Somitogenese im Zebrafisch: Die Interaktion zwischen Wnt und Delta/Notch Signalweg und die ripply vermittelte Termination der "clock" - Eine Untersuchung mittels der her1 und her7 Mutanten

Zusammenfassung Die Somitogenese ist der fundamentale Prozess in der Vertebratenentwicklung, an dem zwei Signalwege -Delta-Notch und Wnt- maßgeblich beteiligt sind. Die Komponenten des Delta- Notch Signalweges sind Bestandteile eines so genannten molekularen Oszillatormechanismus, der die alternierende und somit zyklische Genexpression im präsomitischen Mesoderm (PSM) koordiniert. Im Zebrafisch bilden die zyklisch exprimierten her1 und her7 Gene mit dem Liganden deltaC einen regulatorischen Kreislauf im Sinne eines negativen "feedback loops", der die Grundlage des "prepattering" Mechanismus darstellt. Durch die Analyse der her1 Mutante konnte gezeigt werden, dass das Her1 Protein essentiell an der Bildung der ersten drei Somiten beteiligt ist. Ein Funktionsverlust führt zu deformierten Somitengrenzen, die einhergehen mit einer gestörten rostro-caudalen Polarität. Die räumliche Expression von her1 innerhalb des PSMs wird durch unterschiedliche "clock" Elemente, die auf dem her1 Promotor lokalisiert sind, kontrolliert. Die zyklische Expression im anterioren PSM erfolgt durch die Bindung von Su(H) im anterioren "clock" Element, wohingegen die zyklische Expression im posterioren PSM unabhängig von diesem zu sein scheint. Ergebnisse von her7 knockdown Analysen, die eine gestörte Segmentgrenzenbildung ab etwa Somit zehn andeuteten und als "anterior limit of defect" (ALD) bezeichnet worden sind, können durch die vorliegende her7 Mutantenanalyse dahingehend korrigiert werden, dass etwa ab Somit 17 wildtypische Segmentgrenzen gebildet werden. Dieser mit "posterior limit of defect" (PLD) bezeichnete Defekt, deutet an, dass her7 ähnlich zu her1 eine temporäre Rolle während der Somitogenese ausübt. Gegensätzlich zur beobachteten Situation in Maus konnte für die hes7 Zebrafisch homologen Gene her1 und her7 durchhaus eine regulierende Funktion auf die ripply Transkription festgestellt werden. Die gleichzeitige Inhibition des Wnt Signalweges durch lrp6 Morpholino knockdown in Kombination mit einzelnen Mutanten des Delta-Notch Signalweges, wie deltaC/bea, her1KO oder her7KO konnte eine bislang noch nicht beschriebene Verknüpfung beider Signalwege offen legen. Insbesondere bea und Her7KO zeigen wie auch der lrp6 knockdown im Einzelfall einen posterioren Defekt, der bei einem kombinatorischen Ausfall beider Signalwege zu einem anterioren Defekt führt und damit Entwicklungsprozesse in der frühen Somitogenese so wie die frühe Segmentierungsuhr betrifft. Zusammenfassend kann festgehalten werden, dass der Delta-Notch Signalweg zusammen mit dem Wnt Signalweg in der frühen Somitogenese bzw. im "prepatterning" Mechanismus eine entscheidende Rolle ausübt

Patient Blood Management Bundles to Facilitate Implementation.

More than 30% of the world's population are anemic with serious economic consequences including reduced work capacity and other obstacles to national welfare and development. Red blood cell transfusion is the mainstay to correct anemia, but it is also 1 of the top 5 overused procedures. Patient blood management (PBM) is a proactive, patient-centered, and multidisciplinary approach to manage anemia, optimize hemostasis, minimize iatrogenic blood loss, and harness tolerance to anemia. Although the World Health Organization has endorsed PBM in 2010, many hospitals still seek guidance with the implementation of PBM in clinical routine. Given the use of proven change management principles, we propose simple, cost-effective measures enabling any hospital to reduce both anemia and red blood cell transfusions in surgical and medical patients. This article provides comprehensive bundles of PBM components encompassing 107 different PBM measures, divided into 6 bundle blocks acting as a working template to develop institutions' individual PBM practices for hospitals beginning a program or trying to improve an already existing program. A stepwise selection of the most feasible measures will facilitate the implementation of PBM. In this manner, PBM represents a new quality and safety standard

