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1.  Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli 
PLoS ONE  2015;10(7):e0130446.
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.
doi:10.1371/journal.pone.0130446
PMCID: PMC4492947  PMID: 26147991
2.  A Consanguinity Related Autosomal Translocation which Leads to Premature Ovarian Failure 
The premature ovarian failures with underlying chromosomal abnormalities are normally X-linked, although their associations with the autosomal and the Robertsonian translocations are also possible. Here, we are reporting a case of premature ovarian failure which was associated with a translocation between the long arm of chromosome 7 at q11.23 and the short arm of chromosome 5 at p15.3. The proband was a 26-year-old Malay woman who presented with premature ovarian failure, who was referred for cytogenetic testing due to the suspicion of a chromosomal anomaly. Her physical examination revealed that she had no abdominal or pelvic masses and that she had normal secondary sexual characteristics. Her medical history as well, revealed no points for concern. However, a consanguineous relationship existed, as the patient’s paternal grandmother and maternal grandfather were biological cousins. Our present case indicated that region p15.3 of chromosome 5 and region q11.23 of chromosome 7 possibly carried essential genes for the ovarian function and that they postulated a link between the consanguinity and the chromosomal abnormalities.
doi:10.7860/JCDR/2013/4354.2767
PMCID: PMC3592311  PMID: 23543039
Premature ovarian failure; Chromosomal abnormality; Consanguinity; Translocation; Cytogenetic test
3.  A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study 
PLoS ONE  2016;11(1):e0145986.
There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.
doi:10.1371/journal.pone.0145986
PMCID: PMC4711802  PMID: 26741963
4.  Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines 
Oncology Reports  2015;35(1):13-25.
Natural compounds such as curcumin have the ability to enhance the therapeutic effectiveness of common chemotherapy agents through cancer stem-like cell (CSC) sensitisation. In the present study, we showed that curcumin enhanced the sensitivity of the double-positive (CD166+/EpCAM+) CSC subpopulation in non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) to cisplatin-induced apoptosis and inhibition of metastasis. Our results revealed that initial exposure of NSCLC cell lines to curcumin (10–40 µM) markedly reduced the percentage of viability to an average of ~51 and ~54% compared to treatment with low dose cisplatin (3 µM) with only 94 and 86% in both the A549 and H2170 cells. Moreover, sensitisation of NSCLC cell lines to curcumin through combined treatment enhanced the single effect induced by low dose cisplatin on the apoptosis of the double-positive CSC subpopulation by 18 and 20% in the A549 and H2170 cells, respectively. Furthermore, we found that curcumin enhanced the inhibitory effects of cisplatin on the highly migratory CD166+/EpCAM+ subpopulation, marked by a reduction in cell migration to 9 and 21% in the A549 and H2170 cells, respectively, indicating that curcumin may increase the sensitivity of CSCs to cisplatin-induced migratory inhibition. We also observed that the mRNA expression of cyclin D1 was downregulated, while a substantial increased in p21 expression was noted, followed by Apaf1 and caspase-9 activation in the double-positive (CD166+/EpCAM+) CSC subpopulation of A549 cells, suggested that the combined treatments induced cell cycle arrest, therefore triggering CSC growth inhibition via the intrinsic apoptotic pathway. In conclusion, we provided novel evidence of the previously unknown therapeutic effects of curcumin, either alone or in combination with cisplatin on the inhibition of the CD166+/EpCAM+ subpopulation of NSCLC cell lines. This finding demonstrated the potential therapeutic approach of using curcumin that may enhance the effects of cisplatin by targeting the CSC subpopulation in NSCLC.
doi:10.3892/or.2015.4371
PMCID: PMC4699625  PMID: 26531053
curcumin; cisplatin; cancer stem-like cells; non-small cell lung cancer
5.  Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells 
BMC Cancer  2015;15:84.
Background
Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC).
