Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

BACKGROUND: Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. RESULTS: We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. CONCLUSIONS: Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis

Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene

BACKGROUND: Runx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo. RESULTS: The developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D. CONCLUSIONS: SpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation

CBFbeta is a facultative Runx partner in the sea urchin embryo

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation

CBFbeta is a facultative Runx partner in the sea urchin embryo

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation

Sea urchin vault structure, composition, and differential localization during development

BACKGROUND: Vaults are intriguing ribonucleoprotein assemblies with an unknown function that are conserved among higher eukaryotes. The Pacific coast sea urchin, Strongylocentrotus purpuratus, is an invertebrate model organism that is evolutionarily closer to humans than Drosophila and C. elegans, neither of which possesses vaults. Here we compare the structures of sea urchin and mammalian vaults and analyze the subcellular distribution of vaults during sea urchin embryogenesis. RESULTS: The sequence of the sea urchin major vault protein (MVP) was assembled from expressed sequence tags and genome traces, and the predicted protein was found to have 64% identity and 81% similarity to rat MVP. Sea urchin MVP includes seven ~50 residue repeats in the N-terminal half of the protein and a predicted coiled coil domain in the C-terminus, as does rat MVP. A cryoelectron microscopy (cryoEM) reconstruction of isolated sea urchin vaults reveals the assembly to have a barrel-shaped external structure that is nearly identical to the rat vault structure. Analysis of the molecular composition of the sea urchin vault indicates that it contains components that may be homologs of the mammalian vault RNA component (vRNA) and protein components (VPARP and TEP1). The sea urchin vault appears to have additional protein components in the molecular weight range of 14–55 kDa that might correspond to molecular contents. Confocal experiments indicate a dramatic relocalization of MVP from the cytoplasm to the nucleus during sea urchin embryogenesis. CONCLUSIONS: These results are suggestive of a role for the vault in delivering macromolecules to the nucleus during development

The Burden of Revision Sinonasal Surgery in the UK – Data from the Chronic Rhinosinusitis Epidemiology Study (CRES); a cross sectional study

Objectives/Hypothesis The aim of this study was to investigate the surgical revision rate in patients with Chronic Rhinosinusitis (CRS) in the UK CRS Epidemiology Study (CRES). Previous evidence from national Sinonasal Audit showed that 1459 CRS patients demonstrated a surgical revision rate 19.1% at 5 years, with highest rates seen in those with polyps (20.6%). Setting Thirty secondary care centres around the UK. Participants A total of 221 controls and 1249 patients with CRS were recruited to the study including those with polyps (CRSwNPs), without polyps (CRSsNPs) and with allergic fungal rhinosinusitis (AFRS). Interventions Self-administered questionnaire. Primary outcome measure The need for previous sinonasal surgery. Results A total of 651 patients with CRSwNPs, 553 with CRSsNPs and 45 with AFRS were included. A total of 396 (57%) of patients with CRSwNPs/AFRS reported having undergone previous endoscopic nasal polypectomy (ENP), of which 182 of the 396 (46%) reported having received more than one operation. The mean number of previous surgeries per patient in the revision group was 3.3 (range 2 to 30) and a mean duration of time of 10 years since the last procedure. The average length of time since their first operation up to inclusion in the study was 15.5 years (range 0-74). Only 27.9% of all patients reporting a prior ENP had received concurrent endoscopic sinus surgery (ESS) (n=102). For comparison, surgical rates in patients with CRSsNPs were significantly lower; 13% of cases specifically reported ESS and of those only 30% reported multiple procedures (chi-squared p < 0.001). Conclusions This study demonstrated there is a high burden of both primary and revision surgery in patients with CRS, worst in those with AFRS and least in those with CRSsNPs. The burden of revision surgery appears unchanged in the decade since the Sinonasal Audit

Trigger Point Injections for Headache Disorders: Expert Consensus Methodology and Narrative Review

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109332/1/head12442.pd

The Juvenile Justice Behavioral Health Services Cascade: A New Framework for Measuring Unmet Substance Use Treatment Services Needs Among Adolescent Offenders

Overview—Substance use and substance use disorders are highly prevalent among youth under juvenile justice (JJ) supervision, and related to delinquency, psychopathology, social problems, risky sex and sexually transmitted infections, and health problems. However, numerous gaps exist in the identification of behavioral health (BH) problems and in the subsequent referral, initiation and retention in treatment for youth in community justice settings. This reflects both organizational and systems factors, including coordination between justice and BH agencies. Methods and Results—This paper presents a new framework, the Juvenile Justice Behavioral Health Services Cascade (“Cascade”), for measuring unmet substance use treatment needs to illustrate how the cascade approach can be useful in understanding service delivery issues and identifying strategies to improve treatment engagement and outcomes for youth under community JJ supervision. We discuss the organizational and systems barriers for linking delinquent youth to BH services, and explain how the Cascade can help understand and address these barriers. We provide a detailed description of the sequential steps and measures of the Cascade, and then offer an example of its application from the Juvenile Justice – Translational Research on Interventions for Adolescents in the Legal System project (JJ-TRIALS), a multi-site research cooperative funded by the National Institute on Drug Abuse. Conclusion—As illustrated with substance abuse treatment, the Cascade has potential for informing and guiding efforts to improve behavioral health service linkages for adolescent offenders, developing and testing interventions and policies to improve interagency and cross-systems coordination, and informing the development of measures and interventions for improving the implementation of treatment in complex multisystem service settings

CBFbeta is a facultative Runx partner in the sea urchin embryo

Background: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. Results: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. Conclusion: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation