Ontogenetic variation in the ecological stoichiometry of 10 fish species during early development.

The chemical composition and stoichiometry of vertebrate bodies changes greatly during ontogeny as phosphorus-rich bones form, but we know little about the variation among species during early development. Such variation is important because element ratios in animal bodies influence which element limits growth and how animals contribute to nutrient cycling. We quantified ontogenetic variation from embryos through 2-3 months of age in 10 species of fish in six different families, ranging in adult size from 73 to 720 mm in length. We measured whole-body concentrations (percentage of dry mass) and ratios of carbon (C), nitrogen (N), and phosphorus (P) as fish developed. We also quantified whole-body concentrations of calcium (Ca), because Ca should reflect bone development, and RNA, which can be a major pool of body P. To account for interspecific differences in adult size, we also examined how trends changed with relative size, defined as body length divided by adult length. Ontogenetic changes in body composition and ratios were relatively similar among species and were more similar when expressed as a function of relative size compared to age. Body P increased rapidly in all species (likely because of bone development) from embryos until individuals were ~5%-8% of adult size. Body N also increased, while body C, C:N, C:P, and N:P all decreased over this period. Body Ca increased with development but was more variable among species. Body RNA was low in embryos, increased rapidly in young larvae, then decreased as fish reached 5%-8% of adult size. After fish were about 5%-8% of adult size, changes in body composition were relatively slight for all elements and ratios. These results reveal a consistency in the dynamics of body stoichiometry during early ontogeny, presumably because of similar constraints on the allocation of elements to bones and other body pools. Because most changes occur when individuals are <1 month old (<10% of adult size for that species), early ontogenetic variation in body stoichiometry may be especially important for growth limitation of individuals and ecosystem-level nutrient cycling.

Phytoplankton and cyanobacteria abundances in mid-21st century lakes depend strongly on future land use and climate projections.

Land use and climate change are anticipated to affect phytoplankton of lakes worldwide. The effects will depend on the magnitude of projected land use and climate changes and lake sensitivity to these factors. We used random forests fit with long-term (1971-2016) phytoplankton and cyanobacteria abundance time series, climate observations (1971-2016), and upstream catchment land use (global Clumondo models for the year 2000) data from 14 European and 15 North American lakes basins. We projected future phytoplankton and cyanobacteria abundance in the 29 focal lake basins and 1567 lakes across focal regions based on three land use (sustainability, middle of the road, and regional rivalry) and two climate (RCP 2.6 and 8.5) scenarios to mid-21st century. On average, lakes are expected to have higher phytoplankton and cyanobacteria due to increases in both urban land use and temperature, and decreases in forest habitat. However, the relative importance of land use and climate effects varied substantially among regions and lakes. Accounting for land use and climate changes in a combined way based on extensive data allowed us to identify urbanization as the major driver of phytoplankton development in lakes located in urban areas, and climate as major driver in lakes located in remote areas where past and future land use changes were minimal. For approximately one-third of the studied lakes, both drivers were relatively important. The results of this large scale study suggest the best approaches for mitigating the effects of human activity on lake phytoplankton and cyanobacteria will depend strongly on lake sensitivity to long-term change and the magnitude of projected land use and climate changes at a given location. Our quantitative analyses suggest local management measures should focus on retaining nutrients in urban landscapes to prevent nutrient pollution from exacerbating ongoing changes to lake ecosystems from climate change.

Nutrient excretion by fish supports a variable but significant proportion of lake primary productivity over 15 years.

