Displaying publications 61 - 80 of 305 in total

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  1. Fulltext Sequence-Specific Detection of Aristolochia DNA - A Simple Test for Contamination of Herbal Products
    Sgamma T, Masiero E, Mali P, Mahat M, Slater A
    Front Plant Sci, 2018;9:1828.
    PMID: 30619401 DOI: 10.3389/fpls.2018.01828
    Herbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  2. Fulltext Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach
    Selvarajah GT, Bonestroo FAS, Timmermans Sprang EPM, Kirpensteijn J, Mol JA
    BMC Vet Res, 2017 Nov 25;13(1):354.
    PMID: 29178874 DOI: 10.1186/s12917-017-1281-3
    BACKGROUND: Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines.

    RESULTS: Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.

    CONCLUSIONS: Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods; Real-Time Polymerase Chain Reaction/veterinary
  3. Changes in rumen protozoal community by condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid in vitro
    Saminathan M, Gan HM, Abdullah N, Wong CMVL, Ramiah SK, Tan HY, et al.
    J Appl Microbiol, 2017 Apr 23.
    PMID: 28434189 DOI: 10.1111/jam.13477
    AIMS: To evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community in vitro.

    METHODS AND RESULTS: The effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) on protozoal population and community were evaluated in vitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P real-time PCR analysis showed that the total protozoa was significantly (P 

    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  4. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68
    Samadlouie HR, Hamidi-Esfahani Z, Alavi SM, Varastegani B
    Braz J Microbiol, 2014;45(2):439-45.
    PMID: 25242926
    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  5. Fulltext Providing a laboratory diagnostic service for pandemic SARS-CoV-2 in a developing country
    Sam IC, Chong J, Kamarudin R, Jafar FL, Lee LM, Bador MK, et al.
    Trans R Soc Trop Med Hyg, 2020 08 01;114(8):553-555.
    PMID: 32497211 DOI: 10.1093/trstmh/traa037
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  6. Combinatorial effects of genistein and sex-steroids on the level of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC) and cAMP in the cervix of ovariectomised rats
    Salleh N, Ismail N, Muniandy S, Korla PK, Giribabu N
    Reprod Toxicol, 2015 Dec;58:194-202.
    PMID: 26529183 DOI: 10.1016/j.reprotox.2015.10.017
    The combinatorial effects of genistein and estrogen (E) or estrogen plus progesterone (E+P) on CFTR, AC and cAMP levels in cervix were investigated. Ovariectomised adult female rats received 50 or 100mg/kg/day genistein with E or E followed by E+P [E+(E+P)] for seven consecutive days. Cervixes were harvested and analyzed for CFTR mRNA levels by Real-time PCR. Distribution of AC and CFTR proteins in endocervix were observed by immunohistochemistry. Levels of cAMP were measured by enzyme-immunoassay. Molecular docking predicted interaction between genistein and AC. Our results indicate that levels of CFTR, AC and cAMP in cervix of rats receiving genistein plus E were higher than E-only treatment (p<0.05) while genistein plus [E+(E+P)] were higher than E+(E+P)-only treatment (p<0.05). In conclusions, increased levels of CFTR, AC and cAMP in cervix of E and E+(E+P)-treated rats by genistein could affect the cervical secretory function which could influence the female reproductive processes.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  7. Fulltext Technical aspects in understanding effects of gamma irradiation on flower colour changes in Dendrobium Sonia
    Sakinah Ariffin, Azhar Mohamad, Ratnam, Wickneswari
    Jurnal Sains Nuklear Malaysia, 2012;24(1):91-101.
    MyJurnal
    Colour is one of the most important traits in orchids and has created great interest in breeding programmes. Gamma irradiation is an alternative way for generation of somaclonal variation for new flower colours. Phenotypic changes are usually observed during screening and selection of mutants. Understanding of targeted gene expression level and evaluation of the changes facilitate in the development of functional markers for selection of desired flower colour mutants. Four Dendrobium orchid sequences (NCBI accessions: AM490639, AY41319, FM209429 and DQ462460) were selected to design gene specific primers based on information for chalcone synthase (CHS) from NCBI database. Quantitative real-time PCR (qPCR) was used to understand flower colour expression quantitatively derived from the CHS gene activities in different flower tissues (petal and sepal) from control Dendrobium Sonia (red purple), mutant DS 35-1/M (purple pink) and mutant DS 35-WhiteA. It was found that expression of CHS gene was highest in sepals of white flowers and lowest in both sepals and petals of purple pink flowers. Genomic DNA was amplified and PCR products were sequenced, aligned and compared. Sequence variations of CHS partial gene in Dendrobium Sonia mutants with different flower colour showed that two protein positions have been changed as compared to the control. These non-synonymous mutations may have contributed to the colour alterations in the white and purple pink mutants. This paper describes important procedures to quantify gene expression such as RNA isolation (quantity and quality), cDNA synthesis and primer design steps for CHS genes.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  8. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample
    Saidin S, Yunus MH, Othman N, Lim YA, Mohamed Z, Zakaria NZ, et al.
