Basic & Applied Scienceshttp://ir.kdu.ac.lk/handle/345/58342024-03-28T16:59:23Z2024-03-28T16:59:23ZOptimization of yeGFP Expression as a Reporter for Prospective RNAi Analysis in Pichia pastorisDharmarathna, CSKumar, SGunawardena, YINShttp://ir.kdu.ac.lk/handle/345/62112023-04-26T11:20:18Z2022-01-01T00:00:00ZOptimization 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.
2022-01-01T00:00:00ZA Preliminary Study Conducted on a Traditional Formulation used for the Treatment of DengueHandagala, JKKumarasinghe, NPGoonasekara, CLKuruppu, AIhttp://ir.kdu.ac.lk/handle/345/62102023-04-26T11:18:16Z2022-01-01T00:00:00ZA 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.
2022-01-01T00:00:00ZSurvey on How Many People in Sri Lanka are Interested in Fish Oil Dietary SupplementsHansima, NMGRJayapala, HPSRathnasri, Shttp://ir.kdu.ac.lk/handle/345/62092023-04-26T11:33:24Z2022-01-01T00:00:00ZSurvey 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.
2022-01-01T00:00:00ZEvolutionary and Ecological Characterization of the Laughingthrush of Sri LankaKariyawasam, MGDDSeneviratne, SShttp://ir.kdu.ac.lk/handle/345/62082023-04-26T11:38:45Z2022-01-01T00:00:00ZEvolutionary 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.
2022-01-01T00:00:00Z