Meta-Analysis of Keratoconus Transcriptomic Data Revealed Altered RNA Editing Levels Impacting Keratin Genomic Clusters.

Introduction: Keratoconus (KC) is an ocular disorder with a multifactorial origin. Transcriptomic analyses (RNA-seq) revealed deregulations of coding (mRNA) and non-coding RNAs (ncRNAs) in KC, suggesting that mRNA-ncRNA co-regulations can promote the onset of KC. The present study investigates the modulation of RNA editing mediated by the adenosine deaminase acting on dsRNA (ADAR) enzyme in KC. Materials: The level of ADAR-mediated RNA editing in KC and healthy corneas were determined by two indexes in two different sequencing datasets. REDIportal was used to localize known editing sites, whereas new putative sites were de novo identified in the most extended dataset only and their possible impact was evaluated. Western Blot analysis was used to measure the level of ADAR1 in the cornea from independent samples. Results: KC was characterized by a statistically significant lower RNA-editing level compared to controls, resulting in a lower editing frequency, and less edited bases. The distribution of the editing sites along the human genome showed considerable differences between groups, particularly relevant in the chromosome 12 regions encoding for Keratin type II cluster. A total of 32 recoding sites were characterized, 17 representing novel sites. JUP, KRT17, KRT76, and KRT79 were edited with higher frequencies in KC than in controls, whereas BLCAP, COG3, KRT1, KRT75, and RRNAD1 were less edited. Both gene expression and protein levels of ADAR1 appeared not regulated between diseased and controls. Conclusions: Our findings demonstrated an altered RNA-editing in KC possibly linked to the peculiar cellular conditions. The functional implications should be further investigated.

Breast cancer cells grown on hyaluronic acid-based scaffolds as 3D in vitro model for electroporation.

Nowadays, electroporation (EP) represents a promising method for the intracellular delivery of anticancer drugs. To setting up the process, the EP efficiency is usually evaluated by using cell suspension and adherent cell cultures that are not representative of the in vivo conditions. Indeed, cells are surrounded by extracellular matrix (ECM) whose composition and physical characteristics are different for each tissue. So, various three-dimensional (3D) in vitro models, such as spheroids and hydrogel-based cultures, have been proposed to mimic the tumour microenvironment. Herein, a 3D breast cancer in vitro model has been proposed. HCC1954 cells were seeded on crosslinked and lyophilized matrices composed of hyaluronic acid (HA) and ionic complementary self-assembling peptides (SAPs) already known to provide a fibrous structure mimicking collagen network. Herein, SAPs were functionalized with laminin derived IKVAV adhesion motif. Cultures were characterized by spheroids surrounded by ECM produced by cancer cells as demonstrated by collagen1a1 and laminin B1 transcripts. EP was carried out on both 2D and 3D cultures: a sequence of 8 voltage pulses at 5 kHz with different amplitude was applied using a plate electrode. Cell sensitivity to EP seemed to be modulated by the presence of ECM and the different cell organization. Indeed, cells cultured on HA-IKVAV were more sensitive than those treated in 2D and HA cultures, in terms of both cell membrane permeabilization and viability. Collectively, our results suggest that HA-IKVAV cultures may represent an interesting model for EP studies. Further studies will be needed to elucidate the influence of ECM composition on EP efficiency.

Estudo ecocardiográfico bidimensional em fetos de mäes com cardiopatias congênitas / Two-dimensional echocardiographic study in fetus of mothers with congenital heart diseases

Trinta mulheres com idade gestatória de 14 a 34 semanas (média 25 semanas), portadoras de cardiopatias congênitas diversas, foram submetidas a estudo ecocardiográfico para avaliaçäo da anatomia cardíaca fetal. A posiçäo fetal era determinada pela identificaçäo da cabeça, sacro e coluna vertebral. Em seguida, tentava-se reproduzir cortes equivalentes aos comumente usados na ecocardiografia. Os cortes 4-câmaras, eixo menor e eixo maior foram obtidos em 90%, 85% e 50% dos casos, respectivamente. Os estudos foram satisfatórios em 26 fetos (87%). Os resultados inadequados em 4 fetos foram decorrentes da inexperiência inicial e/ou da dificuldade para obter cortes ecocardiográficos apropriados. Os achados permitiram analisar as cavidades cardíacas, septos ventricular e atrial, valvas atrioventriculares e grandes vasos. Informaçöes adicinais, tais como banda moderadora, veias pulmonares e nível de implantaçäo das valvas atrioventriculares foram frequenemente obtidas. A confirmaçäo dos achados ecocardiográficos foi possível em 25 fetos estudados no período pós-natal. A associaçäo dos cortes obtidos permitiu excluir anomalias intracardíacas graves. Conclui-se que é possível a visibilizaçäo da anatomia cardíaca fetal normal e que o método pode ser útil na detecçäo de algumas das cardiopatias congênitas na fase gestatória