Institute of Biochemistry, Molecular Biology and Biotechnology

BACKGROUND: A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before. METHODS: Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3 days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers. RESULTS: Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3 days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p < 0.05). CONCLUSION: Clinical suspicion overestimates the true number of dengue patients. RT-LAMP is a potentially useful low-cost diagnostic tool for dengue diagnosis. Confirmed dengue patients had significantly higher AST levels and lower lymphocyte counts in early disease compared to others. In confirmed dengue patients, younger age and a higher AST level in early infection were associated with subsequent plasma leakage.

Normal 0 false false false MicrosoftInternetExplorer4 st1\:*{behavior:url(#ieooui) } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} Sixteen SSR markers were used to identify genetic relationships of 43 coconut accessions conserved ex-situ in field gene banks of the Coconut Research Institute of Sri Lanka (CRISL). The 16 SSR markers clearly unveiled the genetic relationships of Sri Lankan coconut populations. Gene diversity and polymorphism information content (PIC) were relatively higher in the common ‘tall’ coconut and Pacific tall coconut than in autogamous dwarf form of coconut. The SSR assessment unveiled the genetic lineages based on evolutionary mechanisms signifying the narrow genetic base of coconut germplasm, with most of the diversity confining to ‘tall’ coconut. The main genetically different coconut groups identified were ‘tall’, ‘San Ramon and alike’ and ‘dwarf’. These have already been utilised in coconut improvement programmes and the study emphasizes the need for enrichment of the gene pool by exotic introductions. The overall results also supports the hypothesis that coconut disseminated from it’s center/s of origin in far east to Indo Atlantic regions via America. Normal 0 false false false MicrosoftInternetExplorer4 st1\:*{behavior:url(#ieooui) } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} Keywords: Coconut, genetic diversity, germplasm, microsatellite doi :10.4038/jnsfsr.v37i2.1065 Normal 0 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} J.Natn.Sci.Foundation Sri Lanka 2009 37 (2):99-109

Purpose: To investigate the anti-proliferative and antioxidant potentials of four different solvent extracts of Phoenix paludosa Roxb leaves.Methods: Four different solvent (hexane, chloroform, ethyl acetate and methanol) leaf extracts of the plant were tested for cytotoxicity against four cancer cells, viz, MCF-7 (oestrogen positive breast cancer cell line), MDA-MB-231 (triple negative breast cancer cell line), SK-BR-3 (breast adenocarcinoma) and ACHN (renal adenocarcinoma) as well as two normal cell lines, namely, HEK-293 (embryonic kidney cells) and MCF-10A (normal mammary epithelial cells)]. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2, 29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assays were used to evaluate the antioxidant activity of the crude extracts.Results: The methanol extract showed the highest antioxidant activity (DPPH, half maximal inhibitory concentration (IC50) = 30.17 ± 6.21 μg/mL) and (ABTS, IC50 = 27.91 ± 3.21 μg/mL). Of the four extracts, methanol extract showed the strongest significant (p &lt; 0.05) cytotoxicity to all four cancer cell lines at 24 and 48 h of incubation followed by the chloroform extract (IC50 of methanol extract (24 and 48 h): 36.71 ± 8.72 and 33.19 ± 5.53 μg/mL (MCF-7), 159.7 ± 32.09 and 141.9 ± 26.2 μg/mL (MDA-MB-231), 103.3 ± 18.9 and 75.39 ± 19.39 μg/mL (SKBR-3), 57.21 ± 3.72 and 43.16 ± 10.25 μg/mL (MCF-10A), 37.48 ± 5.75 and 26.99 ± 1.85 (ACHN) and 66.83 ± 14.26 and 60.34 ± 10.66 μg/mL (HEK-293)). Furthermore, the methanol extract was least cytotoxic to normal cell lines.Conclusion: The results obtained indicate that the methanol leaf extract of P. paludosa exhibit potent antioxidant and cytotoxic activities and has the potential of being developed into an anti-cancer agent.Keywords: Phoenix paludosa, antiproliferative, antioxidant, cytotoxicity

The presence of genetically different strains of malarial parasites in cases of human malaria is a severe drawback in the successful control of the disease. In Sri Lanka, although this species accounts for 60%-80% of all of the cases of clinical malaria that occur each year, the genetic complexity of Plasmodium vivax on the island remains to be elucidated. In recent studies based on PCR-RFLP and the parasites' merozoite-surface-protein-3alpha locus, the genetic structure of 201 clinical isolates of P. vivax, from two malaria-endemic areas and a non-endemic area of the island, was investigated. Although the PCR only produced amplicons of three sizes [1900 (72.6%), 1500 (25.9%) and 1200 (1.5%) bp], the RFLP analysis based on HhaI or AluI digestion yielded 22 and 26 restriction patterns, respectively, with 51 combined patterns recorded. The distribution of the prominent PCR-RFLP haplotypes was area-specific. The probability that an investigated case had a multiple-clone infection (MCI) was higher among the cases from the endemic areas (20.0%) than among those from the non-endemic area (13.8%) but this difference was not statistically significant. Since 17 single-clone isolates produced only 11 different PCR-RFLP haplotypes but (after sequencing) 13 distinct nucleotide haplotypes, it is clear that the results of the PCR-RFLP were not revealing all of the diversity that existed at the nucleotide level. Four mass blood surveys in a malaria-endemic area demonstrated that seasonal changes in the prevalences of human infection with P. vivax may influence the occurrence of MCI.