    Pathog Glob Health, 2017 May;111(3):128-136.
    PMID: 28335696 DOI: 10.1080/20477724.2017.1300421
    Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml-1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml-1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  9. Fulltext Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis
    Sady H, Al-Mekhlafi HM, Ngui R, Atroosh WM, Al-Delaimy AK, Nasr NA, et al.
    Int J Mol Sci, 2015;16(7):16085-103.
    PMID: 26193254 DOI: 10.3390/ijms160716085
    The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  10. Th17 and Treg cells function in SARS-CoV2 patients compared with healthy controls
    Sadeghi A, Tahmasebi S, Mahmood A, Kuznetsova M, Valizadeh H, Taghizadieh A, et al.
    J Cell Physiol, 2021 04;236(4):2829-2839.
    PMID: 32926425 DOI: 10.1002/jcp.30047
    In the course of the coronavirus disease 2019 (COVID-19), raising and reducing the function of Th17 and Treg cells, respectively, elicit hyperinflammation and disease progression. The current study aimed to evaluate the responses of Th17 and Treg cells in COVID-19 patients compared with the control group. Forty COVID-19 intensive care unit (ICU) patients were compared with 40 healthy controls. The frequency of cells, gene expression of related factors, as well as the secretion levels of cytokines, were measured by flow cytometry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay techniques, respectively. The findings revealed a significant increase in the number of Th17 cells, the expression levels of related factors (RAR-related orphan receptor gamma [RORγt], IL-17, and IL-23), and the secretion levels of IL-17 and IL-23 cytokines in COVID-19 patients compared with controls. In contrast, patients had a remarkable reduction in the frequency of Treg cells, the expression levels of correlated factors (Forkhead box protein P3 [FoxP3], transforming growth factor-β [TGF-β], and IL-10), and cytokine secretion levels (TGF-β and IL-10). The ratio of Th17/Treg cells, RORγt/FoxP3, and IL-17/IL-10 had a considerable enhancement in patients compared with the controls and also in dead patients compared with the improved cases. The findings showed that enhanced responses of Th17 cells and decreased responses of Treg cells in 2019-n-CoV patients compared with controls had a strong relationship with hyperinflammation, lung damage, and disease pathogenesis. Also, the high ratio of Th17/Treg cells and their associated factors in COVID-19-dead patients compared with improved cases indicates the critical role of inflammation in the mortality of patients.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  11. Fulltext Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging
    Ruszymah BH, Izham BA, Heikal MY, Khor SF, Fauzi MB, Aminuddin BS
    Med J Malaysia, 2011 Dec;66(5):440-2.
    PMID: 22390097 MyJurnal
    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  12. Fulltext Three-dimensional culture environment increases the efficacy of platelet rich plasma releasate in prompting skin fibroblast differentiation and extracellular matrix formation
    Rothan HA, Djordjevic I, Bahrani H, Paydar M, Ibrahim F, Abd Rahmanh N, et al.
    Int J Med Sci, 2014;11(10):1029-38.
    PMID: 25136258 DOI: 10.7150/ijms.8895
    Platelet rich plasma clot- releasate (PRCR) shows significant influence on tissue regeneration in clinical trials. Although, the mechanism of PRCR effect on fibroblast differentiation has been studied on 2D culture system, a detailed investigation is needed to establish the role of PRCR in cell seeded in 3D scaffolds. Therefore, a study was conducted to evaluate the influence of PRCR in fibroblasts (DFB) differentiation and extracellular matrix formation on both 3D and 2D culture systems. Cell viability was measured using MTT assay and DFB differentiation was evaluated by determining the expression levels of nucleostamin and alpha smooth muscle actin (α-SMA), using indirect immunostaining and Western blotting. The expression levels of extracellular matrix genes (collagen-I, collagen-III, fibronectin and laminin) and focal adhesion formation gene (integrin beta-1) were measured using Real-time PCR. The PRCR at 10% showed significant effect on cells viability compared with 5% and 20% in both culture environments. The decrease in the expression levels of nucleostamin and the increase in α-SMA signify the DFB differentiation to myofibroblast-like cells that was prominently greater in 3D compared to 2D culture. In 3D culture systems, the total collage production, expression levels of the extracellular matrix gene and the focal adhesion gene were increased significantly compared to 2D culture. In conclusion, 3D culture environments enhances the proliferative and differentiation effects of PRCR on DFB, thereby potentially increases the efficacy of DFB for future tissue engineering clinical application.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  13. Fulltext Recombinant human adiponectin as a potential protein for treating diabetic tendinopathy promotes tenocyte progenitor cells proliferation and tenogenic differentiation in vitro
    Rothan HA, Suhaeb AM, Kamarul T
    Int J Med Sci, 2013;10(13):1899-906.
