Browsing by Author "Hapugaswatta, H."

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Differential expression of microRNA, miR-150 and Enhancer of Zeste Homolog 2 (EZH2) in peripheral blood cells as early prognostic markers of severe forms of dengue
(Karger Medical and Scientific Publishers., 2020) Hapugaswatta, H.; Amarasena, P.; Premaratna, R.; Seneviratne, K.N.; Jayathilaka, N.
BACKGROUND: Dengue presents a wide clinical spectrum. Most patients recover following a self-limiting non-severe clinical course. A small proportion of patient’s progress to severe disease, mostly characterized by plasma leakage with or without hemorrhage. Early symptoms of severe dengue (SD) are similar to those of non-severe dengue fever (DF). Severe symptoms manifest after 3-5 days of fever, which can be life threatening due to lack of proper medications and inability to distinguish severe cases during the early stages. Early prediction of SD in patients with no warning signs who may later develop severe infection is very important for proper disease management to alleviate related complications and mortality. microRNA are small non-coding RNA molecules that regulate post-transcriptional gene expression. Due to the remarkable stability and the role of microRNA in gene expression, altered expression of microRNA was evaluated to explore clinically relevant prognostic markers of severe dengue. METHODS: The relative expression of microRNA hsa-let-7e (let-7e), hsa-miR-30b-5p (miR-30b), hsa-miR-30e-3p (miR-30e), hsa-miR-33a (miR-33a), and hsa-miR-150-5p (miR-150) and several putative target genes in peripheral blood cells (PBC) collected from 20 DF and 20 SD positive patients within 4 days from fever onset was evaluated by quantitative reverse transcription PCR (qRT-PCR). RESULTS: miR-150 showed significant (P < 0.01) up regulation in PBC of SD patients compared to DF patients during the acute phase of infection. Expression of enhancer of zeste homolog 2 (EZH2) was significantly (P < 0.01) down regulated indicating that genes involved in epigenetic regulation are also deferentially expressed in SD patients during the early stage of infection. CONCLUSIONS: Differential expression of microRNA miR-150 and the putative target gene EZH2 may serve as reliable biomarkers of disease severity during early stages of dengue infection. KEYWORDS: Acute dengue biomarkers; Dengue; Severe dengue; microRNA.
Effect of chain length and saturation of the fatty acids in dietary triglycerides on lipid metabolism in Wistar rats
(Journal of Food Biochemistry, 2021) Senanayake, C. M.; Hapugaswatta, H.; Samarawickrama, G. R.; Jayathilaka, N.; Seneviratne, K. N.
We investigated the effect of the chain length and the degree of saturation of fatty acids in dietary triglycerides on serum lipid profiles and hepatic lipid metabolism in Wistar rats. Fat component of the basal diet (soybean oil) was replaced with fats with fatty acids of different chain lengths and saturation and the serum lipids were monitored for 150 days. Principal component (PC) analysis of serum lipid components was related to chain length and saturation. The combined effect of chain length and saturation on PC 1 scores was evaluated by multiple regression analysis. The results indicated that average chain length of the fatty acids of triglycerides has a higher influence on the quality of serum lipid parameters than the average degree of saturation. Expression of selected genes responsible for lipid metabolism showed similar trends in medium chain saturated and long chain polyunsaturated diet groups. Practical applications Dietary lipids contain a wide range of saturated and unsaturated fatty acids with different chain lengths. Overall contribution of these different fatty acids decides the health effects of the lipids in the diet. Present study shows that the fats with medium chains and higher degree of saturation and fats with long chains and higher degree of unsaturation (lower degree of saturation) affect serum lipid parameters and expression of hepatic genes involved in the lipid metabolism in a similar manner. Such information is important for physicians to plan dietary schemes to improve the nutritional health and manage the noncommunicable diseases.
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).
Expression of Nitric Oxide synthase and Nitric Oxide levels in peripheral blood cells and oxidized low-density lipoprotein levels in saliva as early markers of severe dengue.
(Hindawi, 2021) Hapugaswatta, H.; Ruwani, L.; Wimalasekara; Perera, S.S.; Premaratna, R.; Seneviratne, K.N.; Jayathilaka, N.
