Tools for live imaging of active Rho GTPases in Xenopus

Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP‐bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well‐suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/136041/1/dvg22998_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/136041/2/dvg22998.pd

Break, Flare, Repair: Rho Flares Locally Reinforce the Tight Junction Barrier

Epithelial tissues serve essential functions, such as preventing infection and water loss, regulating absorption and secretion, and creating specialized compartments within complex multicellular organisms. Epithelia are sheets of cells connected by specialized apical cell-cell junctions. Adherens junctions adhere cells to one another and mechanically integrate cells in the tissue, while tight junctions regulate both how much and what kind of materials can cross through the paracellular space (the space between cells). Both tight junctions and adherens junctions are dynamically regulated by filamentous actin and myosin II (actomyosin), which forms a contractile array near the apical cell-cell junctions. The forces generated by the apical actomyosin array can be transmitted to neighboring cells through adherens junctions to drive cell- and tissue-scale changes. Cellular events, such as epithelial cytokinesis, cell extrusion, and wound healing, alter tension on adherens junctions and the dynamics of the apical actomyosin array. However, very little is known about how these changes in cell shape and actomyosin dynamics influence tight junctions and epithelial barrier function. To better understand how dynamic cell shape change influences epithelial barrier function, we developed a tight junction barrier assay compatible with live imaging. This approach, the Zinc-based Ultrasensitive Microscopic Barrier Assay, or ZnUMBA, allows for the detection of localized, transient leaks. Using this approach in the epithelium of Xenopus laevis gastrula-staged embryos, we discovered that leaks that result from cell shape change are rapidly repaired by transient accumulation of the active conformation of the small GTPase RhoA, or Rho flares. Then, using fluorescently-tagged tight junction proteins, I found that occludin and ZO-1 show localized decline prior to Rho flares and are reinforced afterwards. Using molecular inhibitors of the targets of Rho activity, I concluded that both actin polymerization and Rho Kinase-mediated junction contraction reinforce ZO-1 and occludin, promoting efficient restoration of epithelial barrier function. We hypothesize that Rho flares serve as a rapid repair mechanism to quickly restore barrier function and that this allows epithelial cells to dynamically change shape without prolonged breaches in barrier function. Rho flares are accompanied by an apical protrusion of the plasma membrane. However, both the cause of the membrane protrusion and its purpose are unclear. I examine three potential causes of membrane protrusion and assess each one by reviewing the temporal and spatial accumulation of F-actin and myosin II, as well as several candidate actin nucleators. Based on the data presented, I propose that a bleb-like protrusion mechanism is likely, and I hypothesize that the protrusion acts to temporarily seal the paracellular space while the tight junction is reinforced. The work presented in this dissertation advances tight junction biology in several ways. ZnUMBA is a widely adaptable technique that will allow other researchers to examine changes in barrier function with greater temporal and spatial precision. We hope that this will usher in a better understanding about what causes tight junction leaks and how they are repaired. Finally, I describe a previously unknown mechanism for rapid repair of local breaches in epithelial barrier function by active Rho, adding nuance to our understanding of the many roles this small GTPase plays in epithelial tissues.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/146133/1/restep_1.pdfDescription of restep_1.pdf : Restricted to UM users only

Development and pilot of clinical performance indicators for English ambulance services

Introduction: There is a compelling need to develop clinical performance indicators for ambulance services in order to move from indicators based primarily on response times and in light of the changing clinical demands on services. We report on progress on the national pilot of clinical performance indicators for English ambulance services. Method: Clinical performance indicators were developed in five clinical areas: acute myocardial infarction, cardiac arrest, stroke (including transient ischaemic attack), asthma and hypoglycaemia. These were determined on the basis of common acute conditions presenting to ambulance services and in line with a previously published framework. Indicators were piloted by ambulance services in England and results were presented in tables and graphically using funnel (statistical process control) plots. Results: Progress for developing, agreeing and piloting of indicators has been rapid, from initial agreement in May 2007 to completion of the pilot phase by the end of March 2008. The results of benchmarking of indicators are shown. The pilot has informed services in deciding the focus of their improvement programme in 2008 and 2009 and indicators have been adopted for national performance assessment of standards of prehospital care. Conclusion: The pilot will provide the basis for further development of clinical indicators, benchmarking of performance and implementation of specific evidencebased interventions to improve care in areas identified for improvement. A national performance improvement registry will enable evaluation and sharing of effective improvement methods as well as increasing stakeholder and public access to information on the quality of care provided by ambulance services

Room temperature negative differential resistance of a monolayer molecular rotor device

