Displaying publications 1 - 20 of 24 in total

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  1. Causes of childhood blindness in Malaysia: results from a national study of blind school students
    Reddy SC, Tan BC
    Int Ophthalmol, 2001;24(1):53-9.
    PMID: 11998890
    A national study was conducted in children attending six schools for the blind in Malaysia to identify the anatomical site and underlying causes of blindness (BL) and severe visual impairment (SVI), with a view to determine the potentially preventable and treatable causes so that appropriate control measures can be implemented in the future. The standardized clinical examination of eyes was performed and the findings were recorded on the WHO Prevention of Blindness Programme eye examination record form for children with blindness and low vision. A total of 358 children aged between 7 and 17 years were examined, of whom 332 (92.7%) were blind or severely visually impaired. The results relate to these 332 children. Lens was the major anatomical site (22.3%) of visual loss followed by retina (20.8%), whole globe (17.2%), cornea (15.1%), optic nerve (8.7%) and uvea (5.1%). Glaucoma was responsible for BL/SVI in 7.2% and others in 3.6% of cases. Hereditary diseases were responsible for visual loss in 29.5%, intrauterine factors in 4.5%, perinatal factors in 9% and childhood factors in 7.8% of cases. However, the aetiology was unknown in 49.1% of cases which included congenital anomalies of the globe. Childhood cataract and corneal scarring are major treatable causes of BL/SVI that can benefit from future intervention strategies. Perinatal screening for intrauterine factors and hereditary eye diseases, and appropriate interventional therapy will help in reducing the prevalence of childhood blindness.
  2. Compression neuropathy of the common peroneal nerve secondary to a ganglion cyst
    Yazid Bajuri M, Tan BC, Das S, Hassan S, Subanesh S
    Clin Ter, 2011;162(6):549-52.
    PMID: 22262327
    There are various causes of the common peroneal nerve palsy. However, common peroneal nerve palsy caused by ganglia are uncommon. We hereby present a case of a 55-year-old man with a 1 week history of foot drop and swelling in the region of the right leg. Physical examination and nerve conduction study studies confirmed a diagnosis of common peroneal nerve palsy. Magnetic resonance imaging (MRI) revealed a lobulated, elongated cystic-appearing mass anterior to the head of fibula. Surgical decompression of the nerve with removal of the mass was performed. Surgical pathology reports confirmed the diagnosis of a ganglion cyst. Findings on physical examination, nerve conduction study and MRI results of this interesting case are being discussed. We wish to highlight that even a tumour which is benign and within the nerve sheath can cause compression.
  3. Fulltext Marjolin's ulcer--a near forgotten entity
    Tan BC, Horton TC, Sara Ahmad T
    Med J Malaysia, 2006 Feb;61 Suppl A:91-3.
    PMID: 17042239
    We report a case of a 55-year-old man who presented with a 6-month history of a fungating ulcer on the right hand at the site of a previously healed ulcer that had been present for 40 years. Histopathological examination of four-quadrant biopsy specimens showed a moderately differentiated squamous cell carcinoma (SCC). A transradiocarpal amputation with stump closure using radial flap was performed as it was not possible to achieve a functionally and cosmetically acceptable hand after a wide excision with 2 cm tumour-free margin. It is our intention to highlight this rare condition as reminder to consider this entity as a differential diagnosis of chronic non-healing skin ulcer.
  4. Proteomics in commercial crops: An overview
    Tan BC, Lim YS, Lau SE
    J Proteomics, 2017 10 03;169:176-188.
    PMID: 28546092 DOI: 10.1016/j.jprot.2017.05.018
    Proteomics is a rapidly growing area of biological research that is positively affecting plant science. Recent advances in proteomic technology, such as mass spectrometry, can now identify a broad range of proteins and monitor their modulation during plant growth and development, as well as during responses to abiotic and biotic stresses. In this review, we highlight recent proteomic studies of commercial crops and discuss the advances in understanding of the proteomes of these crops. We anticipate that proteomic-based research will continue to expand and contribute to crop improvement.

