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  1. Eamsobhana P, Yong HS, Song SL, Prasartvit A, Boonyong S, Tungtrongchitr A
    J Helminthol, 2018 Mar;92(2):254-259.
    PMID: 28330511 DOI: 10.1017/S0022149X17000244
    The rat lungworm Angiostrongylus malaysiensis is a metastrongyloid nematode parasite. It has been reported in Malaysia, Thailand, Laos, Myanmar, Indonesia and Japan. In this study, A. malaysiensis adult worms recovered from the lungs of wild rats in different geographical regions/provinces in Thailand were used to determine their haplotype by means of the mitochondrial partial cytochrome c oxidase subunit I (COI) gene sequence. The results revealed high COI haplotype diversity of A. malaysiensis from Thailand. The geographical isolates of A. malaysiensis from Thailand and other countries formed a monophyletic clade distinct from the closely related A. cantonensis. In the present study, five new haplotypes were identified in addition to the four haplotypes reported in the literature. Phylogenetic analysis revealed that four of these five new haplotypes - one from Mae Hong Song (northern region), two from Tak (western region) and one from Phang Nga (southern region) - formed a distinct clade with those from Phatthalung (southern region) and Malaysia. The haplotype from Malaysia was identical to that of Phatthalung (haplotype AM1). In general, the COI sequences did not differentiate unambiguously the various geographical isolates of A. malaysiensis. This study has confirmed the presence of high COI genetic diversity in various geographical isolates of A. malaysiensis. The COI gene sequence will be suitable for studying genetic diversity, population structure and phylogeography.
    Matched MeSH terms: Angiostrongylus/genetics*; Electron Transport Complex IV/genetics*; DNA, Helminth/genetics
  2. Liu Y, Yu Q, Shu YL, Zhao JH, Fang JY, Wu HL
    J Helminthol, 2019 Jul 12;94:e59.
    PMID: 31296272 DOI: 10.1017/S0022149X19000518
    We identified and characterized a new cosmocercid nematode species, Cosmocercoides wuyiensis n. sp., through microscopic examination and sequencing of the partial small ribosomal RNA gene (18S rDNA), internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (COI) genes. The new species was isolated from the intestine of the Asiatic frog Amolops wuyiensis Liu and Hu, 1975 captured from four localities of the Anhui province in south-east China. Among the 25 recorded species of the Cosmocercoides genus, the morphology of C. wuyiensis n. sp. is closest to that of C. kiliwai and C. malayensis, which were isolated from various Mexican frog and Malaysian lizard species, respectively. However, C. wuyiensis n. sp. displayed several distinguishing features, such as small size of the male body, two spicules of unequal lengths in the male, small gubernaculum, pre-, ad- and post-cloacal caudal rosette papillae in the ratio of 18-24:2:6 and simple papillae in the ratio of 14:multiple:4, circle and number of punctation in each rosette at 1:11-16, sharply conical tail-end and the presence of lateral alae and somatic papillae in both sexes. BLAST and the phylogenetic analyses of the 18S rDNA and ITS sequences indicated that C. wuyiensis n. sp. belonged to the genus Cosmocercoides, while that of the COI gene sequence of C. wuyiensis n. sp. showed 16.36% nucleotide divergence with C. pulcher and 47.99% nucleotide divergence with C. qingtianensis. The morphological and molecular characterization of C. wuyiensis n. sp. provides new taxonomic data for this genus.
    Matched MeSH terms: Electron Transport Complex IV/genetics; DNA, Ribosomal/genetics; DNA, Intergenic/genetics
  3. Moon RW, Sharaf H, Hastings CH, Ho YS, Nair MB, Rchiad Z, et al.
    Proc Natl Acad Sci U S A, 2016 Jun 28;113(26):7231-6.
    PMID: 27303038 DOI: 10.1073/pnas.1522469113
    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.
    Matched MeSH terms: Carrier Proteins/genetics*; Protozoan Proteins/genetics*; Plasmodium knowlesi/genetics*
  4. Ariffin N, Newman DW, Nelson MG, O'cualain R, Hubbard SJ
    J Proteome Res, 2024 May 03;23(5):1583-1592.
    PMID: 38651221 DOI: 10.1021/acs.jproteome.3c00675
    MD2 pineapple (Ananas comosus) is the second most important tropical crop that preserves crassulacean acid metabolism (CAM), which has high water-use efficiency and is fast becoming the most consumed fresh fruit worldwide. Despite the significance of environmental efficiency and popularity, until very recently, its genome sequence has not been determined and a high-quality annotated proteome has not been available. Here, we have undertaken a pilot proteogenomic study, analyzing the proteome of MD2 pineapple leaves using liquid chromatography-mass spectrometry (LC-MS/MS), which validates 1781 predicted proteins in the annotated F153 (V3) genome. In addition, a further 603 peptide identifications are found that map exclusively to an independent MD2 transcriptome-derived database but are not found in the standard F153 (V3) annotated proteome. Peptide identifications