siRNAs bearing aromatic residues in the 3’-overhang region

RNA interference (RNAi) is a biological process whereby small interfering RNA (siRNA) and microRNA (miRNA) silence gene expression in a sequence-specific manner. These effectors regulate gene expression through the RNA-induced silencing complex (RISC). It has been suggested that RISC preferentially selects and incorporates one of two strands of the siRNA duplex depending on its thermodynamic features and that the off-target effects of siRNAs can be correlated to the Tm of the duplex. The problem of unwanted incorporation of the passenger strand into RISC could be address altering the thermodynamic asymmetry of the duplex by using specific chemical modifications. Structural studies have revealed that the 3’-overhang region of the guide strand of siRNA is recognized by the PAZ domain and is accommodated into its hydrophobic binding pocket. We expected that aromatic-based modifications in 3’-overhang would enhance RISC selection of antisense strands of siRNA duplexes, reducing off-target effects induced by sense strands. In this study, we report the synthesis of siRNAs bearing diphenylpropylamine, tyramine and tryptamine units at the 3’-end of sense and antisense strands. We found thermodynamic stability of the conjugates was increased by these modifications. Furthermore, but not surprisingly, the modified duplexes were found to retain RNA-like A-type conformation. We also assessed the nuclease resistance of the modified siRNAs and found it was similar to those of unmodified siRNAs. These results prompted us to investigate the silencing activity of the siRNAs possessing the aromatic moiety in the 3’-end by in vitro experiments in mammalian cells

Synthesis, biophysical characterization and anti-HIV activity of d(TG3AG) Quadruplexes bearing hydrophobic tails at the 5'-end

Novel conjugated G-quadruplex-forming d(TG3AG) oligonucleotides, linked to hydrophobic groups through phosphodiester bonds at 50-end, have been synthesized as potential anti-HIV aptamers, via a fully automated, online phosphoramidite-based solid-phase strategy. Conjugated quadruplexes showed pronounced anti-HIV activity with some preference for HIV-1, with inhibitory activity invariably in the low micromolar range. The CD and DSC monitored thermal denaturation studies on the resulting quadruplexes, indicated the insertion of lipophilic residue at the 50-end, conferring always improved stability to the quadruplex complex (20 < DTm < 40 C). The data suggest no direct functional relationship between the thermal stability and anti-HIV activity of the folded conjugated G-quartets. It would appear that the nature of the residue at 50 end of the d(TG3AG) quadruplexes plays an important role in the thermodynamic stabilization but a minor influence on the anti-HIV activity. Moreover, a detailed CD and DSC analyses indicate a monophasic behaviour for sequences I and V, while for ODNs (II–IV) clearly show that these quadruplex structures deviate from simple two-state melting, supporting the hypothesis that intermediate states along the dissociation pathway may exis

Harpin oligonucleotides forming G-quadruplexes: new aptamers with potential anti-HIV activity

Several G-rich synthetic oligodeoxyribonucleotides (ODNs) have shown promising biological properties, ranging from anticancer to anti-HIV activities. G-quadruplex formation was found to be a crucial prerequisite in determining these biological effects. Aptamers exhibiting anti-HIV activity represent an important class of potential therapeutics. Recently we described the synthesis and characterization of new d(TGGGAG) ODNs, conjugated with different aromatic groups at the 5’-end through a phosphodiester bond. The modified sequences showed a parallel stranded tetramolecular G-quadruplexes CD profile and a pronounced anti-HIV-1 activity. Herein, with the aim to use d(TGGGAG) as a lead sequence for a more effective anti-HIV agent, we propose the fully automated synthesis of new ODNs containing two d(TGGGAG) sequences whose 3-ends are joint by an hexaethylenglycole loop. CD analysis was undertaken on the 3’-3’ linked d(TGGGAG) hairpins in comparison with the corresponding unmodified oligomers. Besides, in order to study the influence of the conjugation at the ends of the harpin chains on their ability to stabilize quadruplex structures and on their anti-HIV activity, different conjugated oligomers have been studied

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies

Prevalencia de esofagitis eosinofílica: estudio multicéntrico en población pediátrica evaluada en 36 centros de gastroenterología de América Latina

