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Utilizing Recombinant Reporter Henipaviruses to Conduct Antiviral Screening

Lo MK

Methods Mol Biol, 2023;2682:87-92.
PMID: 37610575 DOI: 10.1007/978-1-0716-3283-3_6

Spillovers of Nipah virus (NiV) from its pteropid bat reservoir into the human population continue to cause near-annual outbreaks of fatal encephalitis and respiratory disease in Bangladesh and India since its emergence in Malaysia over 20 years ago. The current lack of effective antiviral therapeutics against NiV merits further testing of compound libraries against NiV using rapid quantitative antiviral assays. The development of recombinant henipaviruses expressing reporter fluorescence and/or luminescence proteins has facilitated the screening of such libraries. In this chapter, we provide a basic protocol for both types of reporter viruses. Utilizing these live NiV-based reporter assays requires modest instrumentation and sidesteps the labor-intensive steps associated with traditional cytopathic effect or viral antigen-based assays.

Matched MeSH terms: Biological Assay
Fulltext Bioassay-guided fractionation of Melastoma malabathricum Linn. leaf solid phase extraction fraction and its anticoagulant activity

Khoo LT, Abdullah JO, Abas F, Tohit ER, Hamid M

Molecules, 2015 Feb 24;20(3):3697-715.
PMID: 25719740 DOI: 10.3390/molecules20033697

The aims of this study were to examine the bioactive component(s) responsible for the anticoagulant activity of M. malabathricum Linn. leaf hot water crude extract via bioassay-guided fractionation and to evaluate the effect of bioactive component(s) on the intrinsic blood coagulation pathway. The active anticoagulant fraction of F3 was subjected to a series of chromatographic separation and spectroscopic analyses. Furthermore, the effect of the bioactive component(s) on the intrinsic blood coagulation pathway was studied through immediate and time incubation mixing studies. Through Activated Partial Thromboplastin Time (APTT) assay-guided fractionation, Subfraction B was considered the most potent anticoagulant fraction. Characterisation of Subfraction B indicated that anticoagulant activity could partly be due to the presence of cinnamic acid and a cinnamic acid derivative. APTT assays for both the immediate and time incubation mixing were corrected back into normal clotting time range (35.4-56.3 s). In conclusion, cinnamic acid and cinnamic acid derivative from Subfraction B were the first such compounds to be discovered from M. malabathricum Linn. leaf hot water crude extract that possess anticoagulant activity. This active anticoagulant Subfraction B prolonged blood clotting time by causing factor(s) deficiency in the intrinsic blood coagulation pathway.

Matched MeSH terms: Biological Assay/methods*
Fulltext A comparison of assays for accurate copy number measurement of the low-affinity Fc gamma receptor genes FCGR3A and FCGR3B

Haridan US, Mokhtar U, Machado LR, Abdul Aziz AT, Shueb RH, Zaid M, et al.

PLoS One, 2015;10(1):e0116791.
PMID: 25594501 DOI: 10.1371/journal.pone.0116791

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method's performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs.

Matched MeSH terms: Biological Assay/methods*
Fulltext Freshwater-borne bacteria isolated from a Malaysian rainforest waterfall exhibiting quorum sensing properties

Tan WS, Yunos NY, Tan PW, Mohamad NI, Adrian TG, Yin WF, et al.

Sensors (Basel), 2014;14(6):10527-37.
PMID: 24932870 DOI: 10.3390/s140610527

One obvious requirement for concerted action by a bacterial population is for an individual to be aware of and respond to the other individuals of the same species in order to form a response in unison. The term "quorum sensing" (QS) was coined to describe bacterial communication that is able to stimulate expression of a series of genes when the concentration of the signaling molecules has reached a threshold level. Here we report the isolation from aquatic environment of a bacterium that was later identified as Enterobacter sp.. Chromobacterium violaceum CV026 and Escherichia coli [pSB401] were used for preliminary screening of N-acyl homoserine lactone (AHL) production. The Enterobacter sp. isolated was shown to produce two types of AHLs as confirmed by analysis using high resolution tandem mass spectrometry. To the best of our knowledge, this is the first documentation of an Enterobacter sp. that produced both 3-oxo-C6-HSL and 3-oxo-C8-HSL as QS signaling molecules.

