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Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.

The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z’ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

The prevalence of overweight and obesity is increasing globally, driven by the availability of energy-dense palatable foods. Most dietary strategies fail because of hunger generated by calorie restriction, and interventions that specifically control hunger and/or promote fullness may aid success. Current consumers have a limited choice of satiety-enhancing products with proven health benefits, and innovative ways to produce new foods (as structural modification) to enhance satiety/satiation may provide new opportunities. However, this potential is hindered by the cost of product testing. Within the SATIN-SATiety INnovation project-an in vitro platform has been developed to offer a cost-effective means of assessing the potential satiation/satiety effect of novel foods. This combines in vitro technologies to assess changes in colonic bacteria metabolism, appetite hormone release and the stability and bioavailability of active compounds in the new products/ingredients. This article provides a brief review of nutrients for which an impact on short-term appetite regulation has been demonstrated, and a summary of the changes to food structure which can be used to produce a change in appetite expression. Furthermore, the SATIN in vitro platform is discussed as a means of assessing the impact of nutritional and structural manipulations on appetite.

The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput-compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.

The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called “optogenetic” technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group. The most important physiological activator of sGC is nitric oxide, which activates the enzyme upon binding to the heme moiety. By producing the second messenger cGMP, which regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, sGC plays an important role in different physiological processes, thus representing a very attractive pharmacological target. In fact, the pathogenesis of several diseases, especially those of the cardiovascular system, has been linked to inappropriate regulation of sGC. In order to find new modulators for this important enzyme, an innovative cell-based assay has been developed and optimized for the use in high-throughput screening. This luminescent assay, which is suitable for both 96- and 384-well plate formats, has been achieved by stably expressing the alpha and beta subunits of a mutated form of sGC in Chinese hamster ovary cells. The mutated form synthesizes cyclic adenosine 3',5'-monophosphate instead of cGMP, allowing the detection of enzymatic activity by a reporter gene approach. We demonstrated that this cell line responds to compounds typically used in the field of sGC research and that it represents an innovative and robust assay to screen for sGC modulators with high efficiency and high sensitivity by means of standard luminescence readers.

The effect of experimental hypothyroidism on the catabolism of plasma lipoproteins and on the expression of low density lipoprotein receptors by the liver was investigated in rats made hypothyroid by surgery. The animals developed mild hypercholesterolemia, mainly due to an increase of plasma low density lipoprotein, while other lipoprotein classes were only marginally affected. Kinetic studies using [ 125 I]LDL indicated that a decreased fractional catabolic rate of the lipoprotein was responsible for this finding in agreement with the in vitro observation of a reduced binding of lipoproteins to liver membranes from hypothyroid rats and with the demonstration, by ligand blotting analysis, of a decreased expression of lipoprotein receptors in liver membranes. These data suggest that hypothyroidism affects lipoprotein distribution also by decreasing the catabolism of low density lipoproteins by the liver.

A report summarizing results and protocols obtained using fluorescent sensitive dyes and impedance measurements.

A report summarizing results and protocols on measuring SLC-dependent metabolic states through fluorescent sensor proteins.