Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2

AbstractThe yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18Ubc9, the Xenopus homolog of Ubc9p, associates specifically with p88RanGAP1 and p340RanBP2[2]. Ran-binding protein 2 (p340RanBP2) is a nuclear pore protein [3,4], and p88RanGAP1 is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5–7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18Ubc9 acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18Ubc9 interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts

Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo

The Notch ligands Dll1 and Dll3 are coexpressed in the presomitic mesoderm of mouse embryos. Despite their coexpression, mutations in Dll1 and Dll3 cause strikingly different defects. To determine if there is any functional equivalence, we replaced Dll1 with Dll3 in mice. Dll3 does not compensate for Dll1; DLL1 activates Notch in Drosophila wing discs, but DLL3 does not. We do not observe evidence for antagonism between DLL1 and DLL3, or repression of Notch activity in mice or Drosophila. In vitro analyses show that differences in various domains of DLL1 and DLL3 individually contribute to their biochemical nonequivalence. In contrast to endogenous DLL1 located on the surface of presomitic mesoderm cells, we find endogenous DLL3 predominantly in the Golgi apparatus. Our data demonstrate distinct in vivo functions for DLL1 and DLL3. They suggest that DLL3 does not antagonize DLL1 in the presomitic mesoderm and warrant further analyses of potential physiological functions of DLL3 in the Golgi network

Computer-aided assessment of diagnostic images for epidemiological research

<p>Abstract</p> <p>Background</p> <p>Diagnostic images are often assessed for clinical outcomes using subjective methods, which are limited by the skill of the reviewer. Computer-aided diagnosis (CAD) algorithms that assist reviewers in their decisions concerning outcomes have been developed to increase sensitivity and specificity in the clinical setting. However, these systems have not been well utilized in research settings to improve the measurement of clinical endpoints. Reductions in bias through their use could have important implications for etiologic research.</p> <p>Methods</p> <p>Using the example of cortical cataract detection, we developed an algorithm for assisting a reviewer in evaluating digital images for the presence and severity of lesions. Available image processing and statistical methods that were easily implementable were used as the basis for the CAD algorithm. The performance of the system was compared to the subjective assessment of five reviewers using 60 simulated images. Cortical cataract severity scores from 0 to 16 were assigned to the images by the reviewers and the CAD system, with each image assessed twice to obtain a measure of variability. Image characteristics that affected reviewer bias were also assessed by systematically varying the appearance of the simulated images.</p> <p>Results</p> <p>The algorithm yielded severity scores with smaller bias on images where cataract severity was mild to moderate (approximately ≤ 6/16^<it>ths</it>). On high severity images, the bias of the CAD system exceeded that of the reviewers. The variability of the CAD system was zero on repeated images but ranged from 0.48 to 1.22 for the reviewers. The direction and magnitude of the bias exhibited by the reviewers was a function of the number of cataract opacities, the shape and the contrast of the lesions in the simulated images.</p> <p>Conclusion</p> <p>CAD systems are feasible to implement with available software and can be valuable when medical images contain exposure or outcome information for etiologic research. Our results indicate that such systems have the potential to decrease bias and discriminate very small changes in disease severity. Simulated images are a tool that can be used to assess performance of a CAD system when a gold standard is not available.</p

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hyper-trophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocar-diography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Reso-lution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell cycle defects and mitochondrial dys-function. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteosomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteosomal system is activated; a mechanism which has been implicated in cardiomyopathies previously. In parallel, lack of functional al-pha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell cycle defects, the likely cause of death of the embryos. The defects also have wide-ranging morphological consequences

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women

Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5

Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a “shifting” site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity

Maternal iron deficiency perturbs embryonic cardiovascular development in mice

Funder: Novo Nordisk; doi: https://doi.org/10.13039/501100004191Funder: National Heart Foundation of Australia (Heart Foundation); doi: https://doi.org/10.13039/501100001030Funder: NSW Health; doi: https://doi.org/10.13039/501100009287Funder: Oxford University | John Fell Fund, University of Oxford (John Fell OUP Research Fund); doi: https://doi.org/10.13039/501100004789Funder: The Federated FoundationAbstract: Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene–environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women

NAD deficiency, congenital malformations, and niacin supplementation

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice

Evaluation for what purpose? Findings from two stakeholder groups

A host of reasons exist for the pursuit of evidence in the public sector, including to support good governance and policy development. As the expectations for program evaluation from policymakers have evolved, so too has evaluation practice and a great deal of experimentalism has ensued. There is a risk that these developments and the complexity inherent in them, may lead to conflicting expectations about why program evaluation is done, or even a loss of purpose. This prompts the meso-level analysis of two types of stakeholders in a governance network, explored in this chapter. This chapter presents the findings of an ongoing study which explores the perceptions of program evaluators and policy implementers towards the purpose of evidence. The findings suggest that program evaluators and policy implementers have divergent expectations of why and how evaluation data might be used. The findings suggest that program evaluators aspire to support change and enhance the policy domains they serve, whereas policy implementers perceive program evaluation as serving a more governance-/management-orientated role. The chapter demonstrates the complexity of both program evaluation and policy and may have implications for the twin pillars of governance and responsibility at the heart of the book. If governance and responsibility are the twin pillars of sustainability then the complex networks of relationships, expectations, values, and outcomes may need to be considered. The findings also have implications for evaluation commissioners and practitioners, demonstrating the need for the purpose and expectations of program evaluation to be agreed early. The use of program evaluation as a symbolic, aesthetic or structural mechanism also emerges, prompting opportunity for further research, for instance, to explore legitimacy and program evaluation.N/

Search for exotic resonances decaying into WZ/ZZ in pp collisions at √s=7 TeV

Journal of High Energy Physics 2013.2 (2013): 036 reproduced by permission of Scuola Internazionale Superiore di Studi Avanzati (SISSA)Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAMA search for new exotic particles decaying to the VZ final state is performed, where V is either a W or a Z boson decaying into two overlapping jets and the Z decays into a pair of electrons, muons or neutrinos. The analysis uses a data sample of pp collisions corresponding to an integrated luminosity of 5 fb-1 collected by the CMS experiment at the LHC at √s=7 TeV in 2011. No significant excess is observed in the mass distribution of the VZ candidates compared with the background expectation from standard model processes. Model-dependent upper limits at the 95% confidence level are set on the product of the cross section times the branching fraction of hypothetical particles decaying to the VZ final state as a function of mass. Sequential standard model W′ bosons with masses between 700 and 940 GeV are excluded. In the Randall-Sundrum model for graviton resonances with a coupling parameter of 0.05, masses between 750 and 880 GeV are also exclude