Journal/Magazine Articles
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/13
This collection contains original research articles, review articles and case reports published in local and international peer reviewed journals by the staff members of the Faculty of Medicine
Browse
35 results
Search Results
Item Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2(BioMed Central, 2023) Herath, H.M.L.P.B.; de Silva, W.R.M.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Jayasingha, J.R.P.; Gayashan, M.K.; Afonso, L.O.B.; de Silva, K.M.N.BACKGROUND In this study, we designed a novel genetic circuit sensitive to Cd2+, Zn2+ and Pb2+ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfgured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells. RESULTS The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd2+, Zn2+ and Pb2+ either singly or in combination. Further, the same biosensor cells calibrated for fuorescent intensity against heavy metal concentration generated linear graphs for Cd2+, Zn2+ and Pb2+ with the R2 values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specifc metals, Fe3+ (0.0373), AsO4 3− (0.3825) and Ni2+ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1–6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH=7.0 in the medium, resembling the characteristics of wildtype E.coli. CONCLUSION Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1–6 ppb) at normal bacterial physiological conditions.Item Epidemiological evidence of acute transmission of zika virus infection in dengue suspected patients in Sri-Lanka(Elsevier, 2023) Ngwe, T.M.M.; Raini, S.K.; Fernando, L.; Gunawardene, Y.I.N.S.; Inoue, S.; Takamatsu, Y.; Urano, T.; Muthugala, R.; Hapugoda, M.; Morita, K.Background: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. Methods: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. Results: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. Conclusion: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these virusesItem Genome organization, in-silico structure, and cellular localization of putative lipid transporter, ARV1 from parasitic nematode Setaria digitata(Elsevier Inc., 2022) Wickramatunga, P.G.T.S.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Dassanayake, R.S.Setaria digitata, a nematode that lives in the peritoneal cavity of ruminants is the causative agent of cerebrospinal nematodiasis affecting livestock health. The ‘ACAT related enzyme 2 required for viability 1’ (arv-1) gene encodes putative lipid transporter that is essential in eukaryotes. The molecular characterization of nematode arv-1 has scarcely been studied and putative arv-1 isolated from S. digitata was used for this purpose. Docking and computer simulation studies using a modeled 3D structure of S. digitata ARV1 (Sd-ARV-1) with ceramide ligands revealed that the amino acid residues, Ile182, Leu56, Ala61, Gln186 and Gln146 are likely involved in the formation of potential sphingolipid binding sites having the same conserved residues in other nematodes. Sd-arv-1, a single copy gene, genomic region (1676 bp) had five exons encoding 217 amino acids, being interspersed by four introns showing a similar gene organization to other nematodes. Sd-ARV-1 is expressed ubiquitously at all development stages of the S. digitata life cycle. Tissue localization analyses revealed that Sd-ARV-1 was significantly expressed in the longitudinal muscle layer, endodermis, uterine wall, eggs, growing embryos inside the uterus, microfilariae, intestinal wall, esophagus lumen, dorsal nerve cord and ventral nerve cord. Therefore, ARV1 is a structurally conserved, ubiquitously expressed protein, which may be involved in development, reproduction, tissue remodeling, muscle contraction etc., in nematodes.Item Development of the sterile Insect technique to control the dengue vector Aedes aegypti (Linnaeus) in Sri Lanka(Public Library of Science,San Francisco, 2022) Ranathunge, T.; Harishchandra, J.; Maiga, H.; Bouyer, J.; Gunawardene, Y.I.N.S.; Hapugoda, M.Background: The Sterile Insect Technique (SIT) is presently being tested to control dengue in several countries. SIT aims to cause the decline of the target insect population through the release of a sufficient number of sterilized male insects. This induces sterility in the female population, as females that mate with sterilized males produce no offspring. Male insects are sterilized through the use of ionizing irradiation. This study aimed to evaluate variable parameters that may affect irradiation in mosquito pupae. Methods: An Ae. aegypti colony was maintained under standard laboratory conditions. Male and female Ae. aegypti pupae were separated using a Fay and Morlan glass sorter and exposed to different doses of gamma radiation (40, 50, 60, 70 and 80 Gy) using a Co60 source. The effects of radiation on survival, flight ability and the reproductive capacity of Ae. aegypti were evaluated under laboratory conditions. In addition, mating competitiveness was evaluated for irradiated male Ae. aegypti mosquitoes to be used for future SIT programmes in Sri Lanka. Results: Survival