Matched MeSH terms: Biological Assay/instrumentation*
Fulltext An evaluation of dose equivalence between synchrotron microbeam radiation therapy and conventional broad beam radiation using clonogenic and cell impedance assays

Ibahim MJ, Crosbie JC, Yang Y, Zaitseva M, Stevenson AW, Rogers PA, et al.

PLoS One, 2014;9(6):e100547.
PMID: 24945301 DOI: 10.1371/journal.pone.0100547

High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments.

Matched MeSH terms: Biological Assay/methods*
Standardization of a bottle assay--an indigenous method for laboratory and field monitoring of insecticide resistance and comparison with WHO adult susceptibility test

Elamathi N, Barik TK, Verma V, Velamuri PS, Bhatt RM, Sharma SK, et al.

Parasitol Res, 2014 Oct;113(10):3859-66.
PMID: 25098343 DOI: 10.1007/s00436-014-4054-y

The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 μg against An. stephensi and 2 μg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100% mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100% knock-down within 35 min and 100% mortality after 24 h holding period were observed in susceptible population, while in it was 50% knock-down in 1 h and 64% mortality after 24 h holding period for resistant population (50% mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.

Matched MeSH terms: Biological Assay/standards
Fulltext Monitoring resistance gene frequencies in Malaysian Culex quinquefasciatus Say adults using rapid non-specific esterase enzyme microassays

Lee HL, Tadano T

Southeast Asian J. Trop. Med. Public Health, 1994 Jun;25(2):371-3.
PMID: 7855659

The ability to identify the occurrence of different resistance genotypes in field populations of mosquito is considered important for the purpose of optimising chemical control operations. The recent development of rapid microassays of enzymes responsible for resistance has provided a means for rapidly assessing the genetic background of target mosquito populations. This concept is the topic of investigation in this study. Non-specific esterase activity, which is responsible for the resistance to organophosphates in Malaysian Culex quinquefasciatus Say adults, was determined in 3 field populations from Kuala Lumpur City using rapid enzyme assay. The optical density results were used to estimate the genotypic frequencies of the populations. Subsequently, time-dependent changes in the various frequencies were determined. Such techniques allowed rapid assessment of resistance genotypes for decision-making and its possible use in insect control merits further investigation.

Matched MeSH terms: Biological Assay/methods
Fulltext Determination of insecticide susceptibility in Culex quinquefasciatus Say adults by rapid enzyme microassays

Lee HL, Abimbola O, Singh KI

Southeast Asian J. Trop. Med. Public Health, 1992 Sep;23(3):458-63.
PMID: 1488701

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.

Matched MeSH terms: Biological Assay/methods
Analysis of paralytic shellfish poisoning toxin congeners by a sodium channel receptor binding assay

Usup G, Leaw CP, Cheah MY, Ahmad A, Ng BK

Toxicon, 2004 Jul;44(1):37-43.
PMID: 15225560

This study was carried out to characterize the detection and quantitation of several paralytic shellfish poisoning (PSP) toxin congeners using a receptor binding assay (RBA). This involved competitive binding of the toxin congeners against tritium-labeled STX for receptor sites on rat brain sodium channels. Competitive binding curves were described by a four-parameter logistic equation. Half-saturation values (EC(50)) ranged from 4.38 nM for STX to 142 nM for GTX5. Receptor binding affinity was in the order STX>GTX1/4>neoSTX>GTX2/3>dcSTX>GTX5, and this was similar to the order of mouse toxicity of these congeners. Predicted toxin concentrations from observed STXeq values and EC(50) ratios relative to STX were within 20% or better of the actual concentrations used in the assay. In contrast predicted toxin concentrations using mouse toxicity ratios relative to STX did not provide a good match to actual concentrations, except for GTX1/4. This study has shown that the rat brain sodium channel RBA will provide a reliable integration of total toxicity of various PSP toxin congeners present in a sample.

Matched MeSH terms: Biological Assay/methods*
Probe-specific loop-mediated isothermal amplification magnetogenosensor assay for rapid and specific detection of pathogenic Leptospira

Nurul Najian AB, Foo PC, Ismail N, Kim-Fatt L, Yean CY

Mol Cell Probes, 2019 04;44:63-68.
PMID: 30876924 DOI: 10.1016/j.mcp.2019.03.001

This study highlighted the performance of the developed integrated loop-mediated isothermal amplification (LAMP) coupled with a colorimetric DNA-based magnetogenosensor. The biosensor operates through a DNA hybridization system in which a specific designed probe captures the target LAMP amplicons. We demonstrated the magnetogenosensor assay by detecting pathogenic Leptospira, which causes leptospirosis. The color change of the assay from brown to blue indicated a positive result, whereas a negative result was indicated by the assay maintaining its brown color. The DNA biosensor was able to detect DNA at a concentration as low as 200 fg/μl, which is equivalent to 80 genomes/reaction. The specificity of the biosensor assay was 100% when it was evaluated with 172 bacterial strains. An integrated LAMP and probe-specific magnetogenosensor was successfully developed, promising simple and rapid visual detection in clinical diagnostics and service as a point-of-care device.

