Displaying publications 581 - 600 of 3507 in total

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  1. Distinct haplotype profiles and strong linkage disequilibrium at the MDR1 multidrug transporter gene locus in three ethnic Asian populations
    Tang K, Ngoi SM, Gwee PC, Chua JM, Lee EJ, Chong SS, et al.
    Pharmacogenetics, 2002 Aug;12(6):437-50.
    PMID: 12172212
    The MDR1 multidrug transporter plays a key role in determining drug bioavailability, and differences in drug response exist amongst different ethnic groups. Numerous studies have identified an association between the MDR1 single nucleotide polymorphism (SNP) exon 26 3435C>T and differences in MDR1 function. We performed a haplotype analysis of the MDR1 gene in three major ethnic groups (Chinese, Malays and Indians) by examining 10 intragenic SNPs. Four were polymorphic in all three ethnic groups: one occurring in the non-coding region and three occurring in coding exons. All three coding SNPs (exon 12 1236C>T, exon 21 2677G>T/A and exon 26 3435C>T) were present in high frequency in each ethnic group, and the derived haplotype profiles exhibited distinct differences between the groups. Fewer haplotypes were observed in the Malays (n = 6) compared to the Chinese (n = 10) and Indians (n = 9). Three major haplotypes (> 10% frequency) were observed in the Malays and Chinese; of these, two were observed in the Indians. Strong linkage disequilibrium (LD) was detected between the three SNPs in all three ethnic groups. The strongest LD was present in the Chinese, followed by Indians and Malays, with the corresponding LD blocks estimated to be approximately 80 kb, 60 kb and 40 kb, respectively. These data strongly support the hypothesis that strong LD between the neutral SNP exon 26 3435C>T and a nearby unobserved causal SNP underlies the observed associations between the neutral SNP and MDR1 functional differences. Furthermore, strong LD between exon 26 3435T and different unobserved causal SNPs in different study populations may provide a plausible explanation for conflicting reports associating the same exon 26 3435T allele with different MDR1 functional changes.
    Matched MeSH terms: DNA/blood; DNA/genetics; DNA/metabolism; DNA Primers/chemistry
  2. Fulltext Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins
    Fischer H, Tschachler E, Eckhart L
    Apoptosis, 2020 Aug;25(7-8):474-480.
    PMID: 32533513 DOI: 10.1007/s10495-020-01614-4
    The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.
    Matched MeSH terms: DNA/genetics*; DNA/immunology; DNA-Binding Proteins/genetics*; DNA-Binding Proteins/immunology
  3. Predominance of Blastocystis sp. subtype 4 in rural communities, Nepal
    Lee IL, Tan TC, Tan PC, Nanthiney DR, Biraj MK, Surendra KM, et al.
    Parasitol Res, 2012 Apr;110(4):1553-62.
    PMID: 22076050 DOI: 10.1007/s00436-011-2665-0
    Blastocystis sp. is a common intestinal parasite. To date, there have been sporadic and scanty studies on Blastocystis sp. carried out in rural communities in Nepal. We surveyed the prevalence of Blastocystis sp. and its possible associated risk factors, and reported the predominant Blastocystis sp. subtype in two rural communities, Bolde Phediche and Bahunipati, in Nepal. Human faecal samples were collected from 241 participants, cultured using in vitro cultivation and examined for Blastocystis sp. The presence of Blastocystis sp. in faecal samples was further confirmed by polymerase chain reaction (PCR) and subsequently genotyped using subtype-specific sequence tagged site (STS) primers. There were 26.1% (63/241) of the participants that were infected by Blastocystis sp. We detected 84.1% (53/63) of Blastocystis sp. subtype 4 infections in these rural communities. The unusually high prevalence of Blastocystis sp. subtype 4 can be attributed to the rearing of family-owned animals in barns built close to their houses. Eighty one percent (51/63) of the Blastocystis sp. infected participants drank not boiled or unfiltered water. The present study revealed that Blastocystis sp. could pose a health concern to the communities and travellers to the hilly area in Nepal. Infection may be transmitted through human-to-human, zoonotic and waterborne transmissions. We provide recommendations to ensure good public health practices.
    Matched MeSH terms: DNA, Protozoan/isolation & purification; Sequence Analysis, DNA/methods; DNA Primers/isolation & purification
  4. Tropical soil bacterial communities in Malaysia: pH dominates in the equatorial tropics too
    Tripathi BM, Kim M, Singh D, Lee-Cruz L, Lai-Hoe A, Ainuddin AN, et al.