derived from these MD2 transcripts are also cross-referenced to a more recent and complete MD2 genome annotation, resulting in 402 nonoverlapping peptides, which in turn support 30 high-quality gene candidates novel to both pineapple genomes. Many of the validated F153 (V3) genes are also supported by an independent proteomics data set collected for an ornamental pineapple variety. The contigs and peptides have been mapped to the current F153 genome build and are available as bed files to display a custom gene track on the Ensembl Plants region viewer. These analyses add to the knowledge of experimentally validated pineapple genes and demonstrate the utility of transcript-derived proteomics to discover both novel genes and genetic structure in a plant genome, adding value to its annotation.
    Matched MeSH terms: Peptides/genetics; Plant Leaves/genetics; Proteome/genetics
  5. Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y
    J Clin Microbiol, 2010 Oct;48(10):3698-702.
    PMID: 20660217 DOI: 10.1128/JCM.00462-10
    Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.
    Matched MeSH terms: Antigens, Protozoan/genetics; Toxoplasma/genetics; Protozoan Proteins/genetics
  6. Tay ST, Cheah PC, Puthucheary SD
    J Clin Microbiol, 2010 Apr;48(4):1465-7.
    PMID: 20089759 DOI: 10.1128/JCM.01131-09
    Four flagellin allelic types (I to IV) of Burkholderia pseudomallei were identified based on their sequence variation and restriction fragment length polymorphism (RFLP) analysis of the amplified flagellin gene. Flagellin allelic type I was the most predominantly (75.0%) found among the 100 clinical isolates of B. pseudomallei investigated in this study.
    Matched MeSH terms: Bacterial Proteins/genetics*; Flagellin/genetics*; Burkholderia pseudomallei/genetics*
  7. Chook JB, Teo WL, Ngeow YF, Tee KK, Ng KP, Mohamed R
    J Clin Microbiol, 2015 Jun;53(6):1831-5.
    PMID: 25788548 DOI: 10.1128/JCM.03449-14
    Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.
    Matched MeSH terms: Hepatitis B virus/genetics*; Genome, Viral/genetics; DNA Primers/genetics*
  8. Ferdig MT, Taft AS, Severson DW, Christensen BM
    Genome Res, 1998 Jan;8(1):41-7.
    PMID: 9445486
    One of the causative agents of lympahtic filariasis is the nematode parasite Brugia malayi that requires a competent mosquito vector for its development and transmission. Armigeres subalbatus mosquitoes rapidly destroy invading B. malayi microfilariae via a defense response known as melanotic encapsulation. We have constructed a genetic linkage map for this mosquito species using RFLP markers from Aedes aegypti. This heterologous approach was possible because of the conserved nature of the coding sequences used as markers and provided an experimental framework to evaluate the hypothesis that linkage and gene order are conserved between these mosquito species. Of the 56 Ae. aegypti markers tested, 77% hybridize to genomic DNA digests of Ar. subalbatus under stringent conditions, with 53% of these demonstrating strain-specific polymorphisms. Twenty-six Ae. aegypti markers have been mapped using an F2- segregating Ar. subalbatus population derived from a cross of strains originating in Japan and Malaysia. Linear order of these marker loci is highly conserved between the two species. Only 1 of these markers, LF92, was not linked in the manner predicted by the Ae. aegypti map. In addition, the autosomal sex-determination locus that occurs in linkage group 1 in Ae. aegypti resides in group 3 in Ar. subalbatus. The Ar. subalbatus map provides a basic genetic context that can be utilized in further genetic studies to clarify the genetic basis of parasite resistance in this mosquito and is a necessary precursor to the identification of genome regions that carry genes that determine the encapsulation phenotype. [The composite map and sequence database information for Ae. aegypti markers can be retrieved directly from the Ae. aegypti Genome Database through the World Wide Web: http://klab.agsci.colostate.edu.]
    Matched MeSH terms: Aedes/genetics*; Culicidae/genetics*; DNA, Complementary/genetics
  9. Ng KP, Yew SM, Chan CL, Soo-Hoo TS, Na SL, Hassan H, et al.
    Eukaryot Cell, 2012 May;11(5):705-6.
    PMID: 22544899 DOI: 10.1128/EC.00081-12
    Cladosporium sphaerospermum is one of the most widely distributed allergens causing serious problems in patients with respiratory tract disease. We report the 26,644,473-bp draft genome sequence and gene annotation of C. sphaerospermum UM843. Analysis of the genome sequence led to the finding of genes associated with C. sphaerospermum's melanin biosynthesis, allergens, and antifungal drug resistance.
    Matched MeSH terms: Cladosporium/genetics; DNA, Fungal/genetics; Fungal Proteins/genetics
  10. Ng KP, Ngeow YF, Yew SM, Hassan H, Soo-Hoo TS, Na SL, et al.
    Eukaryot Cell, 2012 May;11(5):703-4.
    PMID: 22544898 DOI: 10.1128/EC.00074-12
