PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells

AbstractTo better understand the means by which cells such as human platelets regulate the binding of the integrin αIIbβ3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses αIIbβ3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an αIIbβ3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated αIIbβ3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42MAPK, phospholipase A2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent (‘outside-in’) signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support αIIbβ3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of αIIbβ3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells

"Women's rights, the European Court and Supranational Constitutionalism"

This analysis examines supranational constitutionalism in the European Union. In particular, the study focuses on the role of the European Court of Justice in the creation of women’s rights. I examine the interaction between the Court and member state governments in legal integration, and also the integral role that women’s advocates – both individual activists and groups – have played in the development of EU social provisions. The findings suggest that this litigation dynamic can have the effect of fueling the integration process by creating new rights that may empower social actors and EU organizations, with the ultimate effect of diminishing member state government control over the scope and direction of EU law. This study focuses specifically on gender equality law, yet provides a general framework for examining the case law in subsequent legal domains, with the purpose of providing a more nuanced understanding of supranational governance and constitutionalism

A Collaborative Model for Accelerating the Discovery and Translation of Cancer Therapies

Preclinical studies using genetically engineered mouse models (GEMM) have the potential to expedite the development of effective new therapies; however, they are not routinely integrated into drug development pipelines. GEMMs may be particularly valuable for investigating treatments for less common cancers, which frequently lack alternative faithful models. Here, we describe a multicenter cooperative group that has successfully leveraged the expertise and resources from philanthropic foundations, academia, and industry to advance therapeutic discovery and translation using GEMMs as a preclinical platform. This effort, known as the Neurofibromatosis Preclinical Consortium (NFPC), was established to accelerate new treatments for tumors associated with neurofibromatosis type 1 (NF1). At its inception, there were no effective treatments for NF1 and few promising approaches on the horizon. Since 2008, participating laboratories have conducted 95 preclinical trials of 38 drugs or combinations through collaborations with 18 pharmaceutical companies. Importantly, these studies have identified 13 therapeutic targets, which have inspired 16 clinical trials. This review outlines the opportunities and challenges of building this type of consortium and highlights how it can accelerate clinical translation. We believe that this strategy of foundation-academic-industry partnering is generally applicable to many diseases and has the potential to markedly improve the success of therapeutic development

Oncogenic Deregulation of EZH2 as an Opportunity for Targeted Therapy in Lung Cancer

As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically-engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways leading to epigenetic transformation independent from canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a novel and potent EZH2 inhibitor JQEZ5 that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer

The Lantern, 2011-2012

• Frangipani • A Shadow • Dear Anne, In this Place, Stringbean Girls • Back to a Dandelion • How to Plant a Room • Swimming Pool Poem 30 • The Naming of Daughters • Berman Museum Photographs • Truth or Dare • The Song of Remembrance, L\u27vov, Poland, 1940 • Headlights • Prayer of Thanks • Numbers Game • Pediment • Home Sick • Lust • Sand Lining Instructions • A-A-Ask a Question • Flash Cards • Columbus Day • Mr. Yoest Gives His Report to the Police Officers on Wednesday Night • Gender Trouble • The Internet Connection at Ursinus College • Assuming You\u27ll Still be Here • 10/28/11, Third Poem • October • Actions that Affirm and Confirm Us as a Community • Why I Hate The Lantern • Confessions of an Ex-Vegetarian • Run • Lunch at Caltort • Schemers • You Will Make Beautiful Babies in America • The Black Dirt Region • Il Travatore • Ghost Story • Blue Eyes and Sunny Skies • A Little Sincerity • The Bookstore • The Opposite of Serendipity • The Human Doll • Evil Deeds • Francesca • Sunday Morning • Jersey Aesthetic • Jump! • Behind Reimert • Seaweed in New Zealand • Tombee de L\u27elegance • The Window • Esperando • Rainbow to the Heavens • Encased • In Springtime • A Fiesolan Monk\u27s Room • Inside a Bone • Neon Indian • Moments of Clarity • OneFeral: A Feral Self-Portrait • Cover Image: The Conquerorhttps://digitalcommons.ursinus.edu/lantern/1177/thumbnail.jp

Distress and quality of life characteristics associated with seeking surgical treatment for stress urinary incontinence

<p>Abstract</p> <p>Background</p> <p>Current research focuses on three variables in evaluating the impact of stress urinary incontinence (SUI) on daily living: severity of incontinence, distress or bother resulting from incontinence, and effect on health related quality of life (HRQoL). Understanding the impact of these variables is important as they are the driving force behind women seeking surgical treatment. Given the importance of HRQoL in determining need for treatment, as well as evaluating treatment success, this review provides an assessment of the degree to which HRQoL is impaired in women seeking surgical treatment.</p> <p>Methods</p> <p>PubMed searches for the terms "quality of life and distress and urinary incontinence" and "quality of life and bother and urinary incontinence" were performed with limits of English, human and female subjects through May 2008. All studies using validated instruments were included. No time limit was placed on the search.</p> <p>Results</p> <p>Of 178 articles retrieved, 21 met the inclusion criteria, and 17 reported methods of scoring. The studies used the Urogenital Distress Inventory (UDI) and the Incontinence Impact Questionnaire (IIQ). Wide ranges of mean and individual levels of severity of symptoms, UDI and IIQ scores were seen among women seeking surgical treatment. Fourteen studies reported baseline and post-surgical treatment distress and QoL data. Statistically significant improvements between baseline and post-surgical UDI and IIQ scores were reported in 12 studies. Reported cure rates ranged from 46% to 97%. Satisfaction with the procedure was reported in 4 studies and ranged from 84% to 91%. A minority of studies reported the relationship between reduction in symptoms and change in HRQoL.</p> <p>Conclusion</p> <p>HRQoL is the main reason women seek surgical treatment for incontinence and surgical treatment leads to a significant improvement in mean HRQoL scores. Assessment of HRQoL has proved less useful in identifying why individual women seek treatment for incontinence. Preliminary work has begun to characterize the interaction between severity of symptoms, distress or bother resulting from these urinary symptoms, impact on HRQoL, and treatment seeking behavior, but further research is needed. Greater standardization in the reporting of results of distress or bother and HRQoL would allow for comparison across studies.</p

