Plasmodium ovale is rare and not exactly known to be autochthonous in Malaysia. There are two distinct forms of the parasite, namely P. ovale curtisi (classic form) and P. ovale wallikeri (variant form). Here, the first sequence confirmed case of an imported P. ovale wallikeri infection in Malaysia is presented. Microscopy found Plasmodium parasites with morphology similar to P. ovale or Plasmodium vivax in the blood films. Further confirmation using polymerase chain reaction (PCR) targeting the small-subunit rRNA gene of the parasite was unsuccessful. Genus-specific PCR was then performed and the product was sequenced and analysed. Sequence analyses confirmed the aetiological agent as P. ovale wallikeri. New species-specific primers (rOVA1v and rOVA2v) were employed and P. ovale wallikeri was finally confirmed. The findings highlight the need to look out for imported malaria infections in Malaysia and the importance of a constantly updated and validated diagnostic technique.
In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.
The σ(S) subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.
The brown rat (Rattus norvegicus) is a relatively recent (<300 years) addition to the British fauna, but by association with negative impacts on public health, animal health and agriculture, it is regarded as one of the most important vertebrate pest species. Anticoagulant rodenticides were introduced for brown rat control in the 1950s and are widely used for rat control in the UK, but long-standing resistance has been linked to control failures in some regions. One thus far ignored aspect of resistance biology is the population structure of the brown rat. This paper investigates the role population structure has on the development of anticoagulant resistance. Using mitochondrial and microsatellite DNA, we examined 186 individuals (from 15 counties in England and one location in Wales near the Wales-England border) to investigate the population structure of rural brown rat populations. We also examined individual rats for variations of the VKORC1 gene previously associated with resistance to anticoagulant rodenticides. We show that the populations were structured to some degree, but that this was only apparent in the microsatellite data and not the mtDNA data. We discuss various reasons why this is the case. We show that the population as a whole appears not to be at equilibrium. The relative lack of diversity in the mtDNA sequences examined can be explained by founder effects and a subsequent spatial expansion of a species introduced to the UK relatively recently. We found there was a geographical distribution of resistance mutations, and relatively low rate of gene flow between populations, which has implications for the development and management of anticoagulant resistance.
The cellulose synthase-like gene HvCslF6, which is essential for (1,3;1,4)-β-glucan biosynthesis in barley, collocates with quantitative trait loci (QTL) for grain (1,3;1,4)-β-glucan concentration in several populations, including CDC Bold × TR251. Here, an alanine-to-threonine substitution (caused by the only non-synonymous difference between the CDC Bold and TR251 HvCslF6 alleles) was mapped to a position within HvCSLF6 that seems unlikely to affect enzyme stability or function. Consistent with this, transient expression of full-length HvCslF6 cDNAs from CDC Bold and TR251 in Nicotianabenthamiana led to accumulation of similar amounts of (1,3;1,4)-β-glucan accumulation. Monitoring of HvCslF6 transcripts throughout grain development revealed a significant difference late in grain development (more than 30 days after pollination), with TR251 [the parent with higher grain (1,3;1,4)-β-glucan] exhibiting higher transcript levels than CDC Bold. A similar difference was observed between Beka and Logan, the parents of another population in which a QTL had been mapped in the HvCslF6 region. Sequencing of a putative promoter region of HvCslF6 revealed numerous polymorphisms between CDC Bold and TR251, but none between Beka and Logan. While the results of this work indicate that naturally occurring quantitative differences in (1,3;1,4)-β-glucan accumulation may be due to cis-regulated differences in HvCslF6 expression, these could not be attributed to any specific DNA sequence polymorphism. Nevertheless, information on HvCslF6 sequence polymorphism was used to develop molecular markers that could be used in barley breeding to select for the desired [low or high (1,3;1,4)-β-glucan] allele of the QTL.