Methods
We isolated putative lung CSCs from lung adenocarcinoma cells (A549 and H2170) and normal stem cells from normal bronchial epithelial cells (PHBEC) on the basis of positive expression of stem cell surface markers (CD166, CD44, and EpCAM) using fluorescence-activated cell sorting. The isolated cells were then characterised for their self-renewal characteristics, differentiation capabilities, expression of stem cell transcription factor and in vivo tumouregenicity. The transcriptomic profiles of putative lung CSCs then were obtained using microarray analysis. Significantly regulated genes (p < 0.05, fold change (FC) > 2.0) in putative CSCs were identified and further analysed for their biological functions using the Database for Annotation, Visualization, and Integrated Discovery (DAVID).
Results
The putative lung CSCs phenotypes of CD166+/CD44+ and CD166+/EpCAM+ showed multipotent characteristics of stem cells, including the ability to differentiate into adipogenic and osteogenic cells, self-renewal, and expression of stem cell transcription factors such as Sox2 and Oct3/4. Moreover, the cells also shows the in vivo tumouregenicity characteristic when transplanted into nude mice. Microarray and bioinformatics data analyses revealed that the putative lung CSCs have molecular signatures of both normal and cancer stem cells and that the most prominent biological functions are associated with angiogenesis, migration, pro-apoptosis and anti-apoptosis, osteoblast differentiation, mesenchymal cell differentiation, and mesenchyme development. Additionally, self-renewal pathways such as the Wnt and hedgehog signalling pathways, cancer pathways, and extracellular matrix (ECM)-receptor interaction pathways are significantly associated with the putative lung CSCs.
Conclusion
This study revealed that isolated lung CSCs exhibit the characteristics of multipotent stem cells and that their genetic composition might be valuable for future gene and stem cells therapy for lung cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1086-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1086-3
PMCID: PMC4349658  PMID: 25881239
Cancer stem cells; Non-small cell lung cancer; Cell surface marker; Transcriptome
6.  Molecular basis of Rh blood group system in the Malaysian population 
Background:
Rh molecular studies have been previously mainly conducted in Caucasians and African population. There is a limited data on the molecular basis for Rh genotypes among Asians.
Aims:
This study aims to characterize the Rh genes and frequency of the various RH genotypes among blood donors in National Blood Centre (NBC), Kuala Lumpur.
Materials and Methods:
A total of 1014 blood samples were obtained from blood donors from four different ethnic groups (360 Malays, 434 Chinese, 164 Indians and 56 others). Serological and molecular analysis of all 1014 blood samples were performed. An automated deoxyribonucleic acid sequencing analysis was performed.
Results:
Rh phenotypes and RH genotypes showed heterogeneity and significant association with ethnicities. Discrepancies in allele D, C/c and E/e between phenotypes and genotypes results were observed. Discrepancy results in allele D showed significant association with the ethnic groups of the blood donors in NBC. There were multiple novel mutations (23) and published mutations (5) found in this study. Significant associations between discrepancy results and mutations were found in allele D and C/c.
Conclusion:
Performing RH molecular analysis in Malaysian population provided the basic database for the distribution of Rh genotypes of donors from major ethnic groups in Malaysia.
doi:10.4103/0973-6247.150951
PMCID: PMC4339932  PMID: 25722573
Allele; Rh genotypes; Rh molecular; Rh phenotypes
7.  The effects of virgin coconut oil (VCO) as supplementation on quality of life (QOL) among breast cancer patients 
Background
Breast cancer is the most common cancer amongst Malaysian women. Both the disease and its treatment can disrupt the lives of the woman and adversely affect all aspects of life and thus can alter a woman’s quality of life. The aim of this study was to examine the effect of virgin coconut oil (VCO) on the quality of life (QOL) of patients diagnosed with breast cancer.