    SIGNIFICANCE: Plant proteomics study is a rapidly growing area of biological research that is positively impacting plant science. With the recent advances in new technologies, proteomics not only allows us to comprehensively analyses crop proteins, but also help us to understand the functions of the genes. In this review, we highlighted recent proteomic studies in commercial crops and updated the advances in our understanding of the proteomes of these crops. We believe that proteomic-based research will continue to grow and contribute to the improvement of crops.

  5. Population genetic structure of the tropical moss Acanthorrhynchium papillatum as measured with microsatellite markers
    Leonardía AA, Tan BC, Kumar PP
    Plant Biol (Stuttg), 2013 Mar;15(2):384-94.
    PMID: 22882300 DOI: 10.1111/j.1438-8677.2012.00640.x
    Mosses and other bryophytes are vital components of forests, because they sustain a tremendous diversity of invertebrates and influence significant ecological functions. There have been few studies on moss population diversity in Southeast Asia, despite the escalating deforestation in this region of rich biodiversity. The genetic diversity of the tropical moss Acanthorrhynchium papillatum (Harv.) Fleisch., collected from forested areas in Singapore and Peninsular Malaysia, was elucidated using eight microsatellite markers developed for this species. Significant levels of allelic and haplotypic diversity were observed among clumps of the moss. Differences in allelic richness and genotypic diversity among the populations were higher in less disturbed forests compared to the more disturbed areas, suggesting that genetic diversity is affected by habitat quality. Genetic diversity levels within the clumps studied were low, indicating that vegetative reproduction was more important within clumps than sexual reproduction. However, multilocus genotypes of samples within the clumps studied were not all alike, providing evidence of microsatellite mutation or of occasional sexuality. Despite the isolation of populations, A. papillatum can introduce genetic variability by mutation among vegetatively propagated individuals. This study provides baseline information on the genetic diversity of A. papillatum tropical rain forests.
  6. Delivery of macrosomic babies: management and outcomes of 330 cases
    Lim JH, Tan BC, Jammal AE, Symonds EM
    J Obstet Gynaecol, 2002 Jul;22(4):370-4.
    PMID: 12521456
    This study reviews the deliveries of macrosomic babies and their outcomes. A total of 330 macrosomic (birth weight > or =4 kg) cases were studied retrospectively from July 1999 to December 1999 in the Maternity Hospital of Kuala Lumpur. The variables studied included induction of labour, mode of delivery and the incidence of maternal and perinatal complications. Three hundred and thirty macrosomic infants were delivered during the period of study. Vaginal delivery was achived in 56% of the study cases. The percentage of vaginal delivery was higher among those who had induction of labour (63%) compared to the group without induction of labour (50%). Vaginal delivery was planned in 267 mothers and of these 69% achieved vaginal delivery. Twelve per cent of the macrosomic infants were delivered by elective caesarean section. Shoulder dystocia occurred in 4.9% of vaginal deliveries. Eighty-eight neonates were admitted to the special care nursery unit and 57% of these infants were delivered by elective caesarean section. Perineal trauma occurred in 26% of vaginal deliveries. Post-partum haemorrhage occurred in 32% of caesarean deliveries compared to 4% in vaginal deliveries. Two cases of stillbirths were documented but no maternal death occurred during the period of study. Vaginal delivery is the most frequent mode of delivery for a fetus weighing in excess of 4 kg and vaginal delivery should be attempted in the absence of contraindications, because vaginal delivery has less maternal morbidity compared to caesarean delivery. However, shoulder dystocia remains a significant complication of vaginal delivery for macrosomic fetuses.
  7. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)
    Fonseka M, Ramasamy R, Tan BC, Seow HF
    Cell Biol Int, 2012 Sep;36(9):793-801.
    PMID: 22335239 DOI: 10.1042/CBI20110595
