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Pharmacological and neurobiological studies on Neuropeptide S and its receptor
No full textNeuropeptide S (NPS) is the last neuropeptide identified via reverse pharmacology techniques. NPS
selectively binds and activates a previously orphan GPCR, now named NPSR, producing intracellular Ca2+
mobilization and stimulation of cAMP levels. Biological functions modulated by the NPS/NPSR system
include anxiety, arousal, locomotion, food intake, learning and memory, pain and drug addiction. In our
laboratories we provided further evidence that NPS injected supraspinally in mice acts as a stimulatory
anxiolytic. In fact, in the mouse righting reflex (RR) test, NPS (0.01- 1 nmol, i.c.v.) was able to reduce in a
dose dependent manner the percent of animals losing the RR in response to diazepam (15mg/kg, i.p.) and
their sleep time. Furthermore, NPS in the same range of doses caused a significant increase in locomotor
activity (LA) in mice. These effects were associated with a clear anxiolytic-like action elicited by NPS in the
mouse elevated plus maze (EPM) test, open field (OF) test and stress-induced hyperthermia (SIH) assay.
Thus NPS evokes an unique pattern of behavioural effects: stimulation associated with anxiolysis. To deeply
investigate the biological roles played by the NPS/NPSR system the development of pharmacological (i.e.
selective NPSR ligands, particularly antagonists) and genetic (i.e. receptor knockout animals) tools are
needed. In collaboration with the medicinal chemistry group of the University of Ferrara, we performed a
series of classical structure-activity (SAR) studies on NPS sequence. Specifically, NPS positions 2, 3, 4 and
5 were investigated in details, since they were demonstrated to be crucial for NPS bioactivity. Studies
focussed on NPS position 5 led to the identification and the in vitro and in vivo pharmacological
characterization of the first generation of NPSR peptide antagonists. In vitro, in HEK293 cells stably
expressing the mouse NPSR, [D-Cys(tBu)5]NPS up to 100 μM did not stimulate Ca2+ mobilization but was
able to counteract in a competitive manner the stimulatory action of NPS (pA2: 6.44). In vivo, in the RR test,
[D-Cys(tBu)5]NPS at 10 nmol was inactive per se but dose dependently antagonized the arousal-promoting
action of NPS 0.1 nmol. [D-Val5]NPS acted in vitro as a pure NPSR antagonist, with a pKB of 6.54 in
inhibition experiments. In vivo, in LA test, [D-Val5]NPS at 10 nmol completely blocked the stimulatory
effect evoked by NPS. In a further medicinal chemistry study, the potent NPSR antagonist [tBu-D-Gly5]NPS
was identified. In vitro, [tBu-D-Gly5]NPS did not stimulate calcium mobilization but blocked the stimulant
action of NPS with a pKB of 7.06 7. In vivo, in RR assay, [tBu-D-Gly5]NPS (0.1-10 nmol, i.c.v.) was inactive
per se but dose dependently antagonized the arousal-promoting action of NPS 0.1 nmol. Similarly in the LA
assay [tBu-D-Gly5]NPS (0.1-10 nmol, i.c.v.) was inactive per se but was able to counteract the stimulatory
effect evoked by 0.1 nmol NPS in a dose dependent manner. SHA 68 has been previously identified as the
first non peptide NPSR antagonist. In our laboratories we further assessed the pharmacological profile of
SHA 68 in vitro and in vivo. In vitro SHA 68 was inactive per se up to 10 μM while it antagonized NPSstimulated
Ca2+ mobilization in a competitive manner showing a pA2 value of 8.06. In vivo, in the mouse RR
assay, SHA68 50 mg/kg i.p. fully prevented the arousal promoting action of NPS 0.1 nmol. In LA
experiments, SHA 68 50 mg/kg i.p. was able to partially counteract the stimulant effects elicited by NPS 0.1
nmol. Instead, the anxiolytic-like effects of NPS 0.1 nmol in mouse OF test were slightly reduced by SHA
68. Collectively these data demonstrated the exclusive involvement of NPSR in the arousal promoting and
locomotor stimulant effects of NPS. Finally, we backcrossed on the CD-1 strain the NPSR knockout mice
originally generated on the 129Sv/Ev genetic background. A first phenotype analysis revealed no locomotor
differences between NPSR(+/+) and NPSR(-/-) mice, with the exception of rearing behaviour that was
reduced in knockout animals. Furthermore, the behaviour of NPSR(+/+) and NPSR(-/-) mice in the EPM, OF
and SIH tests is superimposable. Similarly no differences were detected in the novel object recognition,
forced swimming, RR and formalin assays. However, the stimulant actions of 1 nmol NPS in RR and in LA
test could be detected in NPSR(+/+) but not in NPSR(-/-) mice. Collectively these data demonstrated that
endogenous NPS/NPSR system does not play a role in the control of locomotion, anxiety, depression and
memory, at least under the present experimental conditions. These results demonstrated that the NPS
stimulant effects are selectively due to NPSR activation, corroborating the findings obtained with NPSR
