Phytophthora palmivora has caused disease in many crops including oil palm in the South America region. The pathogen has had a significant economic impact on oil palm cultivation in Colombia, and therefore poses a threat to oil palm cultivation in other regions of the World, especially in Southeast Asia, the largest producer of the crop. This study aimed to look at the ability of isolates from Malaysia, Colombia, and other regions to cross-infect Malaysian oil palm, durian, and cocoa and to develop specific biomarkers and assays for identification, detection, and diagnosis of P. palmivora as a key component for the oil palm biosecurity continuum in order to contain the disease especially at the ports of entry. We have developed specific molecular biomarkers to identify and detect Phytophthora palmivora using polymerase chain reaction (PCR) and real-time loop mediated isothermal amplification (rt-LAMP) in various sample types such as soil and plants. The limit of detection (DNA template, pure culture assay) for the PCR assay is 5.94 × 10-2 ng µl-1 and for rt-LAMP is 9.28 × 10-4 ng µl-1. Diagnosis using rt-LAMP can be achieved within 30 min of incubation. In addition, PCR primer pair AV3F/AV3R developed successfully distinguished the Colombian and Malaysian P. palmivora isolates.
A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da. Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4). The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself. In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab. The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH). Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait. At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant. The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH. The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.
The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs - including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2) - at the 'stress-responding' cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'-UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wild-type) or mutant - pGL4.38-Cyp2a5_StREMut and pGL4.38-Cyp2a5_XREMut - reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC50 and EC50 of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wild-type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.
Red palm weevil (RPW), Rhynchophorus ferrugineus, is a polyphagous insect that caused economic damage in various palm species, particularly coconut plantation in Malaysia. Therefore, entomopathogenic fungus Metarhizium anisopliae was being introduced in attempts to control biologically the RPW. The entomopathogenicity of an indigenous (Met-Gra4) and foreign (Met-TH) strains of M. anisopliae isolated from the soil of Malaysia and Thailand, were tested against RPW adults in laboratory bioassays at 50, 70, 90% relative humidity (RH). Bioassays indicate no significance differences in efficacy between both the conidia of M. anisopliae strains against RPW adults. Met-Gra4 showed the highest efficacy at 90% RH (LT50 = 6.17 days). However, LT50 only slightly differed from Met-TH (6.33 days; 90% RH). Scanning el ectron microscopy for the treated RPWs showed that Met-Gra4 (90% RH) was densely sporulated within the abdomen, while Met-TH can be found mainly across cuticular surface of RPW.
White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.
The detection of tetrodotoxin (TTX) and saxitoxin (STX) in dried salted yellow puffer fish (Xenopterus naritus) eggs bought from Satok Market, Kuching, Sarawak was carried out by mouse bioassay method. The amount of TTX and STX detected in the samples ranged from 95.6-195.5 Mouse Unit (MU)/g and 1.72-3.58 MU/g respectively. The results indicate that the dried salted eggs samples were found to contain TTX 9-20 times above the regulatory limit for human consumption (10 MU/g). Although detected, the amount of STX in salted eggs extract was slightly below the accepted threshold limit (4 MU/g). The local public in Sarawak should be educated on the potential danger of consuming dried salted puffer fish eggs in addition to the current warnings on puffer fish.
A cell-based assay to measure cytochrome P450 3A4 (CYP3A4) induction was developed to screen for potential CYP3A4 inducers. This 96-well format assay utilizes HepG2 cells transfected with a gene construct of CYP3A4 proximal promoter linked to green fluorescence protein (GFP) gene, and the expression of the GFP is then measured quantitatively. Bergamottin at 5 to 25 µmol/L produced low induction relative to the positive control. Both curcumin and lycopene were not found to affect the expression of GFP, suggesting no induction properties toward CYP3A4. Interestingly, resveratrol produced significant induction from 25 µmol/L onward, which was similar to omeprazole and may warrant further studies. In conclusion, the present study demonstrated that this cell-based assay can be used as a tool to evaluate the potential CYP3A4 induction properties of compounds. However, molecular docking data have not provided satisfactory pointers to differentiate between CYP3A4 inducers from noninducers or from inhibitors, more comprehensive molecular screening may be indicated.
