A convenient fluorimetry-based degranulation assay using RBL-2H3 cells.

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as ß-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of ß-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation.

Screening for frequently detected quaternary ammonium mixture systems in waters based on frequent itemset mining and prediction of their toxicity.

Screening and prioritizing research on frequently detected mixture systems in the environment is of great significance, as conducting toxicity testing on all mixtures is impractical. Therefore, the frequent itemset mining (FIM) was introduced and applied in this paper to identify variables that commonly co-occur in a dataset. Based on the dataset of the quaternary ammonium compounds (QACs) in the water environment, the four frequent QAC mixture systems with detection rate ≥ 35 % were found, including [BDMM]+Cl--[BTMM]+Cl- (M1), [BDMM]+Cl--[BHMM]+Cl- (M2), [BTMM]+Cl- -[BHMM]+Cl- (M3), and [BDMM]+Cl--[BTMM]+Cl--[BHMM]+Cl- (M4). [BDMM]+Cl-, [BTMM]+Cl-, and [BHMM]+Cl- are benzyl dodecyl dimethyl ammonium chloride, benzyl tetradecyl dimethyl ammonium chloride, and benzyl hexadecyl dimethyl ammonium chloride, respectively. Then, the toxicity of the representative mixture rays and components for the four frequently detected mixture systems was tested using Vibrio qinghaiensis sp.-Q67 (Q67) as a luminescent indicator organism at 0.25 and 12 h. The toxicity of the mixtures was predicted using concentration addition (CA) and independent action (IA) models. It was shown that both the components and the representative mixture rays for the four frequently detected mixture systems exhibited obvious acute and chronic toxicity to Q67, and their median effective concentrations (EC50) were below 7 mg/L. Both CA and IA models predicted the toxicity of the four mixture systems well. However, the CA model had a better predictive ability for the toxicity of the M3 and M4 mixtures than IA at 12 h.

Microplate-Based Cell Viability Assay as a Cost-Effective Alternative to Flow Cytometry for Microalgae Analysis.

Cell viability is a critical indicator for assessing culture quality in microalgae cultivation for biorefinery and bioremediation. Fluorescent dyes that distinguish viable from nonviable cells can enable viability quantification based on the percentage of live cells. However, fluorescence analysis using the typical flow cytometry method is costly and impractical for industrial applications. To address this, we developed new microplate assays utilizing fluorescein diacetate as a live cell stain and erythrosine B as a dead cell stain. These assays provide a low-cost, simple, and reliable method of assessing cell viability. The proposed microplate assays were successfully applied to monitor the viability of the microalgae Dunaliella viridis under carbon and nitrogen limitation stresses and demonstrated good agreement with flow cytometry measurements. We conducted a systematic investigation of the effects of dye concentration, incubation time, and background fluorescence on the microplate assays' performance. Further, we provide a comprehensive review of commonly used fluorescent dyes for microalgae staining, discuss strategies to enhance assay performance, and offer recommendations for dye selection and protocol development. This study presents a comprehensive new method for microplate-based viability analysis, providing valuable insights for future microalgae viability assessments and applications.

New tetrahydropyrimidine-1,2,3-triazole clubbed compounds: Antitubercular activity and Thymidine Monophosphate Kinase (TMPKmt) inhibition.

Two series of new tetrahydropyrimidine (THPM)-1,2,3-triazole clubbed compounds were designed, synthesized and screened for their antitubercular (anti-TB) activity against M. tuberculosis H37Rv strain using microplate alamar blue assay (MABA). The most active compounds 5c, 5d, 5e and 5f were further examined for their cytotoxicity against the growth of RAW 264.7 mouse macrophage cells using MTT assay. The four compounds showed safety profiles better than or comparable to that of ethambutol (EMB). These compounds were evaluated for their inhibition activity against mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt). Compounds 5c and 5e were the most potent exhibiting comparable inhibition activity to that of the natural substrate deoxythymidine monophosphate (dTMP). An in silico study was performed including docking of the most active compounds 5c and 5e into the TMPKmt (PDB: ID 1G3U) binding pocket in addition to prediction of their physicochemical and pharmacokinetic properties to explore the overall activity of these anti-TB candidates. Compounds 5c and 5e are promising anti-TB agents and TMPKmt inhibitors with acceptable oral bioavailability, physicochemical and pharmacokinetic properties.

A novel, quantitative clot retraction assay to evaluate platelet function.