    hUCB-MSC (human umbilical cord blood-derived mesenchymal stem cells) offer an attractive alternative to bone marrow-derived MSC for cell-based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB-MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB-MSC. Co-culturing of hUCB-MSC and K562 resulted in inhibition of proliferation of K562 in a dose-dependent manner. However, the anti-proliferative effect was reduced in transwells, suggesting the importance of direct cell-to-cell contact. hUCB-MSC inhibited proliferation of K562, arresting them in the G0 /G1 phase. NO (nitric oxide) was not involved in the hUCB-MSC-mediated tumour suppression. The presence of IL-6 (interleukin 6) and IL-8 were obvious in the hUCB-MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL-4 and Th17 cytokine, IL-17 were not secreted by hUCB-MSC. There was an increase in the number of hUCB-MSC expressing the latent membrane-bound form of TGFβ1 co-cultured with K562. The anti-proliferative effect of hUCB-MSC was due to arrest of the growth of K562 in the G0 /G1 phase. The mechanisms underlying increased IL-6 and IL-8 secretion and LAP (latency-associated peptide; TGFβ1) by hUCB-MSC remains unknown.
  8. Genetic relatedness of Candida strains isolated from women with vaginal candidiasis in Malaysia
    Chong PP, Lee YL, Tan BC, Ng KP
    J Med Microbiol, 2003 Aug;52(Pt 8):657-66.
    PMID: 12867559
    The aims of this study were to compare the genetic relatedness of: (i) sequential and single isolates of Candida strains from women with recurrent vaginal candidiasis (RVC); and (ii) Candida strains from women who had only one episode of infection within a 1-year period. In total, 87 isolates from 71 patients were cultured, speciated and genotyped by random amplification of polymorphic DNA (RAPD) analysis. Patients were categorized into three groups, namely those with: (i) a history of RVC from whom two or more yeast isolates were obtained (group A); (ii) a history of RVC from whom only a single isolate was obtained (group B); and (iii) a single episode of vaginal candidiasis within a 1-year period (group C). Six yeast species were detected: Candida albicans, Candida glabrata, Candida lusitaniae, Candida famata, Candida krusei and Candida parapsilosis. Interestingly, the prevalence of non-albicans species was higher in group A patients (50 %) than in patients in groups B (36 %) or C (18.9 %). Eighty RAPD profiles were observed, with a total of 61 polymorphic PCR fragments of distinct sizes. Clustering analysis showed that, overall, the majority of patients in group A had recurrent infections caused by highly similar, but not identical, sequential strains [mean pairwise similarity coefficient (S(AB)) = 0.721 +/- 0.308]. The range of mean S(AB) values for intergroup comparisons for C. albicans isolates alone was 0.50-0.56, suggesting that there was no significant relatedness between strains from different groups. Genetic similarity of C. albicans isolates from patients in group A was lower than that of C. albicans isolates from patients in group C (mean S(AB) = 0.532 +/- 0.249 and 0.636 +/- 0.206, respectively); this difference was statistically significant (P = 0.036). These results demonstrate that the cause of recurrent infections varies among individuals and ranges between strain maintenance, strain microevolution and strain replacement; the major scenario is strain maintenance with microevolution. They also show that C. albicans strains that cause recurrent infections are less similar to each other than strains that cause one-off infections, suggesting that the former may represent more virulent subtypes.
  9. Fulltext CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences
    Karlson CKS, Mohd-Noor SN, Nolte N, Tan BC
    Plants (Basel), 2021 Sep 29;10(10).
    PMID: 34685863 DOI: 10.3390/plants10102055
    RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.
  10. Editorial: Genome editing and biotechnological advances for crop improvement and future agriculture
    Hamdan MF, Hensel G, Alok A, Tan BC
    Front Plant Sci, 2023;14:1132821.
    PMID: 36798708 DOI: 10.3389/fpls.2023.1132821
  11. Fulltext Preliminary in vitro cytotoxic evaluation of Uncaria gambier (Hunt) Roxb extract as a potential herbal-based pulpotomy medicament
    Tan BC, Mahyuddin A, Sockalingam SNMP, Zakaria ASI
    BMC Complement Med Ther, 2023 Sep 20;23(1):331.
    PMID: 37730579 DOI: 10.1186/s12906-023-04163-w
    BACKGROUND: The downfall of formocresol as a pulpotomy medicament highlights the importance of cytotoxic evaluation and the establishment of a safe concentration of dental material prior to its usage in the oral cavity. Uncaria gambir is an herbal plant that possesses antimicrobial, anti-inflammatory and antioxidant properties, suggesting its potential as an alternative medicament for pulpotomy. However, there are not many studies published on its cytotoxicity, with some using non-standardised techniques and reported variable outcomes. Here, we investigated the concentration and time-dependent toxicity of Uncaria gambir extract towards the M3CT3-E1 cell line and compared it with the gold standard pulpotomy medicament: mineral trioxide aggregate (MTA).

