1,720,959 research outputs found
Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay b
No full textThe neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests
A method for reproducible measurements of serum BDNF: Comparison of the performance of six commercial assays
Full text linkBrain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKine TM, Promega-Emax ®, R&D-System-Quantikine ®) and one multiplexing assay (Millipore-Milliplex ®). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications
Combined cisplatin and aurora inhibitor treatment increase neuroblastoma cell death but surviving cells overproduce BDNF
Full text linkDrug-resistance to chemotherapics in aggressive neuroblastoma (NB) is characterized by enhanced cell survival mediated by TrkB and its ligand, brain-derived neurotrophic factor (BDNF); thus reduction in BDNF levels represent a promising strategy to overcome drug-resistance, but how chemotherapics regulate BDNF is unknown. Here, cisplatin treatment in SK-N-BE neuroblastoma upregulated multiple BDNF transcripts, except exons 5 and 8 variants. Cisplatin increased BDNF mRNA and protein, and enhanced translation of a firefly reporter gene flanked by BDNF 5′UTR exons 1, 2c, 4 or 6 and 3′UTR-long. To block BDNF translation we focused on aurora kinases inhibitors which are proposed as new chemotherapeutics. NB cell survival after 24 h treatment was 43% with cisplatin, and 22% by cisplatin+aurora kinase inhibitor PHA-680632, while the aurora kinases inhibitor alone was less effective; however the combined treatment induced a paradoxical increase of BDNF in surviving cells with strong translational activation of exon6-3′UTR-long transcript, while translation of BDNF transcripts 1, 2C and 4 was suppressed. In conclusion, combined cisplatin and aurora kinase inhibitor treatment increases cell death, but induces BDNF overproduction in surviving cells through an aurora kinase-independent mechanism
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
Neurotrophic factors and other humoral mediators in chronic stress
Full text link2013/2014Lo stress è stato definito come "una risposta specifica del corpo a qualsiasi richiesta non specifica (stressor) su di esso". Quando lo stress, reale o percepito, è duraturo o la risposta non è appropriata (distress), i cambiamenti fisiologici di breve termine lasciano spazio ad alterazioni di lungo termine che possono causare condizioni patologiche, tra cui malattie mentali. Secondo questo modello di "carico allostatico", i mediatori primari come le catecolamine e gli ormoni steroidei, innescano alterazioni multi-sistemiche su mediatori secondari che portano a manifestazioni negative in diversi sistemi corporei, inclusi quello nervoso e immunitario. Neurotrofine, in particolare il Brain-Derived Neurotrophic Factor (BDNF), e mediatori del sistema immunitario, come citochine e chemochine, sono possibili modulatori della risposta allo stress cronico. Pertanto, ci siamo posti l’obiettivo di valutare i loro livelli circolanti in soggetti sani in condizioni di stress lavoro-correlato. Prima di ciò, poiché la misurazione del BDNF circolante è di grande interesse per la sfera psichiatrica, ma soffre per inconsistenze probabilmente a causa della mancanza di procedure standard di selezione e valutazione, abbiamo cercato di superare queste limitazioni definendo una metodologia standardizzata sia per la raccolta dei campioni che per la misurazione di BDNF. Per fare ciò, abbiamo selezioniamo il siero come fluido elettivo per la quantificazione di BDNF e, utilizzando sieri di 40 soggetti adulti sani, abbiamo confrontato le prestazioni di sei kit di ELISA commerciali specifici per BDNF. Tutti i kit hanno mostrato la capacità di misurare il BDNF nel 100% dei campioni e, con una sola eccezione, range paragonabili. Tuttavia, essi hanno mostrato variazioni inter-assay molto diverse, dal 5% al 38%. Tali variazioni, erano superiori a quelle dichiarate dai produttori fatta eccezione per un kit, che nel complesso ha mostrato le prestazioni migliori. In aggiunta, l’analisi Dot-blot ha rivelato che due kit hanno riconosciuto selettivamente la forma matura di BDNF, mentre gli altri hanno reagito anche con il suo precursore, il pro-BDNF. Occorre notare che, avendo definito una procedura standardizzata che riduce l'elevata variabilità dovuta ai differenti passaggi tecnici nelle fasi pre-analitiche e analitiche, questo studio fornisce la base per ottenere una misura affidabile del BDNF nel siero umano, adatto per potenziali applicazioni cliniche future. Come accennato, il passo successivo è stato quello di valutare il BDNF e i mediatori immunitari circolanti in soggetti con stress lavoro correlato. Abbiamo quindi misurato il BDNF e, utilizzando la tecnologia ELISA multiplex, altri 48 analiti tra citochine, chemochine e fattori di crescita nel siero di 122 soggetti sani (87 femmine e 35 maschi). I partecipanti sono stati arruolati tra assistenti sanitari e valutati per lo stress lavoro-correlato con scale stress psicofisico e burnout estratte da un questionario di auto-valutazione standardizzato per il contesto italiano (test Qu-BO). Lo stress psicofisico è stato valutato con cinque elementi, vale a dire ansia, disturbi emotivi, gastrointestinali, cardiaci e disfunzioni ergonomiche sul posto di lavoro più uno stato generale di stress stimato come la media delle cinque dimensioni; il burnout è stato misurato con tre elementi adattati dal Maslach Burnout Inventory, nello specifico esaurimento emotivo, depersonalizzazione e realizzazione personale. Alti punteggi di score sono associati a situazioni di stress cronico elevato, ad eccezione della scala di realizzazione personale. Abbiamo individuato che le donne avevano punteggi più elevati rispetto agli uomini per tutti gli elementi di stress psicofisico e per lo stato di esaurimento