Potential of the zebrafish model for the forensic toxicology screening of NPS: A comparative study of the effects of APINAC and methiopropamine on the behavior of zebrafish larvae and mice

Abstract The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening

Valutazione della tolleranza di formulazioni standard a confronto con formulazioni personalizzate nella nutrizione parenterale pediatrica

Valutazione della tolleranza al trattamento di nutrizione parenterale di pazienti con età compresa tra 1 e 18 anni (età pediatrica) attraverso l'utilizzo di formulazioni standard sia del commercio sia preparazioni galeniche. Definizione anche di formulazioni a composizione standard per pazienti pediatrici di età inferiore a 1 ann

Modulation of circadian rhythms by glucocorticoids

Il comportamento è concepito come una relazione dipendente stimolo-risposta tra un input sensoriale e una risposta motoria. Nel passaggio da input a output, l’omeostasi interna è continuamente modellata per mantenere un equilibrio ottimale della spesa energetica. Lo scopo ultimo di mantenere l’omeostasi in relazione al mondo circostante viene raggiunto attraverso la produzione di comportamenti adattativi che permettono di incrementare la fitness alla luce della selezione naturale. L’ambiente circostante può essere sia prevedibile sia imprevedibile. La prima condizione ha portato all’evoluzione del ritmo circadiano che promuove la fase di attività durante il momento più favorevole della giornata, mentre la seconda si serve dell’asse dei glucocorticoidi per affrontare le sfide imprevedibili. Quindi, un dialogo tra il sistema circadiano e il sistema dei glucocorticoidi è mantenuto allo scopo di ottenere una regolazione ottimale dell’attività animale. Il mio obiettivo è quello di capire il dialogo tra i due sistemi monitorando il comportamento giornaliero e circadiano, e la sua controparte molecolare, in fase a differenti cicli di luce e cibo. La mia specie modello è lo zebrafish (Danio rerio), in particolare, ho utilizzato un mutante costruito con la tecnica CRISPR/Cas9 che manca della capacità di coordinare la via di trascrizione dei glucocorticoidi a causa della mancata funzionalità del loro recettore cognato tale che l’interazione ligando recettore non è mantenuta. Di conseguenza, i livelli circolanti di glucocorticoidi restano elevati, conferendo al mutante un fenotipo ansioso. Zebrafish gr-/- è stato costruito e gentilmente fornito dal laboratorio della Prof.ssa Luisa Dalla Valle, Università degli studi di Padova. L’analisi sistematica del comportamento in larve e adulti di gr-/- ha mostrato che l’attività locomotoria sincronizzata alla luce mantiene le sue proprietà oscillatorie endogene. Tuttavia, l’attività locomotoria giornaliera insorge con un ritardo di un giorno nei mutanti rispetto ai wild type. Questa insorgenza ritardata è associata a un rallentamento nello sviluppo del tessuto muscolare striato, la normale densità delle fibre muscolari viene ripristinata nei gr-/- al sesto giorno dopo la fertilizzazione. Inoltre, le larve gr-/- hanno mostrato differenze nei livelli di espressione e nelle relative acrofasi di elementi positivi (arntl1a and clock1a) e negativi (per1, per2a and cry1a) dell’orologio molecolare. Al di là degli elementi del cuore dell’orologio circadiano, un’analisi nel fegato di adullti gr-/- rivela un’abolizione dell’espressione di pck2, un gene implicato nella gluconeogenesi. In aggiunta, srebp1 ha un’acrofase anticipata nei mutanti. La sincronizzazione circadiana al cibo fallisce nei gr-/-, sia larve sia adulti producono profili anomali dell’attività locomotoria. L’analisi molecolare non associa la disfunzionalità comportamentale a quella genetica, infatti i geni orologio non mostrano alterate oscillazioni a eccezione di cry1a. Questi dati suggeriscono l’esistenza di un confine sfuocato tra il sistema circadiano e quello dei glucocorticoidi e una complessa organizzazione dei due ha prodotto un alterato output comportamentale negli zebrafish gr-/-. La causa prossima del disallineamento tra lo stimolo alimentare e la locomozione non è stata chiarita sebbene un passo avanti verso una maggiore comprensione del dialogo tra glucocorticoidi e orologio circadiano getta le basi per un’indagine più profonda.Behavior is conceived as a stimulus-response dependent relationship between a sensory input and a motor output. While moving from an input to an output, internal homeostasis is continuously shaped to maintain an optimal energies expenditure balance. The ultimate purpose of enabling animals to adjust their homeostasis with the surrounding world is by producing adaptive behaviors in order to increase their fitness in light of natural selection. The environment can be either predictable or unpredictable. The former condition led to the evolution of the circadian rhythm to promote an active behavior at the time you mostly benefit from, while the latter take advantage of glucocorticoids axis to face sudden challenges. Thus, a crosstalk between the circadian and the glucocorticoid systems allows a fine tuning of animal’s activity. My goal is to understand the circadian-glucocorticoids dialogue by monitoring the locomotor daily/circadian behavior and its molecular oscillation counterpart under differentially phased light and feeding cycle. My model species is the zebrafish, particularly, I utilized a CRISPR/Cas9 mutant lacking the capability to coordinate glucocorticoids transcription because it lacks functional receptors which permit a correct ligand-receptor interaction. As a result, level of circulating glucocorticoids stays raised conferring an anxiety-related phenotype to the mutant. Zebrafish gr-/- has been built and kindly provided by Dr. Luisa Dalla Valle, University of Padua. Systematic behavioral analysis in gr-/- larvae and adults showed that the light entrainable locomotor activity is synchronized to the zeitgeber and maintain its oscillatory properties in absence of any cue. The onset of daily locomotor activity occurred one day later in mutants with respects to the wild type. This delay is linked to the slower striated muscle development in the gr-/- which recover regular fiber density at 6 days post fertilization. Furthermore, gr-/- larvae showed differences in the expression levels or in the peak phase of positive (arntl1a and clock1a) and negative (per1, per2a and cry1a) elements of the molecular clock. Outside the core clock network, an analysis on gr-/- adult livers reported an abolished daily expression of pck2, a gene involved in gluconeogenesis. In addition, srebp1 expression level has an anticipated acrophase in gr-/-. Feeding entrainment fails to occur in the mutants. Larvae and adults produced abnormal profiles of circadian locomotor activity. Further molecular investigation revealed this behavioral disruption wasn’t associated with a breakdown of molecular rhythms in the core clock genes. Nevertheless, the molecular phenotypes observed during feeding entrainment underlined a cry1a lack of rhythmicity. These data suggest the existence of a blurred boundary between the circadian-glucocorticoids crosstalk. A complex organization of the two produces an altered behavioral output in a food entrained schedule in gr-/- zebrafish. The proximate cause of input and output misalignment underlying food entrained locomotion has not been provided, but a step towards a more exhaustive comprehension about the circadian-glucocorticoids interaction paves the way for an in-depth investigation

