Variable parameters in chemiluminescence assay, one of the methods used to assess the functional capacity of neutrophils, were evaluated for suitable adaptation locally. The use of pooled normal human serum as compared to single normal human serum in opsonizing particles for phagocytosis was found to exhibit lower chemiluminescence activity (reduction range of 30%-50%). A similar degree of depression was observed when the particles were opsonized using normal human serum in comparison to that using autologous serum. Different intensity of chemiluminescence was also noted when the opsonized particle used was the Oxford strain of Staphylococcus aureus (NCTC 6571) in contrast to a strain of Staphylococcus aureus isolated from a patient. The results obtained warrant clinicians to deliver appropriate samples as best they can when the chemiluminescence assay is requested.
Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.
In dengue mosquitoes, successful embryonic development and long lifespan are key determinants for the persistence of both virus and vector. Therefore, targeting the egg stage and vector lifespan would be expected to have greater impacts than larvicides or adulticides, both strategies that have lost effectiveness due to the development of resistance. Therefore, there is now a pressing need to find novel chemical means of vector control. Coffee contains many chemicals, and its waste, which has become a growing environmental concern, is as rich in toxicants as the green coffee beans; these chemicals do not have a history of resistance in insects, but some are lost in the roasting process. We examined whether exposure to coffee during embryonic development could alter larval eclosion and lifespan of dengue vectors. A series of bioassays with different coffee forms and their residues indicated that larval eclosion responses of Aedes albopictus and Ae. aegypti were appreciably lower when embryonic maturation occurred in environments containing coffee, especially roasted coffee crude extract (RCC). In addition, the lifespan of adults derived from eggs that hatched successfully in a coffee milieu was reduced, but this effect was less pronounced with roasted and green coffee extracts (RCU and GCU, respectively). Taken together, these findings suggested that coffee and its residues have embryocidal activities with impacts that are carried over onto the adult lifespan of dengue vectors. These effects may significantly reduce the vectorial capacity of these insects. Reutilizing coffee waste in vector control may also represent a realistic solution to the issues associated with its pollution.
Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe, while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L-1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L-1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general.
Dot blot assays have always been associated with antibodies as the main molecular recognition element, which are widely employed in a myriad of diagnostic applications. With the rising of aptamers as the equivalent molecular recognition elements of antibodies, dot blot assays are also one of the diagnostic avenues that should be scrutinized for their amenability with aptamers as the potential surrogates of antibodies. In this review, the stepwise procedures of an aptamer-based dot blot assays are underscored before reviewing the existing aptamer-based dot blot assays developed so far. Most of the applications center on monitoring the progress of SELEX and as the validatory assays to assess the potency of aptamer candidates. For the purpose of diagnostics, the current effort is still languid and as such possible suggestions to galvanize the move to spur the aptamer-based dot blot assays to a point-of-care arena are discussed.
Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.
In this research, we modify a previously developed assay for the quantification molybdenum blue to determine whether inhibitors to molybdate reduction in bacteria inhibits cellular reduction or inhibit the chemical formation of one of the intermediate of molybdenum blue; phosphomolybdate. We manage to prove that inhibition of molybdate reduction by phosphate and arsenate is at the level of phosphomolybdate and not cellular. We also prove that mercury is a physiological inhibitor to molybdate reduction. We suggest the use of this method to assess the effect of inhibitors and activators to molybdate reduction in bacteria.
Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, Km for the laboratory-prepared 10:4-phosphomolybdate is 2.56 +/- 0.25 mM (arbitrary concentration), whereas the apparent V(max) is 99.4 +/- 2.85 nmol Mo-blue min(-1) mg(-1) protein. The apparent Michaelis constant or Km for NADH as the electron donor is 1.38 +/- 0.09 mM, whereas the apparent V(max) is 102.6 +/- 1.73 nmol Mo-blue min(-1) mg(-l) protein. The apparent Km and V(max) for another electron donor, NADPH, is 1.43 +/- 0.10 mM and 57.16 +/- 1.01 nmol Mo-blue min(-1) mg(-1) protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V(max) obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.
A study was conducted on the long term effects of nine heavy metals on the Chironomus plumosus and Culicoides furens larvae. This study tested the effect of the heavy metals on several generations of the larvae to observe the formation of increased hardiness against pollutants present within the aquatic habitat. From this study it was observed that susceptibility or sensitivity to heavy metals decreased with LC50 values becoming larger indicating a decreased toxicity level. Significant variations (p < 0.05) were observed between first generation and third generation culicoides for all metals and at all concentrations. Variations between third and fourth generation culicoides were also significantly different (p < 0.05) with the exception of chromium at 25 degrees C and nickel and lead at every temperature range group. The variation between all generations 4, 5 and 6 was found to be insignificant (p > 0.05). This would indicate that metal tolerance would have occurred in these generations and the effect of metals was less toxic to the culicoides. Generation 9 was found to have LC50 values (p > 0.05) the same as the LC50 values obtained in third generation culicoides. Thus it would appear that heavy metal resistance was developed when the organisms were exposed to prolonged exposure of the heavy metals but was lost when the organisms were bred in non-contaminated water.
Routine surveillance on resistant status of field mosquito populations is important to implement suitable strategies in order to prevent pest outbreaks. WHO test kit bioassay is the most frequent bioassay used to investigate the susceptibility status of field-collected mosquitoes, as it is relatively convenient to be carried out in the field. In contrast, the topical application of active ingredient is less popular in investigating the susceptibility status of mosquitoes. In this study, we accessed the susceptibility status of Aedes albopictus Skuse collected from two dengue hotspots on Penang Island: Sungai Dua and Persiaran Mayang Pasir. Two active ingredients: permethrin and deltamethrin, were used. WHO test kit bioassay showed that both wild strains collected were susceptible to the two active ingredients; while topical application assay showed that they were resistant. This indicated that WHO test kit bioassay less sensitive to low level of resistance compared to topical application assay. Hence, topical application is expected to be more indicative when used in a resistance surveillance programme.
