Basic & Applied Sciences

Optimization of yeGFP Expression as a Reporter for Prospective RNAi Analysis in Pichia pastoris

Sat, 01 Jan 2022 00:00:00 GMT

Optimization of yeGFP Expression as a Reporter for Prospective RNAi Analysis in Pichia pastoris Dharmarathna, CS; Kumar, S; Gunawardena, YINS Green fluorescent protein from the jellyfish Aequorea victoria is a widely used reporter gene in genetic engineering. Yeast codon optimized green fluorescent protein (yeGFP) has been found to have higher expression, and hence it is more reliable as a yeast reporter. Pichia pastoris can be used as a model organism to study gene regulation such as RNA interference (RNAi) because it does not have RNAi, by monitoring the yeGFP expression. This study focuses on the optimization of the expression of yeGFP in the Pichia pastoris GS115 strain, under the control of AOX1 promoter for prospective RNAi studies. yeGFP gene was cloned into the pPICZ A vector to create pPICZ A-yeGFP expression plasmid. P. pastoris GS115 strain was transformed with pPICZ A-yeGFP and positive transformants were selected on Zeocin plates and further confirmed by PCR. Five transgenic colonies were tested for the yeGFP expression by methanol induction in MMH (Minimum Methanol Histidine) media containing 0.5%, 1%, and 2% (v/v) methanol for six days. Fluorescence intensities were measured using SpectraMax M3 spectrophotometer at an excitation wavelength of 395 nm, and emission of 510 nm. According to the Relative fluorescence units of the induced cultures, expression increased with the time till the fifth day and decreased on the sixth day. 0.5% methanol and 2% methanol-induced cultures showed the least and highest fluorescence respectively. Transgenic lines 1 and 4 had the highest yeGFP expression in 2% methanol on the fifth day. For studies on gene regulation by RNAi, optimum yeGFP expression under the AOX1 promoter can be measured in 2% methanol on the fifth day of induction.

A Preliminary Study Conducted on a Traditional Formulation used for the Treatment of Dengue

Sat, 01 Jan 2022 00:00:00 GMT

A Preliminary Study Conducted on a Traditional Formulation used for the Treatment of Dengue Handagala, JK; Kumarasinghe, NP; Goonasekara, CL; Kuruppu, AI Currently, around the globe, there are no effective drugs for the treatment of acute dengue infection. Even though a vaccine was licensed recently, it was found to have limited efficacy. Scientists are currently seeking for a novel treatment for dengue. Bamboo Swarasa is a Sri Lankan traditional medicinal preparation made from shoots of yellow and green common bamboo (Bambusa vulgaris) extracts together with Palmyra sugar. This preparation is currently given to patients with dengue fever. Initially, we assessed the phenolic, flavonoid and antioxidant content of this preparation by Total Phenolic Content (TPC), Total Flavonoid Content (TFC), 1,1-diphenyl- 2-picrylhydrazyl assay (DPPH). We also analysed the anti-inflammatory activity by the human red blood cell membrane stabilization (HRBC), and the egg albumin assays. Finally, the cytotoxicity of this preparation was assessed by the MTT assay. When 75mg/ml of sample preparation was compared to Gallic acid (GA) the total phenolic content was found to be 5mg/g GA equivalent. The total flavonoid content of 75mg/ml sample was 0.02mg/g Quercetin equivalent. For the DPPH assay, the highest percentage radical scavenging value was reported to be 91.79%, at the maximum concentration (75 mg/ml) tested and the IC50 was found as <3.75mg/ml. The anti-inflammatory activity of the preparation was found to be 46.09% inhibition of protein denaturation at 75mg/ml sample while Ibuprofen showed a similar inhibition: 51.30%. The HRBC assay depicted 52.85% inhibition of red cell lysis at 75mg/ml, while Ibuprofen showed 97.20% inhibition. Finally, a moderate cytotoxic effect was noted in Vero cells by the MTT assay where a concentration of 10μg/ml depicted a ~35% cell growth inhibition at 24h. This commercial preparation of Bamboo Swarasa demonstrated excellent antioxidant activity and moderate anti-inflammatory activity at a concentration of 75mg/ml. Viral infections are usually accompanied by oxidative stress, which plays an important role in their pathogenesis, and antioxidants are able to reduce the reactive oxygen species level in infected cells. Thus, this preparation could be an effective medicine for viral diseases such as dengue. This work provides only preliminary scientific evidence, and further research will be conducted to determine its antiviral activity.