    Microb Ecol, 2012 Aug;64(2):474-84.
    PMID: 22395784 DOI: 10.1007/s00248-012-0028-8
    The dominant factors controlling soil bacterial community variation within the tropics are poorly known. We sampled soils across a range of land use types--primary (unlogged) and logged forests and crop and pasture lands in Malaysia. PCR-amplified soil DNA for the bacterial 16S rRNA gene targeting the V1-V3 region was pyrosequenced using the 454 Roche machine. We found that land use in itself has a weak but significant effect on the bacterial community composition. However, bacterial community composition and diversity was strongly correlated with soil properties, especially soil pH, total carbon, and C/N ratio. Soil pH was the best predictor of bacterial community composition and diversity across the various land use types, with the highest diversity close to neutral pH values. In addition, variation in phylogenetic structure of dominant lineages (Alphaproteobacteria, Beta/Gammaproteobacteria, Acidobacteria, and Actinobacteria) is also significantly correlated with soil pH. Together, these results confirm the importance of soil pH in structuring soil bacterial communities in Southeast Asia. Our results also suggest that unlike the general diversity pattern found for larger organisms, primary tropical forest is no richer in operational taxonomic units of soil bacteria than logged forest, and agricultural land (crop and pasture) is actually richer than primary forest, partly due to selection of more fertile soils that have higher pH for agriculture and the effects of soil liming raising pH.
    Matched MeSH terms: DNA, Bacterial/analysis; DNA, Ribosomal/analysis; Sequence Analysis, DNA/methods
  5. Application of multilocus sequence analysis for molecular characterization of enterococci with virulence factors recovered from a tropical recreational beach
    Ahmad A, Dada AC, Usup G
    Southeast Asian J Trop Med Public Health, 2014 May;45(3):700-18.
    PMID: 24974655
    Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.
    Matched MeSH terms: DNA, Bacterial/analysis; DNA, Bacterial/genetics; DNA-Directed RNA Polymerases/genetics*; Sequence Analysis, DNA
  6. Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling
    Learn-Han L, Yoke-Kqueen C, Salleh NA, Sukardi S, Jiun-Horng S, Chai-Hoon K, et al.
    Antonie Van Leeuwenhoek, 2008 Oct;94(3):377-87.
    PMID: 18548329 DOI: 10.1007/s10482-008-9254-y
    Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.
    Matched MeSH terms: DNA, Bacterial/genetics; DNA Fingerprinting; DNA Primers/genetics; Random Amplified Polymorphic DNA Technique
  7. Giardia intestinalis genotypes: Risk factors and correlation with clinical symptoms
    Mohammed Mahdy AK, Surin J, Wan KL, Mohd-Adnan A, Al-Mekhlafi MS, Lim YA
    Acta Trop, 2009 Oct;112(1):67-70.
    PMID: 19560431 DOI: 10.1016/j.actatropica.2009.06.012
    This study was conducted to identify genotypes related risk factors of Giardia intestinalis in an Orang Asli (aboriginal) community in Pahang, Malaysia. Stool samples were collected from 321 individuals aged between 2 and 76 years old, of whom 160 were males and 161 were females. Faecal samples were processed with trichrome staining technique for the primary identification of G. intestinalis. Molecular identification was carried out by the amplification of a partial SSU rRNA gene using nested PCR. PCR products were purified and genotyped. 42 samples successfully amplified from the 76 positive faecal samples, only 1 was Assemblage A, the rest were Assemblage B. Risk analysis based on the detected genotypes of Giardia using univariate analysis and logistic regression identified three significant risk factors of giardiasis caused by assemblage B which included children =12 years (OR=13.56, 95% CI=1.79-102.64, p=0.012), females (OR=2.52, 95% CI=1.11-5.75, p=0.027) and eating fresh fruits (OR=7.78, 95% CI=1.01-60.00, p=0.049). Assemblage B infection was significantly correlated with clinical symptoms of giardiasis (OR=2.4, 95% CI=1.13-5.12, p=0.019). Females infected with Assemblage B were at higher risk of manifesting gastroenteritis signs and symptoms (OR=3.9, 95% CI=1.50-10.31, p=0.004). It has been concluded that giardiasis is still a public health problem in Orang Asli community and most commonly caused by assemblage B. The dynamic of transmission is most probably anthroponotic which is human to human either directly or indirectly through contaminated food. This route of transmission should be considered in the control strategy of the disease. Mass treatment together with health education could be the most practical intervention for reducing the infection. Those at high risk should receive more attention from public health authorities.
