Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

Anatomy and development of the pectoral fin vascular network in the zebrafish.

The pectoral fins of teleost fish are analogous structures to human forelimbs, and the developmental mechanisms directing their initial growth and patterning are conserved between fish and tetrapods. The forelimb vasculature is crucial for limb function, and it appears to play important roles during development by promoting development of other limb structures, but the steps leading to its formation are poorly understood. In this study, we use high-resolution imaging to document the stepwise assembly of the zebrafish pectoral fin vasculature. We show that fin vascular network formation is a stereotyped, choreographed process that begins with the growth of an initial vascular loop around the pectoral fin. This loop connects to the dorsal aorta to initiate pectoral vascular circulation. Pectoral fin vascular development continues with concurrent formation of three elaborate vascular plexuses, one in the distal fin that develops into the fin-ray vasculature and two near the base of the fin in association with the developing fin musculature. Our findings detail a complex, yet highly choreographed, series of steps involved in the development of a complete, functional, organ-specific vascular network.

Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing.

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment.

The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.

How RNA-Binding Proteins Interact with RNA: Molecules and Mechanisms.

RNA-binding proteins (RBPs) comprise a large class of over 2,000 proteins that interact with transcripts in all manner of RNA-driven processes. The structures and mechanisms that RBPs use to bind and regulate RNA are incredibly diverse. In this review, we take a look at the components of protein-RNA interaction, from the molecular level to multi-component interaction. We first summarize what is known about protein-RNA molecular interactions based on analyses of solved structures. We additionally describe software currently available for predicting protein-RNA interaction and other resources useful for the study of RBPs. We then review the structure and function of seventeen known RNA-binding domains and analyze the hydrogen bonds adopted by protein-RNA structures on a domain-by-domain basis. We conclude with a summary of the higher-level mechanisms that regulate protein-RNA interactions.

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling.

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumor's own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.

Miscibility Transition Temperature Scales with Growth Temperature in a Zebrafish Cell Line.

Cells can alter the lipid content of their plasma membranes upon changes in their environment to maintain and adjust membrane function. Recent work suggests that some membrane functions arise because cellular plasma membranes are poised close to a miscibility transition under growth conditions. Here we report experiments utilizing giant plasma membrane vesicles (GPMVs) to explore how membrane transition temperature varies with growth temperature in a zebrafish cell line (ZF4) that can be adapted for growth between 20 and 32°C. We find that GPMV transition temperatures adjust to be 16.7 ± 1.2°C below growth temperature for four growth temperatures investigated and that adjustment occurs over roughly 2 days when temperature is abruptly lowered from 28 to 20°C. We also find that GPMVs have slightly different lipidomes when isolated from cells adapted for growth at 28 and 20°C. Similar to past work in vesicles derived from mammalian cells, fluctuating domains are observed in ZF4-derived GPMVs, consistent with their having critical membrane compositions. Taken together, these experimental results suggest that cells in culture biologically tune their membrane composition in a way that maintains specific proximity to a critical miscibility transition.

Spot size variation FCS in simulations of the 2D Ising model.

Spot variation fluorescence correlation spectroscopy (svFCS) was developed to study the movement and organization of single molecules in plasma membranes. This experimental technique varies the size of an illumination area while measuring correlations in time using standard fluorescence correlation methods. Frequently, this data is interpreted using the assumption that correlation measurements reflect the dynamics of single molecule motions, and not motions of the average composition. Here, we explore how svFCS measurements report on the dynamics of components diffusing within simulations of a 2D Ising model with a conserved order parameter. Simulated correlation functions report on both the fast dynamics of single component mobility and the slower dynamics of the average composition. Over a range of simulation conditions, a conventional svFCS analysis suggests the presence of anomalous diffusion even though single molecule motions are nearly Brownian in these simulations. This misinterpretation is most significant when the surface density of the fluorescent label is elevated, therefore we suggest future measurements be made over a range of tracer densities. Some simulation conditions reproduce qualitative features of published svFCS experimental data. Overall, this work emphasizes the need to probe membranes using multiple complimentary experimental methodologies in order to draw conclusions regarding the nature of spatial and dynamical heterogeneity in these systems.

Influence of leaching solution and catchment location on the fluorescence of water-soluble organic matter.

Organic matter (OM) plays a significant role in biogeochemical processes in soil and water systems. Water-soluble organic matter (WSOM) leached from soil samples is often analyzed as representative of potentially mobile OM. However, there are many WSOM extraction methods in the literature with no clear guidelines for method selection. In this study, four common leaching solutions (0.5 M K2SO4, 0.01 M CaCl2, 2 M KCl, and H2O) were used to extract WSOM from various locations within a forested catchment. Fluorescence spectroscopy was used to analyze the impact of extraction method on WSOM chemistry. While all four methods consistently identified chemical differences between WSOM from a north-facing slope, south-facing slope, and riparian zone, there were clear differences in fluorescence signals between the leaching methods. All three salt solutions contained WSOM with a higher fluorescence index and humification index than WSOM leached with H2O, suggesting the presence of salts releases different fractions of the soil organic matter. A parallel factor analysis (PARAFAC) model developed from the leachates identified a distinctive soil humic fluorophore observed in all samples and fluorescent artifacts present in H2O-leached samples.

