1,721,060 research outputs found
SPHINGOLIPID SIGNALING AS A TARGET IN PHOTORECEPTOR DEGENERATION: AN IN VITRO MODEL FOR THERAPEUTIC STRATEGIES IN RETINITIS PIGMENTOSA
Full text linkIntroduzione: gli Sfingolipidi sono una vasta famiglia di molecole con il duplice ruolo di componenti strutturali di membrane e mediatori intra ed extra cellulari. Sono implicati nel controllo di proliferazione, differenziazione e sopravvivenza cellulare ma anche di fenomeni legati alla morte, tra cui l’apoptosi. Ceramide (Cer), alla base degli sfingolipidi complessi, può andare incontro a deacilazione generando sfingosina e sfingosina-1-fosfato (S1P). Quest’ultimo è considerato un mediatore di sopravvivenza e proliferazione cellulare, in opposizione a Cer, che promuove arresto del ciclo ed apoptosi. La degenerazione retinica ed, in particolare la Retinite Pigmentosa (RP) sono associate all’accumulo di Cer ed all’induzione di morte cellulare. L’inibizione della sintesi e dell’accumulo di Cer, inattivando la serina palmitoiltransferasi (SPT) attraverso l’utilizzo di Miriocina, è stata dimostrata essere coinvolta nel recupero morfologico e funzionale dei fotorecettori retinici, in particolare dei coni, in un modello murino di RP (mutante rd10).
Scopo: Il nostro scopo è di intervenire sul metabolismo degli sfingolipidi per ridurre il danno di fotorecettori retinici, attarveso l’utilizzo di una linea cellulare di fotorecettori coni. In particolare, promuovendo l’aumento di S1P intracellulare, tramite l’inibizione dell’enzima S1P liasi, e la riduzione di Cer attraverso l’inattivazione di SPT.
Metodi: le cellule murine 661W, una linea di fotorecettori (coni), sono state trattate con 2-acetil-4(5)-(1(R),2(S),3(R),4-tetraidrobutil)-imidazolo (THI) 75μM, un inibitore dell’enzima S1Pliasi, per 2 ore; quindi, le cellule sono state sottoposte a deprivazione di siero e trattate con H2O2 1mM per differenti lassi di tempo. La curva di crescita di queste cellule è stata determinata attraverso il saggio MTT e la vitalità con Typan blue. Il saggio TUNEL e il FRAP test sono stati impiegati per verificare, rispettivamente, il grado di apoptosi e il potere antiossidante intrinseco dei fotorecettori. I contenuti intracellulari di sfingolipidi sono stati misurati attraverso l’analisi LC-MS. La tecnica del Western blotting, attraverso l’impiego di anticorpi specifici, è stata utilizzata per valutare la fosforilazione di ERK1/2 e Akt/PKB, il rapporto tra Bcl-2/Bax e l’espressione proteica di Nrf2. Attraverso Real time-PCR, le modulazioni nell’espressione dei trascritti di HO-1 e dei recettori di S1P sono stati valutati in seguito a stress ossidativo e trattamento con THI. S1P esogeno (100nM) e Miriocina (10μM) sono stati impiegati, rispettivamente 1 ora e 5 ore prima H2O2, per antagonizzare l’effetto dello stimolo ossidativo sui fotorecettori. In fine, abbiamo esaminato 30 composti di nuova sintesi per determinare la loro capacità inibitoria su SPT, attraverso un saggio di attività enzimatica.
Risultati: i nostri risultati mostrano che l’aumentata stabilità di S1P, ottenuta tramite l’utilizzo di THI, è in grado di ridurre l’effetto inibitorio sulla proliferazione e la vitalità cellulare, dato dalla deprivazione di siero e dal trattamento con H2O2. Il pretrattamento con THI, infatti, antagonizza la de-fosforilazione di ERK1/2 e la fosforilazione di Akt su Ser473, indotte dagli stimoli di morte utilizzati. Abbiamo, quindi, focalizzato la nostra ricerca sugli effetti dello stress ossidativo, evidenziando che THI si contrappone all’induzione dell’apopstosi tramite l’aumento del rapporto Bcl-2/Bax e influenza il potere antiossidante intrinseco dei fotorecettori modulando la via di segnale di Nrf2 e HO-1. Inoltre, THI aumenta l’espressione dei recettori di S1P, mostrando un effetto maggiore sui recettori S1P4 e S1P5. Inoltre, abbiamo osservato che basse concentrazioni di S1P migliorano la proliferazione, la vitalità e la risposta antiossidante di cellule 661W, mentre alte concentrazioni portano alla saturazione dei recettori.Il pretrattamento con miriocina, sia da solo che in combinazione con THI, protegge i fororecettori dallo stress ossidativo e riduce drasticamente i livelli intracellulari di tutte le specie di Cer. Parallelamente, abbiamo selezionato tre composti con attività inbitoria su SPT, determinando i rispettivi valori di IC50 che sono risultati essere compresi tra 17,71μM e 40,41μM.