Matched MeSH terms: Biological Assay/methods*
Fulltext Evaluation of the bioactivity of novel spiroisoxazoline typecompounds against normal and cancer cell lines

Najim N, Bathich Y, Zain MM, Hamzah AS, Shaameri Z

Molecules, 2010 Dec 17;15(12):9340-53.
PMID: 21169884 DOI: 10.3390/molecules15129340

The aim of this study was to investigate the in vitro cellular activity of novel spiroisoxazoline type compounds against normal and cancer cell lines from lung tissue (Hs888Lu), neuron-phenotypic cells (SH-SY5Y), neuroblastoma (SH-SY5Y), human histiocytic lymphoma (U937), lung cancer (A549), and leukaemia (HL-60). Our bioassay program revealed that the spiroisoxazoline type compounds show cytotoxicity only in lymphoma cell lines, which is in contrast with the pyrrolidine precursor of these spiroisoxazoline compounds, where significant cytotoxicity is seen in all normal and cancer cell lines. These data suggest a tumour-specific mechanism of action. In addition these data also show that spiroisoxazoline compounds are non-toxic in the human neuronphenotypic neuroblastoma SH-SY5Y cell line, and furthermore that they might protect cells from neurodegenerative disease.

Matched MeSH terms: Biological Assay/methods
Antibacterial and Antifouling Properties of the Horseshoe Crab Carcinoscorpius rotundicauda

Mohd Faizal MN, Ismail N, M S Eldeen I, Mariam T

Pak J Biol Sci, 2021 Jan;24(5):579-587.
PMID: 34486333 DOI: 10.3923/pjbs.2021.579.587

Background and Objective: Horseshoe crabs are widely used in both traditional and modern pharmaceutical applications. Most of the previous studies on horseshoe crabs focused on their blood which contains hemolymph and amoebocyte lysate. This study aimed to determine the potential antibacterial and antifouling properties of different extracts from the carapace and the book gills of Carcinoscorpius rotundicauda. Materials and Methods: The crude extracts were subjected to the bioactivity tests using the disc-diffusion and the inhibition of biofilm-formation measurement assays, for both the antibacterial and antifouling activities respectively. Results: The results obtained indicated that the carapace extracts had stronger antibacterial and antifouling effects compared to the book gills extracts. Extracts obtained from the male displayed more activity compared to the extracts from the female with a few exceptions. Methanol and acetone carapace crude extracts showed the best overall performance. A sterol compound was isolated from the carapace acetone extracts of the male of C. rotundicauda. However, the compound did not display strong activity compared to the crude extract. The compound might be contributing to the observed activity with other components through a synergistic effect. Conclusion: The presence of antibacterial and antifouling activities in the carapace and book gills extracts could be added to the complexity of the defence mechanisms of horseshoe crabs. The results of this study, therefore, may contribute to the knowledge of the defence mechanisms of C. rotundicauda. Further research is needed to determine the bioactivities of other parts of the animal and to explore their potential applications.

Matched MeSH terms: Biological Assay/methods
Bioassay-guided detection, identification and assessment of antibacterial and anti-inflammatory compounds from olive tree flower extracts by high-performance thin-layer chromatography linked to spectroscopy

Agatonovic-Kustrin S, Wong S, Dolzhenko AV, Gegechkori V, Morton DW

J Pharm Biomed Anal, 2024 Feb 15;239:115912.
PMID: 38128161 DOI: 10.1016/j.jpba.2023.115912