    METHODS: Uncaria gambir extracts at concentrations ranging from 1000 to 7.8 µg/ml and MTA eluates at 4- and 48 h setting times were prepared. 10% dimethyl sulfoxide (DMSO) and culture media were used as positive and negative controls respectively. Cell viability on days 1, 2, 3 and 7 was analysed using Alamar Blue and Live and Dead Cell assay. Any morphological cellular changes were evaluated using transmission electron microscopes (TEM). Data were analysed using a two-way mixed Analysis of Variance (ANOVA).

    RESULTS: The interaction between the concentration and exposure time on the fluorescence intensity of Uncaria gambir extract and MTA 48 h was found to be statistically significant (p < 0.001). No cytotoxic effects on the cells were exerted by both MTA 48 h and Uncaria gambir extract at a concentration below 500 µg/mL. TEM analysis and Live and Dead Cell assay for both materials were comparable to the negative control. No significant differences in fluorescent intensity were observed between Uncaria gambir extract at 500 µg/mL and MTA 48 h (p > 0.05).

    CONCLUSION: Uncaria gambir extracts at a maximum concentration of 500 μg/mL are non-cytotoxic over time and are comparable to the MTA.

  12. Unearth of small open reading frames (sORFs) in drought stress transcriptome of Oryza sativa subsp. indica
    Ong SN, Tan BC, Hanada K, Teo CH
    Gene, 2023 Aug 20;878:147579.
    PMID: 37336274 DOI: 10.1016/j.gene.2023.147579
    Drought is a major abiotic stress that influences rice production. Although the transcriptomic data of rice against drought is widely available, the regulation of small open reading frames (sORFs) in response to drought stress in rice is yet to be investigated. Different levels of drought stress have different regulatory mechanisms in plants. In this study, drought stress was imposed on four-leaf stage rice, divided into two treatments, 40% and 30% soil moisture content (SMC). The RNAs of the samples were extracted, followed by the RNA sequencing analysis on their sORF expression changes under 40%_SMC and 30%_SMC, and lastly, the expression was validated through NanoString. A total of 122 and 143 sORFs were differentially expressed (DE) in 40%_SMC and 30%_SMC, respectively. In 40%_SMC, 69 sORFs out of 696 (9%) DEGs were found to be upregulated. On the other hand, 69 sORFs out of 449 DEGs (11%) were significantly downregulated. The trend seemed to be higher in 30%_SMC, where 112 (12%) sORFs were found to be upregulated from 928 significantly upregulated DEGs. However, only 8% (31 sORFs out of 385 DEGs) sORFs were downregulated in 30%_SMC. Among the identified sORFs, 110 sORFs with high similarity to rice proteome in the PsORF database were detected in 40%_SMC, while 126 were detected in 30%_SMC. The Gene Ontology (GO) enrichment analysis of DE sORFs revealed their involvement in defense-related biological processes, such as defense response, response to biotic stimulus, and cellular homeostasis, whereas enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that DE sORFs were associated with tryptophan and phenylalanine metabolisms. Several DE sORFs were identified, including the top five sORFs (OsisORF_3394, OsisORF_0050, OsisORF_3007, OsisORF_6407, and OsisORF_7805), which have yet to be characterised. Since these sORFs were responsive to drought stress, they might hold significant potential as targets for future climate-resilient rice development.
  13. Fulltext Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture
    Jalil M, Annuar MS, Tan BC, Khalid N
    Evid Based Complement Alternat Med, 2015;2015:757514.
    PMID: 25767555 DOI: 10.1155/2015/757514
    Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.
  14. Fulltext Basic fibroblast growth factor modulates cell cycle of human umbilical cord-derived mesenchymal stem cells
    Ramasamy R, Tong CK, Yip WK, Vellasamy S, Tan BC, Seow HF
    Cell Prolif, 2012 Apr;45(2):132-9.
    PMID: 22309282 DOI: 10.1111/j.1365-2184.2012.00808.x
    BACKGROUND: Mesenchymal stem cells (MSC) have great potential in regenerative medicine, immunotherapy and gene therapy due to their unique properties of self-renewal, high plasticity, immune modulation and ease for genetic modification. However, production of MSC at sufficient clinical scale remains an issue as in vitro generation of MSC inadequately fulfils the demand with respect to patients.