Animals play an important and sometimes overlooked role in nutrient cycling. The role of animals in nutrient cycling is spatially and temporally variable, but few studies have evaluated the long-term importance of animal-mediated nutrient cycling in meeting nutrient demand by primary producers. We quantified the proportion of phytoplankton nutrient (phosphorus, P) demand met by excretion by gizzard shad (Dorosoma cepedianum) in a eutrophic reservoir where this species dominates fish biomass. From 2000 to 2014, gizzard shad excretion supported a variable proportion of phytoplankton P demand, averaging 7-27% among years over the growing season (spring and summer). Temporal patterns emerged, as gizzard shad consistently supported a higher proportion of demand during summer (mean 31%) than spring (8%). In spring, the proportion of demand met from gizzard shad excretion was best predicted by gizzard shad population biomass, stream discharge, and temperature. In summer, this proportion was best predicted only by biomass of the young-of-year (YOY) gizzard shad. Thus, variation in YOY shad biomass significantly alters nutrient supply, and future studies should explore the long-term role of animal population dynamics in nutrient cycling. Our study shows that several years of data are needed to perform a critical evaluation of the importance of animals in meeting ecosystem nutrient demand.

Temporal trends in methane emissions from a small eutrophic reservoir: the key role of a spring burst.

Waters impounded behind dams (i.e., reservoirs) are important sources of greenhouses gases (GHGs), especially methane (CH4), but emission estimates are not well constrained due to high spatial and temporal variability, limitations in monitoring methods to characterize hot spot and hot moment emissions, and the limited number of studies that investigate diurnal, seasonal, and interannual patterns in emissions. In this study, we investigate the temporal patterns and biophysical drivers of CH4 emissions from Acton Lake, a small eutrophic reservoir, using a combination of methods: eddy covariance monitoring, continuous warm-season ebullition measurements, spatial emission surveys, and measurements of key drivers of CH4 production and emission. We used an artificial neural network to gap fill the eddy covariance time series and to explore the relative importance of biophysical drivers on the interannual timescale. We combined spatial and temporal monitoring information to estimate annual whole-reservoir emissions. Acton Lake had cumulative areal emission rates of 45.6 ± 8.3 and 51.4 ± 4.3 g CH4 m-2 in 2017 and 2018, respectively, or 109 ± 14 and 123 ± 10 Mg CH4 in 2017 and 2018 across the whole 2.4 km2 area of the lake. The main difference between years was a period of elevated emissions lasting less than 2 weeks in the spring of 2018, which contributed 17 % of the annual emissions in the shallow region of the reservoir. The spring burst coincided with a phytoplankton bloom, which was likely driven by favorable precipitation and temperature conditions in 2018 compared to 2017. Combining spatially extensive measurements with temporally continuous monitoring enabled us to quantify aspects of the spatial and temporal variability in CH4 emission. We found that the relationships between CH4 emissions and sediment temperature depended on location within the reservoir, and we observed a clear spatiotemporal offset in maximum CH4 emissions as a function of reservoir depth. These findings suggest a strong spatial pattern in CH4 biogeochemistry within this relatively small (2.4 km2) reservoir. In addressing the need for a better understanding of GHG emissions from reservoirs, there is a trade-off in intensive measurements of one water body vs. short-term and/or spatially limited measurements in many water bodies. The insights from multi-year, continuous, spatially extensive studies like this one can be used to inform both the study design and emission upscaling from spatially or temporally limited results, specifically the importance of trophic status and intra-reservoir variability in assumptions about upscaling CH4 emissions.

Patterns of CO2 concentration and inorganic carbon limitation of phytoplankton biomass in agriculturally eutrophic lakes.

