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Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.

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Plant extracts and pharmacopoeias represent an exceptional breeding ground for the discovery of new antioxidants. Until recently, the antioxidant activity was only measured by chemical hydrogen atom transfer (HAT) and single-electron transfer (SET) cell-free assays that do not inform about the actual effect of antioxidants in living systems. By providing information about the mode of action of antioxidants at the subcellular level, recently developed live cell assays are now changing the game. The idea of this review is to present the different cell-based approaches allowing a quantitative measurement of antioxidant effects of plant extracts. Up to date, only four different approaches have reached a certain degree of standardization: (1) the catalase-like assay using H2O2 as a stressor, (2) the cell antioxidant assay (CAA) using AAPH as a stressor and DCFH-DA as a readout, (3) the AOP1 assay which uses photoinduction to monitor and control cell ROS production, and (4) the Nrf2/ARE gene reporter system. The molecular aspects of these assays are presented in detail along with their features, drawbacks, and benefits. The Nrf2/ARE gene reporter system dedicated to indirect antioxidant effect measurement currently represents the most standardized approach with high-throughput applications. AOP1, the first technology linking a fine-tuning of cell ROS production with a quantitative signal, appears to be the most promising tool for the assessment of direct cellular ROS-scavenging effects at an industrial scale.

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Arrestins (arr) are multifunctional cytosolic adaptors that bind to active and phosphorylated G protein-coupled receptors (GPCRs) via a highly versatile interface. Arrestins stop G protein signaling and trigger other signaling pathways. Recently, 3D structures of arr-GPCR complexes have been solved, which provide a bulk of structural information for understanding the mechanism of arr recruitment and activation. However, many questions about the functional consequences of structural details and the dynamics of the arr-GPCR interaction remain open. A wealth of information about key determinants for the arr-GPCR interaction and their functional relevance, and dynamic insights into the process of arr binding and the functional outcomes of different binding modes have been provided by a series of biochemical methods which we review here. Importantly, most of these methods provide information from the live cell, which is a necessary validation and complement for structural data. With the main focus on the most recent research, we will highlight major findings about arr structure, function, and dynamics derived from mutagenesis studies, cross-linking studies, conformational probes, and sensors, and we summarize available systems to detect arr recruitment. Furthermore, we discuss recent findings and directions of in silico investigations in arr-GPCR complexes.

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The detection and identification of microbial pathogens in meat and fresh produce play an essential role in food safety for reducing foodborne illnesses every year. A new approach based on targeting a specific sequence of the 16S rRNA region for each bacterium is proposed and validated. The probe complex consists of a C60, a conjugated RNA detector which targets a specific 16S rRNA sequence, and a complementary fluorescent reporter. The RNA detectors were designed by integrating NIH nucleotide and Vienna RNA Webservice databases, and their specificities were validated by the RDP database. Probe complexes were synthesized for identifying E. coli K12, E. coli O157: H7, S. enterica, Y. enterocolitica, C. perfringens, and L. monocytogenes. First, under controlled conditions of known bacterial mixtures, the efficiency and crosstalk for identifying the foodborne bacteria were quantified to be above 94% and below 5%, respectively. Second, experiments were designed by inoculating meat products by known numbers of bacteria and measuring the limit of detection. In one experiment, 225 g of autoclaved ground chicken was inoculated with 9 E. coli O157:H7, where 6.8 ± 1.2 bacteria with 95% confidence interval were recovered. Third, by positionally printing probe complexes in microwells, specific microorganisms were identified with only one fluorophore. The proposed protocol is a cell-based system, can identify live bacteria in 15 min, requires no amplification, and has the potential to open new surveillance opportunities.Key points• The identification of foodborne bacteria is enabled in live-cell assays.• The limit of detection for 100 g of fresh chicken breast inoculated with 4 bacteria is 2.7 ± 1.4 with 95% confidence interval.• The identification of five bacteria in a coded microwell chip is enabled with only one fluorophore.

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Activity of acid sphingomyelinase has been implicated in a number of diseases like acute lung injury, sepsis or metastasis of melanoma cells. Here, we present a sphingomyelinase FRET probe based on FAM/BODIPY dyes for real-time monitoring of acid sphingomyelinase. The probe gives rise to a tremendous increase in fluorescence of the fluorescein FRET donor upon cleavage and we show that this is, to a significant part, due to cleavage-associated phase transition, suggesting a more systematic consideration of such effects for future probe development. The probe allows for the first time to monitor relative sphingomyelinase activities of intact living cells by flow cytometry.

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BACKGROUND: Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. METHODS: C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. RESULTS: The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. CONCLUSIONS: The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

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We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

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Optical tweezers have been instrumental in uncovering the mechanisms motor proteins use to generate and react to force. While optical traps have primarily been applied to purified, in vitro systems, emerging methods enable measurements in living cells where the actively fluctuating, viscoelastic environment and varying refractive index complicate calibration of the instrument. Here, we describe techniques to calibrate optical traps in living cells using the forced response to sinusoidal oscillations and spontaneous fluctuations, and to measure the forces exerted by endogenous ensembles of kinesin and dynein motor proteins as they transport cargoes in the cell.

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