Adenosine 5-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F 0 F 1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.Adenosine 5Ј-triphosphate (ATP) is a highly important biomolecule in living cells: It plays a central role in cell energy metabolism and also serves directly or indirectly in a number of cell signaling processes (1-3). The intracellular concentration of ATP is believed to oscillate in some eukaryotic cells, e.g. in ␤-cells (4) and in cells of the yeast Saccharomyces cerevisiae (5). However, changes in cytoplasmic ATP concentration with high time resolution have so far only been measured in a few circumstances (6, 7), mainly because methods for such continuous measurements are not generally available, or, in the case of NMR, require very high densities of cells or tissue (8).The lack of time-resolved measurements has prohibited the understanding of how the level of ATP and other intracellular metabolites are regulated in the cell and how ATP in turn regulates a number of cellular processes. Most current measurements of ATP in cells use extraction of the cell content and measure the concentration of ATP in the extract by various off-line methods such as HPLC (9), luciferase (10), or other enzyme-based methods (11). A few protein-based sensors exist (6,7,(12)(13)(14), which in principle allow for time-resolved measurements of intracellular ATP or ADP, but some of these methods entail expression of the sensor molecule in situ, which is not always possible. Hence, although it is expected that the intracellular concentration of ATP is anything but stationary, this has not been verified by real-time measurements, except in a few cases where ...

The objectives of the study were to describe the distribution of tetrahydrocannabinol (THC)-influenced drivers in a new Danish three-level offence system and discuss the consequences of the changed legislation. MethodsBy request of the police, blood of individuals suspected for driving under the influence of drugs (DUID) in Funen and Southern Jutland was sampled by medical staff and shipped to the Section of Forensic Toxicology of the University of Southern Denmark in Odense. Samples from individuals suspected for driving under the influence of cannabis (DUIC) were stored at 5 °C prior to immediate analysis, and THC content in whole blood samples was established by gas chromatography-mass spectrometry analysis (GC-MS). Quantitative results for blood THC levels were available from 2017 and 2018, resulting in 2206 eligible cases. Data before and after the legal change on the 15 th of December 2017 were extracted from the department's laboratory information management system. ResultsWith the new graduated sanctions introduced in December 2017, 70% of the DUIC suspects faced milder sanctions. The number of DUIC cases has been increasing and has almost doubled in the last four years from 648 cases in 2015 to 1206 in 2018. Correspondingly, the total number of DUID cases increased by 80% from 898 cases in 2015 to 1614 cases in 2018. The concentration of THC in blood was above the legal limit of 0.001 mg/kg in 73% of the cases; 18% had concentrations categorized as low, 32% as medium and 22% as high. ConclusionWe found that more than two-thirds of the THC offenders faced milder sanctions compared to before, suggesting that the new graduated legislation has had a significant impact. The current trend is a steadily increasing number of DUID cases. We believe that the continual increase is caused by a combination of factors including increased police awareness and skill training of police personnel along with improved roadside screening abilities.