Blood platelets are crucial to prevent excessive bleeding following injury to blood vessels. Platelets are crucial for the formation of clots and for clot strength. Platelet activation involves aggregation, attachment to fibrin and clot retraction. Most assays that address platelet function measure platelet aggregation, not clot retraction. Here, we describe a 96-well-based clot retraction assay that requires a relatively short runtime and small sample volume. The assay involves continuous optical density monitoring of platelet-rich plasma that is activated with thrombin. The data can be analyzed using time-series analytical tools to generate quantitative information about different phases of clot formation and clot retraction. The assay demonstrated good repeatability and reproducibility and was robust to different calcium concentrations. Impairment of platelet bioenergetics, actin polymerization, fibrin interaction, and signaling significantly affected clot retraction and was detected and showed good agreement with light transmission aggregometry, suggesting that clot retraction is predictive of platelet function. Using this microplate clot retraction assay, we showed a significant difference in platelets stored in autologous plasma compared with platelet additive solution after 7 days of room temperature storage.

Platelets are cell fragments in the blood that are necessary for clot formation. They are crucial to preventing excessive bleeding following trauma. To form clots, platelets clump (aggregate) and attach to fibrin protein and cells inside the blood vessels to form strong web-like structures. Platelets also contract to pull the edges of the wound close. Most measurements of platelet function involve aggregation. This paper focuses on platelet contraction. Here, we describe a new assay to measure platelets contraction that is repeatable and reproducible. The assay uses standard and common laboratory equipment and can be performed by most laboratory personnel and has the potential to detect clinical pathologies of clot formation. The assay could be developed for bedside patient care where platelet function could be assessed rapidly and assist in the diagnosis of coagulation and platelet disorders.

Hydrogel capsule-based digital quantitative polymerase chain reaction.

Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.

The Antifungal Effect of Pyroligneous Acid on the Phytopathogenic Fungus Botrytis cinerea.

In recent years, climate change has intensified harsh periods of rain alternating with periods of drought, leading to an increase in the presence of phytopathogenic fungi. In this study, we want to analyse the antifungal properties of pyroligneous acid against the fungal phytopathogen Botrytis cinerea. Through the inhibition test, we observed that the application of different dilutions of pyroligneous acid rarefied the growth of the fungal mycelium. Furthermore, we have seen through the metabolic profile that B. cinerea is not able to use pyroligneous acid as a resource or even grow in close contact with this resource. Moreover, we observed that the pre-incubation of the fungus in pyroligneous acid leads to a reduction in biomass production. These results give us hope for the possible use of this natural substance as a possible substance to protect plantations from pathogen attacks.

Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro.

Tissue fibrosis is characterized by chronic fibroblast activation and consequently excessive accumulation of collagen-rich extracellular matrix. In vitro microplate-based assays are essential to investigate the underlying mechanism and the effect of antifibrotic drugs. In this study, in the absence of a gold-standard method, we optimized a simple, cost-effective, Sirius Red-based colorimetric measurement to determine the collagen production of fibroblasts grown on 96-well tissue culture plates. Based on our findings, the use of a serum-free medium is recommended to avoid aspecific signals, while ascorbate supplementation increases the collagen production of fibroblasts. The cell-associated collagens can be quantified by Sirius Red staining in acidic conditions followed by alkaline elution. Immature collagens can be precipitated from the culture medium by acidic Sirius Red solution, and after subsequent centrifugation and washing steps, their amount can be also measured. Increased attention has been paid to optimizing the assay procedure, including incubation time, temperature, and solution concentrations. The resulting assay shows high linearity and sensitivity and could serve as a useful tool in fibrosis-related basic research as well as in preclinical drug screening.

Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.

An optimized protocol for estimating cellulase activity in biological samples.

Cellulase is a microbial enzyme responsible for degrading the ß-1,4 glycoside bond in polysaccharide cellulose, which is abundant in various animal foodstuffs. Cellulase is an important industrial enzyme used for various purposes, including biopolishing textile fibers, softening garments, biostoning denim fabric, and removing excess color from textiles. In the food industry, cellulase is combined with pectinase and hemicellulase. Therefore, the need for a reliable, fast, and inexpensive cellulase activity protocol that could be used with diverse biological and environmental samples is great. This study developed a novel method to quantify cellulase activity using picric acid (PCA), which reacts with generated glucose molecules to produce mahogany red picramic acid. This PCA-cellulase method uses sodium hydroxide instead of sodium carbonate to provide alkalinity in the reaction solution, increasing the stability of picramic acid and the sensitivity and linearity of the reaction. It also overcomes the limitations of previous methods. It is notable for its dependence on few chemicals with low concentrations compared to previous methods that depend on many chemicals with high concentrations. The PCA-cellulase method was optimized using the Box-Behnken design, and its accuracy was determined using a response surface approach. A Bland-Altman cellulase activity graph was used to validate the PCA-cellulase method with a correlation coefficient of 0.9991. Therefore, the novel PCA-cellulase method provides accurate results that are comparable to existing methods.

A low-cost, enzyme-coupled fluorescent assay for rapid quantification of glycolysis rate of cells.

