Fluorescence Plate Reader Research Articles

Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors.

Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and μm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.

The Ixodes scapularis Symbiont Rickettsia buchneri Inhibits Growth of Pathogenic Rickettsiaceae in Tick Cells: Implications for Vector Competence.

Ixodes scapularis is the primary vector of tick-borne pathogens in North America but notably does not transmit pathogenic Rickettsia species. This tick harbors the transovarially transmitted endosymbiont Rickettsia buchneri, which is widespread in I. scapularis populations, suggesting that it confers a selective advantage for tick survival such as providing essential nutrients. The R. buchneri genome includes genes with similarity to those involved in antibiotic synthesis. There are two gene clusters not found in other Rickettsiaceae, raising the possibility that these may be involved in excluding pathogenic bacteria from the tick. This study explored whether the R. buchneri antibiotic genes might exert antibiotic effects on pathogens associated with I. scapularis. Markedly reduced infectivity and replication of the tick-borne pathogens Anaplasma phagocytophilum, R. monacensis, and R. parkeri were observed in IRE11 tick cells hosting R. buchneri. Using a fluorescent plate reader assay to follow infection dynamics revealed that the presence of R. buchneri in tick cells, even at low infection rates, inhibited the growth of R. parkeri by 86–100% relative to R. buchneri-free cells. In contrast, presence of the low-pathogenic species R. amblyommatis or the endosymbiont R. peacockii only partially reduced the infection and replication of R. parkeri. Addition of host-cell free R. buchneri, cell lysate of R. buchneri-infected IRE11, or supernatant from R. buchneri-infected IRE11 cultures had no effect on R. parkeri infection and replication in IRE11, nor did these treatments show any antibiotic effect against non-obligate intracellular bacteria E. coli and S. aureus. However, lysate from R. buchneri-infected IRE11 challenged with R. parkeri showed some inhibitory effect on R. parkeri infection of treated IRE11, suggesting that challenge by pathogenic rickettsiae may induce the antibiotic effect of R. buchneri. This research suggests a potential role of the endosymbiont in preventing other rickettsiae from colonizing I. scapularis and/or being transmitted transovarially. The confirmation that the observed inhibition is linked to R. buchneri's antibiotic clusters requires further investigation but could have important implications for our understanding of rickettsial competition and vector competence of I. scapularis for rickettsiae.

A near-infrared optical nanosensor for measuring aerobic respiration in microbial systems.

We developed a ratiometric oxygen-sensitive nanosensor and demonstrated application in monitoring metabolic oxygen consumption in microbial samples over time. Based on a near-infrared (NIR) emitting oxygen-quenched luminophore, platinum(II) octaethylporphine ketone (PtOEPK), along with a stable dioctadecyl dicarbocyanine reference dye (DiD), this nanosensor system provides an advantageous approach for overcoming imaging issues in biological systems, such as autofluorescence and optical scattering in the visible wavelength region. The dyes are encapsulated within a polymer-based nanoparticle matrix to maintain them at a constant ratio in biological samples, precluding the need for complex synthetic approaches. With this constant ratio of the two dyes, the nanosensor response can be measured as a ratio of their two signals, accounting for nanosensor concentration artifacts in measurements. The nanosensors are reversible, which enabled us to temporally monitor systems in which dissolved oxygen concentrations both increase and decrease. These sensors were applied for the monitoring of oxygen in samples of Saccharomyces cerevisiae (brewing yeast) in a 96-well optical fluorescence plate reader format over 60 h. By mixing the nanosensors directly into the sample well with the yeast, we were able to dynamically track metabolic activity changes over time due to varying cell concentration and exposure to an antimicrobial agent. This system could be a potential platform for high-throughput screening of various species or variants of microbes with unknown metabolic rates in response to external stimuli (antimicrobials, metabolites, etc.).

Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 μM and 42.4 ± 20.9 μM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

Abstract LB103: Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells

Abstract Background: Nuclear events play a well-established role in the cytotoxicity of doxorubicin (DOX). Other reported mechanisms include redox cycling for reactive oxygen species (ROS) production, which is getting recognized for a larger role than previously recognized. In addition, ROS production may be implicated in lysosome membrane permeabilization (LMP) and subsequent cell death. We have previously reported DOX-induced LMP in breast cancer cells. The goal of this study is to establish the role of LMP in the cytotoxic process and whether ROS are the initiator of this LMP. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 h exposure periods in MCF-7 and MDA-MB-231 breast cancer cells. Intracellular localization of DOX was examined by fluorescence microscopy using lysosome and nuclear markers. ROS production was determined by the DCFDA assay using a fluorescence plate reader. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Mitochondrial membrane potential (MMP) was determined by JC-1 assay. Viability was determined by MTT. Cathepsin B activity will be determined by Z-Arg-Arg-AMC cathepsin B substrate. Experiments will be conducted in the presence of E-64 cathepsin B inhibitor and Tiron superoxide scavenger to establish the importance of LMP and ROS towards DOX cytotoxicity. Results: Nuclear uptake was seen in both cell lines as early as 2 h at a concentration of 10 μM. Furthermore, lysosome accumulation of DOX, although small, was visible by 2 h in MDA cells, increasing in degree with time and concentration. ROS levels were found to be highest at 2 h in MCF-7 cells, increasing by up to 100% of the untreated control, and occurring concurrently with LMP at higher concentrations. Preliminary results suggest much lower ROS levels in MDA-MB-231 cells. In MCF-7 cells, ROS production decreased at 6 h, but increased by 24 h with concurrent increases in LMP. Although less than the MCF-7 cells, MDA cells demonstrated notable LMP for the first time at 5 and 10 μM conditions, occurring concurrently with MMP. By 24 h, viability was substantially reduced for both cell lines, with MCF-7s demonstrating a greater reduction in viability. Significant MMP was not observable until 24 h in MCF-7 cells. Conclusions: The early and strong ROS occurring with LMP suggest a previously unrecognized DOX cytotoxic combination in MCF-7 cells. LMP does not appear to result from lysosomal accumulation of DOX. A reduced role of ROS and LMP may account for the lesser cytotoxicity in MDA-MB-231 cells. Both ROS and LMP appear to be necessary for cytotoxicity in MCF-7 cells. Citation Format: Rachel E. Nicoletto, Clyde M. Ofner. Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB103.