emotivo. Oltre a ciò, dopo aver corretto per sesso e indice di massa corporea (IMC), abbiamo cercato di correlare le variabili biologiche con i punteggi delle diverse scale di stress, utilizzando una combinazione di analisi univariate (correlazioni parziali) e multivariate (analisi dei minimi quadrati e fattoriale), per tener conto delle correlazioni tra analiti. Come risultato, non abbiamo trovato alcuna associazione tra le variabili biologiche e lo stato di burnout, mentre, d'altra parte, abbiamo rilevato associazioni negative tra elementi dello stress psicofisico e alcuni marcatori del pattern di chemochine. In particolare, abbiamo osservato che i livelli di MCP-1/CCL2, CTACK/CCL27, RANTES/CCL5 e Eotassina/CCL11 correlano negativamente con diverse scale di stress psicofisico quali ansia, problemi gastrici e cardiaci e disfunzioni ergonomiche sul posto di lavoro. Inoltre, abbiamo osservato che IL-17, una citochina caratteristica del sottotipo T-helper 17 (Th17) e associata a danni a diversi tessuti, incluso quello intestinale, correla positivamente con alti punteggi in soggetti che accusano problemi gastrici (r=0.280, p=0,010). Curiosamente, abbiamo rilevato anche una relazione positiva tra BDNF e il punteggio di disfunzione ergonomica sul posto di lavoro (r=0.284, p=0,009), potenzialmente a causa di meccanismi di compensazione protettivi. Nel loro insieme, i nostri risultati supportano l'ipotesi che lo stress cronico induce una generale soppressione sia risposta immunitaria cellulare e umorale. Tuttavia, le conseguenze sono difficili da prevedere in quanto lo stress è noto per avere un effetto sia soppressivo che stimolante sul sistema immunitario.
Per esplorare ulteriormente il potenziale ruolo protettivo di BDNF in condizioni di stress cronico, a livello cellulare, ci siamo serviti di un modello di stress citotossico in-vitro, consistente in una linea di cellule di neuroblastoma umano, le SK-N-BE, trattata con cisplatino, un farmaco chemioterapico. Da ciò abbiamo osservato che il cisplatino, in particolare dopo 24 di trattamento, aumenta la produzione di BDNF sia a livello trascrizionale che proteico. In aggiunta, lo stimolo citotossico dato dal cisplatino aumenta anche il tasso di trasduzione BDNF. Abbiamo ipotizzato che l'induzione della traduzione di BDNF avvenga attraverso l'attività di Aurora chinasi, come avviene, ad esempio, per la chinasi II dipendente da α-Ca2+/calmodulina (αCaMKII), in quanto si tratta di un meccanismo conservato a partire dagli ovociti di Xenopus fino a livello delle sinapsi neuronali di ippocampo. Pertanto, bloccando l’attività di Aurora chinasi grazie al potente inibitore PHA-680.632, ci aspettavamo di osservare una riduzione nella traduzione di BDNF e un aumento della mortalità cellulare come risultato del diminuito supporto trofico. Sorprendentemente, nonostante un aumento della morte cellulare, ma solo in combinazione con cisplatino, abbiamo osservato una maggiore induzione della traduzione di BDNF, soprattutto da quei trascritti contenenti in particolare l’esone 6 di BDNF (una variante di splicinge della porzione 5’ non tradotta), il cui ruolo è già stato osservato essere cruciale in condizioni di stress citotossico. In conclusione, i nostri risultati delineano un meccanismo di induzione della traduzione degli mRNA di BDNF potenzialmente indipendente da Aurora chinasi, almeno in condizioni di stress citotossico. Sebbene non dimostrato per BDNF, tali meccanismi potrebbero includere il reclutamento dell’unità trascrizionale con modalità indipendenti dalla presenza del cap al 5’ non tradotto degli mRNA. Le suddette osservazioni potrebbero avere importanti implicazioni cliniche nell'uso di inibitori di Aurora come coadiuvanti nel trattamento del neuroblastoma in quanto, nonostante inducano un aumento della mortalità, facilitano la selezione di cellule resistenti che producono una maggiore quantità di BDNF.Stress was defined as “a specific response of the body to any non-specific demand (stressor) made on it”. When the real or perceived stressor is lasting or the response is inappropriate (distress), the physiological short-term changes switch to long-term alterations driving to pathological conditions, including mental illness. According to this “allostatic load” model, primary mediators such as catecholamines and steroid hormones trigger multi-systemic alterations of secondary signaling mediators possibly causing adverse manifestation in different body systems, including nervous and immune ones. Neurotrophins, in particular the Brain-Derived Neurotrophic Factor (BDNF), and immune mediators, like cytokines and chemokines, are possible modulators of the chronic stress response. Therefore, we aimed to assess their circulating levels in healthy persons in conditions of work-related stress. Before that, since measurement of peripheral BDNF is of great interest for psychiatrists but suffers for inconsistencies, possibly due to the lack of consistent procedures for BDNF samples collection and assessment, we aimed to overcome these limitations and defined a standardized methodology for both sample collection and BDNF measurement. Hence, we have selected the serum as the elective body fluid for BDNF quantification and, using sera from 40 healthy adult subjects, we compared the performance of six commercial BDNF ELISA kits. All kits showed 100% of sample recovery and, with one exception, comparable range. However, they exhibited very different inter-assay variations from 5% to 38%. Inter-assay variations, were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. Of note, having defined a standardized procedure which reduces the high variability due to technical, pre-analytical and analytical steps, this study provides the basis to obtain an accurate measure BDNF in human serum, suitable for future clinical applications.