Distress and quality of life characteristics associated with seeking surgical treatment for stress urinary incontinence

<p>Abstract</p> <p>Background</p> <p>Current research focuses on three variables in evaluating the impact of stress urinary incontinence (SUI) on daily living: severity of incontinence, distress or bother resulting from incontinence, and effect on health related quality of life (HRQoL). Understanding the impact of these variables is important as they are the driving force behind women seeking surgical treatment. Given the importance of HRQoL in determining need for treatment, as well as evaluating treatment success, this review provides an assessment of the degree to which HRQoL is impaired in women seeking surgical treatment.</p> <p>Methods</p> <p>PubMed searches for the terms "quality of life and distress and urinary incontinence" and "quality of life and bother and urinary incontinence" were performed with limits of English, human and female subjects through May 2008. All studies using validated instruments were included. No time limit was placed on the search.</p> <p>Results</p> <p>Of 178 articles retrieved, 21 met the inclusion criteria, and 17 reported methods of scoring. The studies used the Urogenital Distress Inventory (UDI) and the Incontinence Impact Questionnaire (IIQ). Wide ranges of mean and individual levels of severity of symptoms, UDI and IIQ scores were seen among women seeking surgical treatment. Fourteen studies reported baseline and post-surgical treatment distress and QoL data. Statistically significant improvements between baseline and post-surgical UDI and IIQ scores were reported in 12 studies. Reported cure rates ranged from 46% to 97%. Satisfaction with the procedure was reported in 4 studies and ranged from 84% to 91%. A minority of studies reported the relationship between reduction in symptoms and change in HRQoL.</p> <p>Conclusion</p> <p>HRQoL is the main reason women seek surgical treatment for incontinence and surgical treatment leads to a significant improvement in mean HRQoL scores. Assessment of HRQoL has proved less useful in identifying why individual women seek treatment for incontinence. Preliminary work has begun to characterize the interaction between severity of symptoms, distress or bother resulting from these urinary symptoms, impact on HRQoL, and treatment seeking behavior, but further research is needed. Greater standardization in the reporting of results of distress or bother and HRQoL would allow for comparison across studies.</p

The role of tyrosine phosphorylation in G protein-coupled receptor mediated signal transduction: Platelets as a model system

Physiological agonists cause platelets to form multicellular aggregates, secrete the contents of their storage granules and spread on extracellular matrix proteins. Many of these agents stimulate G protein-coupled receptors, and have been shown to utilize classical signaling pathways involving phospholipase C and adenylyl cyclase, to transduce their effects. However, platelet activation is also accompanied by a dramatic increase in protein tyrosine phosphorylation, suggesting that these receptors may couple to tyrosine kinases. We have therefore been using platelets, as a model system, to investigate a potential role for tyrosine kinases in G protein-coupled receptor mediated signal transduction, by identifying proteins which become phosphorylated in response to thrombin. Specifically, we have examined phosphorylation events that are independent of platelet aggregation, a process mediated by the \alpha\sb{IIb}\beta\sb3 integrin, in order to define signals that occur as a result of G protein-coupled receptor activation and not as a secondary consequence of integrin engagement. We found that \rm \sb{p}21\sp{ras}GAP, and several associated proteins became phosphorylated on tyrosine in response to thrombin, and that three Src-related kinases Fyn, Lyn and Yes, were associated with GAP in complexes, detectable after agonist stimulation. GAP\u27s phosphorylation and its association with these kinases was not dependent on the formation of platelet aggregates, indicating that (1) thrombin alone is capable of stimulating GAP phosphorylation and that (2) these kinases may be responsible for transducing the effects of thrombin in platelets. To investigate the potential physiological significance of this finding we examined a downstream effector of Ras, MAP kinase, and its reported substrate cPLA\sb2, and found that their phosphorylation patterns correlated with GAP phosphorylation, illustrating one mechanism by which tyrosine kinases may regulate arachidonate formation in platelets. We were also interested in identifying signals involved in platelet aggregation. Since Rho has been shown to be required for this process we examined the phosphorylation of Vav, a putative Rho exchange factor, and found that thrombin rapidly induced its phosphorylation on tyrosine, independent of aggregation. Interestingly, Vav also became phosphorylated as a result of integrin-mediated adhesion, which initiates the process of platelet spreading and the formation of stress fibers. These results demonstrate that thrombin alone can induce protein tyrosine phosphorylation in platelets, suggesting a role for tyrosine kinases in G protein-coupled receptor mediated signal transduction. Furthermore, by identifying these phosphoproteins and potential downstream effectors, we have begun to outline mechanisms by which tyrosine kinases may participate in platelet activation