FC 057SALIVARY POTASSIUM FOLLOWS PLASMA POTASSIUM DYNAMICS IN HEMODIALYSIS PATIENTS

Abstract Background and Aims Hyperkalemia is common in patients on hemodialysis (HD) and in cardiorenal patients on RAAS inhibitors. Frequently, hyperkalemia is a leading reason to withdraw potentially lifesaving therapy in cardiorenal patients. Out-patient monitoring of plasma K+ levels due to preanalytical problems has proven problematic. The present pilot-study examined salivary K+ levels and kinetics based on plasma K+ measurements in HD patients employing a novel class of genetically-encoded fluorescent potassium-ion indicators, the GEPIIs (Bischof et al. Nat. Commun. 2017). HD patients represent a unique population in whom significant K+ derangements and rapid K+ level changes predictably occur and where these phenomena can be safely investigated. Method K+ assessments were performed in healthy individuals (n=20) and HD patients (n=30). Study-related procedures were approved by the local Institutional Review Board. Healthy individuals were recruited on a voluntary basis and provided only saliva samples. HD patients were recruited from our HD unit. Blood and saliva samples for K+ assessment were collected simultaneously during three consecutive HD sessions (HD1-3) prior to and directly after each session (pre/post HD). Blood was drawn in a standardized procedure directly from the HD access line and plasma K+ was measured immediately using standardized ion-selective electrodes (ISE). Saliva samples were gathered in a standardized procedure using a commercially available collection device (SuperSALTM). Of note, gaining adequate amounts of saliva from chronic HD patients, who frequently suffer from xerostomia, has proven challenging in some cases. The K+ content of saliva samples was measured by ISE and GEPII-technique. For the latter, samples were mixed with purified GEPIIs and were inserted into a fluorescent plate-reader. Probes were illuminated at 430 nm and emission light were collected at 475 nm and 525 nm, respectively. The ratio of the fluorescent intensities (F535/F480) after appropriate calibration is a direct measure of the K+ concentration sensed by GEPIIs. Results K+ determination in saliva samples using the GEPII-technique and ISE showed a strong agreement ((Figure 1A), Bias 0,71; 95% limits of agreement from -2.79 to 4.40). Pre-dialytic [K+]saliva of HD patients compared to healthy individuals, was higher (40,64±1,50 vs. 23,15±0,76 mmol/l, p<0.05). As expected, each HD session (HD1-3) led to a significant reduction in [K+]plasma, which is followed by a similar, significant reduction of [K+]saliva (Figure 1B). Dynamics of plasma and salivary [K+] showed a very similar pattern: strong reduction during a HD session followed by a marked increase in the dialysis-free period until the next session 48-72h later (Figure 1C). Although basal [K+]saliva shows individual variations, [K+]Saliva and [K+]plasma exhibited a tendency of linear association (Figure 1D). Correlation analysis in each HD session (HD1-3 pre/post) revealed however no or weak correlation of pre- and post-dialytic saliva and plasma K+ values (Figure 1E). Conclusion The GEPII-technique is an easy to use, reliable and suitable method for salivary K+ determination in healthy individuals and in HD patients with accuracy and precision comparable to that of ISE. Despite heterogeneous baselines, changes of [K+]saliva represent a sensitive marker of K+ derangements as well as hyper- and normokalemia in HD patients. Although we observed that [K+]saliva dynamically follows [K+]plasma , an exact quantification - most likely due to the low number of cases per HD sessions in this pilot-study – was not possible. Additionally, how closely [K+]saliva tracks [K+]plasma in patients with hypokalemia was not addressed in this study. To confidently answer whether [K+]saliva measurement can potentially be used in the care of patients at increased risk of hyperkalemia, further studies in a larger number of patients need to be conducted.

Reactive oxygen species production and the influence of exogenous copper in yeast mitochondria

Mitochondria are essential organelles within eukaryotic cells due to their role in metabolism, ATP production via the oxidative phosphorylation (OXPHOS) pathway, and other regulatory cellular processes. Our aim is to understand how copper in the mitochondria of yeast affects the production of reactive oxygen species due to its influence on the electron transport chain (ETC) and the localization of reactive oxygen species production during yeast lifespan. Reactive oxygen species (ROS) provide signaling in the cells of organisms which is beneficial for cells to maintain homeostasis. However, when there is a surplus of ROS this can lead to harmful and/or lethal results for the cell. To detect ROS, yeast cultures were grown for one to three days and treated with the fluorescent ROS indicator dihydroethidium (DHE), as well as the fluorescent indicators SYTO and DAPI which stain mitochondrial DNA and adenine-thymine rich regions in the DNA respectively. The ROS generated from the yeast was then analyzed via a fluorescence plate reader, fluorescent activated cell sorting (FACS), and fluorescent microscopy to compare methods of determining ROS in the samples. To investigate mitochondrial ROS production, cells were grown in the presence of antimycin A (a Complex III inhibitor), oligomycin (a Complex V inhibitor), or menadione (a futile redox cycling molecule). Detectable increases in ROS were observed through the inhibition of Complex III and in the presence of menadione. We are now using these methods in yeast with a superoxide dismutase 1 (SOD1) gene deletion, to understand the influence of exogenous copper on the generation of ROS. Copper is worth investigating in conjunction with ROS because the mitochondria in mammals and yeast cells contain a labile copper pool located in the matrix, which is used in the metalation of the copper containing enzymes Complex IV, Cytochrome C Oxidase(CcO), and superoxide dismutase (Sod1). It has been established that supplementing cultures with copper increases CcO and Sod1 activity, supporting previous results that exogenous copper contributes to a labile copper pool within the mitochondria to increase enzyme activity. Our results show that small amounts exogenous copper (0.25 mM copper sulfate in restricted nutrient media) extend yeast chronological lifespan, but a dramatic reduction in lifespan is observed when copper levels are increased to 2.0 mM copper sulfate or higher. These data indicates that low levels of exogenous copper in the media is beneficial for yeast in restricted media conditions. Our current work assesses how exogenous copper changes ETC protein complex expression, specifically looking at CcO subunits, and the production of ROS under these conditions.