In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.
The allelochemical 2,4-di-tert-butylphenol (2,4-DTBP) is one of the natural compounds present in medicinal plants.
This compound has been reported to possess herbicidal properties. However, its effect on weed growth parameters is
unknown for it to be utilized in weed management. Hence, the herbicidal potential of the allelochemical 2,4-DTBP on the
root and leaf tissues of the grassy weed, Leptochloa chinensis (L.) Nees and the broadleaf weed, Hedyotis verticillata
(L.) Lam was investigated. After 2,4-DTBP treatment, both bioassay species had abnormal and much shorter root hairs
compared to those of untreated plants. The roots of H. verticillata were severely damaged with the root nodes turned
brown. The phytotoxic effect of 2,4-DTBP on L. chinensis and H. verticillata became apparent at seven days and 14 days
after treatment with symptoms of lamina wilting and necrosis, respectively. These results demonstrated that 2,4-DTBP
could be used as a natural herbicide for the control of L. chinensis and H. verticillata.
The present study focused on investigating the storage stability of oil-in-water (O/W) emulsions with high oil volume fractions prepared with palm olein-based diacylglycerol oil (POL-DAG)/soybean oil (SBO) blends at 25 °C. The incorporation of different ratios of oil blends significantly influenced (p < 0.05) the texture, color, droplet size distribution, and rheological parameters of the emulsions. Only emulsions incorporated with 10% to 20% POL-DAG in oil phase exhibited pseudoplastic behavior that fitted the Power Law model well. Furthermore, the O/W emulsions prepared with POL-DAG/SBO blends exhibited elastic properties, with G' higher than G". During storage, the emulsion was found to be less solid-like with the increase in tan δ values. All emulsions produced with POL-DAG/SBO blends also showed thixotropic behavior. Optical microscopy revealed that the POL-DAG incorporation above 40% caused aggregated droplets to coalesce and flocculate and, thus, larger droplet sizes were observed. The current results demonstrated that the 20% POL-DAG substituted emulsion was more stable than the control emulsion. The valuable insights gained from this study would be able to generate a lot more possible applications using POL-DAG, which could further sustain the competitiveness of the palm oil industry.
Plants have been a good source of therapeutic agents for thousands of years;
an impressive number of modern drugs used for treating human diseases are derived from
natural sources. The Theobroma cacao tree, or cocoa, has recently garnered increasing
attention and become the subject of research due to its antioxidant properties, which are
related to potential anti-cancer effects. In the past few years, identifying and developing
active compounds or extracts from the cocoa bean that might exert anti-cancer effects
have become an important area of health- and biomedicine-related research. This review
provides an updated overview of T. cacao in terms of its potential anti-cancer compounds
and their extraction, in vitro bioassay, purification, and identification. This article also
discusses the advantages and disadvantages of the techniques described and reviews the
processes for future perspectives of analytical methods from the viewpoint of anti-cancer
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.
The resistance status towards permethrin among the laboratory strain, the permethrin-selected strain and four field strains of Culex quinquefasciatus collected in Kuala Lumpur, Malaysia was determined using three standard laboratory methods: WHO larval bioassay, WHO adult bioassay and biochemical microplate assay. Cx. quinquefasciatus permethrin-selected strain larvae were the least susceptible to permethrin with a resistance ratio of 47.28-folds, whereas all field strain larvae of the same species were tolerant to permethrin with resistance ratios of more than 3-folds. In contrast, in adult stage, the permethrin exposed permethrin-selected strain (resistance ratio = 1.27) was found to be more susceptible to permethrin than all permethrin-exposed field strains (resistance ratios = 2.23-2.48). Complete mortalities for all strains of Cx. quinquefasciatus adults proved the effectiveness of the synergist; piperonyl butoxide (PBO). For the biochemical microplate assay, the reduction of the mean optical density of elevated oxidase activity of three field strains upon exposure to PBO confirmed the association between oxidase activity and permethrin tolerance. On the other hand, irregular patterns of the mean optical density of elevated oxidase activity in the laboratory strain, permethrin-selected strain and Jalan Fletcher strain illustrated the gene variation within these mosquito colonies as well as the involvement of other enzyme activities in the permethrin resistance occurred.
Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.
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.
The effect of the oil-spill dispersant Corexit 9527 on egg-hatching rate of Macrobrachium rosenbergii (de Man) was studied by using an innovated flow-through bioassay technique. This bioassay method relies on the fact that M. rosenbergii fertilized eggs when detached from the mother prawn were able to hatch artificially. The flow-through system generated a stable and good water quality environment for hatching the eggs successfully. The Corexit 9527 had a pronounced effect on hatching rate of the M. rosenbergii eggs. In the control, the hatching rate of the eggs was 95.55% +/- 1.74%. However, it was reduced drastically with increasing concentrations of Corexit 9527. A 100% inhibition of egg hatchability was found when the level of Corexit 9527 was higher than 250 mg litre(-1). The EC(50) and the EC(95) values estimated by the probit method were 80.4 +/- 5.5 mg litre(-1) and 193.5 +/- 39.9 mg litre(-1) respectively (P = 0.05). The recommended safety level of Corexit 9527 for M. rosenbergii in Malaysian estuarine waters is below 40 mg litre(-1).