1,720,958 research outputs found
Caratterizzazione funzionale di COQ10A e COQ10B umani, due geni essenziali per la respirazione mitocondriale
No full textIl Coenzima Q10 (CoQ10) è un trasportatore di elettroni essenziale nella catena respiratoria mitocondriale e la carenza di tale coenzima causa gravi malattie multi-sistemiche. La deficienza primaria di coenzima Q10 è una malattia eterogenea sia dal punto di vista clinico sia genetico, dovuta alla riduzione dei livelli di CoQ10 nei tessuti e associata a diverse patologie, quali l’encefalopatia neonatale fatale, la sindrome nefrosica resistente a steroidi (SRNS), la cardiomiopatia ipertrofica (HCM), la retinopatia o atrofia ottica e l’ipoacusia neurosensoriale. I bassi livelli di CoQ10 in organi e tessuti possono essere dovuti alla presenza di mutazioni nei geni chiamati “geni COQ”, i quali codificano per proteine coinvolte nella biosintesi del coenzima Q10. Molti studi riguardanti la biosintesi del coenzima Q sono stati condotti in Saccharomyces cerevisiae, un organismo modello che è stato un punto di partenza essenziale per la caratterizzazione funzionale di molte proteine COQ. In ogni caso, il processo di biosintesi del coenzima Q10, tutte le proteine coinvolte e la loro funzione precisa non sono stati ancora compresi del tutto. Due tra questi geni sono COQ10A e COQ10B (ortologi del gene Coq10 di S. cerevisiae), i quali si ipotizza possano essere coinvolti nella biosintesi del CoQ, ma la cui la funzione in cellule umane è ancora completamente sconosciuta. Lo scopo di questo lavoro è quello di sviluppare un modello efficiente e facile da maneggiare per lo studio della funzione dei geni COQ10A e COQ10B in cellule umane, per comprendere meglio il processo di biosintesi del CoQ10 e possibilmente trovare nuovi target terapeutici per pazienti affetti da deficienza primaria di CoQ10. Per raggiungere questo obbiettivo, abbiamo utilizzato la tecnologia CRISPR/Cas9 in cellule embrionali umane di rene (HEK 293), con l’intento di generare tre linee cellulari “knockout” (KO): COQ10A KO, COQ10B KO e COQ10AB KO, in cui uno o entrambi i geni COQ10 fossero danneggiati, in modo da impedirne la corretta trascrizione e traduzione in proteina funzionante. Le linee KO sono state caratterizzate funzionalmente, in particolare la linea COQ10AB KO, dato che presentava un fenotipo più severo e non vi era il rischio che ci fosse complementazione funzionale tra i due geni. Tutte e tre le linee cellulari KO presentavano livelli fisiologici di Q10 endogeno e un’attività dei complessi respiratori mitocondriali III e II+III leggermente ridotta. Solo le cellule COQ10AB KO mostravano una sottile differenza nell’assemblaggio del complesso III nei supercomplessi mitocondriali. Le cellule COQ10AB KO presentavano inoltre una normale attività in gel del complesso V, alti livelli di specie reattive dell’ossigeno (ROS) e una fosforilazione ossidativa altamente compromessa rispetto alle cellule sane di controllo (wild type). Eccezionalmente, la supplementazione esogena con Q10 non ha portato al recupero della respirazione nelle cellule COQ10AB KO, mentre l’aggiunta di Q6 ne ha ripristinato solo parzialmente la respirazione e invece la supplementazione con Q4 ha portato al completo recupero della respirazione mitocondriale.
