One of the advantages of human adipose-derived stem cells (ASCs) in regenerative medicine is that they can be harvested in abundance. However, the stemness biomarkers, which marked the safety and efficacy of ASCs in accordance with the good manufacturing practice guidelines, is not yet well established. This study was designed to investigate the effect of long-term culture on the stemness properties of ASCs using quantitative real-time polymerase chain reaction and flow cytometry. Results showed the growth rate of ASCs was at its peak when they reached P10 (population doubling; PD = 26) but started to decrease when they were expanded to P15 (PD = 36) and P20 (PD = 46). The ASCs can be culture expanded with minimal alteration in the stemness genes and cluster of differentiation (CD) markers expression up to P10. Expression level of Sox2, Nestin, and Nanog3 was significantly decreased at later passage. CD31, CD45, CD117, and human leukocyte antigen DR, DQ, and DP were lowly expressed at P5 and P10 but their expressions increased significantly at P15 or P20. The differentiation ability of ASCs (adipogenesis, osteogenesis, and neurogenesis) also decreased in long-term culture. Our findings suggested that P10 (PD = 26) should be the "cutoff point" for clinical usage because ASCs at passage 15 onward showed significant changes in the stemness genes, CD markers expression, and differentiation capability.
The catechol-O-methyltransferase (COMT) gene is a candidate gene for schizophrenia as its encoded enzyme is involved in the metabolic inactivation of dopamine and noradrenaline. Several molecular genetic studies thus far have demonstrated that the COMT functional polymorphism of Val158Met is susceptible with schizophrenia. Hence, the present study aims to determine this genetic association of this SNP in the three major ethnic groups of the Malaysian population. A total of 317 patients (79 Malays, 154 Chinese and 84 Indians) meeting DSM-IV criteria for schizophrenia and 417 healthy subjects (160 Malays, 164 Chinese and 93 Indians) were recruited. A PCR-RFLP method was used to determine the genotypes and alleles present. We found a significant association of genotypes within the total pooled samples, as well as in the female subgroup, with a higher frequency of heterozygotes in schizophrenia subjects. However, there were no significant differences in allele and genotype frequency between the schizophrenic patients and normal controls in all three ethnic groups. Our current findings suggest that the Val158Met polymorphism has a weak association with schizophrenia in the Malaysian population and does not play a major role in conferring susceptibility to the schizophrenia in any of the three major local ethnicities.
In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4.
We report for the first time on the prevalence, antibiotic resistance and RAPD types of Campylobacter species in ducks and duck related environmental samples in Malaysia. Samples were examined by enrichment in Bolton Broth followed by plating onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA) and/or plating directly onto mCCDA. A total of 643 samples were screened, and the prevalence of Campylobacter spp. in samples from different sources ranged from 0% to 85%. The method of isolation had a significant (P<0.05) effect on the isolation rate. One hundred and sixteen Campylobacter isolates, comprising of 94 Campylobacter jejuni, 19 Campylobacter coli and three Campylobacter lari, were examined for their sensitivity to 13 antibiotics. Majority of the C. jejuni isolates were resistant to cephalothin (99%), tetracycline (96%), suphamethoxazole/trimethoprim (96%), and very few were resistant to gentamicin (5%), chloramphenicol (7%) and erythromycin (1%). All C. coli isolates were resistant to cephalothin, nalidixic acid, norfloxacin and tetracycline but susceptible to chloramphenicol, erythromycin and gentamicin. The three C. lari isolates were resistant to all the antibiotics tested except chloramphenicol and gentamicin (1/3 and 2/3 susceptible, respectively). Genetic diversity of Campylobacter isolates were determined using random amplification of polymorphic DNA (RAPD). C. jejuni and C. coli isolates belong to fifty-eight and twelve RAPD types, respectively.
Matched MeSH terms: Campylobacter/genetics*; Drug Resistance, Microbial/genetics*; Ducks/genetics*; Campylobacter jejuni/genetics
The present study aimed to evaluate the association between the E-Selectin Ser128Arg polymorphism and breast cancer risk and clinicopathological characteristics of the patients.