Ultraviolet light-emitting diode irradiation induces reactive oxygen species production and mitochondrial membrane potential reduction in HL-60 cells.

ObjectiveUltraviolet light-emitting diode (UV LED) irradiation at 280 nm has been confirmed to induce apoptosis in cultured HL-60 cells, but the underlying mechanisms remain unclear. This study aimed to investigate the effects of 280 nm UV LED irradiation on reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) in HL-60 cells.MethodsHL-60 cells were irradiated with 0, 8, 15, or 30 J/m2 of 280 nm UV LED and incubated for 2 hours. The intracellular ROS levels were assessed using the fluorescent probe 2ʹ-7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA) and a fluorescence plate reader. MMP was determined by flow cytometry using 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazol-carbocyanine iodide (JC-1) staining. The apoptosis-related proteins Bax and Bcl-2 were evaluated by western blot.ResultsUV LED irradiation at 280 nm induced a dose-dependent increase in ROS production and loss of MMP, and it activated apoptosis at irradiation doses of 8 to 30 J/m2. These results were consistent with a previous apoptosis study from the authors’ group.ConclusionEnhanced ROS production and mitochondrial depolarization are two distinct but interacting events, and both are involved in UV LED-induced apoptosis in HL-60 cells.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist S6 Stimulates Insulin Secretion From Rat Islets.

Glucagon-like peptide-1 receptor (GLP-1R) agonist-based therapeutics for type 2 diabetes mellitus have attracted worldwide attention. However, there are challenges in the development of small molecule GLP-1R agonists owing to the complexity of ligand recognition and signal induction mechanisms. Here, we attained S6 using virtual screening and fluorescent imaging plate reader (FLIPR)-based calcium assays. The purpose of this study was to identify and characterize S6, a novel small molecule GLP-1R agonist. Data from cellular thermal shift assay (CETSA) and Bio-Layer Interferometry (BLI) indicated that S6 could bind potently with GLP-1R. Radioimmunoassay data showed that S6 potentiated insulin secretion in a glucose-dependent manner and the insulinotropic effect was mediated by GLP-1R. Calcium imaging techniques suggested that S6 elevated the intracellular calcium concentration [(Ca2+)i] by activating GLP-1R. In patch-clamp experiments, we demonstrated that S6 inhibited voltage-dependent K+ (Kv) channels in a GLP-1R-dependent fashion. Besides, S6 significantly prolonged action potential duration but had no effect on voltage-dependent Ca2+ channels. In summary, these findings indicate that S6 stimulates glucose-dependent insulin secretion mainly by acting on GLP-1R, inhibiting Kv channels, increasing (Ca2+)i. This study will provide direction for the screening and development of novel small-molecule agents targeting GLP-1R in the future.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100nM. Some LECs were pretreated with 1μM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1nM) and CINO (≥ 10nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1μM L-NAME attenuated (p < 0.05) the MPLEC. The β-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes.

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

A Novel High-Throughput FLIPR Tetra-Based Method for Capturing Highly Confluent Kinetic Data for Structure-Kinetic Relationship Guided Early Drug Discovery.

