1,720,978 research outputs found
Study of inborn errors of Coenzyme A biosynthesis using Danio rerio animal models
No full textAnimal models are widely used to investigate the pathogenesis and molecular processes involved in the development and progression of human diseases. The Danio rerio, assumed a predominant role in the last decade due to some characteristics such as ease of use, the high number of embryos obtainable, the possibility to follow embryonic development and a completely sequenced genome. Neurodegeneration with Brain Iron Accumulation (NBIA) is a family of diseases characterized by a peculiar accumulation of iron in the brain. All known forms of NBIA are induced by mutations in single genes that code for proteins involved in different pathways. PKAN and CoPAN are two neuropathologies of this group, respectively caused by mutations in PANK2 and COASY genes, which encode for proteins directly involved in the synthesis of Coenzyme A. The transient downregulation of the two zebrafish orthologues of PANK2 and CoASY, respectively pank2 and coasy, induces the onset of specific phenotypes comparable to human pathologies, suggesting the possibility of using Danio rerio as a model for the study of XXX and a tool to discover potential therapeutic strategies. The study was followed by the generation of Knock-Out models for pank2 and coasy and their subsequent characterization. CRISPR/Cas9 technology was used, which allowed us to obtain mutant lines for pank2 and coasy. Analysis performed on pank2 Knock-Out, revealed no significant changes in gene expression that could compensate the lack of pank2 (pank1a and pank1b), or in genes that could compensate the absence of the protein (coasy). However, Western Blot analysis demonstrated a significant reduction in pank2 levels in mutant embryos. Based on previously obtained data which indicated aberrant neuronal development, evaluation of the nervous system development of mutant embryos and the brains of adult individuals was performed using several techniques. The results obtained do not show significant defects in the development of the main neuronal regions both in embryos and in mutant adults. The analysis of the vascular system instead highlighted a morphological alteration at the level of the caudal venous plexus. This results partially confirms what was found in Knock-Down models, suggesting the importance of pank2 for the correct development of the vessels. Specific stains for primordial germ cells showed a decrease in this population in mutant embryos at early stages of development, defects that are probably implicated in the testicular atrophy observed in adults of 1 year of age. This result is similar to what was observed in the mouse model for PKAN which is mainly characterized by azoospermia. A preliminary analysis of the behaviour of the mutant larvae did not reveal any particular difference, while the study of the stress-induced response in adults showed a decrease in the behaviour associated with stress in the mutants. The results indicate that pank2 is involved in various pathways in zebrafish, specifically the formation of vessels, the correct development of germ cells and in the neural circuits involved in the stress response. Furthermore, we successfully generated a CoPAN model in zebrafish inducing mutations in coasy. To date, the phenotypical analysis of this model demonstrate that individuals carrying homozygotes mutation cannot reach adulthood and die before day 15 of development. We also noted the absence of swimming bladder in mutant individuals at 5 days of development. Further analyses will be carried out to better understand the mechanisms that link the onset of this phenotype and the lack of coasy. In conclusion, we generated two models one for for inborn errors of CoA biosynthesis in Danio rerio, their characterization highlights different phenotypes related to the absence of pank2 and coasy that could be useful to better understand the mechanisms underlying these pathologies and can be used to screen molecules with therapeutic potential.I modelli animali vengono utilizzati per investigare la patogenesi e i processi molecolari coinvolti nell’insorgenza di malattie umane. Danio rerio, ha assunto nell’ultimo decennio un ruolo predominante per via di alcune caratteristiche come la facilità d’utilizzo, l’elevato numero di embrioni ottenibili, la possibilità di seguire lo sviluppo embrionale e un genoma completamente sequenziato. Con il termine Neurodegenerazione con accumulo di ferro cerebrale (NBIA) si indica una famiglia di malattie, caratterizzate da un accumulo di ferro a livello cerebrale. Tutte le forme di NBIA conosciute sono indotte da mutazioni in singoli geni che codificano per proteine coinvolte in diversi processi cellulari. PKAN e CoPAN sono due di queste patologie causate rispettivamente da mutazioni dei geni PANK2 e COASY, che codificano per proteine coinvolte nella sintesi del Coenzima A. La downregolazione dei due ortologhi di zebrafish di PANK2 e CoASY, rispettivamente pank2 e coasy, ha dimostrato l’insorgenza di fenotipi paragonabili alle patologie umane suggerendo la possibilità di usare Danio rerio come modello per lo studio di PKAN e CoPAN. Lo studio è proseguito con la generazione di modelli Knock-Out per pank2 e coasy e dalla loro caratterizzazione. È stata utilizzata la tecnologia CRISPR/Cas9, che ha permesso di ottenere