Matched MeSH terms: Breast Neoplasms/genetics*; E-Selectin/genetics*; Genetic Predisposition to Disease/genetics*; Polymorphism, Single Nucleotide/genetics*
Recent studies associated the HLA-B 1502 allele with carbamazepine (CBZ)-induced Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) in patients from China, Thailand and Malaysia. No association has been found in patients from Europe or Japan. Linkage summary reports from East and South-east Asia predict a highly significant odds ratio (OR) of 84.75 (95% confidence interval [CI]=42.53-168.91; p=8.96×10[-15]) with sensitivity and negative predictive values of 92% and 98%, respectively. The higher prevalence of HLA-B 1502 allele among certain Asian populations (10-15%) compared to Caucasians (1-2%) may explain a 10-fold to 25-fold higher incidence of CBZ-SJS/TEN in patients from Asia. Screening for HLA-B 1502 before using CBZ can prevent SJS/TEN in certain populations, but screening may be less beneficial in populations with low HLA-B 1502 allele frequency and in patients exposed to CBZ for more than 2 months. A retrospective study demonstrated that the costs of HLA-B 1502 screening were less than those of SJS treatment. This article reviews possible benefits and concerns of HLA-B 1502 screening in clinical practice.
Hereditary pancreatitis (HP) is a very rare form of early-onset chronic pancreatitis, which usually begins in childhood with a variable spectrum of severity of disease. HP is commonly caused by variants/mutations in the PRSS1 gene as reported in many studies. Therefore, in this study, we aimed to investigate the possible association of PRSS1 gene variants/mutations in a Malaysian Chinese family with HP.
Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been documented as important events in oral squamous cell carcinoma (OSCC). Five microsatellite markers D3S192, D3S966, D3S647, D3S1228 and D3S659 were selected on chromosome 3p because of high frequency of alterations reported in head and neck squamous cell carcinoma and the involvement of von Hippel Lindau (VHL) at 3p25-26 and the fragile histidine triad (FHIT) at 3p14.2 genes proven in many tumour types. A total of 50 archival tissue samples of OSCC and corresponding normal samples were analyzed for LOH and MSI status. The overall LOH for the markers selected on 3p was 56 out of 189 informative cases (29.6%). The most frequent LOH was identified for the marker D3S966 which was 18/42 (42.8%) of informative cases suggesting the presence of putative tumour suppressor genes (TSGs) in this loci. In this study, high frequency of microsatellite instability was found in D3S966 which was 28.6% of informative cases; this reveals the possibility of mutations of MMR genes in this region. Frequent microsatellite alterations (MA) were observed in 3 markers D3S966 (71.4%), D3S1228 (56.7%) and D3S192 (41.0%). There was no significant association between LOH with gender, tumour stages and differentiation grades. However, there was a significant association between tumour stage and differentiation grades with MSI status in OSCC in Malaysian population with p values of 0.002 and 0.035, respectively. There was also a significant association between MA and differentiation grades (p=0.041).
Matched MeSH terms: Carcinoma, Squamous Cell/genetics*; Mouth Neoplasms/genetics*; Microsatellite Repeats/genetics; Loss of Heterozygosity/genetics*
The output of state-of-the-art reverse-engineering methods for biological networks is often based on the fitting of a mathematical model to the data. Typically, different datasets do not give single consistent network predictions but rather an ensemble of inconsistent networks inferred under the same reverse-engineering method that are only consistent with the specific experimentally measured data. Here, we focus on an alternative approach for combining the information contained within such an ensemble of inconsistent gene networks called meta-analysis, to make more accurate predictions and to estimate the reliability of these predictions. We review two existing meta-analysis approaches; the Fisher transformation combined coefficient test (FTCCT) and Fisher's inverse combined probability test (FICPT); and compare their performance with five well-known methods, ARACNe, Context Likelihood or Relatedness network (CLR), Maximum Relevance Minimum Redundancy (MRNET), Relevance Network (RN) and Bayesian Network (BN). We conducted in-depth numerical ensemble simulations and demonstrated for biological expression data that the meta-analysis approaches consistently outperformed the best gene regulatory network inference (GRNI) methods in the literature. Furthermore, the meta-analysis approaches have a low computational complexity. We conclude that the meta-analysis approaches are a powerful tool for integrating different datasets to give more accurate and reliable predictions for biological networks.
