po 077 identification of dhx30 as an inhibitor of the translation of pro apoptotic mrnas after p53 activation by nutlin

Introduction The transcription factor p53 can be efficiently activated by the small molecule Nutlin-3 without inducing genotoxic stress. Treatment of different cell lines with this small molecule can result in different phenotypes, ranging from cell cycle arrest to apoptosis. HCT116 (colon cancer-derived cells) and SJSA1 (osteosarcoma-derived cells) were used to model the opposite behaviour respectively, by analysing the transcriptional and translational responses after Nutlin-3 treatment. Material and methods Total and polysomal-bound mRNAs were collected and sequenced after 12 hour of 10 uM Nutlin-3 treatment. A bioinformatics analysis of the polysome-enriched mRNAs using Weeder allowed the identification of a 3'UTR motif ('CG-rich') which is enriched in the translationally upregulated genes of SJSA1. The effect of the motif on translation was evaluated after cloning its consensus sequence in the 3'UTR of the b-globin gene, which was put downstream the luciferase reporter. The activity of the construct was evaluated after 12 or 24 hours of Nutlin-3. The same consensus was used for a pull-down experiment followed by mass spectrometry to identify proteins interacting with it. Results and discussions RNA-seq data indicate that HCT116 and SJSA1, although sharing almost completely the transcriptional program lead by p53, show almost no overlap at a translation level. SJSA1 present different pro-apoptotic translationally-upregulated genes after Nutlin-3, which have one or more instances of a CG-rich motif in the 3'UTR. The impact of the motif is to enhance the activity of the luciferase reported when cloned in two copies flanking the 3'UTR of the b-globin gene, but only in SJSA1. A pull-down experiment using an RNA bait with the consensus motif was used to identify interactors, among which DHX30 was deeply studied. DHX30 silencing in HCT116 causes: 1) enhanced the activity of the reporter construct after Nutlin; 2) polysomal association of selected mRNAs containing the motif; 3) induction of apoptosis as assessed by Annexin-V staining. In addition, silencing of DHX30 in U2OS cells decreased their survival after Nutlin-3 treatment. Conclusion We show how a p53-dependent transcriptional program can be shaped at a translational level thanks to the action of a CG-rich motif which is enriched in the 3'UTR of some pro-apoptotic mRNAs and that can be bound by DHX30. This protein acts as a translational repressor of mRNAs containing the motif. The exact mechanism and the generalisation of the model are currently being investigated

Further investigation of confirmed urinary tract infection (UTI) in children under five years: a systematic review.

Background: Further investigation of confirmed UTI in children aims to prevent renal scarring and future complications. Methods: We conducted a systematic review to determine the most effective approach to the further investigation of confirmed urinary tract infection (UTI) in children under five years of age. Results: 73 studies were included. Many studies had methodological limitations or were poorly reported. Effectiveness of further investigations: One study found that routine imaging did not lead to a reduction in recurrent UTIs or renal scarring. Diagnostic accuracy: The studies do not support the use of less invasive tests such as ultrasound as an alternative to renal scintigraphy, either to rule out infection of the upper urinary tract (LR- = 0.57, 95%CI: 0.47, 0.68) and thus to exclude patients from further investigation or to detect renal scarring (LR+ = 3.5, 95% CI: 2.5, 4.8). None of the tests investigated can accurately predict the development of renal scarring. The available evidence supports the consideration of contrast-enhanced ultrasound techniques for detecting vesico-ureteric reflux (VUR), as an alternative to micturating cystourethrography (MCUG) (LR+ = 14.1, 95% CI: 9.5, 20.8; LR- = 0.20, 95%CI: 0.13, 0.29); these techniques have the advantage of not requiring exposure to ionising radiation. Conclusion: There is no evidence to support the clinical effectiveness of routine investigation of children with confirmed UTI. Primary research on the effectiveness, in terms of improved patient outcome, of testing at all stages in the investigation of confirmed urinary tract infection is urgently required

Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold

Elevated maternal lipids in early pregnancy are not associated with risk of intrapartum caesarean in overweight and obese nulliparous women