Guppy_telomere_dataset

Dataset including log muscle and sperm RTL, relative orange size, relative sperm production of male guppies. </p

Dietary stress increases the total opportunity for sexual selection and modifies selection on condition-dependent traits

Although it is often expected that adverse environmental conditions depress the expression of condition-dependent sexually selected traits, the full consequences of environmental change for the action of sexual selection, in terms of the opportunity for total sexual selection and patterns of phenotypic selection, are unknown. Here we show that dietary stress in guppies, Poecilia reticulata, reduces the expression of several sexually selected traits and increases the opportunity for total sexual selection (standardized variance in reproductive success) in males. Furthermore, our results show that dietary stress modulates the relative importance of precopulatory (mating success) and postcopulatory (relative fertilization success) sexual selection, and that the form of multivariate sexual selection (linear vs. nonlinear) depends on dietary regime. Overall, our results are consistent with a pattern of heightened directional selection on condition-dependent sexually selected traits under environmental stress, and underscore the importance of sexual selection in shaping adaptation in a changing world.This work was funded by the University of Padova (Progetto CPDA120105 and Assegno di Ricerca BIRD 2017) to A.P. S.C. was supported by a Postgraduate Scholarship through The University of Padova, and F.G.G. by the Spanish Ministry of Economy (CGL2016-76173-P AEI/FEDER, UE). J.P.E. was supported by a sabbatical grant funded by the University of Western Australia.Peer reviewe

Innovative Renewable Technology Integration for Nearly Zero-Energy Buildings within the Re-COGNITION Project

With the 2010/31/EU directive, all new buildings shall be nearly zero-energy buildings (nZEB) from 2020 onward, with the aim of strongly reducing the energy consumption related to the building sector. To achieve this goal, it is not sufficient to focus on the design of the building envelope; smart and efficient energy management is necessary. Moreover, to ensure the adoption of RES systems in the built environment, innovative technologies need to be further developed in order to increase their cost-effectiveness, energy efficiency and integration capability. This paper proposes a synthesis, design and operation optimization of an integrated multi-energy system composed of traditional and innovative renewable technologies, developed within the European project Re-COGNITION. A biogas-based micro cogeneration unit, lightweight glass-free photovoltaic modules, a passive variable geometry small wind turbine optimized for an urban environment and latent heat thermal storage based on phase change materials are some of the technologies developed within the Re-COGNITION project. The optimization problem is solved to contemporarily evaluate (a) the optimal design and (b) the optimal operations of the set of technologies considering both investment and operating costs, using mixed integer non-linear programming. The optimization is applied to the four pilots that are developed during the project, in various European cities (Turin (Italy), Corby (United Kingdom), Thessaloniki (Greece), Cluj-Napoca (Romania). Simulation results show that the development and optimal exploitation of new technologies through optimization strategies provide significant benefits in terms of cost (between 11% and 42%) and emissions (between 10% and 25%), managing building import/export energy and charge/discharge storage cycles

Extracellular ATP signaling during differentiation of C2C12 skeletal muscle cells: role in proliferation

Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling

Feeding Entrainment of the Zebrafish Circadian Clock Is Regulated by the Glucocorticoid Receptor

Glucocorticoids (GCs) are steroid hormones mainly acting as key regulators of body homeostasis and stress responses. Their activities are primarily based on the binding to the GC receptor (GR), a member of the nuclear receptor family, that regulates tissue-specific sets of genes. GCs secretion follows a circadian rhythmicity with a peak linked to the animal's activity phase. In mammals, GCs are also implicated in feeding entrainment mechanisms as internal zeitgeber. Here, we investigated, by means of behavioural and molecular approaches, the circadian clock and its regulation by light and food in wild-type (WT) and null glucocorticoid receptor (gr-/-) zebrafish larvae, juveniles and adults. In both WT and gr-/- larvae and adults, behavioural activity and clock gene expression were entrained to the light-dark (LD) cycle and rhythmic in constant conditions. Differences in the pattern of clock genes' expression indicated a modulatory role of GCs. A significant role of Gr was detected in the feeding entrainment which was absent or markedly dampened in mutants. Furthermore, the expression of two clock-regulated genes involved in glucidic and lipidic metabolism was altered, highlighting the participation of GCs in metabolic processes also in fish. Taken together, our results confirmed the role of GC-mediated Gr signaling in the feeding entrainment in a non-mammalian species, the zebrafish