An electrically driven molecular rotor device comprised of a monolayer of redox-active ligated copper compounds sandwiched between a gold electrode and a highly doped P+Si substrate was fabricated. Current-voltage spectroscopy revealed a temperature-dependent negative differential resistance (NDR) associated with the device. Time-dependent density functional theory suggests the source of the observed NDR to be redox-induced ligand rotation around the copper metal center, an explanation consistent with the proposed energy diagram of the device. An observed temperature dependence of the NDR behavior further supports this hypothesis

Pelvic circumferential compression devices for prehospital management of suspected pelvic fractures : a rapid review and evidence summary for quality indicator evaluation

Background: Pelvic fractures, especially when unstable, may cause significant haemorrhage. The early application of a pelvic circumferential compression device (PCCD) in patients with suspected pelvic fracture has established itself as best practice. Ambulance services conduct corresponding performance measurement. Quality indicators (QIs) are ideally based on high-quality evidence clearly demonstrating that the desirable effects outweigh the undesirable effects. In the absence of high-quality evidence, best available evidence should be combined with expert consensus. Objectives: The aim of the present study was to identify, appraise and summarize the best available evidence regarding PCCDs for the purpose of informing an expert panel tasked to evaluate the validity of the following QI: A patient with suspected pelvic fracture has a PCCD applied. Methods: A rapid review of four databases was conducted to identify relevant literature published up until 9 June 2020. Systematic reviews, experimental, quasi-experimental and observational analytic studies written in English were included. One author was responsible for study selection and quality appraisal. Data extraction using a priori extraction templates was verified by a second reviewer. Study details and key findings were summarized in tables. Results: A total of 13 studies were assessed to be eligible for inclusion in this rapid review. Of these, three were systematic reviews, one was a randomized clinical trial (crossover design), two were before-after studies, and seven were retrospective cohort studies. The systematic reviews included mostly observational studies and could therefore not be considered as high-level evidence. Overall, the identified evidence is of low quality and suggests that PCCD may provide temporary pelvic ring stabilization and haemorrhage control, although a potential for adverse effects exists. Conclusion: Given the low quality of the best available evidence, this evidence would need to be combined with expert consensus to evaluate the validity of a related quality indicator before its implementation

Illinois State University Wind Symphony, April 6, 2022

Center for the Performing Arts April 6, 2022 Wednesday Evening 8:00 p.m

The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing

The interaction between splicing factors and the transcriptional machinery provides an intriguing link between the coupled processes of transcription and splicing. Here, we show that the two components of the SF3B complex, SF3B3 and SF3B5, that form part of the U2 small nuclear ribonucleoprotein particle (snRNP) are also subunits of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex in Drosophila melanogaster. Whereas SF3B3 had previously been identified as a human SAGA subunit, SF3B5 had not been identified as a component of SAGA in any species. We show that SF3B3 and SF3B5 bind to SAGA independent of RNA and interact with multiple SAGA subunits including Sgf29 and Spt7 in a yeast two-hybrid assay. Through analysis of sf3b5mutant flies, we show that SF3B5 is necessary for proper development and cell viability but not for histone acetylation. Although SF3B5 does not appear to function in SAGA\u27s histone-modifying activities, SF3B5 is still required for expression of a subset of SAGA-regulated genes independent of splicing. Thus, our data support an independent function of SF3B5 in SAGA\u27s transcription coactivator activity that is separate from its role in splicing

Key principles of ecosystem-based management: the fishermen's perspective

Published VersionDespite the growing popularity of ecosystem-based management (EBM) in national legislation and in research and institutional literature, there is often an implementation gap ‘on the ground’, impeding widespread adoption in fisheries. This gap reflects in part the differing understandings of EBM held by fishermen and by management institutions. To explore and seek to close this gap, the underlying principles of EBM considered priorities by fishermen were systematically compared with the priorities identified in the published literature. The fishermen's priorities were determined by asking Atlantic Canadian fishermen to identify the EBM principles they consider most important. Four priority principles were identified: Sustainability, Stakeholder Involvement, Develop Long-Term Objectives and Use of All Forms of Knowledge. The latter two were not frequently noted as priorities in the literature, while some literature priorities were less commonly chosen by fishermen, indicating a significant difference in perspectives on EBM. The rationale for fishermen's choice of priorities was explored by analysing the fishery management issues they raised – many directly connected to the above four priorities. In addition, another principle, Commit to Principles of Equity, often arose as an implicit priority among fishermen. We suggest that success in implementation of EBM may depend on reconciling differing priorities among its underlying principles, and combining knowledge and expertise from fishermen with research and institutional sources. The comparative methodology used here, which could be replicated elsewhere, should lead to better recognition of local challenges in EBM implementation and encourage support for EBM, to further its contribution to sustainable fisheries