    Matched MeSH terms: DNA, Ribosomal/genetics; DNA, Ribosomal/chemistry; DNA, Protozoan/genetics; DNA, Protozoan/chemistry
  8. Langkocyclines: novel angucycline antibiotics from Streptomyces sp. Acta 3034(*)
    Kalyon B, Tan GY, Pinto JM, Foo CY, Wiese J, Imhoff JF, et al.
    J Antibiot (Tokyo), 2013 Oct;66(10):609-16.
    PMID: 23820614 DOI: 10.1038/ja.2013.53
    Langkocyclines A1-A3 and B1 and B2, five new angucycline antibiotics produced by Streptomyces sp. Acta 3034, were detected in the course of our HPLC-diode array screening. The producing strain was isolated from the rhizospheric soil of a Clitorea sp. collected from Burau Bay, Langkawi, Malaysia, and was characterized by morphological, physiological and chemotaxonomic features in addition to 16S ribosomal RNA gene sequence information. Strain Acta 3034 is closely related to Streptomyces psammoticus NBRC 13971(T) and Streptomyces lanatus NBRC 12787(T). Langkocyclines consist of an angular tetracyclic benz[a]anthracene skeleton and hydrolyzable O-glycosidic sugar moieties. The yellow-colored A-type langkocyclines differ in their aglycon from the blue-lilac-colored B-type langkocyclines. The A-type langkocycline aglycon is identical to that of aquayamycin and urdamycin A. The chemical structures of the langkocyclines were elucidated by HR-MS, 1D and 2D NMR experiments. They are biologically active against Gram-positive bacteria and exhibit a moderate antiproliferative activity against various human tumor cell lines.
    Matched MeSH terms: DNA, Bacterial/analysis; DNA, Bacterial/genetics; DNA, Ribosomal/analysis; Sequence Analysis, DNA
  9. Molecular investigation on the occurrence of Toxoplasma gondii oocysts in cat feces using TOX-element and ITS-1 region targets
    Chemoh W, Sawangjaroen N, Nissapatorn V, Sermwittayawong N
    Vet J, 2016 Sep;215:118-22.
    PMID: 27325616 DOI: 10.1016/j.tvjl.2016.05.018
    One of the most important routes of transmission for Toxoplasma gondii infection is the ingestion of foods contaminated with cat feces containing sporulated oocysts. The diagnosis of T. gondii infection by fecal microscopy is complicated, as other similar coccidian oocysts are often present in the same fecal specimen. This study aimed to identify T. gondii oocysts in cat feces using a novel PCR technique. Feline fecal specimens (n = 254) were screened for coccidian oocysts by light microscopy using the Sheather's flotation method. PCR analysis performed on the same specimens targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) regions were used to confirm the presence of Toxoplasma oocysts. By light microscopy, 49/254 (19.3%) of specimens contained coccidian oocysts. PCR analysis demonstrated 2/254 (0.8%) and 17/254 (6.7%) positive results using Tox and ITS-1 primers, respectively. However, coccidian oocysts were not identified on microscopic examination of specimens that were PCR-positive by Tox primers. Coccidian oocysts were identified on microscopic examination of 6/17 (35.3%) of the PCR-positive fecal specimens using ITS-1 primers. The BLAST results of 16 ITS-1 sequences were identified as T. gondii (n = 12; 4.7%) and Hammondia hammondi (n = 4; 1.6%). There was slight agreement between the 529 bp and ITS-1 PCR results (κ = 0.148). This is the first report of the detection of Toxoplasma oocysts using PCR analysis on feline fecal specimens from Southern Thailand. The ITS-1 region has potential as an alternative marker to identify T. gondii oocysts in feline fecal specimens.
    Matched MeSH terms: DNA, Protozoan/analysis; Sequence Analysis, DNA/veterinary; DNA, Ribosomal Spacer/analysis
  10. Fulltext Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7
    Nadzirah Sh, Azizah N, Hashim U, Gopinath SC, Kashif M
    PLoS One, 2015;10(10):e0139766.
