Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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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 Engineering RNA interference-based dengue virus resistance in the mosquito vector aedes aegypti: The current status and future directions(Springer, 2021) Denipitiyage, S.D.; Gunawardene, Y.I.N.S.; Federico, Z.; Dassanayake, R.S.Dengue is an acute, febrile disease caused by the dengue viruses (DENV) comprising four serotypes and transmitted by the mosquito vector Ae. aegypti. DENV are single-stranded, positive-sense RNA viruses of the family Flaviviridae. Dengue is declared as a current significant challenge in the Southeast Asia, imposing growing burden on infected populations. To date, dengue control has mostly relied on vector control strategies which have largely become ineffective. There is, therefore, an urgent need for novel vector control strategies. Development of genetically modified mosquito vectors to manipulate disease-vectoring populations has gathered increased interest in recent time. RNAi-mediated viral resistance contributes to the suppression of viruses, including DENV in the mosquito vector Ae. aegypti. With recent advances in the field of molecular biology, we and other scientists are continuing to engineer genes that confer virus resistance to reduce transmission rates of DENV and introducing these genes into the mosquito genome. Even though scientists successfully generated mosquito refractory to DENV2–4, no mosquito refractory to all four serotypes has been developed to date. This limitation can be overcome by systematic analysis of the molecular mechanisms of RNAi in the mosquito vector Ae. aegypti. An enhanced understanding of RNAi function in the mosquito vector Ae. aegypti will facilitate the application of RNAi to control the transmission of the dengue disease in the future. Here, based on current understanding of the RNAi, we discuss the mechanisms of RNAi in the mosquito vector Ae. aegypti. We also provide guidelines for optimal design of RNAi experiments in Ae. aegypti with the possible risks associated with them along with proposed solutions.Item Advances in Aedes mosquito vector control strategies using CRISPR/Cas9(Springer, 2021) Wickramasinghe, P.D.S.U.; Silva, G.N.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.Advancements in genetic engineering have resulted in the development of mosquitoes with impaired vector competence, thereby limiting acquisition and transmission of pathogens. The main dengue (DENV) vector, Aedes aegypti, is an invasive species that have spread unwittingly across the world as a result of human trade and travel. The Ae. aegypti mosquito species has spread across tropical and subtropical regions, with higher presence in urban regions where rapid breeding patterns have shown in artificial containers. Identification of and treating an adequate number of mosquito breeding sites as a control measure have been done for the past couple of years, and yet improvement is far from the expectations, even with well-funded and well-organized initiatives. In order to stop the pathogen transmission, genetically modified mosquitoes (GMM) needs to be created and released. Despite many Aedes-related achievements, GMM creation has been challenging. The spread of particular genetic elements that impair vector competence, trigger deleterious recessive mutations, or skew a population's sex ratio can be used to prevent the spread of vector disease, or eradicate invasive organisms in a species-specific and eco-friendly manner. In recent years, genome editing strategies have evolved to make use of a variety of nucleases, ranging from sequence-specific zinc finger nucleases to modular TALENs (transcription activator-like effector nucleases) and most recently, RNA-guided nucleases adapted from bacterial adaptive immune systems, dubbed CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated systems). By combining these methods, a new era in gene editing had emerged. Generally, both of these gene editing technologies utilize sequence-specific nucleases to generate double-stranded DNA breaks (or nicks) in the target sequence, resulting in desired DNA modifications using endogenous DNA repair mechanisms. Since cells with DNA lesions are unable to divide further, the nuclease-generated strand breaks must be rapidly repaired by the cell to maintain the viability. CRISPR/Cas has been widely accepted for use in a variety of organisms, including insect species, with only minor optimization steps needed thus far. CRISPR/Cas9 technology transformed the process of engineering nucleases capable of cleaving complex genomic sequences. A complementary guide RNA (gRNA) directs the Cas9 endonuclease's operation to the specific DNA target site, enabling the editing of virtually any DNA sequence without complex protein engineering and selection procedures. Apart from genome editing, the specificity and flexibility of the CRISPR/Cas9 method enables unprecedented rapid development of genetically modified organisms with mutation systems for disease vector insect control. The stability and expression of the gene construct generated by CRISPR/Cas9 or any other method must be addressed before GMM are released, in order to make sure that pathogen transmission and formulation are interrupted robustly and completely. Spreading foreign antipathogen genes through gene drive strategies among wild mosquito populations strengthens the case for a more streamlined approach. Major fields that must be adequately assessed include risk evaluation and management, conducting studies to ensure human and environmental protection, developing effective control strategies built on comprehensive gene-driving systems, and adequately addressing the ethical, legal, and social consequences of GMM release. Although GMM is theoretically feasible as a disease control method, field releases should be made only when strong scientific evidence of human and environmental protection and effectiveness are presented, and public acceptance is addressed appropriately. This chapter discusses the diverse technological advances in generating Ae. aegypti mosquitoes which are resistant to dengue virus (DENV) and other diseases, as well as the biosafety and risk assessment of these procedures. Additionally, the chapter outlines a convincing path forward for developing successful genetic-based DENV control strategies based on CRISPR/Cas9, which could be expanded to control other arboviruses while maintaining biosafety.Item Genetic improvements to the sterile insect technique (SIT) for the control of mosquito population(Springer, 2021) Dilani, P.V.D.