Target engagement by small molecules is necessary for producing a physiological outcome. In the past, a lot of emphasis was placed on understanding the thermodynamics of such interactions to guide structure-activity relationships. It is becoming clearer, however, that understanding the kinetics of the interaction between a small-molecule inhibitor and the biological target [structure-kinetic relationship (SKR)] is critical for selection of the optimum candidate drug molecule for clinical trial. However, the acquisition of kinetic data in a high-throughput manner using traditional methods can be labor intensive, limiting the number of molecules that can be tested. As a result, in-depth kinetic studies are often carried out on only a small number of compounds, and usually at a later stage in the drug discovery process. Fundamentally, kinetic data should be used to drive key decisions much earlier in the drug discovery process, but the throughput limitations of traditional methods preclude this. A major limitation that hampers acquisition of high-throughput kinetic data is the technical challenge in collecting substantially confluent data points for accurate parameter estimation from time course analysis. Here, we describe the use of the fluorescent imaging plate reader (FLIPR), a charge-coupled device (CCD) camera technology, as a potential high-throughput tool for generating biochemical kinetic data with smaller time intervals. Subsequent to the design and optimization of the assay, we demonstrate the collection of highly confluent time-course data for various kinase protein targets with reasonable throughput to enable SKR-guided medicinal chemistry. We select kinase target 1 as a special case study with covalent inhibition, and demonstrate methods for rapid and detailed analysis of the resultant kinetic data for parameter estimation. In conclusion, this approach has the potential to enable rapid kinetic studies to be carried out on hundreds of compounds per week and drive project decisions with kinetic data at an early stage in drug discovery.

Functional characterization of N-octyl 4-methylamphetamine variants and related bivalent compounds at the dopamine and serotonin transporters using Ca2+ channels as sensors

The early characterization of ligands at the dopamine and serotonin transporters, DAT and SERT, respectively, is important for drug discovery, forensic sciences, and drug abuse research. 4-Methyl amphetamine (4-MA) is a good example of an abused drug whose overdose can be fatal. It is a potent substrate at DAT and SERT where its simplest secondary amine (N-methyl 4-MA) retains substrate activity at them. In contrast, N-n-butyl 4-MA is very weak, therefore it was categorized as inactive at these transporters. Here, N-octyl 4-MA and other related compounds were synthesized, and their activities were evaluated at DAT and SERT. To expedite this endeavor, cells expressing DAT or SERT were co-transfected with a voltage-gated Ca2+ channel and, the genetically-encoded Ca2+ sensor, GCaMP6s. Control compounds and the newly synthesized molecules were tested on these cells using an automated multi-well fluorescence plate reader; substrates and inhibitors were identified successfully at DAT and SERT. N-Octyl 4-MA and three bivalent compounds were inhibitors at these transporters. These findings were validated by measuring Ca2+-mobilization using quantitative fluorescence microscopy. The bivalent molecules were the most potent of the series and were further characterized in an uptake-inhibition assay. Compared to cocaine, they showed comparable potency inhibiting uptake at DAT and higher potency at SERT. These observations support a previous hypothesis that amphetamine-related (and, here, N-extended alkyl and) bivalent arylalkylamine molecules are active at monoamine transporters, showing potent activity as reuptake inhibitors, and implicate the involvement of a distant auxiliary binding feature to account for their actions at DAT and SERT.

Establishment of a safe and convenient assay for detection of HA subtype-specific antibodies with PB2 gene-knockout influenza viruses

Monitoring of the epidemic situation is imperative to control the risk of infection with avian influenza H5 and H7 subtype viruses. A microneutralization (MN) assay was employed to detect hemagglutinin (HA) subtype-specific antibodies. However, the conventional MN assay raises biosafety concerns and is labor-intensive and time-consuming. Therefore, a safer and more convenient assay that can be applied in a high-throughput format is warranted. In this study, PB2 knockout (PB2-KO) influenza viruses of H5 and H7 subtypes expressing different colored fluorescent proteins were generated using a reverse genetics system and applied to a novel MN assay for the detection of specific antibodies. The detection sensitivity of our PB2-KO virus-based MN assay was evaluated by observing fluorescent proteins under a fluorescence microscope and measuring fluorescence intensities using a plate reader. In addition, the PB2-KO virus-based MN assay was used for the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Expression of the reporter fluorescent protein from H5 and H7 PB2-KO viruses was restricted toin PB2 protein-expressing cells. The MN titer as determined using fluorescence microscopy and plate reader revealed that the detection sensitivity of our PB2-KO virus-based MN assay was comparable to that of the conventional MN assay. Moreover, H5 and H7 PB2-KO viruses could be usedapplied forto the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Our study demonstrates a safe and convenient assay for the detection of H5 and H7 subtype-specific antibodies.