The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.
Progress in the functional studies of human olfactory receptors has been largely hampered by the lack of a reliable experimental model system. Although transgenic approaches in mice could characterize the function of individual olfactory receptors, the presence of over 300 functional genes in the human genome becomes a daunting task. Thus, the characterization of individuals with a genetic susceptibility to altered olfaction coupled with the absence of particular olfactory receptor genes will allow phenotype/genotype correlations and vindicate the function of specific olfactory receptors with their cognate ligands. We characterized a 118 kb β-globin deletion and found that its 3' end breakpoint extends to the neighboring olfactory receptor region downstream of the β-globin gene cluster. This deletion encompasses six contiguous olfactory receptor genes (OR51V1, OR52Z1, OR51A1P, OR52A1, OR52A5, and OR52A4) all of which are expressed in the brain. Topology analysis of the encoded proteins from these olfactory receptor genes revealed that OR52Z1, OR52A1, OR52A5, and OR52A4 are predicted to be functional receptors as they display integral characteristics of G-proteins coupled receptors. Individuals homozygous for the 118 kb β-globin deletion are afflicted with β-thalassemia due to a homozygous deletion of the β-globin gene and have no alleles for the above mentioned olfactory receptors genes. This is the first example of a homozygous deletion of olfactory receptor genes in human. Although altered olfaction remains to be ascertained in these individuals, such a study can be carried out in β-thalassemia patients from Malaysia, Indonesia and the Philippines where this mutation is common. Furthermore, OR52A1 contains a γ-globin enhancer, which was previously shown to confer continuous expression of the fetal γ-globin genes. Thus, the hypothesis that β-thalassemia individuals, who are homozygous for the 118 kb deletion, may also have an exacerbation of their anemia due to the deletion of two copies of the γ-globin enhancer element is worthy of consideration.
Citrin deficiency is an autosomal recessive disorder caused by mutation in the SLC25AJ3 gene. It has two major phenotypes: adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestatic caused by citrin deficiency (NICCD). NICCD is characterized by neonatal/infantile-onset cholestatic hepatitis syndrome associated with multiple amino acidemia and hypergalactosemia. NICCD is self-limiting in most patients. However, some patients may develop CTLN2 years later, which manifests as fatal hyperammonemia coma. We report three unrelated Malay children with genetically confirmed NICCD characterised by an insertion mutation IVS16ins3kb in SLC25A13 gene. All 3 patients presented with prolonged neonatal jaundice which resolved without specific treatment between 5 to 10 months. Of note was the manifestation of a peculiar dislike of sweet foods and drinks. Elevated plasma citrulline was an important biochemical marker. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Malaysian infants regardless of ethnic origin.
Central corneal thickness (CCT) is a risk factor of glaucoma, the most common cause of irreversible blindness worldwide. The identification of genetic determinants affecting CCT in the normal population will provide insights into the mechanisms underlying the association between CCT and glaucoma, as well as the pathogenesis of glaucoma itself. We conducted two genome-wide association studies for CCT in 5080 individuals drawn from two ethnic populations in Singapore (2538 Indian and 2542 Malays) and identified novel genetic loci significantly associated with CCT (COL8A2 rs96067, p(meta) = 5.40 × 10⁻¹³, interval of RXRA-COL5A1 rs1536478, p(meta) = 3.05 × 10⁻⁹). We confirmed the involvement of a previously reported gene for CCT and brittle cornea syndrome (ZNF469) [rs9938149 (p(meta) = 1.63 × 10⁻¹⁶) and rs12447690 (p(meta) = 1.92 × 10⁻¹⁴)]. Evidence of association exceeding the formal threshold for genome-wide significance was observed at rs7044529, an SNP located within COL5A1 when data from this study (n = 5080, P = 0.0012) were considered together with all published data (reflecting an additional 7349 individuals, p(Fisher) = 1.5 × 10⁻⁹). These findings implicate the involvement of collagen genes influencing CCT and thus, possibly the pathogenesis of glaucoma.
