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Functional Splicing Assay mediante l'utilizzo di minigeni plasmidici nel gene NF1
Full text linkRecent knowledge of the splicing process complexity have unfolded the existence of important regulating elements of the process itself. Variations of these new elements, located both on coding and non-coding gene regions, can exert detrimental effects on the pre-mRNA splicing process. One of the most credited hypotheses in last years is that even silent variations of the reading frame or variations of intronic sequences are to be considered potentially involved in the development of pathologies. The activation of pseudo-exons or of cryptic exons following the creation of new splice sites or the alteration of constitutive splice sites is already known to be causing pathologies related to a defective splicing process. Nevertheless, the informations on genetic pathologies available to date reveal that the knowledge on most of the fundamental mechanisms regulating the pre-mRNA splicing is still very poor.
Splicing defects amount to the 30-50% of NF1 total mutations. About the 30% of these mutations map far away from exon-intron junctions where they can produce new splice sites, activate cryptic sites or lead to the loss or formation of intronic and/or exonic signal sequences important for the messenger recognition by the splicing factors.
Based on these premises, the goal of this study has been to search for mutations potentially responsible of altering the splicing mechanism and to develop a plasmid minigene in order to perform an in vitro characterization of the effect of these genomic variations on the mechanism and its alterations.
Initially, a screening analysis of the NF1 gene mutations has been performed on 310 patients with Neurofibromatosis type 1 received at the University of Padova, Department of Pediatrics – Clinical Genetics and Epidemiology Service. The mutation search has been performed by means of DHPLC and HRMA techniques. The analysis revealed a total of 196 mutations of the NF1 gene, of which 41where implied, or potentially implied, in the alteration of the splicing mechanism.
In the case of de novo mutations, a series of in silico test has been performed, by means of softwares available on the net, such as: BDGP: Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html) and NetGene 2 Server (http://www.cbs.dtu.dk/services/NetGene2), both analysing variations in the splicing consensus sequences GT-AG; Human Splicing Finder Version 2.4 (http://www.umd.be/HSF/) e ESEfinder Release 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home), that based on predictive models, take into account also the alterations of ESE sequences (Exonic Splicing Enhancer), of ESS sequences (Exonic Splicing Silencer), of ISE sequences (Intronic Splicing Enhancer) and of ISS sequences (Intronic Splicing Silencer).
For the in vitro evaluation a b-globin minigene has been built. By cloning the genomic region including the mutation inside this vector, the vector itself become a vehicle to temporarily transfect HeLa cells, in order to extract the transcripts and evaluate the splicing alteration. The analysis of transcription products confirmed the functionality of the system.
In the case of the NF1 IVS 5-5 T>G variation it has been observed that the mutation is responsible of causing the NF1 exon 6 skipping.
In a particular case the method has been used to valuate the effects of the variation CFTR IVS 4+3 A>G. The analysis highlighted that from the mutated construct three transcription products are obtained: a wild type transcript, a transcript bearing the deletion of 93 nucleotides at the 3' end of the CFTR exon 4, and a transcript showing the complete skipping of the CFTR exon 4. In addition, an epidemiological study has been performed, evaluating the occurrence of the aforementioned variation in 250 healthy control individuals by means of the HRMA technique. Frequency computation and transcripts analysis allow to affirm that the variation 621+3 A>G is not to be considered the cause of severe forms of Cystic Fibrosis, differently from what previously reported in the literature. The study has been recently published on the international magazine Journal of Human Genetics.Le recenti conoscenze sulla complessità del processo di splicing, hanno rivelato l’esistenza di importanti elementi di regolazione dello stesso. Variazioni di questi nuovi elementi, che si trovano sia nelle regioni codificanti che non codificanti dei geni, possono manifestarsi con effetti deleteri sullo splicing del pre-mRNA. Forse una delle ipotesi più accreditate di questi anni è che anche variazioni silenti del modulo di lettura o variazioni delle sequenze introniche siano da considerare responsabili di patologie. L'attivazione di pseudo-esoni o di esoni criptici in seguito alla creazione di nuovi siti di splicing o all’alterazione di siti di splicing costitutivi sono eventi già osservati come causa di patologie da difetti dello splicing. Tuttavia, le informazioni attualmente disponibili sulle patologie genetiche rivelano che le conoscenze su molti dei meccanismi fondamentali che regolano lo splicing del pre-mRNA sono ancora molto scarse.