antagonists. In conclusion, the research activity performed during the PhD program led to the identification
of the first generation of NPSR peptide antagonists. The use of these research tools in parallel with knockout
studies generated converging evidence on the biological effects induced by the selective activation of NPSR
Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test
No full textNociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1–1 mg kg−1) and morphine (0.1–10 mg kg−1) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01–0.1 mg kg−1) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Functional Selectivity Does Not Predict Antinociceptive/Locomotor Impairing Potencies of NOP Receptor Agonists
Full text linkNociceptin/orphanin FQ controls several functions, including pain transmission, via stimulation of the N/OFQ peptide (NOP) receptor. Here we tested the hypothesis that NOP biased agonism may be instrumental for identifying innovative analgesics. In vitro experiments were performed with the dynamic mass redistribution label free assay and the NOP non-peptide agonists Ro 65-6570, AT-403 and MCOPPB. In vivo studies were performed in wild type and β-arrestin 2 knockout mice using the formalin, rotarod and locomotor activity tests. In vitro all compounds mimicked the effects of N/OFQ behaving as potent NOP full agonists. In vivo Ro 65-6570 demonstrated a slightly higher therapeutic index (antinociceptive vs. motor impairment effects) in knockout mice. However, all NOP agonists displayed very similar therapeutic index in normal mice despite significant differences in G protein biased agonism. In conclusion the different ability of inducing G protein vs. β-arrestin 2 recruitment of a NOP agonist cannot be applied to predict its antinociceptive vs. motor impairment properties
NOP agonist action of cebranopadol counteracts its liability to promote physical dependence
No full textCebranopadol is a mixed NOP/opioid receptor agonist currently under development as innovative analgesic. In this study the liability of cebranopadol to produce opioid-type physical dependence has been evaluated in comparison with morphine in wild type mice and in mice knockout for the NOP receptor gene (NOP(-/-)). Mice were treated twice a day for 5 days with increasing doses of cebranopadol or morphine (cumulative doses 10.2 and 255 mg/kg, respectively) and the number of jumping in response to naloxone 10 mg/kg were measured after 2 h from the last injection. In wild type mice naloxone evoked a similar withdrawal jumping behavior in animal pretreated with morphine or cebranopadol. In NOP(-/-) mice morphine treatment produced the same signs of withdrawal as in NOP(+/+) animals, while cebranopadol treatment elicited a stronger withdrawal syndrome in NOP(-/-) than of NOP(+/+) mice. These results demonstrated that the activation of the NOP receptor reduces the liability of cebranopadol to produce opioid-like physical dependence. Thus, the simultaneous activation of NOP and opioid receptors can be an effective pharmacological strategy to counteract physical dependence to opioid drugs
Behavioural phenotypic characterization of CD-1 mice lacking the neuropeptide S receptor
No full textNeuropeptide S (NPS) is the endogenous ligand of a previously orphan receptor now named NPSR. In the brain NPS regulates several biological functions including anxiety, arousal, locomotion, food intake, learning and memory, pain and drug abuse. Mice lacking the NPSR gene (NPSR(-/-)) represent an useful tool to investigate the neurobiology of the NPS/NPSR system. NPSR(-/-) mice have been generated in a 129S6/SvEv genetic background. In the present study we generated CD-1 congenic NPSR(+/+) and NPSR(-/-) mice and investigated their phenotype and sensitivity to NPS in various behavioural assays. The phenotype analysis revealed no locomotor differences between NPSR(+/+) and NPSR(-/-) mice. The behaviour of NPSR(+/+) and NPSR(-/-) mice in the righting reflex test was superimposable. No differences were recorded between the two genotypes in the elevated plus maze, open field and stress-induced hyperthermia tests, with the exception of rearing behaviour that was reduced in knockout animals. Moreover the behaviour of NPSR(+/+) and NPSR(-/-) mice in the forced swimming, novel object recognition and formalin assays was similar. The stimulatory effects of NPS in the locomotor activity test and its anxiolytic-like actions in the elevated plus maze and open field assays were evident in NPSR(+/+) but not NPSR(-/-) animals. In conclusion, the present study indicates that the NPS/NPSR system does not tonically control locomotion, sensitivity to diazepam, anxiety, depressive-like behaviours, memory and pain transmission in mice. Furthermore our results clearly show that the product of the NPSR gene represents the mandatory protein for all the NPS biological effects so far described
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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