Lake eutrophication is a pervasive problem globally, particularly serious in agricultural and densely populated areas. Whenever nutrients nitrogen and phosphorus do not limit phytoplankton growth directly, high growth rates will rapidly lead to biomass increases causing self-shading and light-limitation, and eventually CO2 depletion. The paradigm of phytoplankton limitation by nutrients and light is so pervasively established, that the lack of nutrient limitation is ordinarily interpreted as sufficient evidence for the condition of light limitation, without considering the possibility of limitation by inorganic carbon. Here, we firstly evaluated how frequently CO2 undersaturation occurs in a set of eutrophic lakes in the Pampa plains. Our results confirm that conditions of CO2 undersaturation develop much more frequently (yearly 34%, summer 44%) in these agriculturally impacted lakes than in deep, temperate lakes in forested watersheds. Secondly, we used Generalized Additive Models to fit trends in CO2 concentration considering three drivers: total incident irradiance, chlorophyll a concentration, and lake depth; in eight multi-year datasets from eutrophic lakes from Europe, North and South America, Asia and New Zealand. CO2 depletion was more often observed at high irradiance levels, and shallow water. CO2 depletion also occurred at high chlorophyll concentration. Finally, we identified occurrences of light- and carbon-limitation at the whole-lake scale. The different responses of chlorophyll a and CO2 allowed us to develop criteria for detecting conditions of CO2 limitation. For the first time, we provided whole-lake evidence of carbon limitation of phytoplankton biomass. CO2 increases and eutrophication represent two major and converging environmental problems that have additive and contrasting effects, promoting phytoplankton, and also leading to carbon depletion. Their interactions deserve further exploration and imaginative approaches to deal with their effects.

Precipitation, landscape properties and land use interactively affect water quality of tropical freshwaters.

Globally, conversion of pristine areas to anthropogenic landscapes is one of the main causes of ecosystem service losses. Land uses associated with urbanization and farming can be major sources of pollution to freshwaters promoting artificial inputs of several elements, leading to impaired water quality. However, how the effects of land use on freshwater quality are contingent on properties of the local landscape and climate is still poorly understood. The aim of this study was to evaluate the effects of landscape properties (morphometric measurements of lakes and their catchments), precipitation patterns, and land use properties (extent and proximity of the land use to freshwaters) on water quality of 98 natural lakes and reservoirs in northeast Brazil. Water quality impairment (WQI) was expressed as a composite variable incorporating parameters correlated with eutrophication including nitrogen (N), phosphorus (P) and Chlorophyll-a concentration. Regression tree analysis showed that WQI is mainly related to highly impacted "buffer areas". However, the effects of land use in these adjacent lands were contingent on precipitation variability for 13% of waterbodies and on surface area of the buffer in relation to the volume of waterbody (BA:Vol) for 87% of waterbodies. Overall, effects on WQI originating from the land use in the adjacent portion of the lake were amplified by high precipitation variability for ecosystems with highly impacted buffer areas and by high BA:Vol for ecosystems with less impacted buffer areas, indicating that ecosystems subjected to intense episodic rainfall events (e.g. storms) and higher buffer areas relative to aquatic ecosystem size (i.e. small waterbodies) are more susceptible to impacts of land use. Land use at the catchment scale was important for the largest ecosystems. Thus, our findings point toward the need for considering a holistic approach to managing water quality, which includes watershed management within the context of climate change.

Stream Nitrogen and Phosphorus Loads Are Differentially Affected by Storm Events and the Difference May Be Exacerbated by Conservation Tillage.

Storm events disproportionately mobilize dissolved phosphorus (P) compared to nitrogen (N), contributing to reduction in load N:P. In agricultural watersheds, conservation tillage may lead to even further declines in load N:P due to dissolved P accumulation in the top soil layers. Due to an increase in this management activity, we were interested in the impacts of conservation tillage on N and P loads during storm events. Using a 20 year data set of nutrient loads to a hypereutrophic reservoir, we observed disproportionately increasing P loads relative to base flow during storm events, whereas N loads were proportional to discharge. We also observed a change in that relationship, i.e., even greater P load relative to base flow with more conservation tillage in the watershed. This suggests conservation tillage may contribute to significantly reduced N:P loads during storms with potential implications for the water quality of receiving water bodies.

Stream Nitrogen, Phosphorus, and Sediment Concentrations Show Contrasting Long-term Trends Associated with Agricultural Change.