Patient blood management bundles to facilitate implementation

More than 30% of the world's population are anemic with serious economic consequences including reduced work capacity and other obstacles to national welfare and development. Red blood cell transfusion is the mainstay to correct anemia, but it is also 1 of the top 5 overused procedures. Patient blood management (PBM) is a proactive, patient-centered, and multidisciplinary approach to manage anemia, optimize hemostasis, minimize iatrogenic blood loss, and harness tolerance to anemia. Although the World Health Organization has endorsed PBM in 2010, many hospitals still seek guidance with the implementation of PBM in clinical routine. Given the use of proven change management principles, we propose simple, cost-effective measures enabling any hospital to reduce both anemia and red blood cell transfusions in surgical and medical patients. This article provides comprehensive bundles of PBM components encompassing 107 different PBM measures, divided into 6 bundle blocks acting as a working template to develop institutions' individual PBM practices for hospitals beginning a program or trying to improve an already existing program. A stepwise selection of the most feasible measures will facilitate the implementation of PBM. In this manner, PBM represents a new quality and safety standard

Washed cell salvage in surgical patients

Background: Cell salvage is commonly used as part of a blood conservation strategy. However concerns among clinicians exist about the efficacy of transfusion of washed cell salvage. Methods: We performed a meta-analysis of randomized controlled trials in which patients, scheduled for all types of surgery, were randomized to washed cell salvage or to a control group with no cell salvage. Data were independently extracted, risk ratio (RR), and weighted mean differences (WMD) with 95% confidence intervals (CIs) were calculated. Data were pooled using a random effects model. The primary endpoint was the number of patients exposed to allogeneic red blood cell (RBC) transfusion. Results: Out of 1140 search results, a total of 47 trials were included. Overall, the use of washed cell salvage reduced the rate of exposure to allogeneic RBC transfusion by a relative 39% (RR = 0.61; 95% CI 0.57 to 0.65; P < 0.001), resulting in an average saving of 0.20 units of allogeneic RBC per patient (weighted mean differences [WMD] = -0.20; 95% CI -0.22 to -0.18; P < 0.001), reduced risk of infection by 28% (RR = 0.72; 95% CI 0.54 to 0.97; P = 0.03), reduced length of hospital stay by 2.31 days (WMD = -2.31; 95% CI -2.50 to -2.11; P < 0.001), but did not significantly affect risk of mortality (RR = 0.92; 95% CI 0.63 to 1.34; P = 0.66). No statistical difference could be observed in the number of patients exposed to re-operation, plasma, platelets, or rate of myocardial infarction and stroke. Conclusions: Washed cell salvage is efficacious in reducing the need for allogeneic RBC transfusion and risk of infection in surgery

Analysis of her1 and her7 Mutants Reveals a Spatio Temporal Separation of the Somite Clock Module

Somitogenesis is controlled by a genetic network consisting of an oscillator (clock) and a gradient (wavefront). The “hairy and Enhancer of Split”- related (her) genes act downstream of the Delta/Notch (D/N) signaling pathway, and are crucial components of the segmentation clock. Due to genome duplication events, the zebrafish genome, possesses two gene copies of the mouse Hes7 homologue: her1 and her7. To better understand the functional consequences of this gene duplication, and to determine possible independent roles for these two genes during segmentation, two zebrafish mutants her1hu2124 and her7hu2526 were analyzed. In the course of embryonic development, her1hu2124 mutants exhibit disruption of the three anterior-most somite borders, whereas her7hu2526 mutants display somite border defects restricted to somites 8 (+/−3) to 17 (+/−3) along the anterior-posterior axis. Analysis of the molecular defects in her1hu2124 mutants reveals a her1 auto regulatory feedback loop during early somitogenesis that is crucial for correct patterning and independent of her7 oscillation. This feedback loop appears to be restricted to early segmentation, as cyclic her1 expression is restored in her1hu2124 embryos at later stages of development. Moreover, only the anterior deltaC expression pattern is disrupted in the presomitic mesoderm of her1hu2124 mutants, while the posterior expression pattern of deltaC remains unaltered. Together, this data indicates the existence of an independent and genetically separable anterior and posterior deltaC clock modules in the presomitic mesdorm (PSM)