I dati qui presentati mettono in discussione il ruolo dei geni COQ10A e B nella biosintesi del coenzima Q10 e portano ad un’ipotesi completamente nuova riguardo al ruolo delle proteine COQ10A e COQ10B nel metabolismo cellulare.Coenzyme Q10 (CoQ10) is an essential electron shuttle in mitochondrial respiratory chain and its deficiency cause severe multisystemic disorders. Primary CoQ10 deficiency is a clinically and genetically heterogeneous disorder due to reduced levels of CoQ10 in tissues and associated with several diseases, such as fatal neonatal encephalopathy, steroid-resistant nephrotic syndrome (SRNS), hypertrophic cardiomyopathy (HCM), retinopathy or optic atrophy and sensorineural hearing loss. The cause of low CoQ10 levels in tissues and organs could be the presence of mutations in the so called “COQ genes”, which encode for proteins involved in CoQ10 biosynthesis. Many studies about CoQ biosynthesis have been conducted on Saccharomyces cerevisiae, a model organism which have been an essential starting point for functional characterization of many COQ proteins. However, CoQ10 biosynthetic process, all proteins involved, and their precise function have not been fully understood yet. Two of these genes are COQ10A and COQ10B (hortologs of S. cerevisiae Coq10 gene), they are putatively involved in CoQ10 biosynthesis, but their function is still completely unknown in humans. The aim of this work is to develop an efficient and easy-to-handle model to study COQ10A and COQ10B function in human cells, to better clarify CoQ10 biosynthetic process and likely find new therapeutic targets for patients with primary CoQ10 deficiency. To accomplish this, we employed CRISPR/Cas9 technology in Human Embryonic Kidney (HEK) 293 cell line in order to generate three different cellular knockout (KO) cell lines: COQ10A KO, COQ10B KO and COQ10AB KO, in which one or both genes were disrupted to impair their correct transcription and translation into functional proteins. KO cell lines were functionally characterized, in particular COQ10AB KO cell line, since it presented a more severe phenotype and no risk of functional compensation between the two genes. All three KO cell lines presented physiological levels of endogenous CoQ10 and a slightly reduced activity of mitochondrial respiratory complexes III and II+III. Only COQ10AB KO showed a subtle difference in complex III assembly in the analysis of mitochondrial supercomplexes. COQ10AB KO cell line had also normal complex V activity in gel, higher levels of reactive oxygen species (ROS) and highly impaired oxidative phosphorylation compared to wild type cells. Very interestingly, exogenous supplementation with Q10 did not rescue COQ10AB KO cells respiration, while Q6 addition partially restore respiration and supplementation with Q4 leads to a fully recovered mitochondrial respiration.
These data question the role of COQ10A and B in Coenzyme Q10 biosynthesis and lead to a completely new hypothesis about the role of COQ10A and COQ10B in human cells metabolism
Characterization of Two Novel PNKP Splice‐Site Variants in a Proband With Microcephaly, Intellectual Disability, and Multiple Malformations
Full text link: Polynucleotide kinase phosphatase (PNKP), encoded by the PNKP gene, is a DNA processing enzyme involved in double-strand break and single-strand break repair pathways, which are essential for genome stability and for the correct development and maintenance of human nervous system. PNKP biallelic loss-of-function variants have been associated with a broad spectrum of neurological anomalies, ranging from congenital microcephaly with intellectual disability and seizures (MCSZ), to later onset forms of ataxia-oculomotor apraxia (AOA4) or peripheral neuropathy (CMT2B2). We report the atypical clinical manifestations of a patient with severe microcephaly, short stature, developmental delay, conductive hearing loss, and tracheoesophageal malformation, in the absence of seizures. Whole exome sequencing analysis identified two novel, compound heterozygous splice-site variants in the PNKP gene (NM_007254.4): c.1448+1G > A and c.199-8_199-5del. To demonstrate the effect of both variants on the splicing process and prove their pathogenicity, we performed a hybrid minigene assay, which successfully highlighted a deleterious impact on the transcript, particularly regarding the c.199-8_199-5del variant. The uncommon clinical features of the proband and the identification of two newly associated pathogenic variants add further evidence to the allelic and phenotypic heterogeneity of the PNKP locus
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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