As mentioned, the next step was to assess circulating BDNF and immune mediators in subjects with job-related stress. We therefore measured BDNF and, using the multiplex ELISA technology, other 48 analytes among cytokines, chemokines and growth factors in the sera of 122 healthy subjects (87 female and 35 male). The participants were enrolled among healthcare assistants and evaluated for work-related stress using psychophysical stress and burnout scales extracted from a self-assessed questionnaire standardized for the Italian context (Qu-BO test). Psychophysical stress addressed five items, namely anxiety, emotion (depression-like), gastrointestinal disturbances, cardiac disturbances, ergonomic dysfunction at the workplace and an overall stress state as the average of the five items; burnout state has been measured using three cores adapted from the Maslach Burnout Inventory, specifically emotional exhaustion, depersonalization and personal accomplishment. Higher scores mean higher stress status, except for personal accomplishment. We found that female had higher scores than male for all psychophysical stress items and for the emotional exhaustion core. Then, after correcting for gender and body-mass index (BMI), we attempted to correlate the biological variables with the score scales, using a combination of univariate (partial correlations) and multivariate analysis (partial least square and factor analysis) which took in account correlations among analytes. As a result, we found no associations between burnout state and biological variables while, on the other hand, we detected negative associations between psychophysical stress items and some markers of the chemokine profile. In particular, we observed that levels of MCP-1/CCL2, CTACK/CCL27, RANTES/CCL5 and Eotaxin/CCL11 negatively correlate, with different grade, with the scores of anxiety, gastric and cardiac problems and ergonomic dysfunction at the workplace. In addition, we observed IL-17, a feature cytokine of the T-helper subtype 17 (Th17) which have been associated to tissue damage also at intestine level, to be positively associated with score in subjects reporting gastric problems (r=0.280, p=0.010). Intriguingly, we detected also a positive relationship between BDNF and score of ergonomic dysfunction at the workplace (r=0.284, p=0.009), potentially as a result of compensatory protective mechanisms. Taken together, our results support the hypothesis that chronic stress induces suppression of both cellular and humoral response. However, the consequences are hard to predict since stress is known to have both suppressive and enhancing effects on immune system.
To further explore the potential protective role of BDNF in chronic stress conditions, we moved to an in-vitro cellular model of cytotoxic stress, consisting in SK-N-BE human neuroblastoma cell line treated with cisplatin, a chemotherapeutic drug. We found that cisplatin, in particular after 24 of treatment, enhances BDNF production at both transcriptional and protein levels. Of note, it induces also an increment in the transduction rate of BDNF mRNA transcripts. We hypothesized that BDNF translation induction occurs through the activity of Aurora kinase, like what happen for α-Ca2+/calmodulin-dependent protein kinase II (αCaMKII) mRNA, as it is a conserved mechanism from Xenopus oocytes to synapses of hippocampal neurons. Therefore, by blocking Aurora kinase activity thanks to the potent inhibitor PHA-680632, we were expecting to detect a reduction in BDNF translation and increased cell mortality as a result of the decreased trophic support. Surprisingly, despite an improved cell death only in combination with cisplatin, we observed an enhanced BDNF translation induction especially from those transcripts containing exon 6, a splice variant of the 5’ untranslated region, which have been already observed to be crucial in cytotoxic stress conditions. In conclusion, our results pointed toward a BDNF translation induction mechanism that is Aurora kinase independent, at least in conditions of cytotoxic stress. Although not investigated, some of these mechanisms may include 5’cap-independent recruitment of the translational machinery. Furthermore, our observations could have important clinical implications in the use of Aurora inhibitors as adjuvant in neuroblastoma treatment as, even if with a cell mortality increase, they facilitate the selection of resistant cells that further enhance BDNF production.XXVII Ciclo198
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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