1,720,956 research outputs found
RNA editing applied to cystic fibrosis: RESTORE can target G542X CFTR mRNA and revert the nonsense mutation
Full text linkBackground: Nonsense mutations in the CFTR gene are responsible for approximately 8 % of cystic fibrosis (CF)
cases worldwide. The consequent premature termination of translation leads to the production of a truncated and
non-functional CFTR protein. Despite the intensive research in the field, these patients cannot benefit from
specific and approved therapies yet. To address this issue, in this study we evaluated a potential therapeutic
strategy to overcome the nonsense G542X (UGG > UGA) mutation in the CFF-16HBEge human bronchial
epithelial cells by restoring the full-length CFTR protein.
Methods: We applied the RESTORE (Recruiting endogenous ADAR to specific transcripts for oligonucleotidemediated RNA editing) approach, based on specifically designed antisense RNA oligonucleotides (ASOs) to recruit endogenous ADAR (adenosine deaminase acting on RNA) enzymes. The ADAR’s recruitment to the target
CFTR mRNA is expected to promote the deamination of adenosine (A) into inosine (I) within the premature
termination codon (UGA). As the ribosome reads the inosine as guanosine (G), the stop codon could be recoded
as a tryptophan (UGG), thereby allowing the synthesis of a full-length CFTR protein, albeit with a different amino
acid.
Results: Our results indicate that in the CFF-16HBEge G542X cell line, the transfection of a specific ASO allows
the rescue of the CFTR transcript and protein expression, compared to the untransfected mutated cells. Next
generation sequencing of CFTR cDNA also confirmed the occurrence of the expected RNA editing outcome.
Conclusions: The obtained results suggest that the RESTORE approach might be explored as a promising strategy
to treating nonsense mutations in CFTR, potentially contributing to novel therapeutic options for CF patients
Site-Specific RNA Editing of Stop Mutations in the CFTR mRNA of Human Bronchial Cultured Cells
Full text linkIt is reported that about 10% of cystic fibrosis (CF) patients worldwide have nonsense (stop) mutations in the CFTR gene, which cause the premature termination of CFTR protein synthesis, leading to a truncated and non-functional protein. To address this issue, we investigated the possibility of rescuing the CFTR nonsense mutation (UGA) by sequence-specific RNA editing in CFTR mutant CFF-16HBEge, W1282X, and G542X human bronchial cells. We used two different base editor tools that take advantage of ADAR enzymes (adenosine deaminase acting on RNA) to edit adenosine to inosine (A-to-I) within the mRNA: the REPAIRv2 (RNA Editing for Programmable A to I Replacement, version 2) and the minixABE (A to I Base Editor). Immunofluorescence experiments show that both approaches were able to recover the CFTR protein in the CFTR mutant cells. In addition, RT-qPCR confirmed the rescue of the CFTR full transcript. These findings suggest that site-specific RNA editing may efficiently correct the UGA premature stop codon in the CFTR transcript in CFF-16HBEge, W1282X, and G542X cells. Thus, this approach, which is safer than acting directly on the mutated DNA, opens up new therapeutic possibilities for CF patients with nonsense mutations
EVALUATION OF SEQUENCE-SPECIFIC RNA EDITING TOOLS FOR CORRECTING CFTR NONSENSE MUTATIONS IN CYSTIC FIBROSIS THERAPY
No full textBackground. Premature-termination codons (PTCs) in the CFTR mRNA cause theproduction of truncated, non-functional CFTR proteins accounting for approximately 8% ofcystic fibrosis (CF) cases worldwide. Despite the intensive research in the field, these patientscannot benefit from specific and approved therapies yet. To address this issue, we haveexplored RNA-editing approaches to correct PTCs in the CFTR mRNA of CFF-16HBEgehuman bronchial epithelial cells. Methods. These systems exploit ADAR (adenosinedeaminase acting on RNA) enzymes to convert the adenosine (A) within the PTC into inosine(I). As the ribosome reads the inosine as guanosine (G), the stop codon could be recoded as asense codon, thereby allowing the synthesis of a full-length CFTR protein. We used twodifferent approaches according to the use of exogenous or endogenous ADARs. In theminixABE (mini-dCas13X-mediated RNA adenine base editor) system, the ADAR2deaminase domain (ADAR2DD), which is fused to a truncated dCAS13x.1, is recruited to theadenosine within the PTC by specifically designed guide RNAs. Instead, with the RESTORE(Recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNAediting) approach, linear or circular specific antisense RNA oligonucleotides (arRNAs) targetthe CFTR mRNA region surrounding the PTC to promote the endogenous ADAR recruitment.Results. Our results show the rescue of CFTR protein on the cell’s plasma membrane byimmunofluorescence, and the increase of CFTR full-length transcript expression by RT-qPCRin the mutated cell lines when both systems were applied. Moreover, ADAR-mediated RNAediting tools induced the functional rescue of CFTR protein in these cultures, which wasmeasured by electrophysiological methods. Conclusions. The obtained results suggest thatthese RNA editing-based tools mediated by ADARs might be explored as a promising strategyto treating nonsense mutations in CFTR, potentially contributing to novel therapeutic optionsfor CF patients that have nonsense mutations
Specific Irreversible Cell-Cycle Arrest and Depletion of Cancer Cells Obtained by Combining Curcumin and the Flavonoids Quercetin and Fisetin
Full text linkBackground: Induced senescence could be exploited to selectively counteract the proliferation of cancer cells and target them for senolysis. We examined the cellular senescence induced by curcumin and whether it could be targeted by fisetin and quercetin, flavonoids with senolytic activity. Methods: Cell-cycle profiles, chromosome number and structure, and heterochromatin markers were evaluated via flow cytometry, metaphase spreads, and immunofluorescence, respectively. The activation of p21(waf1/cip1) was assessed via RT-qPCR and immunoblotting. Senescent cells were detected via SA-β-Galactosidase staining. Results: We report that curcumin treatment specifically triggers senescence in cancer cells by inducing mitotic slippage and DNA damage. We show that curcumin-induced senescence is p21(waf1/cip1)-dependent and characterized by heterochromatin loss. Finally, we found that flavonoids clear curcumin-induced senescent cancer cells. Conclusions: Our findings expand the characterization of curcumin-induced cellular senescence in cancer cells and lay the foundation for the combination of curcumin and flavonoids as a possible anti-cancer therapy
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
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