of irradiated pupae was reduced by irradiation in a dose-dependent manner but it was invariably greater than 90% in control, 40, 50, 60, 70 Gy in both male and female Ae. aegypti. Irradiation didn't show any significant adverse effects on flight ability of male and female mosquitoes, which consistently exceeded 90%. A similar number of eggs per female was observed between the non-irradiated groups and the irradiated groups for both irradiated males and females. Egg hatch rates were significantly lower when an irradiation dose above 50 Gy was used as compared to 40 Gy in both males and females. Irradiation at higher doses significantly reduced male and female survival when compared to the non-irradiated Ae. aegypti mosquitoes. Competitiveness index (C) scores of sterile and non-sterile males compared with non-irradiated male mosquitoes under laboratory and semi-field conditions were 0.56 and 0.51 respectively at 50 Gy. Signification: Based on the results obtained from the current study, a 50 Gy dose was selected as the optimal radiation dose for the production of sterile Ae. aegypti males for future SIT-based dengue control programmes aiming at the suppression of Ae. aegypti populations in Sri Lanka.Item Molecular characterization of a reemergent Brugia malayi parasite in Sri Lanka, suggestive of a novel strain(Hindawi Pub. Co., 2021) Mallawarachchi, C.H.; Chandrasena, T.G.A.N.; Withanage, G.P.; Premaratna, R.; Mallawarachchi, S.M.N.S.M.; Gunawardene, Y.I.N.S.; Dasanayake, R.S.; Gunarathna, D.; de Silva, N.R.ABSTRACT: Sri Lanka achieved elimination status for lymphatic filariasis in 2016; still, the disease remains a potential public health issue. The present study is aimed at identifying a subperiodic Brugia sp. parasite which has reemerged in Sri Lanka after four decades via molecular-based analysis. Polymerase chain reaction performed with pan-filarial primers specific for the internal transcribed spacer region-2 (ITS-2) of the rDNA of Brugia filarial parasites isolated from human, canine, and feline blood samples yielded a 615 bp band establishing the species identity as Brugia malayi. Comparison of the ITS2 sequences of the reemerged B. malayi isolates with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi with similar phylogenetic evidence. However, the mean interspecies Kimura-2-parameter pairwise divergence between the generated Brugia sequences with B. malayi and B. pahangi was less than 3%. During the analysis of parsimony sites of the new ITS2 sequences, substitutions at A36T, A296G, T373A, and G482A made the sequences different from both B. pahangi and B. malayi suggesting the possibility of a new genetic variant or a hybrid strain of B. malayi and B. pahangi. Mosquito dissections and xenomonitoring identified M. uniformis and M. annulifera as vectors of this novel strain of B. malayi circulating among cats, dogs, and humans in Sri Lanka.Item Voltage-Gated Sodium Channel ( Vgsc) mutation-based pyrethroid resistance in Aedes aegypti populations of three endemic dengue risk areas of Sri Lanka(Hindawi Pub. Co., 2021) Ranathunge, T.; Udayanga, L.; Sarasija, S.; Karunathilaka, S.; Nawarathne, S.; Rathnarajah, H.; Dulficar, F.F.; Shafi, F.N.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.BACKGROUND: Pyrethroid insecticides are widely used in many countries for chemical-based control of Ae. aegypti. Regardless of their efficacy, the constant use of insecticides has induced insecticide resistance mechanisms, such as knockdown resistance (kdr) in mosquitoes. Sri Lankan Vector Controlling Entities (VCE) have been using a variety of pyrethroid insecticides as the primary approach for dengue control. However, development of any resistance among the Aedes mosquitoes has been limitedly studied in the country. Therefore, the current study was conducted to evaluate the prevalence of F1534C, V1016G, and S989P mutations among Ae. aegypti mosquito populations in three dengue endemic high-risk regions of Sri Lanka. Methodology. Immature (both pupae and larvae) stages of Ae. aegypti mosquitoes were collected from Colombo, Gampaha, and Kandy districts of Sri Lanka from February 2018 to December 2019. Polymerase Chain Reaction- (PCR-) based assay for molecular genotyping of mutations was performed to identify the prevalence of kdr mutations in collected Ae. aegypti populations, separately. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy-Weinberg equilibrium. RESULTS: The Ae. aegypti populations from Colombo, Gampaha, and Kandy districts showed 46%, 42%, and 22% of F1534C mutation allele frequencies, along with 15%, 12%, and 6% of V1016G mutation allele frequencies, respectively. The mutation allele frequencies of S989 in Colombo, Gampaha, and Kandy districts were 9.5%, 8.5%, and 4.5%, respectively. The wild-type (PP) genotype remained predominant within all the three districts, whereas the homogenous (QQ) mutation genotype occurred only in minority. The abundance of Q allele frequency in Ae. aegypti mosquitoes was relatively higher for all the three mutations in Colombo. CONCLUSIONS: The findings clearly indicate that long-term insecticide applications and multiple use of pyrethroids have led to the acquisition of kdr mutations, leading to the development of insecticide resistance among local Ae. aegypti populations, especially in the Colombo and Gampaha districts. Therefore, evaluation of the prevalence levels of these kdr mutations highlights the necessity for shifting towards novel vector control strategies.Item Assessment of developmental and reproductive fitness of dengue-resistant transgenic Aedes aegypti and Improvement of fitness using antibiotics(Hindawi Pub. Co., 2021) Ramyasoma, H.P.B.K.