Determination of plasma levels of the active thiol form of the direct-acting PrC-210 ROS-scavenger using a fluorescence-based assay

PrC-210 is a direct-acting ROS-scavenger. It's active when administered orally, IV, or topically; it has none of the nausea/emesis nor hypotension side effects that have precluded human amifostine use. PrC-210 confers 100% survival to mice and rats that received an otherwise 100% lethal radiation dose and 36% reduction of ischemia-reperfusion-induced mouse myocardial infarct damage, and thus is a viable candidate to prevent human ROS-induced ischemia-reperfusion and ionizing radiation toxicities. We report the first assay for the pharmacologically active PrC-210 thiol in blood. PrC-210 has no double-bonds nor light absorption, so derivatizing the thiol with a UV-absorbing fluorochrome enables quantification. This assay: i) is done on the benchtop; it's read with a fluorescence plate reader, ii) provides linear product formation through 60 min, iii) quantifies μM to low mM rodent blood levels of PrC-210 that confer complete radioprotection, iv) accurately reflects PrC-210 thiol formation of mixed disulfides with other thiols in blood, and v) shows excellent between-day assay outcome with very low standard deviation and coefficient of variation. A fluorescence assay quantifying formation of a PrC-210 thiol-bimane adduct enables measurement of blood PrC-210 thiol. A blood assay will help in the development of PrC-210 for use in the human clinical setting.

Time-Resolved Fluorescence Assay for Measuring Oxygen Consumption Rates in Staphylococcus aureus.

Oxygen consumption is a fundamental characteristic of staphylococcal physiology reflecting the energy and metabolic state of the bacterial cell. During aerobic growth, oxygen consumption rates (OCR) depend on nutrient availability and vary at different growth stages. The measurement of oxygen consumption rates provides a versatile tool to characterize the impact of various mutations, environmental cues, and antibiotics on bacterial growth and fitness. In this chapter, we describe a MitoXpress® Xtra-based oxygen consumption assay for fast and reliable determination of respiration rates in Staphylococcus aureus. This highly reproducible and simple method requires a minimal set of reagents and allows rapid screening of multiple samples through real-time determination of the OCR with an oxygen-sensing probe and fluorescence plate reader.

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts.

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses.

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.

Fluorescent indolizine derivative YI-13 detects amyloid-β monomers, dimers, and plaques in the brain of 5XFAD Alzheimer transgenic mouse model.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the aberrant production and accumulation of amyloid-β (Aβ) peptides in the brain. Accumulated Aβ in soluble oligomer and insoluble plaque forms are considered to be a pathological culprit and biomarker of the disorder. Here, we report a fluorescent universal Aβ-indicator YI-13, 5-(4-fluorobenzoyl)-7,8-dihydropyrrolo[1,2-b]isoquinolin-9(6H)-one, which detects Aβ monomers, dimers, and plaques. We synthesized a library of 26 fluorescence chemicals with the indolizine core and screen them through a series of in vitro tests utilizing Aβ as a target and YI-13 was selected as the final imaging candidate. YI-13 was found to stain and visualize insoluble Aβ plaques in the brain tissue, of a transgenic mouse model with five familial AD mutations (5XFAD), by a histochemical approach and to label soluble Aβ oligomers within brain lysates of the mouse model under a fluorescence plate reader. Among oligomers aggregated from monomers and synthetic dimers from chemically conjugated monomers, YI-13 preferred the dimeric Aβ.