Methods
This was a prospective study of breast cancer patients admitted into the Oncology Unit of Hospital Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia. The sample consisted of 60 patients with stage III and IV breast cancer allocated to either an intervention group (n = 30) or a control group (n = 30) using a simple random table. QOL was evaluated from the first cycle of chemotherapy to the sixth cycle, and data were collected using a validated Bahasa Malaysia version of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Breast Cancer Module (EORTC QLQ-C30) and its breast-specific module (QLQ-BR 23).
Results
The mean age of breast cancer patients was 50.2 (SD = 13.5) years. There were significant mean score differences for functioning and global QOL between groups (α < 0.01). The intervention group also had better scores for symptoms including fatigue, dyspnea, sleep difficulties, and loss of appetite compared to the control group. Although there are deteriorations for sexual enjoyment, the intervention group exhibited improvement in breast functioning and symptom scores for body image, sexual function, future perspective, breast symptoms, and systemic therapy side effects.
Conclusion
VCO consumption during chemotherapy helped improve the functional status and global QOL of breast cancer patients. In addition, it reduced the symptoms related to side effects of chemotherapy.
doi:10.1186/1476-511X-13-139
PMCID: PMC4176590  PMID: 25163649
Virgin coconut oil (VCO); Quality of life (QOL); Breast cancer patient; Chemotherapy
8.  Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study 
PLoS ONE  2014;9(1):e83818.
Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 107 K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.
doi:10.1371/journal.pone.0083818
PMCID: PMC3883656  PMID: 24409284
9.  Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli 
BMC Biotechnology  2013;13:107.
Background
HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA.
Result
In this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L-1 (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L-1 HIV-1 CA. A two-step purification strategy (chitin beads + IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay.
Conclusions
By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies.
doi:10.1186/1472-6750-13-107
PMCID: PMC4235032  PMID: 24304876
11.  Chromosomal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case report 
Introduction
Chromosomal aberrations of chromosome 16 are uncommon and submicroscopic deletions have rarely been reported. At present, a cytogenetic or molecular abnormality can only be detected in 55% of Rubinstein-Taybi syndrome patients, leaving the diagnosis in 45% of patients to rest on clinical features only. Interestingly, this microdeletion of 16 p13.3 was found in a young child with an unexplained syndromic condition due to an indistinct etiological diagnosis. To the best of our knowledge, no evidence of a microdeletion of 16 p13.3 with contiguous gene deletion, comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes, has been described in typical Rubinstein-Taybi syndrome.
Case presentation
We present the case of a three-year-old Malaysian Chinese girl with a de novo microdeletion on the short arm of chromosome 16, identified by oligonucleotide array-based comparative genomic hybridization. Our patient showed mild to moderate global developmental delay, facial dysmorphism, bilateral broad thumbs and great toes, a moderate size atrial septal defect, hypotonia and feeding difficulties. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XX karyotype. Further investigation by high resolution array-based comparative genomic hybridization revealed a 120 kb microdeletion on chromosomal band 16 p13.3.
Conclusion
A mutation or abnormality in the cyclic adenosine monophosphate-response element-binding protein has previously been determined as a cause of Rubinstein-Taybi syndrome. However, microdeletion of 16 p13.3 comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes is a rare scenario in the pathogenesis of Rubinstein-Taybi syndrome. Additionally, due to insufficient coverage of the human genome by conventional techniques, clinically significant genomic imbalances may be undetected in unexplained syndromic conditions of young children. This case report demonstrates the ability of array-based comparative genomic hybridization to offer a genome-wide analysis at high resolution and provide information directly linked to the physical and genetic maps of the human genome. This will contribute to more accurate genetic counseling and provide further insight into the syndrome.
doi:10.1186/1752-1947-6-30
PMCID: PMC3295639  PMID: 22269667
12.  Quality indicators for discarding blood in the National Blood Center, Kuala Lumpur 
Background and Objective:
The implementation of quality system and continuous evaluation of all activities of the Blood Transfusion Services (BTS) can help to achieve the maximum quantity and quality of safe blood. Optimizing blood collection and processing would reduce the rate of discard and improve the efficiency of the BTS. The objective of this study is to determine the rate of discard of blood and blood component and identify its reasons at the National Blood Centre (NBC), Kuala Lumpur, during the year of 2007 in order to introduce appropriate intervention.
