Protein–protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and Mdm4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53–Mdm2 inhibitors. However, none exhibited intracellular activity on p53–Mdm4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53–Mdm2 interaction as well as p53–Mdm4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances’ dual Mdm2–Mdm4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.

Differences in autonomic nerve activity between athletes and controls during supine rest and standing were investigated by recording the cardiac rhythm in 18 professional cyclists and 11 controls. We computed four indexes of autonomic control: the standard deviation (SD) of the interbeat intervals, the coefficient of variance (CV) of the interbeat intervals, the percentage of successive intervals differing by more than 50 ms (pNN50), and the fraction low-frequency (0.07-0.14 Hz) spectral power (LF), and we also measured the mean interbeat interval (MI). Significant differences (Student's t-test, P < 0.005) between the athletes and the controls in the supine position were found for pNN50 [mean 52.6 (SEM 2.5) vs 37.1 (SEM 3.4)%], LF [mean 32.2 (SEM 1.6) vs 40.7 (SEM 2.1) normalized units], and MI [mean 1241 (SEM 20) vs 1021 (SEM 25) ms]. A significant difference between the athletes and the controls in the standing position was found for MI [mean 888 (SEM 13) vs 801 (SEM 23) ms]. These results would suggest that there is a parasympathetic predominance in athletes in the supine, but not in the standing position. The finding that pNN50 and LF, but not SD and CV, differed between the athletes and the controls, would seem to demonstrate that the differences in autonomic control between the athletes and the controls are reflected in the quality (balance between slow and fast heart rate fluctuations) rather than in the quantity of heart rate variability.

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In haemodialysis patients the desferrioxamine (DFO) test is an accepted method for estimating the body content of aluminium. However, toxic side effects have been frequently reported with the high-dose (30 or 40 mg/kg) DFO tests. To study if a low-dose (500 mg) DFO test was also useful and free of side effects, we compared it with a high-dose DFO test in 22 patients on regular haemodialysis. During the first two hours of dialysis, 500 mg DFO in 100 ml 0.9% NaCl were administered intravenously. Before dialysis and after 48 hours aluminium concentration was determined in serum. In 14 patients the low-dose DFO test was considered positive. In 11 of those 14 patients the high-dose (30 mg/kg) DFO test was positive too. Analysis of the results showed a significant correlation (p < 0.05) between the outcome of the two DFO tests in the same patient. Compared to the high-dose DFO test, the low-dose test revealed a sensitivity of 79% and a specificity of 63%. In contrast with the high-dose DFO test might be a reliable and safe method for the estimation of the body content of aluminium in patients on regular haemodialysis.

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