linee mutanti per pank2 e coasy. L’analisi di espressione genica sui KO di pank2 non ha rilevato cambiamenti significativi in geni che potrebbero sopperire alla funzione di pank2 (pank1a e pank1b), o in geni coinvolti nella sintesi del CoA (coasy). Analisi dei livelli proteici hanno dimostrato una significativa riduzione dei livelli di pank2 negli embrioni mutanti. Sulla base dei dati ottenuti in precedenza che indicavano uno sviluppo neuronale aberrante, è stata eseguita una valutazione dello sviluppo del sistema nervoso degli embrioni e del cervello di individui adulti utilizzando diverse tecniche. I risultati ottenuti dimostrano che sia negli embrioni che negli adulti mutanti non sono presenti rilevanti difetti nello sviluppo delle principali aree neuronali. L’analisi del sistema vascolare ha evidenziato un’alterazione morfologica del plesso venoso causale, risultato che conferma in parte quello visto in modelli KO per pank2 suggerendo ulteriormente l’importanza di pank2 nello sviluppo dei vasi. È stata osservata una diminuzione del numero di cellule primordiali germinali negli embrioni, difetti che probabilmente è implicato nell’atrofia testicolare osservata negli individui adulti. Questo risultato è in linea con quanto documentato nel modello murino per PKAN che è caratterizzato soprattutto per azoospermia. Un’analisi comportamentale delle larve non ha rilevato alcuna differenza, mentre lo studio della risposta indotta da stress nell’adulto ha dimostrato una diminuzione del comportamento associato a stress nei mutanti. I risultati ottenuti ci hanno permesso di dimostrare che pank2 è implicato in diversi pathways in zebrafish, nello specifico la formazione di vasi, nel corretto sviluppo di cellule germinali e nei circuiti neurali implicati nella risposta allo stress. Il secondo obiettivo della nostra analisi è stata la generazione di una linea mutante per coasy, creando un modello di CoPAN in zebrafish. La caratterizzazione di questo modello a livello fenotipo ha dimostrato che individui omozigoti per la mutazione non riesco a raggiungere lo stadio adulto ma muoiono entro il 15 giorno di sviluppo, inoltre è stata documentata l’assenza della vescia natatoria in individui mutanti a 5 giorni. Ulteriori analisi andranno effettuate per meglio comprendere i meccanismi che legano questo fenotipo alla mancanza di coasy. I risultati ottenuti evidenziano differenze rispetto ai controlli che potrebbero essere utili per comprendere i meccanismi alla base di queste patologie e/o per lo screening di potenziali target terapeutici
The Downregulation of c19orf12 Negatively Affects Neuronal and Musculature Development in Zebrafish Embryos
Full text linkMitochondrial membrane Protein Associated Neurodegeneration (MPAN) is a rare genetic disorder due to mutations in C19orf12 gene. In most cases, the disorder is transmitted as an autosomal recessive trait and the main clinical features are progressive spastic para/tetraparesis, dystonia, motor axonal neuropathy, parkinsonisms, psychiatric symptoms, and optic atrophy. Besides iron accumulation in the globus pallidus and substantia nigra, the neuropathology shows features also observed in Parkinson’s Disease brains, such as α-synuclein-positive Lewy bodies and hyperphosphorylated tau. Mutations in the gene have been found in other neurodegenerative disorders, including PD, hereditary spastic paraplegia, pallido-pyramidal syndrome, and amyotrophic lateral sclerosis. The biological function of C19orf12 gene is poorly defined. In humans, it codes for two protein isoforms: the longer one is present in mitochondria, endoplasmic reticulum, and contact regions between mitochondria and ER. Mutations in the gene appear to be linked to defects in mitochondrial activity, lipid metabolism and autophagy/mitophagy. To increase the available tools for the investigation of MPAN pathogenesis, we generated a new animal model in zebrafish embryos. The zebrafish genome contains four co-orthologs of human C19orf12. One of them, located on chromosome 18, is expressed at higher levels at early stages of development. We downregulated its expression by microinjecting embryos with a specific ATG-blocking morpholino, and we analyzed embryonal development. Most embryos showed morphological defects such as unsettled brain morphology, with smaller head and eyes, reduced yolk extension, tilted and thinner tail. The severity of the defects progressively increased and all injected embryos died within 7 days post fertilization. Appropriate controls confirmed the specificity of the observed phenotype. Changes in the expression and distribution of neural markers documented a defective neuronal development, particularly evident in the eyes, the optic tectum, the midbrain-hindbrain boundary; Rohon Beard and dorsal root ganglia neurons were also affected. Phalloidin staining evidenced a significant perturbation of musculature formation that was associated with defective locomotor behavior. These data are consistent with the clinical features of MPAN and support the validity of the model to investigate the pathogenesis of the disease and evaluate molecules with potential therapeutic effect
Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish
Full text linkSphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches
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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