Preferential conversion of azathioprine or 6-mercaptopurine into methylated metabolites is a major cause of thiopurine resistance. To seek potentially Mendelian causes of thiopurine hypermethylation, we recruited 12 individuals who exhibited extreme therapeutic resistance while taking azathioprine or 6-mercaptopurine and performed whole-exome sequencing (WES) and copy-number variant analysis by array-based comparative genomic hybridisation (aCGH). Exome-wide variant filtering highlighted four genes potentially associated with thiopurine metabolism (ENOSF1 and NFS1), transport (SLC17A4) or therapeutic action (RCC2). However, variants of each gene were found only in two or three patients, and it is unclear whether these genes could influence thiopurine hypermethylation. Analysis by aCGH did not identify any unusual or pathogenic copy-number variants. This suggests that if causative mutations for the hypermethylation phenotype exist they may be heterogeneous, occurring in several different genes, or they may lie within regulatory regions not captured by WES. Alternatively, hypermethylation may arise from the involvement of multiple genes with small effects. To test this hypothesis would require recruitment of large patient samples and application of genome-wide association studies.
This study revealed the biotic and abiotic parameters driving the variations in microcystins (MCs) biodegradability of a practical biological treatment facility (BTF). Results showed that similar trends of seasonal variation were seen for microcystin-LR (MCLR) biodegradability of biofilms on the BTF and indigenous MCLR-degrader population, where both peaks co-occurred in October, following the peaks of natural MCLR concentration and water temperature observed in August. The lag period might be required for accumulation of MCLR-degraders and MCLR-degrading enzyme activity. The MCLR-degrader population was correlated to temperature, MCLR and chlorophyll-a concentration in water where the biofilms submerged, indicating that these abiotic and biotic parameters exerted direct and/or indirect influences on seasonal variation in MCLR-biodegradability. In comparison, no effect of other co-existing MCs on biodegradation of one MC was observed. However, proliferation of MC-degraders along biodegradation processes positively responded to total amount of MCs, suggesting that multiple MCs contributed additively to MC-degrader proliferation.
In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).
The complete mitochondrial genome of the Bass yabby Trypaea australiensis was obtained from a partial genome scan using the MiSeq sequencing system. The T. australiensis mitogenome is 16,821 bp in length (70.25% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a putative 1977 bp non-coding AT-rich region. This Trypaea mitogenome sequence is the 5th for the family Callianassidae and represents a new gene order for the Decapoda involving protein-coding, rRNA and tRNA genes and the control region.
Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91-98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamyssabanus, Rattustiomanicus and Sundamysmuelleri), shrews (Tupaiaglis) and mammals (Tapirusindicus and Prionailurusbengalensis), supported by 82-88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks' blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.
The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods.
Matched MeSH terms: DNA Mutational Analysis/methods*; DNA, Neoplasm/genetics
Papaya (Carica papaya L.) is one of the major tropical fruit crops worldwide, but it is limited throughout its range by papaya ringspot virus type P (PRSV-P). Previous genetic studies identified a functional PRSV-P resistance marker in a mapping population of F2 plants of Vasconcellea pubescens (resistant to PRSV-P) × Vasconcellea parviflora (susceptible to PRSV-P) and showed that the marker exhibited homology to a serine threonine protein kinase (STK) gene. Full length cDNAs of putative PRSV-P resistance genes designated CP_STK from C. papaya and VP_STK1 and VP_STK2 from V. pubescens were cloned by rapid amplification of cDNA ends (RACE). Due to a frame-shift mutation, the two homologous sequences are transcribed and edited differently such that the gene product in V. pubescens is two separate transcripts, whereas in C. papaya they are fused into a single message. A peroxisomal targeting signal (PTS2) present in VP_STK2 but absent in the other transcripts may be the functional source of PRSV resistance in V. pubescens. The STK gene from V. pubescens may have been derived from an alternative splicing to confer resistance. The putative resistance gene, VP_STK2, that was identified in this study is a potential new source of PRSV-P resistance for papaya genotypes.
Genetic diversity and differentiation of 50 Colletotrichum spp. isolates from legume crops studied through multigene loci, RAPD and ISSR analysis. DNA sequence comparisons by six genes (ITS, ACT, Tub2, CHS-1, GAPDH, and HIS3) verified species identity of C. truncatum, C. dematium and C. gloeosporiodes and identity C. capsici as a synonym of C. truncatum. Based on the matrix distance analysis of multigene sequences, the Colletotrichum species showed diverse degrees of intera and interspecific divergence (0.0 to 1.4%) and (15.5-19.9), respectively. A multilocus molecular phylogenetic analysis clustered Colletotrichum spp. isolates into 3 well-defined clades, representing three distinct species; C. truncatum, C. dematium and C. gloeosporioides. The ISSR and RAPD and cluster analysis exhibited a high degree of variability among different isolates and permitted the grouping of isolates of Colletotrichum spp. into three distinct clusters. Distinct populations of Colletotrichum spp. isolates were genetically in accordance with host specificity and inconsistent with geographical origins. The large population of C. truncatum showed greater amounts of genetic diversity than smaller populations of C. dematium and C. gloeosporioides species. Results of ISSR and RAPD markers were congruent, but the effective maker ratio and the number of private alleles were greater in ISSR markers.
