Use of a conditional Ubr5 mutant allele to investigate the role an N-end rule ubiquitin-protein ligase in Hedgehog signalling and embryonic limb development

Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling

The National Early Warning Score and its subcomponents recorded within ±24 hours of emergency medical admission are poor predictors of hospital-acquired acute kidney injury

YesBackground: Hospital-acquired Acute Kidney Injury (H-AKI) is a common cause of avoidable morbidity and mortality. Aim: To determine if the patients’ vital signs data as defined by a National Early Warning Score (NEWS), can predict H-AKI following emergency admission to hospital. Methods: Analyses of emergency admissions to York hospital over 24-months with NEWS data. We report the area under the curve (AUC) for logistic regression models that used the index NEWS (model A0), plus age and sex (A1), plus subcomponents of NEWS (A2) and two-way interactions (A3). Likewise for maximum NEWS (models B0,B1,B2,B3). Results: 4.05% (1361/33608) of emergency admissions had H-AKI. Models using the index NEWS had the lower AUCs (0.59 to 0.68) than models using the maximum NEWS AUCs (0.75 to 0.77). The maximum NEWS model (B3) was more sensitivity than the index NEWS model (A0) (67.60% vs 19.84%) but identified twice as many cases as being at risk of H-AKI (9581 vs 4099) at a NEWS of 5. Conclusions: The index NEWS is a poor predictor of H-AKI. The maximum NEWS is a better predictor but seems unfeasible because it is only knowable in retrospect and is associated with a substantial increase in workload albeit with improved sensitivity.The Health Foundatio

<i>Ubr5</i> is required for embryonic mid-gestational viability—E13.5 analysis.

<p><i>Ubr5</i> is required for embryonic mid-gestational viability—E13.5 analysis.</p

E9.5 <i>Ubr5</i>^mt/mt embryos are developmentally abnormal.

<p>Optical projection tomography images of <i>Ubr5</i>^<i>+/+</i> (Con) (A,B, E-H) and <i>Ubr5</i>^<i>mt/mt</i> (C,D, I-L) E9.5 embryos. Both <i>Ubr5</i>^<i>mt/mt</i> and Con embryos formed a neural floorplate (A,C arrows) and separated the plates of the diencephalic-mesencephalic junction (B,D asterisks). In comparison to control embryos, E9.5 <i>Ubr5</i>^<i>mt/mt</i> embryos exhibited numerous developmental defects: an open posterior neuropore (J dashed line), irregular somites that were also reduced in number (compare G,H with J, arrowheads); lordotic curvature (L) and only one pair of pharyngeal arches (compare E,H with K,L arrows). n = 1. Scale bar (A-C) = 200μm and (E-L) = 1mm.</p

Retinoic Acid induces <i>Shh</i> and suppresses UBR5 expression.

<p>Murine E14 ES cells were treated with (A) the indicated concentration of RA, or (B-D) 0.1μM RA or DMSO (Vehicle) and analysed by RT-PCR at (A) 24hrs and (B) at the indicated times post-RA-treatment. (C) SDS-PAGE and Western blotting determined UBR5 expression over the indicated RA time course. An asterisk denotes an uncharacterised, faster migrating UBR5 antibody reactive species. Tubulin was used as a loading control. (D) qRT-PCR of <i>Ubr5</i> expression normalised against <i>β-actin</i> in mES cells 96hrs post-RA-treatment (n = 3, s.e.m indicated). Statistical analysis by Students t-test. ns = not significant.</p

UBR5 is required for RA-mediated induction of <i>Shh</i>.

<p>Pools of murine E14 mES cells expressing either scrambled control (SCR) or <i>Ubr5</i> shRNAi were tested for (A) UBR5 expression by SDS-PAGE and Western blotting, using Tubulin as a loading control; (B) RA-mediated <i>Shh</i> induction by qRT-PCR (n = 3, relative expression levels to β-actin, s.e.m indicated) or (C) expression analysis of the indicated ES cell pluripotentcy markers in shRNAi pools or parental ES cells by sqRT-PCR. For (B) comparison of all matched time points post-RA-treatment between control and <i>Ubr5</i> shRNAi pools revealed statistically significant differences (p = <0.01), apart from the comparison between control and <i>Ubr5</i>.<i>2</i> at 24 hours, which was not significant (ns). Statistical analysis by one-way ANOVA and Tukey multiple comparison test.</p

Loss of <i>Ubr5</i> at E11.5 does not affect <i>Shh</i> or <i>Ptch1</i> expression domains.

<p>In situ hybridisation for expression of Hh signalling components <i>Ptch1</i> (A,B) and <i>Shh</i> (C,D) in E11.5 embryos of <i>Prx1-Cre</i> (Con) and <i>Prx1-Cre;Ubr5</i>^{<i>mt*/mt*</i>} (<i>Ubr5</i>^{<i>mt*/mt*</i>}) embryos. Analysis revealed no significant effects on either <i>Shh</i> or <i>Ptch1</i> expression patterns in the <i>Ubr5</i>^{<i>mt*/mt*</i>} embryos (representative image from n = >4 of each genotype). Arrowheads indicate <i>Shh</i> ZPA expression domains. Scale bar = 0.5mm.</p

<i>Ubr5</i> is required for embryonic mid-gestational viability—E15.5 analysis.

<p><i>Ubr5</i> is required for embryonic mid-gestational viability—E15.5 analysis.</p

E11.5 <i>Ubr5</i>^mt/mt embryos are not viable.

<p>E11.5 <i>Ubr5</i>^mt/mt embryos are not viable.</p