    PMID: 24324367 DOI: 10.7150/ijms.6774
    Adiponectin is an adipocyte-secreting hormone that increases cell sensitivity to insulin. It has been previously demonstrated that this hormone protects against Type II Diabetes and, is found to concurrently promote cell proliferation and differentiation. It is postulated that diabetic patients who suffer from tendinopathy may benefit from using adiponectin, which not only improves the metabolism of diabetic ridden tenocytes but also promotes progenitor cell proliferation and differentiation in tendons. These changes may result in tendon regeneration, which, in diabetic tendinopathy, is difficult to treat. Considering that such findings have yet to be demonstrated, a study was thus conducted using diabetic ridden human tenocyte progenitor cells (TPC) exposed to recombinant adiponectin in vitro. TPC were isolated from tendons of diabetic patients and exposed to 10 μg/ml adiponectin. Cell proliferation rate was investigated at various time points whilst qPCR were used to determine the tenogenic differentiation potential. The results showed that adiponectin significantly reduced blood glucose in animal models. The proliferation rate of adiponectin-treated TPCs was significantly higher at 6, 8 and 10 days as compared to untreated cells (p<0.05). The levels of tenogenic genes expression (collagen I, III, tenomodulin and scleraxis) were also significantly upregulated; whilst the osteogenic (Runx2), chondrogenic (Sox9) and adipogenic (PPARУγ) gene expressions remained unaltered. The results of this study suggest that adiponectin is a potential promoter that not only improves diabetic conditions, but also increases tendon progenitor cell proliferation and differentiation. These features supports the notion that adiponectin may be potentially beneficial in treating diabetic tendinopathy.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  14. Fulltext Screening of antiviral activities in medicinal plants extracts against dengue virus using dengue NS2B-NS3 protease assay
    Rothan HA, Zulqarnain M, Ammar YA, Tan EC, Rahman NA, Yusof R
    Trop Biomed, 2014 Jun;31(2):286-96.
    PMID: 25134897 MyJurnal
    Dengue virus infects millions of people worldwide and there is no vaccine or anti-dengue therapeutic available. Screening large numbers of medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds. Therefore, this study was designed to identify anti-dengue activities in nineteen medicinal plant extracts that are used in traditional medicine. Local medicinal plants Vernonia cinerea, Hemigraphis reptans, Hedyotis auricularia, Laurentia longiflora, Tridax procumbers and Senna angustifolia were used in this study. The highest inhibitory activates against dengue NS2B-NS3pro was observed in ethanolic extract of S. angustifolia leaves, methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems. These findings were further verified by in vitro viral inhibition assay. Methanolic extract of V. cinerea leaves, ethanol extract of T. procumbens stems and at less extent ethanolic extract of S. angustifolia leaves were able to maintain the normal morphology of DENV2-infected Vero cells without causing much cytopathic effects (CPE). The percentage of viral inhibition of V. cinerea and T. procumbens extracts were significantly higher than S. angustifolia extract as measured by plaque formation assay and RT-qPCR. In conclusion, The outcome of this study showed that the methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems possessed high inhibitory activates against dengue virus that worth more investigation.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  15. Fulltext Flavonoid biosynthesis genes putatively identified in the aromatic plant Polygonum minus via Expressed Sequences Tag (EST) analysis
    Roslan ND, Yusop JM, Baharum SN, Othman R, Mohamed-Hussein ZA, Ismail I, et al.
    Int J Mol Sci, 2012;13(3):2692-706.
    PMID: 22489118 DOI: 10.3390/ijms13032692
    P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  16. Fulltext Association and interaction effect between VEGF receptor-2 (VEGFR-2) gene polymorphisms and dietary pattern on blood uric acid in Malays and Indians
    Roseline YW, Shidoji Y, Hon WM, Masaki M
    Malays J Nutr, 2012 Dec;18(3):307-17.