Conclusioni: possiamo quindi concludere che la stabilizzazione di S1P e, più in generale, l’azione sul metabolismo degli sfingolipidi è da considerarsi un target terapeutico per promuovere la sopravvivenza fotorecettoriale in differenti condizioni di stress, tra cui lo stress ossidativo.Background: Sphingolipids are a broad class of molecules with the double role of cell membranes components and intra/extra cellular signal mediators, controlling proliferation, differentiation, stress survival and apoptosis. Ceramide (Cer), the core of complex sphingolipids, can undergo deacylation giving rise to sphingosine and sphingosine-1-phosphate (S1P). This latter exerts a pro-survival and proliferative activity, as opposed to Cer, which promotes cell cycle arrest and apoptosis. Retinal degeneration and in particular Retinitis Pigmentosa (RP) are associated to Cer accumulation and cell death induction. In a murine model of RP (rd10 mutant mice), it has been demonstrated that inhibition of Cer synthesis and accumulation, blocking serine palmitoyltransferase (SPT) with Myriocin, rescues retinal photoreceptors, especially cones, from degeneration.
Aim: Our aim is to target sphingolipid metabolism to reduce retinal photoreceptor damage, in a cone photoreceptors cell line. In particular, promoting S1P intracellular increase by S1Plyase inhibition and Cer depletion through SPT inactivation.
Methods: Murine 661W cone-like cell line were treated with 75μM 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI), an inhibitor of S1Plyase, for 2 hours; next, cells were starved and treated with 1mM H2O2 for different times. Cell growth curve was determined by MTT assay and viability with Trypan blue. TUNEL assay and FRAP test were employed to verify, respectively, apoptosis degree and antioxidant-intrinsic power. Sphingolipid intracellular amounts were measured through LC-MS analysis. ERK1/2 and Akt/PKB phosphorylation, Bcl-2/Bax ratio and Nrf2 expression was evaluated by Western blotting with specific antibodies. Real time-PCR was performed to establish HO-1 and S1P receptors transcript changes upon THI and oxidative stress treatments. Exogenous S1P (100nM) and Myriocin (10μM) were also employed, respectively 1 hour and 5 hours before H2O2, to antagonize oxidative stress effect on photoreceptors. Lastly, we screened 30 new synthetic compounds to determine their ability in inhibiting SPT, through an enzymatic activity assay.
Results: We show that enhanced stability of S1P, obtained through THI administration, reduces inhibitory starvation and H2O2 effect on cell proliferation and viability. In particular, through THI ability to reverse stresses-induced ERK1/2 dephosphorylation and Akt phosphorylation on Ser473. We focused our investigations on oxidative stress, finding that THI counteracts H2O2-induced apoptosis increasing Bcl-2/Bax ratio and antioxidant-intrinsic power modulating Nrf2/HO-1 pathway. Furthermore, THI differentially induces S1P receptors transcript expression, showing the major effect on S1P4 and S1P5. In addition, low exogenous S1P improves 661W proliferation, viability and antioxidant response, whereas higher concentration leads to S1P receptors saturation. Myriocin treatment, either alone or in combination with THI, rescues photoreceptors from H2O2-induced oxidative stress and drastically reduces all Cer pools. Simultaneously, we selected three compounds with inhibitory activity on SPT, showing an IC50 values ranging from 17.71μM to 40.41μM.
Conclusions: We conclude that S1P stabilization and, in general, sphingolipid metabolism manipulation can be considered a therapeutic target in order to promote photoreceptors survival under different stress conditions, such as oxidative stress
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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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