvent system (70% v/v) and the total phenolic content was measured by Folin Ciocalteu method. HEp-2 cells at 80% confluence were treated with 0.5 mg/ml CMPA overnight. Oxidative stress in HEp-2 cells was induced by exposing cells to H2O2 (10, 50, 100, 250 and 500 μM) for 1hr. The maximum concentration of H2O2 that does not affect the cell viability (>99%) was 100 μM as measured by Cell-Titer Glo Luminescent Cell viability assay (Promega). Glutathione (GSH) to oxidized glutathione (GSSG) ratio (GSH/GSSG) was measured by GSH/GSSG Glo assay (Promega). To evaluate the mt-DNA damage, total DNA of high purity (A260nm/ A280nm >1.8) was extracted from stressed and unstressed cells pretreated with CMPA using HiPurATM Blood Genomic DNA Miniprep Purification Kit. Two primer sets that amplify a short and long fragment of mt-DNA in the D-loop region were selected to assess damage in the D-loop region that is known to be more prone to DNA damage. Quantitative Real time PCR was carried using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions and the amplification was monitored with StepOne quantitative thermal cycler (Applied Bio). The ratio of intact DNA was calculated according to the following equation; Lesion rate [Lesions per 10kb DNA] = (1-2-( Δ long- Δ short)) x (10000 bp/Size of long fragment bp). Total CMPA was 1892 ± 51 mg/kg as gallic acid equivalents . Two sample t-test was carried out for the determination of significant differences (p ≤ 0.05) between the mean values .The GSH/GSSG ratio of the samples pretreated with CMPA, subjected to H2O2 oxidative stress (100 μM) (5.01±0.08 (was significantly higher )P<0.05 (compared to that of samples subjected to H2O2 oxidative stress without pretreatment )2.19±0.04) while GSH/GSSG ratio of control cells without any treatment was 5.19±0.20. In mt-DNA damage assay, the samples pretreated with CMPA, subjected to H2O2 oxidative stress (100 μM) showed significantly less (P<0.05) lesions (7.65±0.06 lesions/10 kb DNA) compared to samples subjected to H2O2 oxidative stress without pretreatment (9.38±0.60 lesions/10 kb DNA). Thus, CMPA can inhibit glutathione oxidation and mt-DNA damage in HEp-2 cells suggesting that CMPA may help reduce health conditions associated with oxidative stressItem Expression Changes in Putative Target Genes of Differentially Expressed miRNA as Early Biomarkers for Severe Dengue(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Hapugaswatta, H.; Seneviratne, K.N.; Perera, H.S.S.; Premaratna, R.; Jayathilaka, N.Dengue fever is caused by a flavivirus transmitted by mosquitoes. Primary infection of dengue mostly causes mild dengue fever (DF) characterized by headache, retro orbital pain, body pain, nausea, vomiting, joint pains and weakness. Severe manifestations of dengue, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) also shows similar symptoms during the early stages of infection. After 3-5 days from fever onset, DHF patients manifest plasma leakage, elevated hematocrit and pleural effusions. Lack of proper medication or vaccines for dengue fever and inability to distinguish severe dengue from DF during the early stages of infection renders this disease life threatening. Early diagnosis and disease management can alleviate DHF related complications. Therefore, biomarkers that distinguish DHF during the acute phase of infection can help reduce mortality. In our previous studies, we evaluated the differential expression of five miRNAs during the acute phase of infection including hsa-miR-150, which showed significant (p<0.05) expression changes with the disease severity. Since the main function of miRNA is to regulate target gene expression at post-transcriptional level, we evaluated the expression levels of four target genes of those miRNA in peripheral blood cells (PBC) collected from 20 DF (male-70% and female-30%) and 20 DHF (male-85% and female-15%) patients (based on evidence of plasma leakage by ultrasonography) who tested positive for NS1 antigen within four days of fever onset (acute phase) by qRT-PCR. Relative expression of EZh2, ABCA1, DNMT3a and RIP140 were evaluated against GAPDH as the reference gene. EZh2 showed over 2-fold downregulation (P<0.05) in DHF patients compared to DF patients. Based on logistic regression analysis of ΔCq values, EZh2 expression within 4 days from fever onset may be useful as a biomarker for progression from DF to DHF with an area under the receiver operating characteristic curve (AUC) of 0.76, sensitivity of 0.80 and specificity of 0.65 at 2.69 (P<0.05). DNMT3a, RIP140 and ABCA1 did not show significant differential expression during the acute phase of infection between DF and DHF patient samples. EZh2 also showed significant (P<0.05) downregulation within 4 days from fever onset in patients with platelet count <100,000 cells/mm3 (n=31) compared to those with platelet count >100,000 cells/mm3 (n=9) during the course of infection. Therefore, EZh2 expression may also serve as a biomarker for disease severity marked by low platelet count. This analysis is limited by relatively small sample size and a disproportionate number of male subjects. However, the calculated sample size with 95% CI at 80% power for EZh2 expression as a marker to predict disease outcome is 34 (17 each). The data was confirmed normally distributed based on q-q plot and Shapiro-Wilk test (P>0.05).