Matched MeSH terms: Collagen/genetics*; Glaucoma/genetics*; Collagen Type VIII/genetics; Asian Continental Ancestry Group/genetics*
CYP1A2 play an important role in the metabolism of many carcinogens and clinically important drugs. CYP1A2 activity has been found to be influenced by the presence of polymorphic variants which were reported to display wide interethnic variation. This study investigates the frequency distribution and linkage disequilibrium patterns of CYP1A2 genetic polymorphisms, and characterize their haplotype structures in three healthy Asian populations in Singapore (Chinese, Malay, and Indian). The entire CYP1A2 gene was screened in 126 healthy subjects from all three ethnic groups (N=42 each). A total of 25 polymorphisms was identified, of which nine were novel. The polymorphisms, -2467delT and -163C>A were detected at high frequencies in all Asian ethnic groups. Significant interethnic differences were observed in the genotypic frequency distribution of IVS2-99G>A (P<0.01) and 1548C>T (P=0.05) across the three ethnic groups while -163C>A (P=0.02) was found to differ between Chinese and Malays. Haplotype analyses revealed four to six major haplotypes in each ethnic population which accounted for more than 60% of the cumulative haplotype frequencies. Future studies should be done to investigate the functional roles of these haplotypes.
Matched MeSH terms: Ethnic Groups/genetics; Cytochrome P-450 CYP1A2/genetics*; European Continental Ancestry Group/genetics*; Asian Continental Ancestry Group/genetics*
The Lancefield group C alpha-hemolytic Streptococcus dysgalactiae ssp. dysgalactiae (GCSD) causes systemic granulomatous inflammatory disease and high mortality rates in infected fish. Superantigen and streptolysin S genes are the most important virulence factors contributing to an invasive streptococcal infection. PCR amplification revealed that all strains isolated from moribund fish harbored the streptolysin S structural gene (sagA). GCSD fish isolates were PCR negative for emm, speA, speB, speC, speM, smeZ, and ssa. However, the size of the streptococcal pyrogenic exotoxin G (spegg) locus, a superantigen, in positive S. dysgalactiae fish and pig strains was variable. The ORF of the spegg locus of 26 GCSD fish strains and one GCSD pig strain was inserted with IS981SC. Interestingly, the ORF of the spegg locus of two fish strains of GCSD collected in Malaysia was inserted with an IS981SC-IS1161 hybrid IS element. The hybrid IS element was found in all of the GCSD fish isolates and one GCSD pig through PCR screening. Although no insertion sequence (IS) was detected in the spegg locus of S. dysgalactiae ssp. equisimilis (GCSE) strains, a five-nucleotide deletion mutation was detected in the ORF of the spegg locus of one GCSE strain at the supposed site of IS981SC insertion, resulting in a frameshift mutation.
A new electrochemical biosensor is described for voltammetric detection of gene sequence related to Trichoderma harzianum. The sensor involves immobilization of a 20 base single-stranded probe (ssDNA), which is complementary to a specific gene sequence related to T. harzianum on a gold electrode through specific adsorption. The DNA probe was used to determine the amount of target gene in solution using methylene blue (MB) as the electrochemical indicator. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the electrode. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE and the probe-modified AuE which localized to the affinity of MB. Control experiments with the non-complementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. DNA biosensor also able to detect microorganism at the species levels without nucleic acid amplification. The redox current was linearly related to the concentration of target oligonucleotide DNA, ranged from 1-20 ppm. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time.
