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Sensitivity of two in vitro assays for evaluating plant activity against the infective stage of Haemonchus contortus strains

Al-Rofaai A, Rahman WA, Abdulghani M

Parasitol Res, 2013 Feb;112(2):893-8.
PMID: 22961237 DOI: 10.1007/s00436-012-3113-5

The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.

Matched MeSH terms: Biological Assay/methods
HPTLC based approach for bioassay-guided evaluation of antidiabetic and neuroprotective effects of eight essential oils of the Lamiaceae family plants

Romero Rocamora C, Ramasamy K, Meng Lim S, Majeed ABA, Agatonovic-Kustrin S

J Pharm Biomed Anal, 2020 Jan 30;178:112909.
PMID: 31618702 DOI: 10.1016/j.jpba.2019.112909

A high-performance thin-layer chromatography (HPTLC) method combined with effect-directed-analysis (EDA) was developed to screen the antioxidant, neuroprotective and antidiabetic effects in essential oils derived from lavender flower, lemon myrtle, oregano, peppermint, sage, and rosemary leaves (Lamiaceae family). HPTLC hyphenated with microchemical (DPPH•, p-anisaldehyde, and ferric chloride) derivatizations, was used to evaluate antioxidant activity, presence of phytosterols and terpenoids, and polyphenolic content, while the combination with biochemical (α-amylase and acetylcholine esterase (AChE) enzymatic) derivatizations was used to asses α-amylase and AChE inhibitory activities. The superior antioxidant activity of oregano leaf extract is attributed to the presence of high levels of aromatic compounds, like polyphenolic acids. The strongest α-amylase inhibition was observed in lemon myrtle and rosemary plus extracts due to the presence of monoterpenes. Rosemary and sage extracts exhibit the highest AChE inhibition activity, with 1 μL essential oils being more potent than the recommended daily dose of donepezil. This superior neuroprotection was attributed to the presences of di- and triterpenes that displayed strong AChE inhibition and antioxidant potential in DPPH• free radical assay. Antioxidant activity was related to phenolic content (R = 0.49), while α-amylase inhibitory activity was positively related to antioxidant activity (R = 0.20) and terpenoid/sterol content (R = 0.31). AChE inhibitory activity was correlated (R = 0.80) to the combined effect of phenolics and terpenoids. Thus, the superior AChE inhibitory and neuroprotection potential of rosemary and sage essential oils could be attributed to joint effects of main phenolic and terpene constituents. The hyphenated HPTLC method provided rapid bioanalytical profiling of highly complex essential oil samples.

Matched MeSH terms: Biological Assay/methods
Discovery of new depigmenting compounds and their efficacy to treat hyperpigmentation: Evidence from in vitro study

Lajis AFB, Ariff AB

J Cosmet Dermatol, 2019 Jun;18(3):703-727.
PMID: 30866156 DOI: 10.1111/jocd.12900

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.

Matched MeSH terms: Biological Assay/methods*
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
Fulltext Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour

Kumarasingha R, Karpe AV, Preston S, Yeo TC, Lim DSL, Tu CL, et al.

Int J Parasitol Drugs Drug Resist, 2016 12;6(3):171-178.
PMID: 27639945 DOI: 10.1016/j.ijpddr.2016.08.002

Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.

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*
Effect of copper and cadmium on three Malaysian tropical estuarine invertebrate larvae

Ramachandran S, Patel TR, Colbo MH

Ecotoxicol Environ Saf, 1997 Mar;36(2):183-8.
PMID: 9126437

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.

Matched MeSH terms: Biological Assay/methods*
Fuzzy logic and adaptive neuro-fuzzy inference system for characterization of contaminant exposure through selected biomarkers in African catfish

Karami A, Keiter S, Hollert H, Courtenay SC

Environ Sci Pollut Res Int, 2013 Mar;20(3):1586-95.
PMID: 22752811 DOI: 10.1007/s11356-012-1027-5

This study represents a first attempt at applying a fuzzy inference system (FIS) and an adaptive neuro-fuzzy inference system (ANFIS) to the field of aquatic biomonitoring for classification of the dosage and time of benzo[a]pyrene (BaP) injection through selected biomarkers in African catfish (Clarias gariepinus). Fish were injected either intramuscularly (i.m.) or intraperitoneally (i.p.) with BaP. Hepatic glutathione S-transferase (GST) activities, relative visceral fat weights (LSI), and four biliary fluorescent aromatic compounds (FACs) concentrations were used as the inputs in the modeling study. Contradictory rules in FIS and ANFIS models appeared after conversion of bioassay results into human language (rule-based system). A "data trimming" approach was proposed to eliminate the conflicts prior to fuzzification. However, the model produced was relevant only to relatively low exposures to BaP, especially through the i.m. route of exposure. Furthermore, sensitivity analysis was unable to raise the classification rate to an acceptable level. In conclusion, FIS and ANFIS models have limited applications in the field of fish biomarker studies.

Matched MeSH terms: Biological Assay/methods
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 Inhibition of oxidative stress and lipid peroxidation by anthocyanins from defatted Canarium odontophyllum pericarp and peel using in vitro bioassays

Khoo HE, Azlan A, Ismail A, Abas F, Hamid M

PLoS One, 2014;9(1):e81447.
PMID: 24416130 DOI: 10.1371/journal.pone.0081447

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD(+) and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection.

Matched MeSH terms: Biological Assay/methods*
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
A method to study the effects of chemical and biological reduction of molybdate to molybdenum blue in bacteria

Shukor Y, Shamsuddin B, Mohamad O, Ithnin K

Pak J Biol Sci, 2008 Feb 15;11(4):672-5.
PMID: 18817148

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.

Matched MeSH terms: Biological Assay/methods*
Cyp2a5 Promoter-based Gene Reporter Assay: A Novel Design of Cell-based Bioassay for Toxicity Prediction

Abu-Bakar A, Hu H, Lang MA

Basic Clin Pharmacol Toxicol, 2018 Sep;123 Suppl 5:72-80.
PMID: 29788535 DOI: 10.1111/bcpt.13046

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.

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
Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach

Arbab IA, Abdul AB, Sukari MA, Abdullah R, Syam S, Kamalidehghan B, et al.

J Ethnopharmacol, 2013 Jan 9;145(1):343-54.
PMID: 23178663 DOI: 10.1016/j.jep.2012.11.020

Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.

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
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*
Evaluation of variable parameters in neutrophil function test using chemiluminescence assay

Noah RM, Jais MR, Noh LM

Malays J Pathol, 1994 Dec;16(2):157-60.
PMID: 9053565

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.

Matched MeSH terms: Biological Assay/methods*
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*
Quantitative Flow Cytometry-Based Assays for Measuring Constitutive Secretion

Gordon DE, Shun-Shion AS, Asnawi AW, Peden AA

Methods Mol Biol, 2021;2233:115-129.
PMID: 33222131 DOI: 10.1007/978-1-0716-1044-2_8

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.

Matched MeSH terms: Biological Assay/methods*

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