Purpose: To isolate active cytotoxic compounds from the hexane and chloroform extracts of the leaves of the mangrove plant, Scyphiphora hydrophyllacea C.F. Gaertn (Rubiaceae), grown in Sri Lanka.Methods: Dried pulverized leaves of S. hydrophyllacea were extracted with hexane and chloroform. Vacuum liquid chromatography (VLC), column chromatography (size exclusion chromatography, Sephadex LH-20) and reversed phase preparative recycling high performance liquid chromatography (HPLC) techniques were used to isolate three compounds (compounds 1, 2 and 3). The structures of the isolated compounds were established with the aid of 1H, 13C and two-dimensional nuclear magnetic resonance (2D-NMR) and electron ionization-mass spectrometry (EI-MS) techniques. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects of the compounds on oestrogen receptor positive breast (MCF-7) and non-small cell lung (NCIH- 292) cancer cells.Results: The isolated compounds were identified as oleanolic acid (1), ursolic acid (2) and eichlerianic acid (3). Ursolic acid and eichlerianic acid showed strong cytotoxic effects {IC50- ursolic acid: 8.47 μg/mL (24 h, MCF-7), 7.78 μg/mL (24 h, NCI-H292) and eichlerianic acid: 8.86 μg/mL (24 h, MCF-7), 10.15 μg/mL (24 h, NCI-H292)} in MCF-7 and NCI-H292 cancer cells at 24, 48 and 72 h post-incubation periods.Conclusion: Hexane and chloroform extracts of the leaves of S. hydrophyllacea yielded three compounds namely oleanolic acid, eichlerianic acid and ursolic acid. Ursolic acid and eichlerianic acid have been isolated for the first time from the leaves of S. hydrophyllacea grown in Sri Lanka and demonstrate in-vitro cytotoxic effects in oestrogen receptor positive (MCF-7) and non-small lung cancer (NCI-H-292) cells.Keywords: Scyphiphora hydrophyllacea, eichlerianic acid, ursolic acid, oleanolic acid, MCF-7, NCI-H-292

BACKGROUND: Antacids, anticholinergic drugs, histamine H2- receptor antagonists and irreversible proton pump inhibitors have been used for the treatment of gastric ulcers. However, prolonged use of these drugs may lead to series of adverse effects such as diarrhea, headache, rash, hypertension, muscular and joint pain. Therefore, there is an urgent need of more effective and safer treatments with fewer side effects. The aim of the present study was to scientifically evaluate the gastroprotective activity of fractions of the hot water extract of Trichosanthes cucumerina Linn (Family: Cucurbitaceae) aerial parts with a view to identifying the fraction with the best gastroprotective activity and the possible mechanism/s by which this fraction exert gastroprotection. METHODS: Gastroprotective activity of hexane fraction (HF), ethyl acetate fraction (EF), butanol fraction (BF) and aqueous fraction (AF) were evaluated by the assessment of ability to reduce the ulcer index in ethanol-induced rat model and the mode of action by which the most active fraction mediating gastroprotection. RESULTS: EF showed the maximum gastroprotection effect followed by BF and AF. EF (75 mg/kg) exhibited significantly higher gastroprotection compared to the reference drugs. Further investigations with two lower doses of EF confirmed that EF can mediated a significant and dose dependent gastroprotection. The rats treated with the EF showed significant reduction in free acidity (45%), total acidity (by 48%) in the gastric juice, increased the amount of mucus produced by the rat gastro mucosa and potent antihistamine activity (by 25.6%). EF was also rich in phenolic compounds and flavonoids. CONCLUSION: Gastroprotective mechanism of EF is possibly involves inhibition of acidity, elevation in mucus content, inhibition of histamine and antioxidant mechanisms.

Journal of Pharmaceutical Research International (ISSN: 2456-9119) is dedicated to publish high quality papers in all areas of pharmaceutical Science including pharmaceutical drugs, community pharmacy, hospital pharmacy, clinical pharmacy, compounding pharmacy, consultant pharmacy, internet pharmacy, veterinary pharmacy, nuclear pharmacy, military pharmacy, pharmacy informatics, pharmaceutics, medicinal chemistry, pharmacognosy, pharmacotherapy, pharmacodynamics, pharmacokinetics, clinical pharmacology, neuropharmacology, psychopharmacology, pharmacogenetics, pharmacogenomics, pharmacoepidemiology, toxicology, theoretical pharmacology, posology, pharmacognosy, behavioral pharmacology, environmental pharmacology, medicine development and safety testing, drug legislation and safety, pharmaceutical microbiology, pharmaceutical molecular biology, pharmaceutical biotechnology. The journal also encourages the submission of useful reports of negative results. This is a quality controlled, OPEN peer reviewed, open access INTERNATIONAL journal.