Survey on How Many People in Sri Lanka are Interested in Fish Oil Dietary Supplements

Sat, 01 Jan 2022 00:00:00 GMT

Survey on How Many People in Sri Lanka are Interested in Fish Oil Dietary Supplements Hansima, NMGR; Jayapala, HPS; Rathnasri, S Consumption of fish or fish oil containing ω-3 polyunsaturated fatty acids (PUFAs) reduces the risk of coronary heart diseases, decreases mild hypertension, lowers the incidents of diabetics, and is important during pregnancy as a critical building block of foetus brain and retina. Therefore, fish oil dietary supplements have attracted more attention from people around the world recently. The present study attempts to gain an understanding of Sri Lankan people's interest in and awareness of fish oil dietary supplements. A questionnaire was used to collect the most reliable and accurate data from 200 individuals. In order to gather useful and relevant information, the questionnaire contained various components including general information about participants, fish consumption pattern, fish oil dietary supplement usage, diseases, etc. A recent survey indicates that Sri Lankan people have generally less interest in fish oil dietary supplements. According to the data analysis, only 10% of people use fish oil dietary supplements and 85% of them are between 20-30 age limits. Moreover, most of them eat fish usually in their diets and non-fish eaters do not take both fish and fish oil dietary supplements. Omega-3 fatty acids play a vital role in the human body. It has been suggested that nonconsumers of fish should take fish oil as a dietary supplement to fulfill their omega- 3 requirement. Based on the findings, this research can contribute towards informing the general public about fish oil dietary supplements and their accurate usage.

Evolutionary and Ecological Characterization of the Laughingthrush of Sri Lanka

Sat, 01 Jan 2022 00:00:00 GMT

Evolutionary and Ecological Characterization of the Laughingthrush of Sri Lanka Kariyawasam, MGDD; Seneviratne, SS The Ashy-headed Laughingthrush, an endemic bird to Sri Lanka, has been historically considered a laughingthrush in the genus Garulax. However, recent phylogenetic studies have suggested a new classification for the species under the genus Argya (true babblers). However, as the name suggests, the Ashy-headed laughingthrush shows remarkable morphological similarities to other laughingthrushes, despite the genetic signal showing evolutionary affinity to true babblers. This study attempted to address these seemingly divergent signals in the Ashy-headed Laughingthrush by reconstructing the molecular phylogeny of babblers and laughingthrushes of the Indo-Himalayan region and comparing it with key morphological, plumage features of the two groups. The phylogenetic relationships were reconstructed using the Bayesian inference using four mitochondrial and seven nuclear gene regions. Hierarchical clustering was performed to assess the phenotypic characters. The phylogenetic tree placed the Ashy-headed Laughingthrush with the genus Argya, with the A. malcolmi (Great Grey Babbler) of India being the sister taxa. Morphometrics and plumage placed Ashy-headed Laughingthrush with laughingthrushes in the phenotypic clustering. Therefore, a discordance is evident between phenotype and phylogeny. This discordance suggests a rapid phenotypic divergence from babblers and convergent patterns of evolution with laughingthrushes of the Indo-Himalayan region. Our findings agree with the recent placement of Ashy-headed Laughingthrush in Argya based on phylogenetic evidence despite seemingly divergent phenotypic signals. We think that the convergence of phenotypic evolution has historically confused the taxonomic delimitation of the Ashy-headed Laughingthrush of Sri Lanka.