Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms

Toothbrushing: to the best of one’s abilities is possibly not good enough

Abstract Background Weaknesses in toothbrushing performance can be seen when young adults are instructed to perform habitual toothbrushing. Nothing is known about toothbrushing behavior when instructed to perform to the best abilities. The present study analyzes such behavior and compares it to habitual behavior. Methods A random sample of N = 98 young adults born in 1995 was examined in 2014/2015.They were asked to perform oral hygiene to the best of their abilities in front of a camera. Videos were analyzed regarding details of brushing behavior. A quality index was developed which describes the extent of the neglect of brushing on palatinal and vestibular surfaces. Data were compared to those of an earlier study of young adults (born in 1992, examined in 2011, N = 101) who were asked to perform oral hygiene as they habitually do. Results The 1995 cohort (best abilities) brushed their teeth significantly longer than the 1992 cohort (habitual brushing). This was due to significant longer brushing at vestibular and occlusal surfaces. Neglect of palatinal surfaces was similar in both cohorts. Groups did not differ regarding brushing movements. 40% of the brushing time on lateral surfaces was spent with scrubbing movements despite opposing advice in common oral hygiene instructions. Conclusions Toothbrushing to the best of one’s abilities might still not be good enough. Young adults apparently lack a reasonable concept of what is meant by high quality toothbrushing. More efforts should thus be undertaken to explain them (and adults) this concept

Applying label-free dynamic mass redistribution technology to frame signaling of G protein–coupled receptors noninvasively in living cells

Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein-coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca<sup>2+</sup> or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1-2