Olive trees are one of the most widely cultivated fruit trees in the world. The chemical compositions and biological activities of olive tree fruit and leaves have been extensively researched for their nutritional and health-promoting properties. In contrast, limited data have been reported on olive flowers. The present study aimed to analyse bioactive compounds in olive flower extracts and the effect of fermentation-assisted extraction on phenolic content and antioxidant activity. High-performance thin-layer chromatography (HPTLC) hyphenated with the bioassay-guided detection and spectroscopic identification of bioactive compounds was used for the analysis. Enzymatic and bacterial in situ bioassays were used to detect COX-1 enzyme inhibition and antibacterial activity. Multiple zones of antibacterial activity and one zone of COX-1 inhibition were detected in both, non-fermented and fermented, extracts. A newly developed HPTLC-based experimental protocol was used to measure the high-maximal inhibitory concentrations (IC50) for the assessment of the relative potency of the extracts in inhibiting COX-1 enzyme and antibacterial activity. Strong antibacterial activities detected in zones 4 and 7 were significantly higher in comparison to ampicillin, as confirmed by low IC50 values (IC50 = 57-58 µg in zone 4 and IC50 = 157-167 µg in zone 7) compared to the ampicillin IC50 value (IC50 = 495 µg). The COX-1 inhibition by the extract (IC50 = 76-98 µg) was also strong compared to that of salicylic acid (IC50 = 557 µg). By comparing the locations of the bands to coeluted standards, compounds from detected bioactive bands were tentatively identified. The eluates from bioactive HPTLC zones were further analysed by FTIR NMR, and LC-MS spectroscopy. Multiple zones of antibacterial activity were associated with the presence of triterpenoid acids, while COX-1 inhibition was related to the presence of long-chain fatty acids.

Matched MeSH terms: Biological Assay/methods
Fulltext Bioassay-Guided Different Extraction Techniques of Carica papaya (Linn.) Leaves on In Vitro Wound-Healing Activities

Soib HH, Ismail HF, Husin F, Abu Bakar MH, Yaakob H, Sarmidi MR

Molecules, 2020 Jan 24;25(3).
PMID: 31991676 DOI: 10.3390/molecules25030517

Herbal plants are traditionally utilized to treat various illnesses. They contain phytochemicals that can be extracted using conventional methods such as maceration, soxhlet, and boiling, as well as non-conventional methods including ultrasonic, microwave, and others. Carica papaya leaves have been used for the treatment of dengue, fungal, and bacterial infections as well as an ingredient in anti-aging products. Phytochemicals analysis detected the presence of kaempferol, myricetin, carpaine, pseudocarpaine, dehydrocarpaine I and II, ferulic acid, caffeic acid, chlorogenic acid, β-carotene, lycopene, and anthraquinones glycoside. Conventional preparation by boiling and simple maceration is practical, simple, and safe; however, only polar phytochemicals are extracted. The present study aims to investigate the effects of three different non-conventional extraction techniques (ultrasonic-assisted extraction, reflux, and agitation) on C. papaya phytochemical constituents, the antioxidant capacity, and wound-healing activities. Among the three techniques, the reflux technique produced the highest extraction yield (17.86%) with the presence of saponins, flavonoids, coumarins, alkaloids, and phenolic metabolites. The reflux technique also produced the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging with an IC50 value of 0.236 mg/mL followed by ultrasonic-assisted extraction (UAE) (IC50: 0.377 mg/mL) and agitation (IC50: 0.404 mg/mL). At tested concentrations (3.125 µg/mL to 500 µg/mL), all extracts do not exhibit a cytotoxicity effect on the human skin fibroblast, HSF1184. Interestingly, reflux and UAE were active fibroblast proliferators that support 85% (12.5 µg/mL) and 41% (6.25 µg/mL) better cell growth, respectively. Additionally, during the early 24 h of the scratch assay, the migration rate at 12.5 µg/mL was faster for all extracts with 51.8% (reflux), 49.3% (agitation), and 42.5% (UAE) as compared to control (21.87%). At 48 h, proliferated cells covered 78.7% of the scratch area for reflux extract, 63.1% for UAE, 61% for agitation, and 42.6% for control. Additionally, the collagen synthesis was enhanced for 31.6% and 65% after 24 and 48 h of treatment for reflux. An HPLC-MS/MS-QTOF (quadruple time-of-flight) analysis of reflux identified nine phytochemicals, including carpaine, kaempferol 3-(2G-glucosylrutinoside), kaempferol 3-(2″-rhamnosylgalactoside), 7-rhamnoside, kaempferol 3-rhamnosyl-(1->2)-galactoside-7-rhamnoside, luteolin 7-galactosyl-(1->6)-galactoside, orientin 7-O-rhamnoside, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, palmitic amide, and 2-hexaprenyl-6-methoxyphenol. The results suggested that reflux was the best technique as compared to ultrasonic and agitation.