    PMID: 26445455 DOI: 10.1371/journal.pone.0139766
    Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system's physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10(-13)M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.
    Matched MeSH terms: DNA, Bacterial/analysis; DNA, Bacterial/genetics*; DNA, Single-Stranded/genetics; DNA, Single-Stranded/chemistry
  11. Overexpression of DNA methyltransferase 1 (DNMT1) protein in astrocytic tumour and its correlation with O6-methylguanine-DNA methyltransferase (MGMT) expression
    Rahman WF, Rahman KS, Nafi SN, Fauzi MH, Jaafar H
    Int J Clin Exp Pathol, 2015;8(6):6095-106.
    PMID: 26261487
    The relationship between DNA methyltransferase (DNMT) and O6-methylguanine-DNA methyltransferase (MGMT) in mediating tumorigenesis is still poorly understood. This study was carried out to investigate a correlation between DNMT1 and MGMT immunoexpression in astrocytic tumour samples.
    Matched MeSH terms: DNA Modification Methylases/analysis*; DNA Repair Enzymes/analysis*
  12. DNA barcoding and phylogenetic analysis of Malaysian groupers (Subfamily: Epinephelinae) using mitochondrial Cytochrome c oxidase I (COI) gene
    Aziz NMA, Esa Y, Arshad A
    J Environ Biol, 2016 07;37(4 Spec No):725-33.
    PMID: 28779732
    The present study was carried out to examine the species identification and phylogenetic relationships of groupers in Malaysia using mitochondrial Cytochrome c Oxidase I (COI) gene, commonly known as barcoding gene. A total of 63 individuals comprising 10 species from three genera were collected from the coastal areas of Johor, Kelantan, Pahang, Perak, Selangor and Terengganu. All the individuals were morphologically identified and molecular works involved polymerase chain reaction (PCR) and sequencing of COI barcoding fragment (655 base pairs). Results from the BLAST search showed that 55 sequences could be assigned to 10 grouper species with high percentage identity index (≥95% to 100%), while eight grouper individuals showed discrepancies in their taxonomic identification based on the morphology and the COI barcoding results. The histogram of distances showed that there was a clear-cut barcode gap present in the sequences indicating a clear separation between intraspecific and interspecific distances. The pairwise genetic distances showed lowest pairwise distance between P. leopardus and P. maculatus (4.4%), while the highest pairwise distance was between E. bleekeri and P. maculatus (23.5%), supporting their morphological and habitat similarities and differences. Phylogenetic analysis (Neighbor-Joining) showed the presence of two major clades (1) genus Epinephelus vs (2) genus Plectropomus and Cephalopholis). In conclusion, the present study has managed to show the accuracy of DNA barcoding method for species identification, and utilization of COI gene for phylogenetic study among groupers. ?
    Matched MeSH terms: DNA Barcoding, Taxonomic*
  13. DNAzyme Based Amplified Biosensor on Ultrasensitive Fluorescence Detection of Pb (II) Ions from Aqueous System
    Ravikumar A, Panneerselvam P, Radhakrishnan K, Morad N, Anuradha CD, Sivanesan S
    J Fluoresc, 2017 Nov;27(6):2101-2109.
    PMID: 28819702 DOI: 10.1007/s10895-017-2149-4
    A label -free DNAzyme amplified biosensor is found to be highly selective and sensitive towards fluorescent detection of Pb2+ ions in aqueous media. The DNAzyme complex has designed by the hybridization of the enzyme and substrate strand. In the presence of Pb2+, the DNAzyme activated and cleaved the substrate strand of RNA site (rA) into two oligonucleotide fragments. Further, the free fragment was hybridized with a complementary strand on the surface of MBs. After magnetic separation, SYBER Green I was added and readily intercalate with the dsDNA to gives a bright fluorescence signal with intensity directly proportional to the concentration of Pb2+ions. A detection limit of 5 nM in Pb2+ the detection range 0 to 500 nM was obtained. This label- free fluorescent biosensor has been successfully applied to the determination of environmental water samples. Then results open up the possibility for real-time quantitative detection of Pb2+ with convenient potential applications in the biological and environmental field. Graphical Abstract.