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.; Dassanayake, R.S.BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.Item Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A;-B;-C;-DRB1 and-DQB1 loci using sequencing-based typing.(National Science Foundation, 2019) Perera, P.C.D.; Upamali, B.D.N.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.ABSTRACT: Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing that is currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature. © 2019, National Science Foundation. All rights reserved.Item Optimization of Aedes albopictus rearing procedures for combined sterile insect techniques (SIT) and Wolbachia-based laboratory studies in Sri Lanka(Springer Science and Business Media Deutschland GmbH, 2020) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Abeyewickreme, W.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Manamperi, A.The establishment of a laboratory colony is an essential first step for conduction of laboratory studies on the biology and control of Aedes albopictus mosquitoes. Therefore, with the objective of generating high quality mosquitoes as research material for utilization in ongoing vector control studies and to disseminate authenticated, high-quality Ae. albopictus mosquito rearing information to the research community, maintenance of an Ae. albopictus mosquito colony was initiated at the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka. A self-mating colony was established from eggs of the F1 progeny of individuals collected as free-living larvae in Narahenpita (Western Province), Sri Lanka. The mean temperature of 28 °C (± 2 °C) and relative humidity of 80% (± 5%) was constantly maintained inside the insectary. Lighting was provided by fluorescent lights, regulated with 12:12 h continuous dark and light period. Pest insects were controlled manually. Mosquitoes were maintained on bovine blood provided via an artificial membrane feeding system and a continuous supply of 10% sugar supplements. Larvae were maintained in deoxygenate water and fed with International Atomic Energy Agency (IAEA) recommended diet of tuna meal, bovine liver powder, and brewery yeast in a ratio of 37.5:27:10.5 in 1 L. Data on fecundity, fertility, larvae death, pupation, adult emergence, adult mosquito longevity were recorded. Adhering to bio-safety, all discarded materials were boiled thoroughly and incinerated if required. This report on the establishment and maintenance of a laboratory colony of Ae. albopictus will be of value for identifying the critical requirements essential under artificial conditions.Item RNAi-mediated silencing of ARV1 in Setaria digitata impairs in-vitro microfilariae release, embryogenesis and adult parasite viability(Elsevier Scientific Pub. Co., 2020) Wickramatunga, P.G.T.S.; Gunawardene, Y.I.N.S.; Wijesinghe, K.J.; Ellepola, A.N.B.; Dassanayake, R.S.ABSTRACT: Setaria digitata is a nematode that resides in the peritoneal cavity of ruminants causing cerebrospinal nematodiasis disease affecting livestock and inflicting significant economic forfeitures in Asia. Further, this nematode can infect humans, causing abscesses, allergic reactions, enlarged lymph nodes, eye lesions and inflammation of the lungs. The 'ARE2 required for viability1' (ARV1) encodes for putative lipid transporter localized in the endoplasmic reticulum (ER) and Golgi complex membrane in humans and yeast. In the present study, the functional role of S. digitata ARV1 (SD-ARV1) was investigated using RNA interference (RNAi) reverse genetic tool. The targeted silencing SD-ARV1 transcripts by siRNA mediated RNAi resulted in a dramatic reduction of SD-ARV1 gene and protein expressions in S. digitata, which in turn modulated the parasitic motility, its production of eggs and microfilaria viability. Further, the same silencing caused severe phenotypic deformities such as distortion of eggs and embryonic development arrest in the intrauterine stages of adult female S. digitata. These results suggest that SD-ARV1 plays a pivotal role in worm embryogenesis, adult parasite motility and microfilariae viability. Finally, the ubiquitous presence of ARV1 in human filarial nematodes, its crucial functional roles in nematode biology and its remarkable diversity in primary protein structure compared to homologues in their hosts warrants further investigations to ascertain its candidacy in anthelmintic drug development. KEYWORDS: ARV1; Morphological deformities; RNA interference; Setaria digitata.