Matched MeSH terms: DNA, Fungal/genetics; Genes, Fungal/genetics*; Trichoderma/genetics*; DNA Probes/genetics
17 Y-STRs (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635 or Y-GATA C4, DYS392, Y-GATA H4, DYS437, DYS438 and DYS448) have been analyzed in 320 male individuals from Sarawak, an eastern state of Malaysia on the Borneo island using the AmpFlSTR Y-filer (Applied Biosystems, Foster City, CA). These individuals were from three indigenous ethnic groups in Sarawak comprising of 103 Ibans, 113 Bidayuhs and 104 Melanaus. The observed 17-loci haplotypes and the individual allele frequencies for each locus were estimated, whilst the locus diversity, haplotype diversity and discrimination capacity were calculated in the three groups. Analysis of molecular variance (AMOVA) indicated that 87.6% of the haplotypic variation was found within population and 12.4% between populations (fixation index F(ST)=0.124, p=0.000). This study has revealed that the indigenous populations in Sarawak are distinctly different to each other, and to the three major ethnic groups in Malaysia (Malays, Chinese and Indians), with the Melanaus having a strikingly high degree of shared haplotypes within. There are rare unusual variants and microvariants that were not present in Malaysian Malay, Chinese or Indian groups. In addition, occurrences of DYS385 duplications which were only noticeably present in Chinese group previously was also observed in the Iban group whilst null alleles were detected at several Y-loci (namely DYS19, DYS392, DYS389II and DYS448) in the Iban and Melanau groups.
Breast cancer is a leading cause of worldwide mortality in females. In Malaysia, breast cancer is the most commonly diagnosed cancer in women. Of these, the Chinese had the most number of breast cancer cases, followed by the Indian and the Malay. The most common type of breast cancer is infiltrating ductal carcinoma (IDC). A proteomic approach was used to identify protein profile changes in cancerous tissues compared with the normal tissues, the tissues were collected from patients of three different ethnicities, i.e. Chinese, Malay and Indian. Ten differentially expressed hydrophobic proteins were identified. We had evaluated the potential of these proteins as biomarker for infiltrating ducal carcinoma (IDC) and the ethnic-specific expression of these proteins was also determined. The data showed that peroxiredoxin-2, heat shock protein 60, protein disulfide isomerase and calreticulin may serve as ethnic-related potential markers for either one or combination of Chinese, Malay and Indian cohorts as their expression levels were significantly high in the cancerous tissues compared to the normal tissues in the ethnic group tested.
Publication year=2010-2011
PURPOSE: to investigate genetic polymorphisms in GSTM1, GSTT1 and CYP1A1 and the association with the risk of oral cancer in the Jakarta population.
METHOD: A total of 81 cases and 162 controls matched for age and sex were selected from 5 hospitals in Jakarta. Sociodemographic data using questionnaires were obtained and peripheral blood samples were collected with informed consent for PCR-RFLP assay. Conditional logistic regression analysis was performed to obtain the association between the risk of oral cancer and GSTM1, GSTT1 and CYP1A1 polymorphisms.
RESULTS: GSTM1 and GSTT1 null were slightly overrepresented among cases (60.5% and 45.7% respectively) compared to controls (55.6% and 41.4% respectively), but no statistically significant differences were observed. In contrast, the distribution of CYP1A1 polymorphism was higher among controls compared to cases (52.5 % versus 42.4 %). The odds ratio of null GSTM1 and GSTT1 genotypes was slightly higher compared to wild type genotypes (OR 1.19, 95% CI 0.70-2.02 and OR 1.19, 95% CI 0.72-2.05 respectively). Furthermore, the presence of CYP1A1 polymorphism did not increase the risk of oral cancer (OR 0.70, 95% 0.39-1.25).
CONCLUSION: Genetic polymorphisms of GSTM1, GSTT1 and CYP1A1 may not be risk factors for oral cancer in the Jakarta population.
A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).