    PMID: 24568071 MyJurnal
    INTRODUCTION: Gout and hyperuricaemia attributed to genetic and lifestyle factors have been associated with several chronic diseases. This study aimed to determine the association and interaction effects between vascular endothelial growth factor receptor-2 (VEGFR-2) gene polymorphisms (rs1870377 and rs2071559) and dietary patterns on blood uric acid in Malay and Indian adults.
    METHODS: Dietary intakes of 153 Malays and 177 Indians were obtained using a food frequency questionnaire for the construction of dietary patterns using factor analysis. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Anthropometric measurements, body mass index (BMI) and blood pressure and biomarkers, uric acid, glycated haemoglobin A1c (HbA1c), and blood lipids were determined.
    RESULTS: There were significant differences in the mean values for HbA1c (41±-12 vs 45±-8 mmol/mol, p<0.001) and blood lipids levels (p<0.05) between Malays and Indians. Significant correlations were obtained between uric acid with selected blood lipids (p<0.05) and BMI in Malays (r=0.362, p<0.001) and Indians (r=0.212, p<0.01). Four dietary patterns were extracted from dietary intakes of all subjects: ‘Vegetables diet’; ‘Fruits diet’ (FD); ‘Animal protein and rice diet’; and ‘Fast foods and preserved foods diet’. There were no significant associations between dietary patterns (p=0.054-0.609) and VEGFR-2 gene polymorphisms (p=0.348-0.778) with uric acid. In Malay subjects, the interaction of rs2071559 and FD had a borderline effect (p=0.05) on blood uric acid after adjusting for potential confounders.
    CONCLUSION: The associations and gene-diet interactions involving VEGFR-2 gene polymorphisms and FD on uric acid provide new information on gout and hyperuricaemia risks in Malays.
    Keywords: Gene-diet interaction, VEGFR-2 gene polymorphisms, dietary pattern, uric acid, Malaysians
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  17. Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane
    Rohaina CM, Then KY, Ng AM, Wan Abdul Halim WH, Zahidin AZ, Saim A, et al.
    Transl Res, 2014 Mar;163(3):200-10.
    PMID: 24286920 DOI: 10.1016/j.trsl.2013.11.004
    The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; β1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  18. The L-cell isolation from heterogonous population of intestinal cell line using antibiotic selection method
    Rasouli M, Abbasi S, Sarsaifi K, Hani H, Ahmad Z, Omar AR
    Appl Biochem Biotechnol, 2014 Jan;172(1):394-404.
    PMID: 24081707 DOI: 10.1007/s12010-013-0514-6
    Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  19. Eleusine indica inhibits early and late phases of herpes simplex virus type 1 replication cycle and reduces progeny infectivity
    Rashidah Iberahim, Norefrina Shafinaz Md. Nor, Wan Ahmad Yaacob, Nazlina Ibrahim
    Sains Malaysiana, 2018;47:1431-1438.
    The present study was aimed at determining the compounds available in Eleusine indica methanol extract and the effects on
    herpes simplex virus type 1 (HHV1) replication cycle and progeny infectivity. Twelve compounds mostly from the flavonoid
    and phenolic groups were identified by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. The
    effect on replication phases of HHV1 was determined by time-of-addition, time-removal and virus yield reduction assays
    with expression of selected genes analysed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The extract
    inhibited plaque formation the most during the first 2 h and at 24 h of infection. Plaque formation inhibition was also
    noted at all other time points but at lesser percentage. Treatment with E. indica reduced progeny infectivity when treated
    for 10 h and was dose-dependent. E. indica methanol extract inhibited immediate early, early and late phases of HHV1
    replication cycle by modifying the expression of UL
    54, UL
    27 and UL
    30 genes during the infection. Immunostaining of
    infected cells confirmed that E. indica inhibited mainly Glycoproteins B but not Glycoprotein C and D. Thus, the methanol
    extract of E. indica has the ability to alter HHV1 replication cycle at almost all stages and reduce progeny infectivity.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  20. Fulltext Droplet digital PCR application for the detection of SARS-CoV-2 in air sample
    Rashid SA, Nazakat R, Muhamad Robat R, Ismail R, Suppiah J, Rajendran K, et al.
    Front Public Health, 2023;11:1208348.
    PMID: 37965510 DOI: 10.3389/fpubh.2023.1208348
    Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
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