Matched MeSH terms: Biological Assay*
The screening of extracts from Goniothalamus scortechinii, Aralidium pinnatifidum and Andrographis paniculata for anti-malarial activity using the lactate dehydrogenase assay

Siti Najila MJ, Noor Rain A, Mohamad Kamel AG, Syed Zahir SI, Khozirah S, Lokman Hakim S, et al.

J Ethnopharmacol, 2002 Oct;82(2-3):239-42.
PMID: 12242001

Goniothalamus scortechinii, Andrographis paniculata and Aralidium pinnatifidum were selected for the study based on their ethnomedicinal values. They were screened for anti-malarial activity towards Plasmodium falciparum in vitro using the lactate dehydrogenase (LDH) assay. The crude extract of G. scortechinii exhibited the most potent schizonticidal activity compared to the other extracts. It is effective against both the chloroquine resistant isolate, Gombak A and the sensitive strain, D10 of Plasmodium falciparum. Furthermore a better IC(50) value was obtained against the resistant strain, (9 microg/ml) compared to the sensitive strain, 40 microg/ml. When the crude extract was fractionated into 3 fractions, the chloroform fraction yielded the best activity, exhibiting equipotency against both strains of parasite used; IC(50) of 23.53 microg/ml against Gombak A and 21.06 microg/ml against D10.

Matched MeSH terms: Biological Assay/methods; Biological Assay/statistics & numerical data
Fulltext Voltage-sensitive sodium channel (Vssc) mutations associated with pyrethroid insecticide resistance in Aedes aegypti (L.) from two districts of Jeddah, Kingdom of Saudi Arabia: baseline information for a Wolbachia release program

Endersby-Harshman NM, Ali A, Alhumrani B, Alkuriji MA, Al-Fageeh MB, Al-Malik A, et al.

Parasit Vectors, 2021 Jul 12;14(1):361.
PMID: 34247634 DOI: 10.1186/s13071-021-04867-3

BACKGROUND: Dengue suppression often relies on control of the mosquito vector, Aedes aegypti, through applications of insecticides of which the pyrethroid group has played a dominant role. Insecticide resistance is prevalent in Ae. aegypti around the world, and the resulting reduction of insecticide efficacy is likely to exacerbate the impact of dengue. Dengue has been a public health problem in Saudi Arabia, particularly in Jeddah, since its discovery there in the 1990s, and insecticide use for vector control is widespread throughout the city. An alternative approach to insecticide use, based on blocking dengue transmission in mosquitoes by the endosymbiont Wolbachia, is being trialed in Jeddah following the success of this approach in Australia and Malaysia. Knowledge of insecticide resistance status of mosquito populations in Jeddah is a prerequisite for establishing a Wolbachia-based dengue control program as releases of Wolbachia mosquitoes succeed when resistance status of the release population is similar to that of the wild population.
METHODS: WHO resistance bioassays of mosquitoes with deltamethrin, permethrin and DDT were used in conjunction with TaqMan® SNP Genotyping Assays to characterize mutation profiles of Ae. aegypti.
RESULTS: Screening of the voltage-sensitive sodium channel (Vssc), the pyrethroid target site, revealed mutations at codons 989, 1016 and 1534 in Ae. aegypti from two districts of Jeddah. The triple mutant homozygote (1016G/1534C/989P) was confirmed from Al Safa and Al Rawabi. Bioassays with pyrethroids (Type I and II) and DDT showed that mosquitoes were resistant to each of these compounds based on WHO definitions. An association between Vssc mutations and resistance was established for the Type II pyrethroid, deltamethrin, with one genotype (989P/1016G/1534F) conferring a survival advantage over two others (989S/1016V/1534C and the triple heterozygote). An indication of synergism of Type I pyrethroid activity with piperonyl butoxide suggests that detoxification by cytochrome P450s accounts for some of the pyrethroid resistance response in Ae. aegypti populations from Jeddah.
CONCLUSIONS: The results provide a baseline for monitoring and management of resistance as well as knowledge of Vssc genotype frequencies required in Wolbachia release populations to ensure homogeneity with the target field population. Vssc mutation haplotypes observed show some similarity with those from Ae. aegypti in southeast Asia and the Indo-Pacific, but the presence of the triple mutant haplotype in three genotypes indicates that the species in this region may have a unique population history.