Stream water quality can be greatly influenced by changes in agricultural practices, but studies of long-term dynamics are scarce. Here we describe trends over 21 yr (1994-2014) in nutrients and suspended sediments in three streams in a Midwestern US agricultural watershed. During this time, the watershed experienced substantial changes in agricultural practices, most importantly a pronounced shift from conventional to conservation tillage. In the 1990s and early 2000s, NH, soluble reactive P, and suspended sediment concentrations (standardized for discharge and season) each declined significantly (>4-12% per year) in at least two of the three streams ( < 0.01), whereas NO changed relatively little. However, since the early 2000s, declines in NH and sediment concentrations have slowed, soluble reactive P concentrations have not declined and may actually have increased, and NO concentrations have declined sharply. The more recent lack of decline in soluble reactive P coincides with a plateau in the prevalence of conservation tillage and may be because of increased soil P stratification due to long-term reduced tillage. The more recent decline in NO may be due to improved efficiency of N fertilizer use, increased soil denitrification, and/or declines in atmospheric N deposition. Our study shows that stream concentrations of N, P, and sediment can respond in contrasting ways to changes in agriculture, and that temporal trends can moderate, accelerate, or reverse over decadal timescales. Management strategies must consider contrasting temporal responses of water quality indicators and may need to be adaptively adjusted at scales of years to decades.

Assessing uncertainty in annual nitrogen, phosphorus, and suspended sediment load estimates in three agricultural streams using a 21-year dataset.

Accurate estimation of constituent loads is important for studies of ecosystem mass balance or total maximum daily loads. In response, there has been an effort to develop methods to increase both accuracy and precision of constituent load estimates. The relationship between constituent concentration and stream discharge is often complicated, potentially leading to high uncertainty in load estimates for certain constituents, especially at longer-term (annual) scales. We used the loadflex R package to compare uncertainty in annual load estimates from concentration vs. discharge relationships in constituents of interest in agricultural systems, including ammonium as nitrogen (NH4-N), nitrate as nitrogen (NO3-N), soluble reactive phosphorus (SRP), and suspended sediments (SS). We predicted that uncertainty would be greatest in NO3-N and SS due to complex relationships between constituent concentration and discharge. We also predicted lower uncertainty with a composite method compared to regression or interpolation methods. Contrary to predictions, we observed the lowest uncertainty in annual NO3-N load estimates (relative error 1.5-23%); however, uncertainty was greatest in SS load estimates, consistent with predictions (relative error 19-96%). For all constituents, we also generally observed reductions in uncertainty by up to 34% using the composite method compared to regression and interpolation approaches, as predicted. These results highlight differences in uncertainty among different constituents and will aid in model selection for future studies requiring accurate and precise estimates of constituent load.

A global database of nitrogen and phosphorus excretion rates of aquatic animals.

Animals can be important in modulating ecosystem-level nutrient cycling, although their importance varies greatly among species and ecosystems. Nutrient cycling rates of individual animals represent valuable data for testing the predictions of important frameworks such as the Metabolic Theory of Ecology (MTE) and ecological stoichiometry (ES). They also represent an important set of functional traits that may reflect both environmental and phylogenetic influences. Over the past two decades, studies of animal-mediated nutrient cycling have increased dramatically, especially in aquatic ecosystems. Here we present a global compilation of aquatic animal nutrient excretion rates. The dataset includes 10,534 observations from freshwater and marine animals of N and/or P excretion rates. These observations represent 491 species, including most aquatic phyla. Coverage varies greatly among phyla and other taxonomic levels. The dataset includes information on animal body size, ambient temperature, taxonomic affiliations, and animal body N:P. This data set was used to test predictions of MTE and ES, as described in Vanni and McIntyre (2016; Ecology DOI: 10.1002/ecy.1582).

Consumer-driven nutrient dynamics in freshwater ecosystems: from individuals to ecosystems.