Matched MeSH terms: Random Amplified Polymorphic DNA Technique
The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.
The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5' EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.
Congracilaria babae was first reported as a red alga parasitic on the thallus of Gracilaria salicornia based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of C. babae on a Hydropuntia species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on Hydropuntia sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes (nuclear ITS region, mitochondrial cox1 gene and plastid rbcL gene). Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from Hydropuntia sp. and C. babae, suggesting their conspecificity. The parasite from Hydropuntia sp. has a DNA signature characteristic to C. babae in having plastid rbcL gene sequence identical to G. salicornia. C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host Hydropuntia sp. We also recommend the transfer of C. babae to the genus Gracilaria and propose a new combination, G. babae, based on the anatomical observations and molecular data.
A total of 30 specimens belonging to five species, namely; Cryptozona siamensis, Sarika resplendens and Sarika sp. from the family Ariophantidae as well as Quantula striata and Quantula sp. from the family Dyakiidae were collected from the Langkawi Island in Northern Peninsular Malaysia. All specimens were identified through comparisons of shell morphology and amplification of a 500 bp segment of the 16S rRNA mtDNA gene. To assess phylogenetic insights, the sequences were aligned using ClustalW and phylogenetic trees were constructed. The analyses showed two major lineages in both Maximum Parsimony and Neighbour Joining phylogenetic trees. Each putative taxonomic group formed a monophyletic cluster. Our study revealed low species and intraspecies genetic diversities based on the 16S rRNA gene sequences. Thus, this study has provided an insight of land snail diversity in populations of an island highly influenced by anthropogenic activities through complementary use of shell morphological and molecular data.
Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.
: The distinctive properties of graphene, characterized by its high carrier mobility and biocompatibility, have stimulated extreme scientific interest as a promising nanomaterial for future nanoelectronic applications. In particular, graphene-based transistors have been developed rapidly and are considered as an option for DNA sensing applications. Recent findings in the field of DNA biosensors have led to a renewed interest in the identification of genetic risk factors associated with complex human diseases for diagnosis of cancers or hereditary diseases. In this paper, an analytical model of graphene-based solution gated field effect transistors (SGFET) is proposed to constitute an important step towards development of DNA biosensors with high sensitivity and selectivity. Inspired by this fact, a novel strategy for a DNA sensor model with capability of single-nucleotide polymorphism detection is proposed and extensively explained. First of all, graphene-based DNA sensor model is optimized using particle swarm optimization algorithm. Based on the sensing mechanism of DNA sensors, detective parameters (Ids and Vgmin) are suggested to facilitate the decision making process. Finally, the behaviour of graphene-based SGFET is predicted in the presence of single-nucleotide polymorphism with an accuracy of more than 98% which guarantees the reliability of the optimized model for any application of the graphene-based DNA sensor. It is expected to achieve the rapid, quick and economical detection of DNA hybridization which could speed up the realization of the next generation of the homecare sensor system.
Conservation is imperative for the Asian snakeheads Channa striata, as the species has been overfished due to its high market demand. Using maternal markers (mitochondrial cytochrome c oxidase subunit 1 gene (COI)), we discovered that evolutionary forces that drove population divergence did not show any match between the genetic and morphological divergence pattern. However, there is evidence of incomplete divergence patterns between the Borneo population and the populations from Peninsular Malaysia. This supports the claim of historical coalescence of C. striata during Pleistocene glaciations. Ecological heterogeneity caused high phenotypic variance and was not correlated with genetic variance among the populations. Spatial conservation assessments are required to manage different stock units. Results on DNA barcoding show no evidence of cryptic species in C. striata in Malaysia. The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provide information relevant to identification of species.