    Matched MeSH terms: DNA, Catalytic/chemistry*
  14. Detection of somatic mutations in the mitochondrial DNA control region D-loop in brain tumors: The first report in Malaysian patients
    Mohamed Yusoff AA, Mohd Nasir KN, Haris K, Mohd Khair SZN, Abdul Ghani ARI, Idris Z, et al.
    Oncol Lett, 2017 Nov;14(5):5179-5188.
    PMID: 29098023 DOI: 10.3892/ol.2017.6851
    Although the role of nuclear-encoded gene alterations has been well documented in brain tumor development, the involvement of the mitochondrial genome in brain tumorigenesis has not yet been fully elucidated and remains controversial. The present study aimed to identify mutations in the mitochondrial DNA (mtDNA) control region D-loop in patients with brain tumors in Malaysia. A mutation analysis was performed in which DNA was extracted from paired tumor tissue and blood samples obtained from 49 patients with brain tumors. The D-loop region DNA was amplified using the PCR technique, and genetic data from DNA sequencing analyses were compared with the published revised Cambridge sequence to identify somatic mutations. Among the 49 brain tumor tissue samples evaluated, 25 cases (51%) had somatic mutations of the mtDNA D-loop, with a total of 48 mutations. Novel mutations that had not previously been identified in the D-loop region (176 A-deletion, 476 C>A, 566 C>A and 16405 A-deletion) were also classified. No significant associations between the D-loop mutation status and the clinicopathological parameters were observed. To the best of our knowledge, the current study presents the first evidence of alterations in the mtDNA D-loop regions in the brain tumors of Malaysian patients. These results may provide an overview and data regarding the incidence of mitochondrial genome alterations in Malaysian patients with brain tumors. In addition to nuclear genome aberrations, these specific mitochondrial genome alterations may also be considered as potential cancer biomarkers for the diagnosis and staging of brain cancers.
    Matched MeSH terms: DNA Mutational Analysis; DNA, Mitochondrial
  15. Fulltext Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness
    Yu EPK, Reinhold J, Yu H, Starks L, Uryga AK, Foote K, et al.
    Arterioscler Thromb Vasc Biol, 2017 12;37(12):2322-2332.
    PMID: 28970293 DOI: 10.1161/ATVBAHA.117.310042
    OBJECTIVE: Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis.

    APPROACH AND RESULTS: Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE-/-) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE-/- mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE-/-). Tw+/ApoE-/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw+/ApoE-/- mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE-/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis.

    CONCLUSIONS: Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.

    Matched MeSH terms: DNA Damage*; DNA Helicases/genetics; DNA Helicases/metabolism; DNA, Mitochondrial/genetics; DNA, Mitochondrial/metabolism*
  16. Fulltext Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
    Hassan MI, McSorley FR, Hotta K, Boddy CN
    J Vis Exp, 2017 06 27.
    PMID: 28715370 DOI: 10.3791/55187
    Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.
    Matched MeSH terms: DNA-Directed RNA Polymerases/genetics*
  17. Tracking the southern river terrapin (Batagur affinis) through environmental DNA: prospects and challenges
    Wilson JJ, Sing KW, Chen PN, Zieritz A
    Mitochondrial DNA A DNA Mapp Seq Anal, 2018 08;29(6):862-866.
    PMID: 28885060 DOI: 10.1080/24701394.2017.1373109
    Environmental DNA detection has emerged as a powerful tool to monitor aquatic species without the need for capture or visual identification and is particularly useful for rare or elusive species. Our objective was to develop an eDNA approach for detecting the southern river terrapin (Batagur affinis) in Malaysia. We designed species-specific primers for a fragment of B. affinis mtDNA and evaluated their effectiveness in silico, in vitro and in situ. The primers amplified 110 bp of the cytochrome b mtDNA sequence of B. affinis from aquarium water samples housing nine juvenile B. affinis. We also successfully detected B. affinis eDNA from river samples taken from a site where turtles were known to be in the vicinity. Prospects and challenges of using an eDNA approach to help determine the distribution of B. affinis, essential information for an effective conservation plan, are discussed.
    Matched MeSH terms: DNA, Mitochondrial/genetics*
  18. Fulltext Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly
    Tan MH, Austin CM, Hammer MP, Lee YP, Croft LJ, Gan HM
    Gigascience, 2018 03 01;7(3):1-6.
    PMID: 29342277 DOI: 10.1093/gigascience/gix137
    Background: Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics.

    Results: We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches.