The role of animals in modulating nutrient cycling [hereafter, consumer-driven nutrient dynamics (CND)] has been accepted as an important influence on both community structure and ecosystem function in aquatic systems. Yet there is great variability in the influence of CND across species and ecosystems, and the causes of this variation are not well understood. Here, we review and synthesize the mechanisms behind CND in fresh waters. We reviewed 131 articles on CND published between 1973 and 1 June 2015. The rate of new publications in CND has increased from 1.4 papers per year during 1973-2002 to 7.3 per year during 2003-2015. The majority of investigations are in North America with many concentrating on fish. More recent studies have focused on animal-mediated nutrient excretion rates relative to nutrient demand and indirect impacts (e.g. decomposition). We identified several mechanisms that influence CND across levels of biological organization. Factors affecting the stoichiometric plasticity of consumers, including body size, feeding history and ontogeny, play an important role in determining the impact of individual consumers on nutrient dynamics and underlie the stoichiometry of CND across time and space. The abiotic characteristics of an ecosystem affect the net impact of consumers on ecosystem processes by influencing consumer metabolic processes (e.g. consumption and excretion/egestion rates), non-CND supply of nutrients and ecosystem nutrient demand. Furthermore, the transformation and transport of elements by populations and communities of consumers also influences the flow of energy and nutrients across ecosystem boundaries. This review highlights that shifts in community composition or biomass of consumers and eco-evolutionary underpinnings can have strong effects on the functional role of consumers in ecosystem processes, yet these are relatively unexplored aspects of CND. Future research should evaluate the value of using species traits and abiotic conditions to predict and understand the effects of consumers on ecosystem-level nutrient dynamics across temporal and spatial scales. Moreover, new work in CND should strive to integrate knowledge from disparate fields of ecology and environmental science, such as physiology and ecosystem ecology, to develop a comprehensive and mechanistic understanding of the functional role of consumers. Comparative and experimental studies that develop testable hypotheses to challenge the current assumptions of CND, including consumer stoichiometric homeostasis, are needed to assess the significance of CND among species and across freshwater ecosystems.

Predicting nutrient excretion of aquatic animals with metabolic ecology and ecological stoichiometry: a global synthesis.

The metabolic theory of ecology (MTE) and ecological stoichiometry (ES) are both prominent frameworks for understanding energy and nutrient budgets of organisms. We tested their separate and joint power to predict nitrogen (N) and phosphorus (P) excretion rates of ectothermic aquatic invertebrate and vertebrate animals (10,534 observations worldwide). MTE variables (body size, temperature) performed better than ES variables (trophic guild, vertebrate classification, body N:P) in predicting excretion rates, but the best models included variables from both frameworks. Size scaling coefficients were significantly lower than predicted by MTE (<0.75), were lower for P than N, and varied greatly among species. Contrary to expectations under ES, vertebrates excreted both N and P at higher rates than invertebrates despite having more nutrient-rich bodies, and primary consumers excreted as much nutrients as carnivores despite having nutrient-poor diets. Accounting for body N:P hardly improved upon predictions from treating vertebrate classification categorically. We conclude that basic data on body size, water temperature, trophic guild, and vertebrate classification are sufficient to make general estimates of nutrient excretion rates for any animal taxon or aquatic ecosystem. Nonetheless, dramatic interspecific variation in size-scaling coefficients and counter-intuitive patterns with respect to diet and body composition underscore the need for field data on consumption and egestion rates. Together, MTE and ES provide a powerful conceptual basis for interpreting and predicting nutrient recycling rates of aquatic animals worldwide.

Climate and land use interactively affect lake phytoplankton nutrient limitation status.

Climate-change models predict more frequent and intense summer droughts for many areas, including the midwestern United States. Precipitation quantity and intensity in turn drive the rates and ratios at which nitrogen (N) and phosphorus (P) are exported from watersheds into lakes, but these rates and ratios are also modulated by watershed land use. This led us to ask the question, is the effect of precipitation on phytoplankton nutrient limitation dependent on watershed land use? Across 42 lakes, we found that phytoplankton in lakes in agricultural landscapes were usually P limited but shifted to strong N limitation under increased drought intensity, and that droughts promoted N-fixing cyanobacteria. In contrast, phytoplankton in lakes with forested watersheds were consistently N limited, regardless of drought status. This climate-land use interaction suggests that droughts may increase the incidence of N limitation in agriculturally impacted lakes. N limitation would likely impair valuable ecosystem services such as drinking water, fisheries, and recreation by promoting the occurrence and severity of cyanobacterial blooms.