    Daldinia eschscholzii is an invasive endophyte that is most commonly found in plant tissues rich in secondary metabolites. We report the draft genome sequence of D. eschscholzii isolated from blood culture. The draft genome is 35,494,957 bp in length, with 42,898,665 reads, 61,449 contigs, and a G+C content of 46.8%. The genome was found to contain a high abundance of genes associated with plant cell wall degradation enzymes, mycotoxin production, and antifungal drug resistance.
    Matched MeSH terms: Ascomycota/genetics*; DNA, Fungal/genetics*; Endophytes/genetics*
  11. Chan GF, Bamadhaj HM, Gan HM, Rashid NA
    Eukaryot Cell, 2012 Nov;11(11):1419-20.
    PMID: 23104371 DOI: 10.1128/EC.00245-12
    Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.
    Matched MeSH terms: Ascomycota/genetics*; Fungal Proteins/genetics; RNA, Fungal/genetics*
  12. Xing J, Watkins WS, Witherspoon DJ, Zhang Y, Guthery SL, Thara R, et al.
    Genome Res, 2009 May;19(5):815-25.
    PMID: 19411602 DOI: 10.1101/gr.085589.108
    We report an analysis of more than 240,000 loci genotyped using the Affymetrix SNP microarray in 554 individuals from 27 worldwide populations in Africa, Asia, and Europe. To provide a more extensive and complete sampling of human genetic variation, we have included caste and tribal samples from two states in South India, Daghestanis from eastern Europe, and the Iban from Malaysia. Consistent with observations made by Charles Darwin, our results highlight shared variation among human populations and demonstrate that much genetic variation is geographically continuous. At the same time, principal components analyses reveal discernible genetic differentiation among almost all identified populations in our sample, and in most cases, individuals can be clearly assigned to defined populations on the basis of SNP genotypes. All individuals are accurately classified into continental groups using a model-based clustering algorithm, but between closely related populations, genetic and self-classifications conflict for some individuals. The 250K data permitted high-level resolution of genetic variation among Indian caste and tribal populations and between highland and lowland Daghestani populations. In particular, upper-caste individuals from Tamil Nadu and Andhra Pradesh form one defined group, lower-caste individuals from these two states form another, and the tribal Irula samples form a third. Our results emphasize the correlation of genetic and geographic distances and highlight other elements, including social factors that have contributed to population structure.
    Matched MeSH terms: Ethnic Groups/genetics; Genetics, Population; Polymorphism, Single Nucleotide/genetics*
  13. Alwi AR, Mahat NA, Mohd Salleh F, Ishar SM, Kamaluddin MR, A Rashid MR, et al.
    Leg Med (Tokyo), 2024 May;68:102416.
    PMID: 38325234 DOI: 10.1016/j.legalmed.2024.102416
    X-chromosome short tandem repeats (X-STRs) are useful for human identification, especially in complex kinship scenarios. Since forensic statistical parameters vary among populations and the X-STRs population data for the diverse population of Peninsular Malaysia's are unavailable, this attempt for Indians (n = 201) appears forensically relevant to support the 12 X-STRs markers' evidential value for human identification in Malaysia. The Qiagen Investigator® Argus X-12 QS kit showed that DXS10135 was the most polymorphic locus with high genetic diversity, polymorphism information richness, heterozygosity, and exclusion power. Based on allele frequencies, the strength of discrimination and mean exclusion chance (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) values for the Malaysian Indians were ≥0.999997790686228. As for haplotype frequencies, the overall discrimination power and mean exclusion probability (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) were ≥0.9999984801951. The genetic distance, neighbor-joining phylogenetic tree, and principal component analysis also supported the evidential value of the 12 X-STRs markers for forensic practical caseworks in Malaysia.
    Matched MeSH terms: Genetics, Population/methods; Forensic Genetics/methods
  14. Ramzi NH, Chahil JK, Lye SH, Munretnam K, Sahadevappa KI, Velapasamy S, et al.
    Indian J Med Res, 2014 Jun;139(6):873-82.
    PMID: 25109722
    Colorectal cancer (CRC) is second only to breast cancer as the leading cause of cancer-related deaths in Malaysia. In the Asia-Pacific area, it is the highest emerging gastrointestinal cancer. The aim of this study was to identify single nucleotide polymorphisms (SNPs) and environmental factors associated with CRC risk in Malaysia from a panel of cancer associated SNPs.
    Matched MeSH terms: Cytochrome P-450 Enzyme System/genetics*; Colorectal Neoplasms/genetics*; Polymorphism, Single Nucleotide/genetics
  15. Ang KC, Bogdanova N, Markoff A, Ch'ng ES, Tang TH
    Fertil Steril, 2019 05;111(5):971-981.e2.
    PMID: 30922645 DOI: 10.1016/j.fertnstert.2019.01.015
    OBJECTIVE: To ascertain the magnitude and precision of the association between M2/ANXA5 haplotype and repeated pregnancy loss (RPL).