BACKGROUND: Severe dengue (SD), experienced by only a fraction of dengue patients, can be lethal. Due to the lack of early markers that can predict the evolution of SD, all dengue patients have to be monitored under hospital care. We discovered early oxidative stress markers of SD to identify patients who can benefit from early intervention before the symptoms appear. METHODS: The expression of inducible nitric oxide synthase (iNOS) in peripheral blood cells (PBC), nitric oxide (NO), and oxidized lowdensity lipoprotein (oxLDL) levels in plasma and saliva collected at early stages of dengue infection from 20 nonsevere dengue fever (DF) patients and 20 patients who later developed SD were analyzed in a retrospective nested case-control study. RESULTS: The expression of iNOS is significantly (P < 0:05) lower in patients who developed SD than in DF patients at admission within 4 days from fever onset. Median plasma NO concentration within 4 days from fever onset is also significantly (P < 0:05) lower in patients who developed SD (17:9±1:6 μmol/L) than DF (23:0±2:1 μmol/L). Median oxLDL levels in plasma within 3 days from fever onset is significantly (P < 0:05) lower in patients who developed SD (509:4 ± 224:1 ng/mL) than DF (740:0 ± 300:0 ng/mL). Median salivary oxLDL levels are also significantly (P < 0:05) lower in patients who developed SD (0:8±0:5 ng/mL) than DF (3:6±2:6 ng/mL) within 4 days from fever onset. CONCLUSIONS: These findings suggest that the expression of iNOS (73% sensitivity, 86% specificity) and plasma NO (96% sensitivity, 61% specificity at 22.3 μmol/L; P < 0:05) may serve as early markers of SD within 3 days from fever onset. Salivary oxLDL levels may serve as early noninvasive markers of SD with a sensitivity and specificity, respectively, of 57% and 91% at 0.9 ng/mL; 76% and 55% at 2.3 ng/mL; and 100% and 50% at 4.6 ng/mL (P < 0:05) within 4 days from fever onset
In vitro antioxidant, anti-inflammatory and anti-cancer activities of plant extracts used in Ayurvedic medicine
(Faculty of Science, University of Kelaniya, Sri Lanka, 2021) Munasinghe, C. C. J.; Hapugaswatta, H.; Seneviratne, K. N.; Jayathilaka, N.
Traditional medicine may provide leads for potential new therapeutics. Sri Lanka is home to numerous plant species with reported anti-cancer properties according to traditional medical practices. Further, polyphenolic compounds extracted from various plant products have been reported to exhibit antioxidant, anti-inflammatory and anticancer activity. In this study we assessed the antioxidant, anti-inflammatory and anti-cancer potential against acute myelogenous leukemia (AML) in several ayurvedic plant species; Nigella sativa (Kaluduru) seeds, Hemidesmus indicus (Iramusu) roots, Adenanthera pavonina (Madatiya) seeds and Murraya koenigii (Curry leaves). These plant species have already reported of possessing anti-cancer activity against different cancer types. The main objective of this study is to determine the anti-cancer activity of theses plant extracts against AML. All the plant parts were thoroughly cleaned, dried, grounded into fine powder and bioactive compounds were fractionated into different solvents. The Total phenolic content (TPC), antioxidant activity, anti-inflammatory activity and cytotoxicity against AML were evaluated using Folin-Ciocalteu assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, Ferric reducing power antioxidant assay, protein denaturation inhibition assay and Cell TiterGo viability assay using THP-1 cells as a model for AML. N. sativa (kaluduru) aqueous extract showed the highest TPC; 3.14 ± 0.01 mg GAE/g dry weight (after tannin removal) and 2.92 ± 0.01 mg GAE/g dry weight (after protein removal), H. indicus (iramusu) aqueous extract recorded the highest DPPH radical scavenging activity and ferric reducing power and N. sativa (aqueous extract), A. pavonina (madatiya) ethanol extract and M. koenigii (curry leaves) methanol extract showed significantly high values (p<0.05) for the anti-inflammatory activity compared to other tested extracts. H. indicus (aqueous extract) showed the highest cytotoxicity against THP-1 cells with the lowest IC50 value (0.37 ± 0.01 mg/mL). All the tested extracts showed more than 50% inhibition of THP-1 cells at the highest tested concentration (15 mg/mL). All the tested plant extracts showed presence of polyphenols with antioxidant, anti-inflammatory and cytotoxic properties. However, the TPC as determined by Folin Ciocalteu assay does not explain the resulting cytotoxicity against AML cells. Further studies are needed to assess whether the observed anti-leukemic activity is due to cytotoxicity that would affect healthy cells and to assess whether the observed activities are specific against AML.
Phenolic extracts of the leaves of Psidium guineense Sw. improve the shelf life of sunflower oil and baked cake and antioxidant status of Wistar rats
(J Food Biochem, 2018) Senanayake, C.M.; Hapugaswatta, H.; Jayathilaka, N.; Seneviratne, K.N.
