F11R expression upon hypoxia is regulated by RNA editing.

F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54(nrb). We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress

Role of vasopressin and terlipressin in refractory shock compared to conventional therapy in the neonatal and pediatric population: a systematic review, meta-analysis, and trial sequential analysis

Abstract Background Vasopressin (AVP) and terlipressin (TP) have been used as last-line therapy in refractory shock in children. However, the efficacy and safety profiles of AVP and TP have not been determined in pediatric refractory shock of different origins. We aimed to assess the efficacy and safety of the addition of AVP/TP therapy in pediatric refractory shock of all causes compared to conventional therapy with fluid resuscitation and vasopressor and inotropic therapy. Methods We conducted a systematic review, meta-analysis, and trial sequential analysis (TSA) comparing AVP and TP to conventional therapy. MEDLINE, EMBASE, Cochrane Library, and ClinicalTrials.gov were searched up to February 2016. The systematic review included all reports of AVP/TP use in the pediatric population. Reports of clinical trials were pooled using random-effects models and TSA. Main outcomes were mortality and tissue ischemia. Results Three randomized controlled trials and five \u201cbefore-and-after clinical\u201d trials (without comparator) met the inclusion criteria. Among 224 neonates and children (aged 0 to 18\ua0years) with refractory shock, 152 received therapy with AVP or TP. Pooled analyses showed no association between AVP/TP treatment and mortality (relative risk (RR),1.19; 95% confidence interval (CI), 0.71\u20132.00), length of stay in the pediatric intensive care unit (PICU) (mean difference (MD), \u20133.58\ua0days; 95% CI, \u20139.05 to 1.83), and tissue ischemia (RR, 1.48; 95% CI, 0.47\u20134.62). In TSA, no significant effect on mortality and risk for developing tissue ischemia was observed with AVP/TP therapy. Conclusion Our results emphasize the lack of observed benefit for AVP/TP in terms of mortality and length of stay in the PICU, and suggest an increased risk for ischemic events. Our TSA suggests that further large studies are necessary to demonstrate and establish benefits of AVP/TP in children. PROSPERO registry: CRD4201603587

Percentage of average RNA editing in pre- and mature- F11R mRNA .

<p>Pre- or mature- F11R mRNA from control and DFO-treated cells was amplified using specific primers. Significant average RNA editing was seen only in mature F11R mRNA which was further increased upon DFO treatment. Error bars indicate standard deviation (±SD, **p≤ 0.01) .</p

A proposed model for the regulation of F11R expression following hypoxic stress.

<p>Upon normoxia, F11R mRNA is transcribed and a fraction of the molecules are exported to the cytoplasm for translation. Upon hypoxia, editing of F11R is increased. The majority of the hyperedited molecules binds p54^nrb and is retained in the nucleus in large amounts. Translation of the small number of edited transcripts which escape nuclear retention, is attenuated by a yet to be determined mechanism. We propose that when oxygen levels return to normal, a large amount of F11R hyperedited molecules disengage from p54^nrb and thus are released to the cytoplasm for translation enabling protein levels to return to normal very quickly.</p

Protein expression of F11R upon hypoxia.

<p>Western blot analysis was performed on protein extracts from LB cells 24h following DFO treatment or without treatment. Numbers under the F11R panel represent the relative quantification of the amount of protein. Actin was used as a loading control.</p

RNA immunoprecipitation with an anti-p54^nrb antibody.

<p>(A) PCR amplification with F11R-specific primers on RNA extracted from a RIP experiment conducted with an anti-p54^nrb antibody on fractionated control cells and fractionated cells treated with DFO. PCR amplification was obtained in the RNA extracted from p54^nrb-precipitated RNA from nuclear extracts of DFO-treated cells. As a positive control PCR was performed on total RNA (B) Percentage of RNA average editing of F11R RNA that was bound to p54^nrb. High levels of editing were obtained in the RNA that was extracted from the p54^nrb-bound molecules. </p

Knock-down of ADAR2.

<p>LB cells were transfected with si-ADAR2 or with a control si molecule (siControl). Cells were either treated with DFO 16h post-transfection or untreated. RNA and proteins of all cells were extracted 40h post-transfection (24h post-DFO treatment). (A) Relative quantification of mRNA. mRNA was quantified relatively to the siControl sample which was set as 1. Error bars indicate standard deviation (±SD, *p≤ 0.05, **p≤ 0.01) Silenced cells showed low expression of ADAR2. No effect of the silencing was seen on F11R mRNA which was induced upon hypoxia similarily to the control-transfected cells. (B) Percentage of average RNA editing in F11R upon ADAR2 silencing with or without DFO treatment. ADAR2-silenced cells did not show a change in editing. Editing in the control-transfected cellswas induced upon DFO treatment. </p

Treatment with α–amanitin.

<p>LB cells were treated for 24h with α–amanitin with or without DFO treatment. (A) Relative quantification of mRNA. mRNA was quantified relatively to the non-treated sample which was set as 1. Error bars indicate standard deviation (±SD, *p≤ 0.05, **p≤ 0.01). ADARs and F11R expression was reduced upon α–amanitin treatment. Treatment with both α–amanitin and DFO showed an increase only in F11R mRNA when compared to the amount obtained with α–amanitin treatment only. (B) Relative quantification of mRNA following cell fractionation. mRNA was quantified relatively to the cytoplasmic control sample which was set as 1. Error bars indicate standard deviation (±SD, *p≤ 0.05, **p≤ 0.01). F11R mRNA was higher in the nuclear fractions. α–amanitin treatment reduced the amount of F11R transcripts and the addition of DFO induced them. (C) Percentage of average RNA editing in F11R. Cells treated with α–amanitin showed high levels of RNA editing in F11R. RNA extracted from the cytoplasm showed higher levels of editing when compared to those obtained in the RNA extracted from the nucleus. Error bars indicate standard deviation (±SD, **p≤ 0.01) (D) Percentage of RNA editing in F11R at additional sites. Treatment with α–amanitin revealed significant editing at seven additional sites. Upper panel: editing at these sites in the nuclear extracts following treatment. Lower panel: editing at these sites in the cytoplasmic extracts. Error bars indicate standard deviation (±SD). All the results obtained for the α–amanitin and the α-amanitin+DFO treatments have a p≤ 0.01. (E) Western blot analysis. Numbers in between rows show the relative quantification of the amount of protein set at 1 in the non-treated sample. No F11R protein was seen in the α–amanitin-treated cells. Low F11R expression was seen upon α-amanitin + DFO treatment. Actin was used as a loading control.</p