    DESIGN: Meta-analysis of odds ratios.

    SETTING: Not applicable.

    PATIENT(S): Subjects were women with RPL and their partners.

    INTERVENTION(S): Not applicable.

    MAIN OUTCOME MEASURE(S): The association between M2/ANXA5 haplotype and RPL was evaluated in a meta-analysis of odds ratios. We further scrutinized this association according to [1] the sequence of miscarriages, [2] the number of consecutive losses, [3] the extent of excluding other pathologies of RPL, and [4] the timing of fetal loss.

    RESULT(S): Fourteen individual studies (n = 4,664 subjects) were included in this meta-analysis. The results show that women with the M2/ANXA5 haplotype have 1.54 times (95% confidence interval, 1.08-2.20) the odds of having associated RPL compared with women with the normal haplotype, regardless of consecutive or nonconsecutive pregnancy losses. Acknowledging the clinical heterogeneity among the studies, this significant association comes with a caveat that the lower bound of the confidence interval is close to unity. In couple populations (n = 2,449), M2/ANXA5 haplotype subjects have an odds ratio of 1.48 (95% confidence interval, 1.14-1.91) of experiencing RPL, which suggests contributions from paternal M2/ANXA5 carriers in RPL.

    CONCLUSION(S): This meta-analysis ascertains that women with the M2/ANXA5 haplotype have a higher risk of experiencing RPL, especially consecutive early idiopathic RPL. Male partners with the M2/ANXA5 haplotype partly contribute to this risk. Hence, screening for the M2/ANXA5 haplotype as a panel of laboratory investigations for RPL is recommended.

    Matched MeSH terms: Abortion, Habitual/genetics*; Haplotypes/genetics*; Annexin A5/genetics*
  16. Ugai T, Milne RL, Ito H, Aronson KJ, Bolla MK, Chan T, et al.
    Mol Genet Genomic Med, 2019 Jun;7(6):e707.
    PMID: 31066241 DOI: 10.1002/mgg3.707
    BACKGROUND: Epidemiological studies consistently indicate that alcohol consumption is an independent risk factor for female breast cancer (BC). Although the aldehyde dehydrogenase 2 (ALDH2) polymorphism (rs671: Glu>Lys) has a strong effect on acetaldehyde metabolism, the association of rs671 with BC risk and its interaction with alcohol intake have not been fully elucidated. We conducted a pooled analysis of 14 case-control studies, with individual data on Asian ancestry women participating in the Breast Cancer Association Consortium.