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.Mosquito-borne diseases are becoming a major health problem worldwide. At present, the principal method of controlling these diseases entirely depends on the mosquito vector control strategies. However, traditional control methods which are focussed on reducing mosquito populations through environmental management and the application of insecticides are largely ineffective. Hence, various control methods, including the release of sterile insect technique (SIT), have been proposed for the reduction of the mosquito population. As a species-specific control strategy, SIT offers considerable environmental benefits and a chemical-free option for insect control. However, the application of the SIT to mosquito control consistently suffered from lack of efficient sexing system, high fitness cost and operational difficulty in ionizing radiation, density-dependent nature of the target mosquito population and various other technical issues. The intervention of genetic engineering has led to several improvements in the operation or security of SIT programmes. The advent of mosquito transgenesis has paved the way for novel approaches in mosquito control. One possibility is a release of insects carrying dominant lethal (RIDL) strategy by engineering self-limiting gene, which offers solutions for many drawbacks of traditional SIT by providing genetic sterilization, genetic sexing, genetic containment and provision of genetic markers while maintaining its environmentally benign and species-specific utility. The success of this strategy often depends on how genetic modification affects the fitness of the mosquitoes. With several improvements and modifications allowing minimum fitness load, RIDL is now available for a wide range of mosquitoes such as Aedes aegypti, Aedes albopictus and Anopheles stephensi with field-testing possibilities. However, with solid epidemiological evidence and community support, widespread implementation of these strategies might reverse the current alarming global mosquito vector-borne diseases.Item Hemocup blood feeder: An affordable and simplified blood-feeding device for maintenance of aedes aegypti mosquito colonies in Sri Lanka(Academic Press, 2023) Dilani, P.V.D.; Wickramasinghe, P.D.S.U.; Lakshman, G.V.C.P.; Ranathunge, T.; Dassanayake, R.S.; Silva, G.Y.I.N.Dengue is a mosquito-borne viral disease mainly transmitted by Aedes aegypti and disease control is primarily reliant on mosquito vector control strategies. In the failure of conventional vector control strategies, new strategies are being developed which specifically require the maintenance of mosquito colonies in the laboratories. Blood-feeding is an essential part of the routine colony maintenance of Ae. aegypti. Therefore, the current study was focused on developing a simplified artificial membrane-feeding device, "Hemocup" feeder out of affordable material. viz., plastic cups, styrofoam insulation system, parafilm-M, and preheated water to facilitate the Ae. aegypti artificial blood feeding. The performance of the device was compared to that of a commercially available blood-feeding device, "Hemotek", by assessing the blood-feeding rate, fecundity, and egg hatchability. Similar blood feeding rates were observed for Hemocup and Hemotek methods (91.8 ± 1.6 and 94.3 ± 1.6 respectively>0.05) as well as comparable fecundity between the two methods (20.8 ± 0.7 and 22.0 ± 1.5 respectively; p > 0.05). Furthermore, there was no statistically significant difference in egg hatchability between the two methods (91.9 ± 1.4 and 93.8 ± 1.4, respectively; p > 0.05). The results indicate that this simple Hemocup blood-feeding system can be used for routine colonization of laboratory strains of Ae. aegypti and for mass-rearing purposes.Item 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 Reconstruction of Metabolic Pathways for the Setaria digitata Whole Genome(Sri Lanka Medical Association, 2020) Rashanthy, N.; Kothalawala, M.S.A.; Mugunamalwaththa, T.S.; Darshika, W.A.S.; Lakmali, G.L.Y.; de Zoysa, K.; Chandrasekharan, N.V.; Gunawardene, Y.I.N.S.; Suravajhala, P.; Dassanayake, R.S.INTRODUCTION AND OBJECTIVES: Setaria digitata is a Wolbachia-free filarial parasite that resides in the abdominal cavity of ungulates. It can cause cerebrospinal nematodiasis (CNS) in unnatural hosts such as sheep, goats, which causes a serious threat to livestock farming. Furthermore, S. digitata can also infect humans causing several conditions showing a gradual adaption to humans. METHODS: Despite, to date, complete a metabolic pathway reconstruction of S. digitata has not been undertaken and therefore, in this study the latter analyses were carried out using BLAST2GO software. RESULTS: Metabolic pathway analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified 111 enzymes found in total of 246 contigs that involve in 95 metabolic pathways, in which the most over-represented pathways are Biosynthesis of antibiotics, Phosphatidylinositol signaling system and Purine metabolism. Since S. digitata does not harbor Wolbachia endosymbiont, it was theorized that the S. digitata genome must encodes genes to carryout haem, riboflavin and nucleotides pathways, otherwise encoded by Wolbachia genome, potentially through lateral transfer of Wolbachia to an ancestor of S.digitata. Here, KEGG analysis identified 16 enzyme coding genes involve in nucleotide biosynthesis and one enzyme involve in riboflavin biosynthesis pathway. Although studies have revealed that FAD and glutathione pathways are complete in all nematode genomes, the genes encoding FAD and glutathione pathways were not found in the S. digitata. Moreover, complete nucleotide synthesis pathway and haem synthesis pathway were not found. CONCLUSION: This suggests that S. digitata may have evolved its own sequences to encode those biosynthetic pathways and hence calling for investigations to undertake characterization of genes involved in these pathways.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.