    Conclusions: We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.

    Matched MeSH terms: Sequence Analysis, DNA/methods*
  19. Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids
    Gomez-Eichelmann MC, Holz G, Beach D, Simpson AM, Simpson L
    Mol Biochem Parasitol, 1988 Jan 15;27(2-3):143-58.
    PMID: 3344003
    Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species [L. hoogstrali, L. adleri, L. agamae and Leishmania sp. LizS], with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircle sequences and in terms of nuclear chromosome patterns on orthogonal gel electrophoresis. The L. tarentolae strains fell into two major groups, one (group A) consisting of the L. tarentolae strains, UC, Krassner and Trager, derived from an Algerian gecko isolate and the other (group B) consisting of five L. tarentolae LEM strains isolated from geckos in southern France. T. platydactyli TPCL2, which was postulated by Wallbanks et al. to represent the lizard form of a French L. tarentolae strain, was closely related to the UC strain and not to the LEM strains, in all respects analyzed. Leishmania sp. LizS from a Mongolian gecko and L. hoogstrali from a Sudanese gecko showed some sequence similarities to the L. tarentolae strains, but the leishmanias said to be L. adleri from a Kenyan lacertid and L. agamae from an Israeli agamid showed no minicircle sequence similarities with lizard Leishmania and in fact were probably the same species. The maxicircle divergent region was larger in the group B strains than in the group A strains, but there were sequences in common with both groups, and not with L. hoogstrali and L. major. Four strains of L. tarentolae, the four other supposed saurian Leishmania species, three mammalian leishmanias, T. platydactyli and four other trypanosomes, T. cyclops (Malaysian macaque), T. conorrhini (Hawaiian reduviid bug), T. cruzi (man) and T. lewisi (feral rat) were analyzed for their contents of sterols and phosphoglyceride fatty acyl groups. T. platydactyli TPCL2 contained a sterol (5-dehydroepisterol), a phosphatidylcholine fatty acyl group (alpha-linolenic acid) and a phosphatidylethanolamine fatty acyl group (dihydrosterculic acid) characteristic of members of the genus Leishmania and not the genus Trypanosoma. The proportions of those lipids in the free sterol and phosphoglyceride fractions of T. platydactyli TPCL2 most closely resembled those seen in the Leishmania strains from Algerian, French, Mongolian and Sudanese geckos.
    Matched MeSH terms: DNA, Circular/analysis
  20. Fulltext Genetic variability in selected date palm (Phoenix dactylifera L.) cultivars of United Arab Emirates using ISSR and DAMD markers
    Purayil FT, Robert GA, Gothandam KM, Kurup SS, Subramaniam S, Cheruth AJ
    3 Biotech, 2018 Feb;8(2):109.
    PMID: 29430370 DOI: 10.1007/s13205-018-1108-3
    Nine (9) different date palm (Phoenix dactylifera L.) cultivars from UAE, which differ in their flower timings were selected to determine the polymorphism and genetic relationship between these cultivars. Hereditary differences and interrelationships were assessed utilizing inter-simple sequence repeat (ISSR) and directed amplification of minisatellite DNA region (DAMD) primers. Analysis on eight DAMD and five ISSR markers produced total of 113 amplicon including 99 polymorphic and 14 monomorphic alleles with a polymorphic percentage of 85.45. The average polymorphic information content for the two-marker system was almost similar (DAMD, 0.445 and ISSR, 0.459). UPGMA based clustering of DAMD and ISSR revealed that mid-season cultivars, Mkh (Khlas) and MB (Barhee) grouped together to form a subcluster in both the marker systems. The genetic similarity analysis followed by clustering of the cumulative data from the DAMD and ISSR resulted in two major clusters with two early-season cultivars (ENg and Ekn), two mid-season cultivars (MKh and MB) and one late-season cultivar (Lkhs) in cluster 1, cluster 2 includes two late-season cultivars, one early-season cultivar and one mid-season cultivar. The cluster analysis of both DAMD and ISSR marker revealed that, the patterns of variation between some of the tested cultivars were similar in both DNA marker systems. Hence, the present study signifies the applicability of DAMD and ISSR marker system in detecting genetic diversity of date palm cultivars flowering at different seasons. This may facilitate the conservation and improvement of date palm cultivars in the future.
    Matched MeSH terms: DNA; DNA Primers
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