    OBJECTIVES: This study has aimed to establish optimum conditions to generate and characterize MSC from human umbilical cord (UC-MSC).

    MATERIALS AND METHODS: To optimize MSC population growth, basic fibroblast growth factor (bFGF) was utilized in culture media. Effects of bFGF on expansion kinetics, cell cycle, survival of UC-MSC, cytokine secretion, expression of early stem-cell markers and immunomodulation were investigated.

    RESULTS: bFGF supplementation profoundly enhanced UC-MSC proliferation by reducing population doubling time without altering immunophenotype and immunomodulatory function of UC-MSC. However, cell cycle studies revealed that bFGF drove the cells into the cell cycle, as a higher proportion of cells resided in S phase and progressed into M phase. Consistent with this, bFGF was shown to promote expression of cyclin D proteins and their relevant kinases to drive UC-MSC to transverse cell cycle check points, thus, committing the cells to DNA synthesis. Furthermore, supplementation with bFGF changed the cytokine profiles of the cells and reduced their apoptotic level.

    CONCLUSION: Our study showed that bFGF supplementation of UC-MSC culture enhanced the cells' growth kinetics without compromising their nature.

  15. Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method
    Tong CK, Vellasamy S, Tan BC, Abdullah M, Vidyadaran S, Seow HF, et al.
    Cell Biol Int, 2011 Mar;35(3):221-6.
    PMID: 20946106 DOI: 10.1042/CBI20100326
    MSCs (mesenchymal stem cells) promise a great potential for regenerative medicine due to their unique properties of self-renewal, high plasticity, modulation of immune response and the flexibility for genetic modification. Therefore, the increasing demand for cellular therapy necessitates a larger-scale production of MSC; however, the technical and ethical issues had put a halt on it. To date, studies have shown that MSC could be derived from human UC (umbilical cord), which is once considered as clinical waste. We have compared the two conventional methods which are classic enzymatic digestion and explant method with our newly tailored enzymatic-mechanical disassociation method to generate UC-MSC. The generated UC-MSCs from the methods above were characterized based on their immunophenotyping, early embryonic transcription factors expression and mesodermal differentiation ability. Our results show that enzymatic-mechanical disassociation method increase the initial nucleated cell yield greatly (approximately 160-fold) and maximized the successful rate of UC-MSC generation. Enzymatic-mechanical disassociation-derived UC-MSC exhibited fibroblastic morphology and surface markers expression of CD105, CD73, CD29, CD90 and MHC class I. Furthermore, these cells constitutively express early embryonic transcription factors (Nanog, Oct-4, Sox-2 and Rex-1), as confirmed by RT-PCR, indicating their multipotency and high self-renewal capacity. They are also capable of differentiating into osteoblasts and adipocytes when given an appropriate induction. The present study demonstrates a new and efficient approach in generating MSC from UC, hence serving as ideal alternative source of mesenchymal stem cell for clinical and research use.
  16. Fulltext Iodine deficiency disorders among pregnant women in Sarawak, Malaysia
    Lim KK, Chan YY, Mahmud NA, Ismail H, Tan BC, Chua BK, et al.
    Int J Public Health Res, 2018;8(2):980-986.
    MyJurnal
    Introduction Iodine deficiency disorders (IDD) during pregnancy may impair the neurological development of the fetus. The aim of this study is to determine the iodine status among pregnant women (PW) in Sarawak after introduction of mandatory universal salt iodisation (USI) for seven years.