Introducción y objetivo: La esofagitis eosinofílica es una enfermedad crónica, mediada inmunológicamente, descrita en series y publicaciones alrededor del mundo. En los últimos 20 años diversos estudios han intentado evaluar la incidencia y prevalencia de la enfermedad. El objetivo del presente trabajo es estimar la prevalencia de esofagitis eosinofílica en un grupo de niños atendidos en 36 centros de gastroenterología pediátrica de 10 países latinoamericanos. Materiales y métodos: A través de un protocolo multicéntrico, observacional y transversal se estimó la prevalencia de período de esofagitis eosinofílica entre los niños atendidos en consulta externa y sometidos a endoscopia superior diagnóstica por cualquier motivo en 36 centros de 10 países latinoamericanos durante un período de 3 meses. Resultados: Entre abril y junio de 2016 108 casos de esofagitis eosinofílica fueron evaluados. Asimismo, un promedio de 29,253 consultas ambulatorias y 4,152 endoscopias superiores de carácter diagnóstico fueron realizadas en los 36 centros participantes. La tasa de prevalencia de esofagitis eosinofílica en la población estudiada (n = 29,253) fue de 3,69 casos × 1,000 (IC 95%: 3.04 a 4.44) y entre los niños sometidos a endoscopia superior de rutina (n = 4,152) fue de 26 x 1,000 (IC 95%: 22.6 a 29.4). Conclusión: La tasa general de prevalencia de período de esofagitis eosinofílica en un grupo de niños evaluados en 36 centros latinoamericanos de gastroenterología pediátrica resultó de 3,69 × 1,000, y entre aquellos sometidos a endoscopia fue de 26 × 1,000. La prevalencia mostró una importante variabilidad entre los países y centros participantes. Este es el primer estudio de prevalencia de esofagitis eosinofílica pediátrica en Latinoamérica. Abstract: Introduction and objective: Eosinophilic esophagitis is a chronic, immune-mediated disease described in case series and publications worldwide. Over the past twenty years, the authors of different studies have attempted to evaluate its incidence and prevalence. The objetive of the present study was to estimate the prevalence of eosinophilic esophagitis in a group of children seen at 36 pediatric gastroenterology centers in ten Latin American countries. Materials and methods: A multicenter, observational, and cross-sectional study was conducted that estimated the period prevalence of eosinophilic esophagitis in children seen at outpatient consultation and that underwent diagnostic upper gastrointestinal endoscopy for any indication at 36 centers in 10 Latin American countries, within a 3-month time frame. Results: Between April and June 2016, 108 cases of eosinophilic esophagitis were evaluated. Likewise, an average of 29,253 outpatient consultations and 4,152 diagnostic upper gastrointestinal endoscopies were carried out at the 36 participating centers. The period prevalence of eosinophilic esophagitis in the population studied (n = 29,253) was 3.69 cases × 1,000 (95% CI: 3.04 to 4.44), and among the children that underwent routine upper gastrointestinal endoscopy (n = 4,152), it was 26 x 1,000 (95% CI: 22.6 to 29.4). Conclusions: The general period prevalence of eosinophilic esophagitis in a group of children evaluated at 36 Latin American pediatric gastroenterology centers was 3.69 × 1,000, and in the children that underwent endoscopy, it was 26 × 1,000. There was important prevalence variability between the participating countries and centers. The present analysis is the first study conducted on the prevalence of pediatric eosinophilic esophagitis in Latin America. Palabras clave: Esofagitis, Eosinofílica, Niños, Prevalencia, Latinoamérica, Keywords: Esophagitis, Eosinophilic, Children, Prevalence, Latin Americ

Il cibo dalla Costituzione alle pratiche educative nel quadro delle politiche per la sostenibilit\ue0

Il cibo, anche a seguito di recenti contenziosi, sta assumendo sempre pi\uf9 una rilevanza costituzionalistica. Oltre a tutelare il diritto al sostentamento, la Costituzione ne presidia la libert\ue0 di scelta, che trova ancoraggio nelle libert\ue0 personali, di coscienza e religiosa. Il cibo \ue8 altres\uec connesso al diritto alla salute: da questo punto di vista, emerge il problema costituzionale del costo del cibo salubre, che non dovrebbe produrre disuguaglianze. Le questioni vanno inquadrate considerando i diritti delle generazioni future, a cui l\u2019attuale comunit\ue0 sociale \ue8 legata dal dovere di solidariet\ue0. In questo contesto, si colloca l\u2019educazione alimentare, riconosciuta nei programmi dell\u2019Agenda 2030 dell\u2019Onu e nelle Linee Guida per l\u2019Educazione Alimentare del Miur, in cui prende forma un\u2019idea di azione globale integrata fra tutti i soggetti chiamati a promuovere corrette abitudini sul cibo. Tuttavia, su questo versante, recenti studi hanno evidenziato alcune debolezze nella cooperazione per programmi comuni e unitari verso un\u2019educazione alimentare promossa sul piano nazionale

Recent Progress in Chemically Modified siRNAs

RNA interference technology has become a powerful laboratory tool to study gene function. Small interfering RNAs (siRNAs) have provided unprecedented opportunities for the development of new therapeutics in human diseases. Unfortunately, siRNA duplexes are not optimal drug-like molecules. The problems for their effective application are fundamentally delivery, stability and off-target effects. Chemical modification provides solutions to many of the challenges facing siRNA therapeutics. In this review, we recapitulate and discuss the development of the latest described chemical modifications of siRNAs, with a special focus on novel chemical modifications of siRNA structure, architecture and siRNA conjugates