Rapid enzyme microassays for the detection of resistance due to organophosphate and carbamate in individual field-collected strains of Culex quinquefasciatus adults were conducted. These tests allowed accurate differentiation by eye, on the basis of color changes of susceptible and resistant individuals. Two separate tests were conducted for the biochemical assays. In the insensitive acetylcholinesterase (AChE) test, acetylthiocholine iodide (ACTH) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were used as substrate and coupling agent respectively. The resulting yellow chromophore indicated AChE activity. Test results showed that the color intensity decreased as increasing concentrations of propoxur were added, thereby confirming the susceptibility of the enzyme to inhibitor. Assay of non-specific esterase however, indicated elevated levels which were correlated with degree of malathion resistance. Electrophoretic data revealed the presence of 2 esterase bands in all strains. It was concluded that such a pattern was not contributory to malathion resistance in adults.
Some chemicals in the environment possess the potential to interact with the endocrine system in the human body. Multiple receptors are involved in the endocrine system; estrogen receptor α (ERα) plays very important roles in endocrine activity and is the most studied receptor. Understanding and predicting estrogenic activity of chemicals facilitates the evaluation of their endocrine activity. Hence, we have developed a decision forest classification model to predict chemical binding to ERα using a large training data set of 3308 chemicals obtained from the U.S. Food and Drug Administration's Estrogenic Activity Database. We tested the model using cross validations and external data sets of 1641 chemicals obtained from the U.S. Environmental Protection Agency's ToxCast project. The model showed good performance in both internal (92% accuracy) and external validations (∼ 70-89% relative balanced accuracies), where the latter involved the validations of the model across different ER pathway-related assays in ToxCast. The important features that contribute to the prediction ability of the model were identified through informative descriptor analysis and were related to current knowledge of ER binding. Prediction confidence analysis revealed that the model had both high prediction confidence and accuracy for most predicted chemicals. The results demonstrated that the model constructed based on the large training data set is more accurate and robust for predicting ER binding of chemicals than the published models that have been developed using much smaller data sets. The model could be useful for the evaluation of ERα-mediated endocrine activity potential of environmental chemicals.
In this paper, we propose an easy-to-implement passive liquid valve (PLV) for the microfluidic compact-disc (CD). This valve can be implemented by introducing venting chambers to control the air flow of the source and destination chambers. The PLV mechanism is based on equalizing the main forces acting on the microfluidic CD (i.e., the centrifugal and capillary forces) to control the burst frequency of the source chamber liquid. For a better understanding of the physics behind the proposed PLV, an analytical model is described. Moreover, three parameters that control the effectiveness of the proposed valve, i.e., the liquid height, liquid density, and venting chamber position with respect to the CD center, are tested experimentally. To demonstrate the ability of the proposed PLV valve, microfluidic liquid switching and liquid metering are performed. In addition, a Bradford assay is performed to measure the protein concentration and evaluated in comparison to the benchtop procedure. The result shows that the proposed valve can be implemented in any microfluidic process that requires simplicity and accuracy. Moreover, the developed valve increases the flexibility of the centrifugal CD platform for passive control of the liquid flow without the need for an external force or trigger.
Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.
To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.