    Methods A total of 508 first trimester PW attending government Maternal and Child Health Care clinics in all 11 divisions in Sarawak between 1st April and 15th June 2015 were recruited. Urine samples were obtained and analysed for urinary iodine concentration (UIC) using the modified Sandell-Kolthoff reaction method. For pregnant women, an adequate iodine intake was defined as a median UIC between 150-249 µg/L according to the WHO/UNICEF/ICCIDD’s criterion. For further analyses, the 11 divisions were then combined into 3 regions, namely Northern (Miri, Bintulu, Limbang), Central (Kapit, Mukah, Sibu, Sarikei, Betong) and Southern (Kota Samarahan, Kuching, Sri Aman).
    Results TThe median UIC of the PW in Sarawak was 105.6 µg/L, indicating iodine deficiency. A total of 330 (65.0%) PW had UIC<150 µg/L. In terms of urinary iodine levels by region, the median UIC in Northern, Central and Southern regions were 136.3 µg/L, 85.5 µg/L and 97.4 µg/L respectively. The differences in median UIC between regions were significant. In addition, the Northern region (p = 0.001), Malay/Melanau ethnicity (p = 0.015) and parous parity (p = 0.014) were significantly associated with higher median UIC. No significant association was found for locality, age nor gravida.
    Conclusions This study indicates inadequate iodine status among PW in Sarawak despite seven years of mandatory USI. In fact, the majority of PW appear not to be protected against IDD and its consequences. In future, a comprehensive study should be carried out to determine the levels of iodine in salt at the retail outlets, villages and households in Sarawak.
    Keywords Iodine deficiency disorders - Pregnant women - Mandatory USI - Sarawak
  17. A New Plant Expression System for Producing Pharmaceutical Proteins
    Abd-Aziz N, Tan BC, Rejab NA, Othman RY, Khalid N
    Mol Biotechnol, 2020 Apr;62(4):240-251.
    PMID: 32108286 DOI: 10.1007/s12033-020-00242-2
    In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
  18. Fulltext Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda
    Liew YJM, Lee YK, Khalid N, Rahman NA, Tan BC
    PeerJ, 2020;8:e9094.
    PMID: 32391211 DOI: 10.7717/peerj.9094
    Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.
  19. Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda
    Liew YJM, Lee YK, Khalid N, Rahman NA, Tan BC
    Mol Biotechnol, 2021 Apr;63(4):316-326.
    PMID: 33565047 DOI: 10.1007/s12033-021-00304-z
    Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.
  20. Fulltext Plant Nitric Oxide Signaling under Drought Stress
    Lau SE, Hamdan MF, Pua TL, Saidi NB, Tan BC
    Plants (Basel), 2021 Feb 13;10(2).
    PMID: 33668545 DOI: 10.3390/plants10020360
    Water deficit caused by drought is a significant threat to crop growth and production. Nitric oxide (NO), a water- and lipid-soluble free radical, plays an important role in cytoprotection. Apart from a few studies supporting the role of NO in drought responses, little is known about this pivotal molecular amendment in the regulation of abiotic stress signaling. In this review, we highlight the knowledge gaps in NO roles under drought stress and the technical challenges underlying NO detection and measurements, and we provide recommendations regarding potential avenues for future investigation. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool with which plant fitness can be improved under adverse growth conditions.
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