Matched MeSH terms: Biological Assay/methods; Biological Assay/statistics & numerical data
Turning cigarette butt waste into an alternative control tool against an insecticide-resistant mosquito vector

Dieng H, Rajasaygar S, Ahmad AH, Ahmad H, Rawi CS, Zuharah WF, et al.

Acta Trop, 2013 Dec;128(3):584-90.
PMID: 23999373 DOI: 10.1016/j.actatropica.2013.08.013

Annually, 4.5 trillion cigarette butts (CBs) are flicked into our environment. Evidence exists that CB waste is deadly to aquatic life, but their lethality to the aquatic life of the main dengue vector is unknown. CBs are full of toxicants that occur naturally, during planting and manufacturing, which may act as larvicidal agents. We assessed Aedes aegypti vulnerability to Marlboro butts during its development. Overall, CBs showed insecticidal activities against larvae. At early phases of development, mortality rates were much higher in two CBs solution (2CBSol) and 3CBSol microcosms (MICRs). Larval survival gradually decreased with development in 1CBSol-MICRs. However, in great presence of CBs, mortality was high even for the late developmental stages. These results suggest that A. aegypti larvae are vulnerable to CB presence in their habitats, but this effect was seen most during the early developmental phases and in the presence of increased amounts of cigarette remnants. CB filters are being used as raw material in many sectors, i.e., brick, art, fashion, plastic industries, as a practical solution to the pollution problem, the observed butt waste toxicity to mosquito larvae open new avenues for the identification of novel insecticide products.

Matched MeSH terms: Biological Assay
Fulltext Microsphere integrated microfluidic disk: synergy of two techniques for rapid and ultrasensitive dengue detection

Hosseini S, Aeinehvand MM, Uddin SM, Benzina A, Rothan HA, Yusof R, et al.

Sci Rep, 2015;5:16485.
PMID: 26548806 DOI: 10.1038/srep16485

The application of microfluidic devices in diagnostic systems is well-established in contemporary research. Large specific surface area of microspheres, on the other hand, has secured an important position for their use in bioanalytical assays. Herein, we report a combination of microspheres and microfluidic disk in a unique hybrid platform for highly sensitive and selective detection of dengue virus. Surface engineered polymethacrylate microspheres with carefully designed functional groups facilitate biorecognition in a multitude manner. In order to maximize the utility of the microspheres' specific surface area in biomolecular interaction, the microfluidic disk was equipped with a micromixing system. The mixing mechanism (microballoon mixing) enhances the number of molecular encounters between spheres and target analyte by accessing the entire sample volume more effectively, which subsequently results in signal amplification. Significant reduction of incubation time along with considerable lower detection limits were the prime motivations for the integration of microspheres inside the microfluidic disk. Lengthy incubations of routine analytical assays were reduced from 2 hours to 5 minutes while developed system successfully detected a few units of dengue virus. Obtained results make this hybrid microsphere-microfluidic approach to dengue detection a promising avenue for early detection of this fatal illness.

Matched MeSH terms: Biological Assay
Development and Validation of Decision Forest Model for Estrogen Receptor Binding Prediction of Chemicals Using Large Data Sets

Ng HW, Doughty SW, Luo H, Ye H, Ge W, Tong W, et al.

Chem Res Toxicol, 2015 Dec 21;28(12):2343-51.
PMID: 26524122 DOI: 10.1021/acs.chemrestox.5b00358

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.

Matched MeSH terms: Biological Assay
Improved sensitivity of lateral flow assay using paper-based sample concentration technique

Tang R, Yang H, Choi JR, Gong Y, Hu J, Feng S, et al.

Talanta, 2016 May 15;152:269-76.
PMID: 26992520 DOI: 10.1016/j.talanta.2016.02.017

Lateral flow assays (LFAs) hold great promise for point-of-care testing, especially in resource-poor settings. However, the poor sensitivity of LFAs limits their widespread applications. To address this, we developed a novel device by integrating dialysis-based concentration method into LFAs. The device successfully achieved 10-fold signal enhancement in Human Immunodeficiency Virus (HIV) nucleic acid detection with a detection limit of 0.1nM and 4-fold signal enhancement in myoglobin (MYO) detection with a detection limit of 1.56ng/mL in less than 25min. This simple, low-cost and portable integrated device holds great potential for highly sensitive detection of various target analytes for medical diagnostics, food safety analysis and environmental monitoring.

Matched MeSH terms: Biological Assay

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