Natural products continue to play an important role as a source of biologically active substances for the development of new drug. Streptomyces, Gram-positive bacteria which are widely distributed in nature, are one of the most popular sources of natural antibiotics. Recently, by using a bioassay-guided fractionation, an antimalarial compound, Gancidin-W, has been discovered from these bacteria. However, this classical method in identifying potentially novel bioactive compounds from the natural products requires considerable effort and is a time-consuming process. Metabolomics is an emerging "omics" technology in systems biology study which integrated in process of discovering drug from natural products. Metabolomics approach in finding novel therapeutics agent for malaria offers dereplication step in screening phase to shorten the process. The highly sensitive instruments, such as Liquid Chromatography-Mass Spectrophotometry (LC-MS), Gas Chromatography-Mass Spectrophotometry (GC-MS), and Nuclear Magnetic Resonance ((1)H-NMR) spectroscopy, provide a wide range of information in the identification of potentially bioactive compounds. The current paper reviews concepts of metabolomics and its application in drug discovery of malaria treatment as well as assessing the antimalarial activity from natural products. Metabolomics approach in malaria drug discovery is still new and needs to be initiated, especially for drug research in Malaysia.
Human skin pigmentation is a result of constitutive and facultative pigmentation. Facultative pigmentation is frequently stimulated by UV radiation, pharmacologic drugs, and hormones whereby leads to the development of abnormal skin hyperpigmentation. To date, many state-of-art depigmenting compounds have been studied using in vitro model to treat hyperpigmentation problems for cosmetic dermatological applications; little attention has been made to compare the effectiveness of these depigmenting compounds and their mode of actions. In this present article, new and recent depigmenting compounds, their melanogenic pathway targets, and modes of action are reviewed. This article compares the effectiveness of these new depigmenting compounds to modulate several melanogenesis-regulatory enzymes and proteins such as tyrosinase (TYR), TYR-related protein-1 (TRP1), TYR-related protein-2 (TRP2), microphthalmia-associated transcription factor (MITF), extracellular signal-regulated kinase (ERK) and N-terminal kinases (JNK) and mitogen-activated protein kinase p38 (p38 MAPK). Other evidences from in vitro assays such as inhibition on melanosomal transfer, proteasomes, nitric oxide, and inflammation-induced melanogenesis are also highlighted. This article also reviews analytical techniques in different assays performed using in vitro model as well as their advantages and limitations. This article also provides an insight on recent finding and re-examination of some protocols as well as their effectiveness and reliability in the evaluation of depigmenting compounds. Evidence and support from related patents are also incorporated in this present article to give an overview on current patented technology, latest trends, and intellectual values of some depigmenting compounds and protocols, which are rarely highlighted in the literatures.
Tocotrienols and tocopherols are isoforms of vitamin E. Vitamin E may exhibit antioxidant, prooxidant and non-antioxidant activities depending upon circumstances. In this study, the effect of tocotrienols and α-tocopherol on the activities of HMG CoA reductase and cholesterol 7 α-hydroxylase was investigated. Pure tocotrienols were isolated from palm fatty acid distillate and pure α-tocopherol was obtained commercially. Guinea pigs were treated with different dosages of tocotrienols and α-tocopherol. After the treatment period, animals were sacrificed and liver microsomes were prepared. HMG CoA reductase and cholesterol 7α-hydroxylase were assayed using tracer techniques. Our results showed that the effects of tocotrienols and α-tocopherol on the activities of both the enzymes were dose-dependent. At low dosages, both tocotrienols and α-tocopherol exhibited an inhibitory effect on both the enzymes. Moreover, tocotrienols were a much stronger inhibitors than α-tocopherol. At high dosages, on the other hand, tocotrienols and α-tocopherol showed opposite effects on the enzymes. While tocotrienols continued to exhibit an inhibitory effect, α-tocopherol actually exhibited a stimulatory effect on both the enzymes. A possible explanation for this observation is suggested.
Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL-1. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.
