The concentration of dissolved oxygen in aquatic systems helps regulate biodiveristy 1, 2 , nutrient biogeochemistry 3 , greenhouse gas emissions 4 , and drinking water quality 5 . The long-term declines in dissolved oxygen concentrations in coastal and ocean waters have been linked to climate warming and human activity 6, 7 , but little is known about changes in dissolved oxygen concentrations in lakes. While dissolved oxygen solubility decreases with increasing water temperatures, long-term lake trajectories are not necessarily predictable. Oxygen losses in warming lakes may be amplified by enhanced decomposition and stronger thermal stratification 8, 9 or they may increase as a result of enhanced primary production 10 . Here we analyse 45,148 dissolved oxygen and temperature profiles from 393 temperate lakes spanning 1941-2017. We find that a decline in dissolved oxygen is widespread in surface and deep-water habitats. The decline in surface waters is primarily associated with reduced solubility under warmer water temperatures, although surface dissolved oxygen increased in a subset of highly-productive warming lakes, likely due to increasing phytoplankton production. In contrast, the decline in deep waters is associated with stronger thermal stratification and water clarity losses, but not with changes in gas solubility. Our results suggest that climate change and declining water clarity have altered the physical and chemical environment of lakes. Freshwater dissolved oxygen losses are 2.5-10 times greater than observed in the world's oceans 6, 7 and could threaten essential lake ecosystem services 2,3,5,11 .

The concentration of dissolved oxygen (DO) is an important attribute of aquatic ecosystems, influencing habitat, drinking water quality, biodiversity, nutrient biogeochemistry, and greenhouse gas emissions. While average summer DO concentrations are declining in lakes across the temperate zone, much remains unknown about seasonal factors contributing to deepwater DO losses. It is unclear whether declines are related to increasing rates of seasonal DO depletion or changes in seasonal stratification that limit re-oxygenation of deep waters. Furthermore, despite the presence of important biological and ecological DO thresholds, there has been no large-scale AnalysisSample size Median number yearsChanges in seasonal DO depletion rates 128 23Changes in stratification duration 57 23Changes in proportion of water column < DO thresholds (5, 4, 3, 2, 0.5 mg/L)

Split fluorescent proteins have been engineered for various purposes, in each case signaling their spontaneous reconstitution by fluorescence. By combining split protein reconstitution and computational protein design, we have constructed a circularly permuted and truncated variant of green fluorescent protein (GFP) in which the seventh beta strand has been left out and the sites around it computationally designed to accommodate a peptide from influenza hemagglutinin. We call this a ''leave-one-out'' GFP biosensor (LOO-GFP). A LOO-GFP was designed using DEEdesign, selected by plate screening a bacterial library in the presence of the influenza peptide target, and was found to have seven point mutations. But binding was weak (9mM) and was at the expense of stability. The weakened, partially folded protein aggregated in the absence of its target. Furthermore, the aggregated biosensor fluoresced more than its monomeric peptide-bound form. In this work, the LOO-GFP was rationally redesigned to fold more robustly and bind the target tighter. Modeling of the GFP folding pathway suggested that one of the seven mutations, F83W, interfered with the closing of the beta barrel. Mutating this residue back to a F indeed, along with several other rationally justified changes followed by re-screening, produced several biosensor sequences with slower unfolding rates, a positive binding signal, and higher chromophore maturation efficiency. We also observed a blue-shift in the excitation spectrum, and lower background fluorescence in the unbound state. Kd was unchanged. In parallel experiments, LOO-GFP biosensors were genetically fused to fibers formed by the Drosophila protein ultrabithorax (Ubx), and were found to be absent any background fluorescence in the unbound state, but recovered fluorescence when exposed to the target peptide. Implications for the design of biosensing materials are discussed. Glucokinase (GCK) is the rate-limiting enzyme for glucose metabolism in glucose-sensing tissues, including hypothalamic nuclei. Post-translational regulation of GCK by receptor-mediated signaling pathways has been identified in the liver and the pancreas, and is critical for the maintenance of glucose homeostasis; yet, it is unclear if a similar regulation of GCK occurs in hypothalamic neurons. Using the hypothalamically-derived, glucosensing GT1-7 neuronal cell line, the existence of a receptor-mediated signaling cascade culminating in the S-nitrosylation and activation of GCK is demonstrated. GCK activity is assessed by either measuring NAD(P)H autofluorescence while raising extracellular glucose, or through expression of a FRET GCK biosensor. Treatment of GT1-7 cells with isoproterenol, a G s GPCR agonist, augments a glucose-dependent rise in NAD(P)H autofluorescence, and activates the GCK biosensor. Applying the nitric oxide synthase inhibitor L-NAME impedes the metabolic effect of isoproterenol. Additionally, incorporation of an S-nitrosylation-blocking V367M mutation into the biosensor prevents GCK activation by isoproterenol....