    METHODS: We included 12,595 invasive BC cases and 12,884 controls for the analysis of rs671 and BC risk, and 2,849 invasive BC cases and 3,680 controls for the analysis of the gene-environment interaction between rs671 and alcohol intake for BC risk. The pooled odds ratios (OR) with 95% confidence intervals (CI) associated with rs671 and its interaction with alcohol intake for BC risk were estimated using logistic regression models.

    RESULTS: The Lys/Lys genotype of rs671 was associated with increased BC risk (OR = 1.16, 95% CI 1.03-1.30, p = 0.014). According to tumor characteristics, the Lys/Lys genotype was associated with estrogen receptor (ER)-positive BC (OR = 1.19, 95% CI 1.05-1.36, p = 0.008), progesterone receptor (PR)-positive BC (OR = 1.19, 95% CI 1.03-1.36, p = 0.015), and human epidermal growth factor receptor 2 (HER2)-negative BC (OR = 1.25, 95% CI 1.05-1.48, p = 0.012). No evidence of a gene-environment interaction was observed between rs671 and alcohol intake (p = 0.537).

    CONCLUSION: This study suggests that the Lys/Lys genotype confers susceptibility to BC risk among women of Asian ancestry, particularly for ER-positive, PR-positive, and HER2-negative tumor types.

    Matched MeSH terms: Breast Neoplasms/genetics*; Asian Continental Ancestry Group/genetics; Aldehyde Dehydrogenase, Mitochondrial/genetics*
  17. Parsons MT, Tudini E, Li H, Hahnen E, Wappenschmidt B, Feliubadaló L, et al.
    Hum Mutat, 2019 Sep;40(9):1557-1578.
    PMID: 31131967 DOI: 10.1002/humu.23818
    The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.
    Matched MeSH terms: Neoplasms/genetics; BRCA1 Protein/genetics*; BRCA2 Protein/genetics*
  18. Lee PY, Yong VC, Rosli R, Gam LH, Chong PP
    Protein Expr Purif, 2014 Feb;94:15-21.
    PMID: 24184232 DOI: 10.1016/j.pep.2013.10.012
    Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.
    Matched MeSH terms: Pichia/genetics*; Farnesyl-Diphosphate Farnesyltransferase/genetics*; Candida tropicalis/genetics
  19. Mirzadeh A, Saadatnia G, Golkar M, Babaie J, Noordin R
    Protein Expr Purif, 2017 05;133:66-74.
    PMID: 28263855 DOI: 10.1016/j.pep.2017.03.001
    SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
    Matched MeSH terms: Escherichia coli/genetics; Recombinant Proteins/genetics; Toxoplasma/genetics*
  20. Chan MK, Lim SK, Miswan N, Chew AL, Noordin R, Khoo BY
    Protein Expr Purif, 2018 Jan;141:52-62.
    PMID: 28893606 DOI: 10.1016/j.pep.2017.09.003
    This study described the isolation of the coding region of human topoisomerase I (TopoI) from MDA-MB-231 and the expression of multiple copy recombinant genes in four Pichia pastoris strains. First, polymerase chain reaction (PCR)-amplification of the enzyme coding region was performed. The PCR fragment was cloned into pPICZ-α-A vector and sequenced. It was then transformed into X33, GS115, SMD1168H and KM71H strains of Pichia. PCR-screening for positive clones was performed, and estimation of multiple copy integrants in each Pichia strain was carried out using agar plates containing increasing concentrations of Zeocin(®). The selected clones of multiple copy recombinant genes were then induced for TopoI expression in shaker flasks. GS115 and SMD1168 were found to be better Pichia strains to accommodate the recombinant gene for the expression of TopoI extracellularly. However, the DNA relaxation activity revealed that only the target enzyme in the culture supernatants of GS115-pPICZ-α-A-TopoI exhibited consistent enzyme activity over the cultivation time-points. Active enzyme activity was inhibited by Camptothecin. The enzyme produced can be used for in-house gel-based DNA relaxation assay development in performing high throughput screening for target-specific growth inhibitors that display similar effect as the TopoI inhibitors. These inhibitors may contribute to the improvement of the treatment of cancer patients.
    Matched MeSH terms: DNA Topoisomerases, Type I/genetics; Pichia/genetics*; Recombinant Proteins/genetics
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