Pten function in zebrafish : Anything but a fish story

Zebrafish is an excellent model system for the analysis of gene function. We and others use zebrafish to investigate the function of the tumor suppressor, Pten, in tumorigenesis and embryonic development. Zebrafish have two pten genes, ptena and ptenb. The recently identified N-terminal extension of human PTEN that may facilitate cell membrane transfer, appears not to be conserved in zebrafish Ptena or Ptenb. Mutants that retain a single wild type pten allele develop tumors, predominantly hemangiosarcomas. Homozygous double mutants are embryonic lethal. Zebrafish embryos lacking functional Pten display enhanced proliferation of endothelial cells, resulting in hyperbranching of blood vessels. In addition, ptena-/-ptenb-/- mutant embryos display enhanced proliferation of hematopoietic stem and progenitor cells and concomitant arrest of differentiation, although Pten-deficient cells commit to all blood cell lineages. Zebrafish is an ideal model for intravital imaging and future work using ptena-/-ptenb-/- mutants will enhance our understanding of the function of Pten in vivo. (C) 2014 Elsevier Inc. All rights reserved

Analysis of in vivo Function of Predicted Isoenzymes-A Metabolomic Approach

Isoenzymes occur in all organisms. Often, they are regulated with respect to environmental perturbations to assure metabolic flexibility. In bioinformatics-driven functional genome annotations, the presence of isoenzymes is frequently predicted. It is desirable to verify the functions of putative isoenzymes experimentally for a correct estimation of the metabolic capacities in a given organism. Using metabolome analysis, we investigated two knockout mutants of putative shikimate dehydrogenases (SDH) in Corynebacterium glutamicum (C. glutamicum). Here, we show that different metabolic responses to defined gene deactivations of the putative SDHs clearly indicate different functions of these enzymes. Our investigation revealed that the gene product of open reading frame Cg1835 is the essential SDH in C. glutamicum, whereas it is more likely that the gene product of the open reading frame Cg1283 belongs to the SDH-like enzyme family (SDH-L). The results of the metabolome analysis are verified with two independent methods. First, by in vitro characterization of kinetic constants after heterologous expression, and second, by measurement of SDH activity in raw cell extracts

Deriving cell lines from zebrafish embryos and tumors

Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months

Anaesthesia and orphan disease: management of a case of Nicolaides-Baraitser syndrome undergoing cleft palate surgery

Background: Nicolaides-Baraitser syndrome (NCBRS) is a rare disease caused by mutations in the SMRCA2 gene, which affects chromatin remodelling and leads to a wide range of symptoms including microcephaly, distinct facial features, recurrent seizures, and severe mental retardation. Until now, less than 100 cases have been reported. Case presentation: A 22-month old male infant with NCBRS underwent elective cleft palate surgery. The anaesthetists were challenged by the physiological condition of the patient: narrow face, very small mouth, mild tachypnea, slight sternal retractions, physical signs of partial monosomy 9p, and plagiocephalus, midface hypoplasia, V-shaped cleft palate, enhanced muscular hypotension, dysplastic kidneys (bilateral, estimated GFR: approx. 40 ml/m2), nocturnal oxygen demand, and combined apnea. In addition, little information was available about interaction of the NCBRS displayed by the patient and anaesthesia medications. Conclusions: The cleft palate was successfully closed using the bridge flap technique. Overall, we recommend to perform a trial video assisted laryngoscopy in the setting of spontaneous breathing with deep inhalative anaesthesia before administration of muscle relaxation to detect any airway difficulties while remaining spontaneoues breathing and protective reflexes