I difetti di splicing costituiscono il 30-50% del totale di mutazioni del gene NF1. Circa il 30% di queste mappa lontano dalle giunzioni esone-introne dove possono causare la creazione di nuovi siti di splicing, attivare siti cripitici, o portare alla perdita o formazione di sequenze segnale esoniche e/o introniche, importanti per il riconoscimento del messaggero da parte dei fattori di splicing.
Sulla base di queste premesse l’obbiettivo di questo lavoro è stato di ricercare mutazioni potenzialmente alteranti il meccanismo di splicing e di sviluppare un minigene plasmidico per caratterizzare in vitro l’effetto di queste variazioni genomiche sul meccanismo e sulle sue alterazioni.
Inizialmente è stata effettuata un’analisi di screening di mutazioni nel gene NF1 su 310 pazienti affetti da Neurofibromatosi di tipo 1 afferiti presso il servizio di Genetica Clinica ed Epidemiologia del Dipartimento di Pediatria dell’Università degli Studi di Padova. La ricerca di mutazione è stata eseguita mediante DHPLC e HRMA. Ne sono emerse 196 mutazioni a carico del suddetto gene di cui 41 che alterano, o potrebbero alterare, il meccanismo di splicing.
Nel caso delle mutazioni osservate de novo sono stati eseguiti dei test in silico mediante l'utilizzo di software disponibili in rete come: BDGP: Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html) e NetGene 2 Server (http://www.cbs.dtu.dk/services/NetGene2) che analizzano variazioni a carico delle sequenze consensus GT-AG dello splicing, Human Splicing Finder Version 2.4 (http://www.umd.be/HSF/) e ESEfinder Release 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) che, basandosi su modelli di calcolo predittivi, prendono in considerazione anche le alterazioni delle sequenze ESE (Exonic Splicing Enhancer), ESS (Exonic Splicing Silencer), ISE (Intronic Splicing Enhancer) e ISS (Intronic Splicing Silencer).
Per la valutazione in vitro è stato costruito un b-globin minigene. Clonando all’interno di questo vettore la regione genomica interessata dalla mutazione, questo diventa un veicolo per trasfettare in modo transiente cellule HeLa in modo da poter estrarre i trascritti e valutare l’alterazione dello splicing.
L’analisi dei prodotti di trascrizione ha confermato la funzionalità del sistema. Nel caso della variazione NF1 IVS 5-5 T>G si è potuto osservare che tale mutazione provoca lo skipping dell’esone 6 del gene NF1.
In un caso particolare è stata utilizzata la metodica per valutare gli effetti della variazione: CFTR IVS 4+3 A>G. L’analisi ha evidenziato che dal costrutto mutato si ottengono tre prodotti di trascizione: un trascritto wild type, un trascritto che presenta la delezione di 93 nucleotidi all’estremità 3’ dell’esone 4 di CFTR e un trascritto che presenta il completo skipping dell’esone 4 di CFTR. In aggiunta sono stati effettuati anche degli studi epidemiologici valutando mediante HRMA la presenza di tale variante in 250 individui sani di controllo. Il calcolo delle frequenze alleliche e l’analisi dei trascritti ha portato ad affermare che la variazione 621+3 A>G non sia da considerarsi la causa di forme gravi di Fibrosi Cistica, andando a smentire quanto precedentemente riportato in letteratura. Il lavoro è stato di recente pubblicato sulla rivista internazionale Journal of Human Genetics
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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