The potential of the ethanolic extract of Psidium guineense Sw. leaves (PGLE) to protect food from oxidation was evaluated using sunflower oil and baked cake as food models. The nutritional quality of PGLE was evaluated by feeding Wistar rats with PGLE for 150 days. Psidium guineense Sw. leaves contain 195.25 ± 9.56 mg g–1 phenolic substances, 51% of which are o‐diphenols. Protection factor, the ability of Psidium guineense Sw. leaves to protect sunflower oil against oxidation (1.82 ± 0.13), was not significantly affected by heat treatment compared to BHT. Formation of oxidation products, peroxide and hexanal in PGLE‐ and BHT‐added cake was significantly lower (<5 ppm over 28 days) compared to control with no added antioxidants. Therefore, addition of PGLE improved the shelf life of sunflower oil and oxidative stability of baked cake. PGLE also improves the serum antioxidant capacity and inhibits lipid and protein oxidation in Wistar rats. Practical applications PGLE is a rich source of phenolic substances. Due to high antioxidant activity, pleasant sensory quality and high thermal stability, PGLE can be used to improve the shelf life of baked cake and edible oils. As PGLE also improves serum and plasma antioxidant properties without causing any toxicity, nutritional food supplements can be developed based on PGLE.
Protective Effect of Coconut Oil Meal Phenolic Antioxidants against Macromolecular Damage: In Vitro and In Vivo Study
(Journal of Chemistry, 2020) Karunasiri, A.N.; Senanayake, C.M.; Hapugaswatta, H.; Jayathilaka, N.; Seneviratne, K.N.
Coconut oil meal, a cheap by-product of coconut oil production, is a rich source of phenolic antioxidants. Many age-related diseases are caused by reactive oxygen species- (ROS-) induced damage to macromolecules such as lipids, proteins, and DNA. In the present study, the protective effect of the phenolic extract of coconut oil meal (CMPE) against macromolecular oxidative damage was evaluated using in vitro and in vivo models. Sunflower oil, bovine serum albumin (BSA), and plasmid DNA were used in the in vitro study, and thiobarbituric acid reactive substances (TBARS), protein carbonyl, and nicked DNA were evaluated as oxidation products. The inhibitory effect of CMPE against H2O2-induced macromolecular damage was evaluated using cultured HEp-2 cells. The results indicate that CMPE inhibits macromolecular damage both in vitro and in vivo. In addition, CMPE regulates redox status of HEp-2 cells under oxidative stress conditions by maintaining higher reduced glutathione levels. There was no significant difference in the expression of glutathione peroxidase in stressed and unstressed cells suggesting that CMPE regulates the cellular oxidative stress responses without affecting the expression of oxidative stress response genes. Oral feeding of Wistar rats with CMPE improves the serum and plasma antioxidant status without causing any toxic effects.
Use of miR-33a in human plasma as a potential biomarker for severe Dengue infection
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Hapugaswatta, H.; Seneviratne, K.N.; Jayathilaka, N.
MicroRNAs (miRNAs) are small noncoding RNAs about 22 nucleotides long that can regulate the gene expression at mRNA level by RNA silencing. As such, differential expression of miRNA leading to regulation of gene expression during various infections has been investigated as potential biomarkers for many diseases. Perturbations in lipid homeostasis and cellular imbalance of cholesterol and fatty acid metabolism has been implicated in Dengue infections. Intronic microRNA, miR33a, located within the sterol regulatory element-binding protein-2 and -1 genes, has been shown to regulate cholesterol homeostasis in concert with their host genes. In fact, miR33a has shown differential expression in cultured cells infected with Dengue virus. Therefore, we evaluated the expression level of miR33a in human plasma to evaluate its potential as a biomarker for severe Dengue infection. Total RNA was purified from plasma from six EDTA blood samples collected with consent from healthy people using mirVana microRNA isolation kit (Applied Biosciences). Plasma was separated within one hour of sample collection and was stored at -80 °C. Purified RNA was used for subsequent 3’polyadenylation of the mature microRNA and cDNA synthesis with oligo-dT primers with a universal tag sequence using miScript II RT Kit (Qiagen). Similarly, negative control experiments for genomic DNA were carried out with same amount of RNA without any reverse transcriptase in the cDNA synthesis reaction (-RT). Presence of miR33a in plasma was confirmed by quantitative real-time PCR using the mature miRNA33a nucleotide sequence with thiamidine in place of uracils as the forward primer and a universal primer with universal tag sequence in the oligo-dT primer as the reverse primer. Target specificity of the miR33a specific primer was confirmed by NCBI BLASTN suite. Quantitative real-time PCR using miScript SYBR Green PCR Kit (Qiagen) was performed using diluted cDNA at an annealing temperature of 58 °C for 40 cycles followed by melting curve analysis. Negative control reactions were carried out in parallel with same volume of water and -RT as template. miR33a in human plasma was detected above threshold 1.0 at cycle number (Ct) 35. The Ct number is higher due to the low abundance of RNA in plasma. miR33a was consistently detected above threshold at the given Ct values while no amplification was detected in the negative control experiments containing water or -RT as template, indicating the absence of primer dimer formation and genomic DNA. Presence of a single peak in the melting curve confirmed single product amplification. Therefore, plasma is a good source for detection of miR33a to evaluate differential expression in severe Dengue.