Ontogenetic variation in the body stoichiometry of two fish species.

One of the central questions of ecological stoichiometry theory is to what extent animal species maintain constant elemental composition in their bodies. Although several recent studies demonstrate intraspecific variation in animal elemental composition, relatively little is known about ontogenetic changes in vertebrates, especially during early life stages. We studied the intraspecific and interspecific ontogenetic variation in the body stoichiometry of two fish species in two different orders; fathead minnow (Pimephales promelas) and sheepshead minnow (Cyprinodon variegatus), reared under controlled laboratory conditions. During ontogeny, we measured the chemical composition of fish bodies, including carbon (C), nitrogen (N), phosphorus (P), calcium (Ca), and ribonucleic acid (RNA) contents. We found that N and RNA contents were relatively high in early life stages and declined substantially during development. In contrast, body C and C:N ratios were relatively low in embryos, post-embryos and larvae, and increased remarkably thereafter. Concentrations and ratios of some elements (e.g., Ca, P, Ca:P) did not exhibit consistent ontogenetic trends, but fluctuated dynamically between consecutive developmental stages in both species. Specific growth rates correlated significantly with RNA contents in both species. Analyses of the relative importance of different P pools at each developmental stage revealed that RNA was a considerable P pool in post-embryos, while bone-associated P was the dominant body P pool in later stages. Our results suggest that the elemental composition of fish bodies changes considerably during ontogeny. Each ontogenetic stage has its own stoichiometric signature, but the timing, magnitude and direction of ontogenetic changes can vary substantially between taxa.

When are fish sources vs. sinks of nutrients in lake ecosystems?

Animals can be important in nutrient cycling through a variety of direct and indirect pathways. A high biomass of animals often represents a large pool of nutrients, leading some ecologists to argue that animal assemblages can represent nutrient sinks within ecosystems. The role of animals as sources vs. sinks of nutrients has been debated particularly extensively for freshwater fishes. We argue that a large pool size does not equate to a nutrient sink; rather, animals can be nutrient sinks when their biomass increases, when emigration rates are high, and/or when nutrients in animal carcasses are not remineralized. To further explore these ideas, we use a simple model to evaluate the conditions under which fish are phosphorus (P) sources or sinks at the ecosystem (lake) level, and at the habitat level (benthic and water column habitats). Our simulations suggest that, under most conditions, fish are sinks for benthic P but are net P sources to the water column. However, P source and sink strengths depend on fish feeding habits (proportion of P consumed from the benthos and water column), migration patterns, and especially the fate of carcass P. Of particular importance is the rate at which carcasses are mineralized and the relative importance of benthic vs. pelagic primary producers in taking up mineralized P (and excreted P). Higher proportional uptake of P by benthic primary producers increases the likelihood that fish are sinks for water column P. Carcass bones and scales are relatively recalcitrant and can represent a P sink even if fish biomass does not change over time. Thus, there is a need for better documentation of the fraction of carcass P that is remineralized, and the fate of this P, under natural conditions. We urge a more holistic perspective regarding the role of animals in nutrient cycling, with a focus on quantifying the rates at which animals consume, store, release, and transport nutrients under various conditions.

Quinone-induced activation of Keap1/Nrf2 signaling by aspirin prodrugs masquerading as nitric oxide.

The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.

Inhibition of amyloidogenesis by nonsteroidal anti-inflammatory drugs and their hybrid nitrates.