Glycolysis is the predominant energy-yielding metabolic pathway in most cancer cells and rapidly proliferating cells. Currently available methods for glycolysis rate analysis are either time-consuming or cost-intensive/specialized equipment-dependent. The present study demonstrates a convenient, fast, and low-cost enzyme-coupled fluorometric assay for rapid quantification of glycolysis rate in small amount of cells. This assay involves the oxidation of cell-secreted lactate to produce hydrogen peroxide (H2O2) and subsequent conversion of Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) to fluorometric resorufin, in the presence of lactate oxidase (LOx) and peroxidase. High detection sensitivity and stability were realized by optimization of assay medium composition, enzyme and substrate concentration, and assay procedure. The lower limit of detection on HeLa cells was achieved on 50 cells per sample and the optimized linear range of the detection was 250-7000 cells per sample (r2 = 0.9842). The repetitive intraday and interday measurements of HeLa cell provided small variance and were highly agreeable with the results of endpoint method, which is a conventional validated method but detects lactate in relatively long time of larger cell population. The present assay was successfully applied on measuring the glycolytic parameters of human cancer cells (HeLa, HepG2) and mouse immune cells (T cells, macrophages), indicating great potential for wide application in cancerous and immunological research.

Non-destructive investigation of extracellular enzyme activities and kinetics in intact freshwater biofilms with mineral beads as carriers.

Current procedures for fluorometric detection of extracellular hydrolytic enzyme activities in intact aquatic biofilms are very laborious and insufficiently standardized. To facilitate the direct determination of a multitude of enzymatic parameters without biofilm disintegration, a new approach was followed. Beads made of different mineral materials were subjected to biofilm growth in various aquatic environments. After biofilm coating, the beads were singly placed in microplate wells, containing the required liquid analytical medium and a fluorogenic substrate. Based on fluorometric detection of the enzymatically generated reaction products, enzyme activities and kinetics were determined. Mean enzymatic activities of ceramic bead-attached biofilms grown in a natural stream followed the decreasing sequence L-alanine aminopeptidase > L-leucine aminopeptidase > phosphomonoesterase > ß-glucosidase > phosphodiesterase > α-glucosidase > sulfatase. After one week of exposure, the relative standard deviations of enzyme activities ranged from 21 to 67%. Sintered glass bead-associated biofilms displayed the lowest standard deviations ranging from 19 to 34% in all experiments. This material proved to be suitable for short-time experiments in stagnant media. Ceramic beads were stable during more than three weeks of exposure in a natural stream. Biofilm formation was inhomogeneous or poorly visible on glass and lava beads accompanied by high variations of enzyme activities. The applicability of the method to study enzyme inhibition reactions was successfully proven by the determination of inhibition effects of caffeine on biofilm-associated phosphodiesterase.Key points• Optimized method to determine enzymatic parameters in aquatic biofilms• Direct investigation of bead-bound biofilms without biofilm disintegration• Fluorometric detection offers high sensitivity and sample throughput.

Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

MPH-GST sensing microplate for easy detection of organophosphate insecticides.

OBJECTIVE: To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. RESULTS: With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1 µM and the linear range of 0.1-50 µM. Despite its highest stability at 4 °C, it was considerably stable at 25 °C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. CONCLUSIONS: All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus.

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.

Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP.

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the ß-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants.

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein-protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy-based in vitro binding assay system to screen for the ligands of the COI1-JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader-based assays of both agonists and antagonists of COI1-JAZ co-receptors.

Drug Permeability Profiling Using the Novel Permeapad® 96-Well Plate.

PURPOSE: Here, first experiences with a prototype tool for high throughput (passive) permeability profiling, a 96-well plate comprising the Permeapad® membrane, are reported. The permeabilities of a set of drugs were determined and compared to published measures of oral absorption, such as human fraction absorbed (Fa) and in vitro permeability values obtained using other tools. METHODS: The tool consists of a 96-well bottom and screen plate with the artificial, phospholipid-based barrier (Permeapad®) mounted between the plates' lower and upper compartments. The permeability of 14 model compounds including high- and low-absorption drugs, cationic, anionic, zwitterionic and neutral molecules, was determined by quantifying the compounds' transport over time, deriving the steady-state flux from the linear part of the cumulative curves and calculating the apparent permeability (Papp). The membrane structure was investigated in a high-resolution digital light microscope. RESULTS: The Permeapad® 96-well plate was found suited to distinguish high and low absorption drugs and yielded a hyperbolic correlation to Fa. The Papp values obtained were congruent with those determined with in-house prepared Permeapad® in the Franz cell set-up. Furthermore, good to excellent correlations were seen with Caco-2 permeability (R2 = 0.70) and PAMPA permeability (R2 = 0.89). Microscopic investigation of the Permeapad® barrier revealed the formation of phospholipid vesicles and myelin figures in aqueous environment. CONCLUSION: The Permeapad® 96-well plate permeation set-up is a promising new tool for rapid and reproducible passive permeability profiling.