Three species of tropical estuarine invertebrates were exposed to copper sulfate and cadmium chloride to investigate their potential as test specimens for sediment toxicity assays in the South-east Asian regions. The larvae of the reef sea urchin (Diadema setosum), the oyster (Crassostrea iradalei), and the mud crab (Scylla seratta Forskall) were used in the 48-hr assays with copper and cadmium as reference toxicants. In addition the sea urchin were tested for end point measurements at different stages of the larval development and a 60-min sperm bioassay. The study revealed that the sea urchin first cleavage, which is an assay end point and which takes place about 1 hr after fertilization, was the most sensitive stage for both toxicants, with copper being more toxic than cadmium. Sensitivity comparisons between the three invertebrate larvae revealed the mud crab zoea larvae to be most sensitive for cadmium with an LC50 value of 0.078 microgram/ml, while the sea urchin was more sensitive for copper, with EC50 values of 0.01 microgram/ml at the first cleavage stage and 0.04 microgram/ml at the pluteus larva stage. All the invertebrates tested gave responses that made them suitable test organisms for metal bioassays in the tropical estuarine environment.
Heterotrigona itama is a Malaysian stingless bee species that actively reared for meliponiculture. This stingless bee is cultivated in a commercial scale for its honey production, propolis and among the greatest commercial potential as crop pollinators. However, this species has been potentially exposed to agronomic practices, among which the use of synthetic insecticides against pests.The indirect toxicity effect of the post-insecticide had affected the mortalities of H. itama especially, to the foragers. Due to that, a study has been conducted to determine the lethal concentration of 50% (LC50) and 95% (LC95) of the selected insecticides against stingless bee forager workers through residual exposure. The bioassay test was conducted to the local stingless bee H. itama at Agricultural Research Station, Tenom. Four commonly used insecticides in crop protection; Deltamethrin, Chlorpyrifos, Cypermethrin and Malathion were tested at five concentrations that diluted with 500 ml of distilled water in three replications for each insecticide. Lethal concentrations (LC50 and LC95) were obtained from probit analysis after 1-hour dry residues exposure and 24-hour mortality observation. The result shows that; all four tested insecticides were harmful to H. itama through dry residue. Deltamethrin shows the higher value of LC50 (1.256 ml) and LC95 (3.582ml) that make it less toxic to the H. itama than cypermethrin, malathion, and chlorpyrifos, however, as the concentration gets higher it becomes more toxic.
T4 genotype Acanthamoeba are opportunistic pathogens that cause two types of infections, including vision-threatening Acanthamoeba keratitis (AK) and a fatal brain infection known as granulomatous amoebic encephalitis (GAE). Due to the existence of ineffective treatments against Acanthamoeba, it has become a potential threat to all contact lens users and immunocompromised patients. Metal nanoparticles have been proven to have various antimicrobial properties against bacteria, fungi, and parasites. Previously, different types of cobalt nanoparticles showed some promise as anti-acanthamoebic agents. In this study, the objectives were to synthesize and characterize the size, morphology, and crystalline structure of cobalt phosphate nanoparticles, as well as to determine the effects of different sizes of cobalt metal-based nanoparticles against A. castellanii. Cobalt phosphate octahydrate (CHP), Co3(PO4)2•8H2O, was synthesized by ultrasonication using a horn sonicator, then three different sizes of cobalt phosphates Co3(PO4)2 were produced through calcination of Co3(PO4)2•8H2O at 200 °C, 400 °C and 600 °C (CP2, CP4, CP6). These three types of cobalt phosphate nanoparticles were characterized using a field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analysis. Next, the synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. The overall results showed that 1.30 ± 0.70 µm of CHP microflakes demonstrated the best anti-acanthemoebic effects at 100 µg/mL, followed by 612.50 ± 165.94 nm large CP6 nanograins. However, amongst the three tested cobalt phosphates, Co3(PO4)2, the smaller nanoparticles had stronger antiamoebic effects against A. castellanii. During cell cytotoxicity analysis, CHP exhibited only 15% cytotoxicity against HeLa cells, whereas CP6 caused 46% (the highest) cell cytotoxicity at the highest concentration, respectively. Moreover, the composition and morphology of nanoparticles is suggested to be important in determining their anti-acathamoebic effects. However, the molecular mechanisms of cobalt phosphate nanoparticles are still unidentified. Nevertheless, the results suggested that cobalt phosphate nanoparticles hold potential for development of nanodrugs against Acanthamoeba.