"Trichosanthes cucumerina Linn. is one of the medicinal plants that is often used in Sri Lankan traditional systems of medicine for the preparation of formulations to treat a variety of disease conditions. However, the toxic effects of T. cucumerina are not known. The aims of the present study were to (a) standardize hot water (HWE) and cold ethanolic (CEE) extracts of T. cucumerina aerial parts, and (b) evaluate toxic potential of the plant extracts. Both extracts were standardized by developing their densitograms and HPLC fingerprints and determination of physico - chemical parameters such as total ash, water soluble ash and acid insoluble ash. Administration of the HWE or CEE to mice did not result in acute or chronic toxic effects as evident from their effects on (a) liver and kidney functions and (c) hematological parameters and (d) fertility of male or female mice.In conclusion, the results of this study have revealed that standardized extracts of T. cucumerina at the doses tested do not produce any serious toxic side effects."

Malaria, a life-threatening disease prevalent in tropical regions, primarily affects infants, children under five, pregnant women, travelers, and individuals with HIV/AIDS. This study utilized an immunoinformatics approach to design a peptide-based malaria vaccine targeting antigenic proteins, including Apical Membrane Antigen 1, Knob-Associated Histidine-Rich Protein, Merozoite Surface Protein 1, and Sporozoite Surface Protein 2. Antigenic protein sequences were screened for antigenicity, allergenicity, toxicity, and immune responses involving CTLs, B-cells, and HTLs. Selected epitopes were linked with appropriate linkers and an adjuvant to enhance immunogenicity, forming a vaccine construct. The construction, comprising 1473 amino acids, exhibited a molecular weight of 15.21 kDa, a theoretical pI of 8.94, an aliphatic index of 60.01, and an instability index of 31.66, indicating stability. It was hydrophilic (GRAVY: –0.385) with favorable half-lives in mammalian, yeast, and E. coli systems. Docking studies showed strong binding affinity to human TLR2 and TLR4. In silico cloning indicated a CAI value of 0.92 and a GC content of 59.31%. Further studies are needed to validate its efficacy and safety.

Abstract Background Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53 . We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. Results Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS ; three of them were on codon 12 and one was on codon 61. Conclusion The prevalence of pathogenic somatic TP53 variants was 31% and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5 – 8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

Abstract Background Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. Results Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. Conclusion The prevalence of pathogenic somatic TP53 variants was 31% and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5 – 8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

Neutrophils are one of the key cells that actively participate in the innate immune reactions. Sequences of event of pathogen elimination by neutrophils are initiated with the activation of resting neutrophils. Pathogen is then eliminated by oxygen-dependent killing mechanism or oxygen-independent killing mechanism. This process includes margination, diapedesis, chemotaxis, phagocytosis and degranulation of neutrophils. Although, these mechanisms of the neutrophils are targeted to eliminate the pathogens and protect the body, excessive production of inflammatory mediators can cause host tissue damage. These harmful effects of neutrophils initiate some detrimental diseases like arthritis. Therefore, different drugs which can modulate the neutrophil functions are used in the medicinal practices as targeted therapy. From the ancient time, medicinal plant extracts have been used in traditional medicine in many different regions of the world to achieve the same effect. Ixora coccinea is one such medicinal plant used for the treatment of various diseased conditions and as an anti-inflammatory agent. Modulation of neutrophil function is one of an important underlying mechanism that contributes to the anti-inflammatory activity of Ixora coccinea. Keywords: Ixora coccinea, super oxide production, anti-inflammation, phagocytes, oxygen-dependent killing, chemotaxis Cite this Article N.K. Rachitha D. Wickramasinghe and Shiroma M. Handunnetti. Role of Human Neutrophils in Inflammation and its Modulation by Ixora coccinea . Research and Reviews: Journal of Immunology . 2016; 6(1):  32–48p.

Three cloned highly repetitive DNA sequences Rp36, Rp217 and Rp234 isolated from An. culicifacies Giles, sensu lato were developed as non radioactive DNA probes by using a biotinylated labeling and colorimetric detection system. These non radioactive DNA probes distinguish sibling species A from species B and C of the An. culicifacies complex in a dot blot hybridization assay using single mosquito DNA extracts diluted 50 fold. The biotinylated Rp217 probe was further assayed in a more simple procedure which involves the hybridization of blots prepared from squashed mosquito heads. This technique avoids the separate extraction of mosquito DNA and facilitates a number of samples to be processed rapidly while also allowing several field analyses to be carried out on one mosquito specimen.