Study Designs and Methods:
Data on the number of discarded whole blood units and its components, reasons for discard, and the number of blood components processed as well as the number of collected blood units were obtained from the Blood Bank Information System - NBC database. These were analyzed.
Results:
The total number of blood units collected in 2007 was 171169 from which 390636 units of components were prepared. The total number of discarded whole blood units and its components was 8968 (2.3%). Platelet concentrate recorded the highest of discard at 6% (3909) followed by whole blood at 3.7% (647), fresh frozen plasma (FFP) at 2.5% (2839), and cryoprecipitate at 2% (620). The rate of discarded packed red blood cells RBCs, plasma aphaeresis, and PLT aphaeresis was less than 1% at 0.6% (902), 0.6% (37), and 0.29% (14), respectively. RBC contamination of PLT and plasma were the major cause of discard at 40% (3558). Other causes include leakage (26% - 2306), lipemia (25% - 2208), and underweight (4% - 353).
Conclusion:
Good donor selection, training and evaluation of the staff, as well as implementation of automation will help to improve processes and output of BTS. This would reduce discard of blood components and wastage caused by non conformance.
doi:10.4103/0973-6247.95045
PMCID: PMC3353623  PMID: 22623837
Discard blood; National Blood Centre Kuala Lumpur; quality indicators
13.  Association of ABO blood groups with diabetes mellitus 
The Libyan Journal of Medicine  2010;5:10.3402/ljm.v5i0.4847.
Objective
So far no studies have been performed in Malaysia to look at association of diabetes mellitus (DM) with blood groups. We studied the association of ABO blood groups with DM type 2.
Patients and methodology
It was a case control study conducted at Kepala Batas Hospital Batas, Penang, Malaysia in the year 2009, involving 70 patients with DM type 2 and 140 healthy controls. Ethical approval was obtained from Universiti Sains Malaysia. Blood samples were collected from the patients after consent. Samples were tested for ABO blood groups using ID-Card gel method.
Results
Chi-square test results showed that there was an association between the ABO blood groups and DM type 2. It was found that A and O blood groups were negatively associated with DM type 2 (P<0.05) with higher percentage of A and O groups individuals were non-diabetic. No significant association was noted between DM type 2 and blood groups B (P=0.423) and AB (P=0.095). It was also noted that B blood group was distributed with highest percentage among patients with DM type 2 (53.71%) compared to controls (22.52%), but no statistical significance achieved.
Conclusion
The results obtained suggest that there was a negative association between ABO blood groups A and O with DM type 2, with A and O group having less chances of diabetes. Large studies in other ethnic groups are needed to confirm these results.
doi:10.3402/ljm.v5i0.4847
PMCID: PMC3071167  PMID: 21483592
blood group A; blood group O; diabetes mellitus
14.  Hereditary Spherocytosis in a Malay Patient with Chronic Haemolysis 
This case report describes a 35-year-old lady who presented with generalized weakness and lethargy of two weeks duration and jaundice of more than 20 years duration. Her initial workup was suggestive of haemolysis and blood film showed a leucoerythoblastic picture with moderate microspherocytes. She was finally diagnosed as a case of hereditary spherocytosis after ruling out other possible causes of chronic haemolysis and supported by an abnormal osmotic fragility test, although family members refused for screening. Hereditory spherocytosis is uncommon in Malay population and presentation with jaundice of 20 years duration with leucoerythroblastic picture on blood film were interesting features in this case. Patient is being followed closely for need of splenectomy in near future as per severity of haemolysis and currently being managed with folic acid supplement.
PMCID: PMC3442627  PMID: 22993492
Hereditary spherocytosis; Jaundice; Chronic haemolysis; Leucoerythroblastic picture; Microspherocytes

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