Symposium on Dengue - 2015

Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/10378

Papers presented at the Symposium on Current Research Activities on Dengue, organized by the Molecular Medicine Unit, on 13th of November 2015

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Author: search.filters.author.Athapaththu, A.M.M.H.Has files: No

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    Synthesis of Fe2O3 nanoparticles for the development of a rapid diagnostic test kit for dengue detection
    (Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Jayarathna, I.P.L.; Gunathilaka, P.A.D.H.N.; Athapaththu, A.M.M.H.; Abeyewickreme, W.
    BACKGROUND: Hybrid nanoparticles have great potential for biotechnological and biomedical applications. It was recently proposed that biopolymer/co-shell nano-materials could be easily obtained by adapting traditional routes used in pharmaceutical science to design drug delivery system. Most of the case, super-paramagnetic nanoparticles of iron oxides, magnetite (Fe3O4) and maghemite (ᵧ -Fe2O3) have been employed and these interesting magnetic properties are due to finite-size effects and high surface/volume ratio. METHOD: Iron oxide was synthesized by using modified co-precipitation method and resulting particles were characterized using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Diffused Reflectance Fourier Transform-Infrared Spectroscopy (DRIFT-IR). RESULTS: The XRD pattern matches well that of ᵧ -Fe2O3. Six characteristic peaks for ᵧ -Fe2O3 (2θ = 31.7°, 36.7°, 41.1°, 53.4°, 57.0° and 62.6°) marked by their Miller indices [(220), (311), (400), (422), (511) and (440)] were observed for sample. The TEM images reveal that the particles are in 5 – 20 nm range, and well fitted with spaniel cubic structure, but when particles are dry it prefer to agglomerate with neighboring particles to reduce their surface charges. The spectrum of ᵧ -Fe2O3 nanoparticles shows a characteristic broad band at 3410 cm-1 is due to the stretching vibration of H2O molecules. The band corresponding to the bending vibrations of H2O molecules is positioned at 1633 cm-1. Two intense IR bands at 627 and 451 cm-1 are typical for ferrihydrite or ᵧ -Fe2O3 nanoparticles. The spectrum of this sample showed the presence of carbonate groups on the basis of IR bands at 1508, 1340 and 1069 cm-1. The presence of carbonate groups is due to the adsorption of atmospheric carbon dioxide by ᵧ -Fe2O3 nanoparticles. The bands of Fe–O stretching vibrations of ᵧ -Fe2O3 appeared at 627 and 451 cm-1 and the bands at 892 and 796 cm-1 can be assigned to Fe-OH···H bending vibrations. CONCLUSION: Magnetic ᵧ -Fe2O3 nanoparticles were synthesized by the co-precipitation method and this work confirmed that magnetic ᵧ -Fe2O3 nanoparticles are in nano-scale and well matches with spaniel cubic structure
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    Designing of immunogenic peptides from Dengue Virus NS1 region for production of monoclonal antibodies as diagnostic intermediates
    (Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Munasinghe, M.M.E.; Chandrasekharan, N.V.; Korbakis, D.; Soosaipillai, A.; Diamandis, E.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
    BACKGROUND: Small peptide antigens have become an essential tool for antibody production in the recent life science research applications. The immunogenicity of peptide antigens is a critical factor to induce the immune response in order to produce desired antibodies. METHODS: In the current study, we have previously determined four Dengue (DEN) serotype specific peptides, containing 28 Amino Acid (AA) residues were re-designed. The peptides were re-designed considering many factors, for instance, sequence of the Sri Lankan isolates, abundance of Cysteine residues, solubility and the length of the peptide, carrier protein to be used and several other factors such as the N-terminal and C- terminal AAs and multiple AA residues. The peptide sequences were analysed using Antigen Profiler Peptide Tool (Thermo-scientific), Peptide Property Calculator (Genscript) and Swiss-Model (Biozentrum). RESULTS: The protein sequence of the peptides were changed according to the Sri Lankan isolates (AEB98757.1, ACS32038.1, AHG23239.1 and AHN50410.1). Oxidation of Cysteine residues results in significant conformational changes. Replacement of Cysteine with Serine prevents such oxidation reactions and it often retains full biological activity. Generally, peptides with a high number of hydrophobic AA (>50%) may result insoluble peptides. Similarly, to obtain a soluble peptide, it is important to contain at least one charged AA in every five AAs. Hence, the number of hydrophobic residues in the peptides were maintained below 50% and ensured that one out of every five amino acids is charged. The length of the peptide is an important factor as long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic. In order to obtain both highly conserved and variable regions among four serotypes, the peptide length was determined as 29 residues. A terminal Cysteine was added to allow peptide conjugation with carrier protein. Keyhole Limpet Hemocyanin was selected as the carrier protein due to its higher immunogenicity. N-terminal Glutamine or Aspargine and C-terminal Proline or Glycine in the sequences were avoided. Finally, the peptides sequences were determined as: DEN1; CPESSDDQRA WNIWEVEDYGFGIFTTNIW,DEN2; CAESPN TNRA WNSLEVEDYGFGVFTTNIW, DEN3;CPESPSASRAWNVWEVEDYGFGVFTTNIW and .DEN4;CSESPNERRAWNSLEVEDYGFGMFTTNIW. CONCLUSION: These peptides have a high potential to be used as peptide antigens for Monoclonal Antibody production.