The face of sleepiness: improvement in appearance after treatment of sleep apnea.

STUDY OBJECTIVES: Anecdote but no formal evidence suggests that facial appearance improves after hypersomnolent patients with obstructive sleep apnea are treated. We investigated whether masked volunteer raters can identify post- rather than pre-treatment images as looking more alert, and whether impressions are predicted by any objective changes on highly precise 3-dimensional digital photogrammetry. METHODS: Participants included 20 adults with obstructive sleep apnea on polysomnography and excessive sleepiness on Epworth Sleepiness Scales. Photogrammetry was performed before and after ≥ 2 months of adherent use of positive airway pressure. Twenty-two raters then assessed pre- and post-treatment facial images, paired side-by-side in random order. RESULTS: Subjects included 14 men and 6 women, with mean age 45 ± 11 (SD) years and mean baseline apnea/hypopnea index of 26 ± 21. The 22 raters twice as often identified post-treatment rather than pre-treatment images to look more alert (p = 0.0053), more youthful (p = 0.026), more attractive (p = 0.0068), and more likely to reflect the treated state (p = 0.015). Photogrammetry documented post-treatment decreases in forehead surface volume and decreased infraorbital and cheek redness, but no narrowing of the interpalpebral fissure. Decreased deep NREM sleep at baseline, and pre- to post-treatment decrements in facial redness showed promise as predictors of improved subjective ratings for alertness. CONCLUSIONS: Patients with obstructive sleep apnea are perceived to appear more alert, more youthful, and more attractive after adherent use of positive airway pressure. Objective changes in facial surface volume and color were identified. Post-treatment decrements in redness may inform subjective impressions of improved alertness.

Development of a caregiver empowerment model to promote positive outcomes.

Family members caring for aging parents experience both negative and positive outcomes from providing care. Theoretical explanations for negative outcomes have been developed. There is need for models that explain and predict positive outcomes. This article describes the evolution of the Caregiver Empowerment Model (CEM) to explain and predict positive outcomes of family caregiving. Although empirical findings support positive outcomes of family caregiving, less attention has been given to theoretical rationale for positive effects. The CEM predicts that, in the presence of filial values and certain background variables, caregiving demands are appraised as challenges instead of stressors. Appraising caregiving demands as a challenge, finding meaning, and using certain types of coping strategies are posited to be associated with growth and well-being. The CEM extends our understanding of the complexity of the caregiving experience, and can serve as a framework to guide in developing and testing theory-based interventions to promote positive outcomes.

Use of evidence for prevention of work-related musculoskeletal injuries.

Work-related injuries among nurses are a complex and costly problem. To thoughtfully approach this issue, one medical center developed a set of injury-reduction strategies and related tools based on a review of research literature and locally obtained evidence. This article describes the evidence-based approach to prevention of work-related musculoskeletal injuries. It also covers research-based observations about prevention strategies; provides a description of selected interventions, such as a patient handling guide; and includes sample tools that enable collection of meaningful data about local patient handling practices.

Structure and process of outcomes research for nurse practitioners.

PURPOSE: To present a framework for nurse practitioners (NPs) who wish to engage in outcomes research. DATA SOURCE: Review of scientific literature on the design of outcomes research. CONCLUSION: The structure and process of outcomes research includes both the research protocol and the decisions related to designing and implementing a study. Each identified step of the presented framework incorporates issues related to decision making relevant for that specific component of the research protocol. IMPLICATIONS FOR PRACTICE: Practical advice guides the NP in beginning to identify researchable problems within the clinical setting suited to outcomes research. Using a framework to design an outcomes study assists the NP in making key study decisions.

Challenges of outcomes research for nurse practitioners.

PURPOSE: To describe the numerous methodological challenges nurse practitioners (NPs) face in designing and conducting outcomes research and provide practical tips to implement an outcomes study within an institution. DATA SOURCES: Review of world wide scientific literature on outcomes research. CONCLUSIONS: Nurse practitioners must be aware of the challenges of conducting outcomes research. Challenges associated with variable definition, designing outcomes studies, use of data sets, and instrument development and selection must be understood prior to undertaking an outcome study. IMPLICATIONS FOR PRACTICE: Valuable studies have laid a foundation for evidence of quality care provision by NPs. Now is the time to measure patient outcomes of the NP care longitudinally over significant periods of time.