Background: Maternal overweight and obesity are associated with slower labour progress and increased caesarean delivery for failure to progress. Obesity is also associated with hyperlipidaemia and cholesterol inhibits myometrial contractility in vitro. Our aim was, among overweight and obese nulliparous women, to investigate 1. the role of early pregnancy serum cholesterol and 2. clinical risk factors associated with first stage caesarean for failure to progress at term. Methods: Secondary data analysis from a prospective cohort of overweight/obese New Zealand and Australian nullipara recruited to the SCOPE study. Women who laboured at term and delivered vaginally (n=840) or required first stage caesarean for failure to progress (n=196) were included. Maternal characteristics and serum cholesterol at 14–16 weeks’ of gestation were compared according to delivery mode in univariable and multivariable analyses (adjusted for BMI, maternal age and height, obstetric care type, induction of labour and gestation at delivery ≥41 weeks). Results: Total cholesterol at 14–16 weeks was not higher among women requiring first stage caesarean for failure to progress compared to those with vaginal delivery (5.55 ± 0.92 versus 5.67 ± 0.85 mmol/L, p= 0.10 respectively). Antenatal risk factors for first stage caesarean for failure to progress in overweight and obese women were BMI (adjusted odds ratio [aOR (95% CI)] 1.15 (1.07-1.22) per 5 unit increase, maternal age 1.37 (1.17-1.61) per 5 year increase, height 1.09 (1.06-1.12) per 1cm reduction), induction of labour 1.94 (1.38-2.73) and prolonged pregnancy ≥41 weeks 1.64 (1.14-2.35). Conclusions: Elevated maternal cholesterol in early pregnancy is not a risk factor for first stage caesarean for failure to progress in overweight/obese women. Other clinically relevant risk factors identified are: increasing maternal BMI, increasing maternal age, induction of labour and prolonged pregnancy ≥41 weeks’ of gestation.Elaine M Fyfe, Karen S Rivers, John MD Thompson, Kamala PL Thiyagarajan, Katie M Groom, Gustaaf A Dekker, Lesley ME McCowan and On behalf of the SCOPE consortiu

3D finite compartment modeling of formation and healing of bruises may identify methods for age determination of bruises

Simulating the spatial and temporal behavior of bruises may identify methods that allow accurate age determination of bruises to assess child abuse. We developed a numerical 3D model to simulate the spatial kinetics of hemoglobin and bilirubin during the formation and healing of bruises. Using this model, we studied how skin thickness, bruise diameter and diffusivities affect the formation and healing of circular symmetric bruises and compared a simulated bruise with a natural inhomogeneous bruise. Healing is faster for smaller bruises in thinner and less dense skin. The simulated and natural bruises showed similar spatial and temporal dynamics. The different spatio-temporal dynamics of hemoglobin and bilirubin allows age determination of model bruises. Combining our model predictions with individual natural bruises may allow optimizing our model parameters. It may particularly identify methods for more accurate age determination than currently possible to aid the assessment of child abuse

Effect of Restricted Preen-Gland Access on Maternal Self Maintenance and Reproductive Investment in Mallards

As egg production and offspring care are costly, females should invest resources adaptively into their eggs to optimize current offspring quality and their own lifetime reproductive success. Parasite infections can influence maternal investment decisions due to their multiple negative physiological effects. The act of preening--applying oils with anti-microbial properties to feathers--is thought to be a means by which birds combat pathogens and parasites, but little is known of how preening during the reproductive period (and its expected disease-protecting effects) influences maternal investment decisions at the level of the egg.Here, we experimentally prevented female mallards (Anas platyrhynchos) from accessing their preen gland during breeding and monitored female immunoresponsiveness (e.g., plasma lysozyme concentration) as well as some egg traits linked to offspring quality (e.g., egg mass, yolk carotenoid content, and albumen lysozyme levels). Females with no access to their preen gland showed an increase in plasma lysozyme level compared to control, normally preening females. In addition, preen-gland-restricted females laid significantly lighter eggs and deposited higher carotenoid concentrations in the yolk compared to control females. Albumen lysozyme activity did not differ significantly between eggs laid by females with or without preen gland access.Our results establish a new link between an important avian self-maintenance behaviour and aspects of maternal health and reproduction. We suggest that higher yolk carotenoid levels in eggs laid by preen-gland-restricted females may serve to boost health of offspring that would hatch in a comparatively microbe-rich environment