INTRODUCTION: Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.
Molecular characterization of the compound heterozygous condition - (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia - in four families showing mild beta-thalassemia intermedia was carried out using DNA amplification techniques. Using the Amplification Refractory Mutation System (ARMS) to confirm the beta-mutations and DNA amplification to detect the 100-kb Chinese-specific (G)gamma((A)gammadeltabeta)(o)-deletion, ()two families were confirmed to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia with the IVSII No. 654 beta(+)-allele. In the third family, the (G)gamma((A)gammadeltabeta)(o)-deletion was confirmed in the father and the mother was a beta-thalassemia carrier with the cd 41-42 beta(o)-allele. Their affected child with (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia was found to be transfusion dependent. The same (G)gamma((A)gammadeltabeta)(o)-deletion and beta-thalassemia (cd 41-42) was also confirmed in a fourth family. In addition, the mother was also diagnosed with Hb H disease (genotype -alpha(3.7)/-(SEA)). Both the children were found to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia but they were not transfusion dependent and this could be due to co-inheritance of alpha-thalassemia-2 (genotype-alpha(3.7)/alphaalpha) in the children together with their compound heterozygous condition.
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.
The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.
The MDR1 multidrug transporter represents one of the better characterized drug transporters that play an important role in protecting the body against xenobiotic insults. Single nucleotide polymorphisms (SNPs) and SNP haplotypes within this gene have been variously associated with differences in MDR1 expression/function, drug response as well as disease susceptibility. Nonetheless, the effect of polymorphisms at the MDR1 promoter region on its promoter activity remains less characterized. Through the examination of approximately 1.5 kilobases of MDR1 promoter region from five populations, including the Chinese, Malays, Indians, European Americans, and African Americans, we identified eight low-frequency SNPs, of which only two were polymorphic in at least four of the five populations examined. The other SNPs are mainly population-specific, the majority of which occur only in the African-American population. Recapitulation of the various combinations of SNP haplotypes in vitro in promoter-reporter assays revealed a few notable trends. The African and European American-specific haplotypes tended to result in enhanced MDR1 promoter activity only in the human embryonic kidney (HEK) 293 cell line. Haplotype GCTAACC, which occurs at variable frequencies in all the populations examined, with Asians having much lower frequencies (<2%) compared with the European Americans/African Americans (>4%), affected MDR1 promoter activity differently in different cell lines. Compared with the commonest haplotype, GCTA-ACC haplotype resulted in a significant decrease in MDR1 promoter activity in HeLa cells (P < 0.05) but a significant increase in the same promoter activity in HEK293 cells (P < 0.05). These results suggest that the MDR1 promoter region is largely invariant but that different haplotypes have differential effects on the MDR1 promoter activity in different cell lines.
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.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman((R)) MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.
The inherent ability of nucleic acids to recognize a complementary pair has gained wide popularity in DNA sensor applications. DNA molecules can be produced in bulk and easily incorporated with various nanomaterials for sensing applications. More complex designs and sophisticated DNA sensors have been reported over the years to allow DNA detection in a faster, cheaper, and more convenient manner. Here, we report a DNA sensor designed to function like a switch to turn "on" silver nanocluster (AgNC) generation in the presence of a specific DNA target. By defining the probe region sequence, we are able to tune the color of the AgNC generated in direct relation to the different targets. As a proof of concept, we used dengue RNA-dependent RNA polymerase conserved sequences from all four serotypes as targets. This method was able to distinguish each dengue serotype by generating the serotype-respective AgNCs. The DNA switch was also able to identify and amplify the correct target in a mixture of targets with good specificity. This strategy has a detection limit of between 1.5 and 2.0 µM depending on the sequence of AgNC. The DNA switch approach provides an attractive alternative for single-target or multiplex DNA detection.
Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains - a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.
Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.
Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.
To investigate RCL2 as a fixative for tissue fixation in routine histopathological examination and to assess tissue suitability for ancillary investigations.
Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.
The realization that ancient biomolecules are preserved in "fossil" samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia's indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller et al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.
Iranihindia martellata (Senior-White, 1924) is recorded from peninsular Malaysia for the first time. Male and female specimens in the recent collections of forensically important sarcophagid flies were examined and identified based on morphology and DNA sequencing analysis. Male genitalia offer unambiguous species identification characteristics in the traditional taxonomy of flesh flies but the female flies are very similar to one another in general morphology. Female of I. martellata was determined by DNA sequencing (COI and COII) and PCR-RFLP (COI) analysis. Identified females were carefully examined and compared with the morphologically similar species, Liopygia ruficornis (Fabricius, 1794). Female genitalia are re-described and illustrated in this paper.
The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.
High-quality DNA extracts are imperative for downstream applications in molecular identification. Most processed food products undergo heat treatments causing DNA degradation, which hampers application of DNA-based techniques for food authentication. Moreover, the presence of inhibitors in processed food products is also problematic, as inhibitors can impede the process of obtaining high qualities and quantities of DNA. Various approaches in DNA extraction and factors in structure and texture of various food matrices affecting DNA extraction are explained in this review.
The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.