Poor blood-brain barrier penetration of nonsteroidal anti-inflammatory drugs (NSAIDs) has been blamed for the failure of the selective amyloid lowering agent (SALA) R-flurbiprofen in phase 3 clinical trials for Alzheimer's disease (AD). NO-donor NSAIDs (NO-NSAIDs) provide an alternative, gastric-sparing approach to NSAID SALAs, which may improve bioavailability. NSAID analogues were studied for anti-inflammatory activity and for SALA activity in N2a neuronal cells transfected with human amyloid precursor protein (APP). Flurbiprofen (1) analogues were obtained with enhanced anti-inflammatory and antiamyloidogenic properties compared to 1, however, esterification led to elevated Aß(1-42) levels. Hybrid nitrate prodrugs possessed superior anti-inflammatory activity and reduced toxicity relative to the parent NSAIDs, including clinical candidate CHF5074. Although hybrid nitrates elevated Aß(1-42) at higher concentration, SALA activity was observed at low concentrations (≤1 µM): both Aß(1-42) and the ratio of Aß(1-42)/Aß(1-40) were lowered. This biphasic SALA activity was attributed to the intact nitrate drug. For several compounds, the selective modulation of amyloidogenesis was tested using an immunoprecipitation MALDI-TOF approach. These data support the development of NO-NSAIDs as an alternative approach toward a clinically useful SALA.

Synthesis and biological evaluation of 2'-oxo-2,3-dihydro-3'H- spiro[chromene-4,5'-[1,3]oxazolidin]-3'yl]acetic acid derivatives as aldose reductase inhibitors.

Aldose reductase (ARL2) is the first enzyme in the polyol pathway which catalyzes the NADPH-dependent reduction of glucose to sorbitol. Its involvement on diabetic complications makes this enzyme a challenge therapeutic target widely investigated to limit and/or prevent them. On this basis, a limited series of 4-spiro-oxazolidinone-benzopyran derivatives (1-7) were synthesized to evaluate them as potential ARL2 inhibitors. The activity was determined spectrophotometrically by monitoring the oxidation of NADPH catalyzed by ALR2. Within the series of compounds, the 4-methoxy derivative 1b showed to be the most active compound, exhibiting inhibitory levels in the submicromolar range. In addition, the activity against the aldehyde reductase isoform (ARL1) was also evaluated. Unlike sorbinil (reference drug) that lack of selectivity towards the two enzyme all the tested compounds resulted to be devoid of ARL1 inhibitory activity (IC(50) > 10 µM), thus proving to be selective.

Synthesis and biological evaluation of 5-membered spiro heterocycle-benzopyran derivatives against myocardial ischemia.

The activation of ATP-sensitive potassium channels (K(ATP)), play a key role in an endogenous "self-defence" mechanism, known as ischemic preconditioning (IPC), which is fundamentally involved in the protection of the heart against the ischemia/reperfusion injury. Presently, it is widely accepted that IPC is mainly (albeit not exclusively) mediated by the activation of K(ATP) channels expressed in the mitochondrial inner membrane (mito-K(ATP)) rather than the sarcoplasmatic ones (sarc-K(ATP)). Consistently, exogenous activation of K(ATP) channels by pharmacological tools can be viewed as one of the most promising strategies for the therapy of myocardial ischemia. As part of our research program devoted to the synthesis and the evaluation of new cardioprotective agents, we extensively studied several six-membered spiro-heterocycle-benzopyran compounds endowed of a significant anti-ischemic activity. The positive results obtained, prompted us to further explore the influence on the biopharmacological effects, of the spiro-substitution at C4 benzopyran nucleus by replacing the six-membered spirocycle of the most active compounds with 5-membered-one. The preliminary evaluation of the new compounds on cultured H9c2 cardiomyoblasts exposed to anoxia/reperfusion and on Langendorff-perfused rat hearts submitted to ischemia/reperfusion cycles, showed that some of them can exert a cardioprotective effect. This anti-ischemic activity was antagonized by 5-hydroxydecanoic acid, a selective blocker of mito-K(ATP) channels, confirming the involvement of this channel in the cardioprotective activity.