Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states

Inventing Modernity in Medieval European Thought ca. 1100–ca. 1550

One of the most challenging problems in the history of Western ideas stems from the emergence of Modernity out of the preceding period of the Latin Middle Ages. This volume develops and extends the insights of the noted scholar Thomas M. Izbicki into the so-called medieval/modern divide. The contributors include a wide array of eminent international scholars from the fields of History, Theology, Philosophy, and Political Science, all of whom explore how medieval ideas framed and shaped the thought of later centuries. This sometimes involved the evolution of intellectual principles associated with the definition and imposition of religious orthodoxy. Also addressed is the Great Schism in the Roman Church that set into question the foundations of ecclesiology. In the same era, philosophical and theoretical innovations reexamined conventional beliefs about metaphysics, epistemology and political life, perhaps best encapsulated by the fifteenth-century philosopher, theologian and political theorist Nicholas of Cusa.https://scholarworks.wmich.edu/mip_smemc/1003/thumbnail.jp

Ornithine-δ-aminotransferase is essential for Arginine Catabolism but not for Proline Biosynthesis

<p>Abstract</p> <p>Background</p> <p>Like many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu).</p> <p>Results</p> <p>We show here that ornithine-δ-aminotransferase (δOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of δ<it>OAT</it>, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of δ<it>OAT </it>expression.</p> <p>Conclusion</p> <p>Our findings indicate that δOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism.</p

A murine model of variant late infantile ceroid lipofuscinosis recapitulates behavioral and pathological phenotypes of human disease.

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6(nclf)) mouse line to validate its utility for translational research. We demonstrate that this Cln6(nclf) mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6(nclf) mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics

Activity of metazoa governs biofilm structure formation and enhances permeate flux during Gravity-Driven Membrane (GDM) filtration

The impact of different feed waters in terms of eukaryotic populations and organic carbon content on the biofilm structure formation and permeate flux during Gravity-Driven Membrane (GDM) filtration was investigated in this study. GDM filtration was performed at ultra-low pressure (65 mbar) in dead-end mode without control of the biofilm formation. Different feed waters were tested (River water, pre-treated river water, lake water, and tap water) and varied with regard to their organic substrate content and their predator community. River water was manipulated either by chemically inhibiting all eukaryotes or by filtering out macrozoobenthos (metazoan organisms). The structure of the biofilm was characterized at the meso- and micro-scale using Optical Coherence Tomography (OCT) and Confocal Laser Scanning Microscopy (CLSM), respectively. Based on Total Organic Carbon (TOC) measurements, the river waters provided the highest potential for bacterial growth whereas tap water had the lowest. An increasing content in soluble and particulate organic substrate resulted in increasing biofilm accumulation on membrane surface. However, enhanced biofilm accumulation did not result in lower flux values and permeate flux was mainly influenced by the structure of the biofilm. Metazoan organisms (in particular nematodes and oligochaetes) built-up protective habitats, which resulted in the formation of open and spatially heterogeneous biofilms composed of biomass patches. In the absence of predation by metazoan organisms, a flat and compact biofilm developed. It is concluded that the activity of metazoan organisms in natural river water and its impact on biofilm structure balances the detrimental effect of a high biofilm accumulation, thus allowing for a broader application of GDM filtration. Finally, our results suggest that for surface waters with high particulate organic carbon (POC) content, the use of worms is suitable to enhance POC removal before ultrafiltration units

Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20°C) mainly affects protein metabolism

<p>Abstract</p> <p>Background</p> <p>Temperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated <it>Daphnia pulex</it>.</p> <p>Results</p> <p>Specific sets of proteins were found to be differentially expressed in 10°C or 20°C acclimated <it>D. pulex</it>. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases). The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific), and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or down-regulated in the cold.</p> <p>Conclusion</p> <p>Specific sets of cold-repressed and cold-induced proteins in <it>D. pulex </it>can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10°C and 20°C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animal's body. Conversely, the increase of actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30) in the <it>D. pulex </it>genome suggests large-scaled gene family expansions that might reflect specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment.</p

Kontrolle des Roggenschwarzrostes, Puccinia graminis f. sp. secalis, im Ökologischen Landbau durch Züchtung resistenten Roggens

Der Ökologische Landbau (ÖLB) ist von der zunehmenden Ausbreitung des Roggenschwarz-rostes, Puccinia graminis f. sp. secalis, besonders betroffen, da resistente Roggensorten bisher nicht zur Verfügung stehen. Zur Analyse der Virulenzsituation des Roggenschwarzrostes wurden 389 Einpustelisolate (EPI) hergestellt, von denen 323 mit einem Differentialsortiment aus 15 Inzuchtlinien getestet wurden. Die EPI konnten 226 Pathotypen zugeordnet werden, von denen nur 56 Pathotypen häufiger als einmal vorkamen. Die Mehrzahl der Isolate wies in den Jahren 2011, 2012 und 2013 eine Komplexität von sieben, sechs bzw. fünf auf. Keine der 15 Differentiallinien reagierte vollständig resistent. Mit einem Simpson-Wert von 0,99 zeigt sich eine maximale Diversität der deutschen Schwarzrostpopulation. Das im Rahmen des Projektes entwickelt Isolatesortiment deckt das aktuelle Virulenzspektrum der Schwarzrostpopulationen ab und kann auch zukünftig genutzt werden, um die Wirksamkeit von Schwarzrostresistenzen zu prüfen. Außerdem kann das Auftreten neuer Virulenzen in der Schwarzrostpopulation jetzt rechtzeitig erkannt und wirtschaftlicher Schaden abgewendet werden. Zur Ermittlung der Adultpflanzen-Resistenz wurden bis zu 70 Genetische Ressourcen sowie adaptiertes, selbstfertiles Roggenmaterial unter ökologischen Bedingungen an fünf Feldstandorten je Jahr angebaut. Das Inokulum für die künstlichen Inokulationen wurde am JKI produziert und die Inokulation fand im Entwicklungsstadium BBCH37 (Mai 2013 und Mai 2014) statt. Die im Ökologischen Landbau verwendeten Sorten Recrut, Conduct, Amilo, Firmament®, Lichtkornroggen®, Rolipa und Lautenbacher waren nach künstlicher Infektion hoch anfällig gegenüber Schwarzrost mit Befallsstärken von über 50%. Nach drei Versuchs-jahren konnten insgesamt 17 Populationssorten aus Österreich (Tiroler, Kärntner, Oberkärnt-ner), Russland (Hy75/81, Hy2407/87, Talwoskaja 29, Hy9a/86, Talowskija, Zidlochowicke rane, Instituckie Wcz), den USA (Wheeler, Elbon, Wrens Abruzzi, Gator, Alfa), und Argentinien (Manfredi), mit mittleren Befallswerten von ≤30 % für die Züchtung bereitgestellt werden. Das Projekt leistete damit einen erheblichen Beitrag zur Nutzung der Biodiversität des Roggens und zur Optimierung der Resistenzzüchtung im ÖLB. Resistente Roggensorten werden in Zukunft eine effiziente Kontrolle des Roggenschwarzrostes ermöglichen, die den Grundsätzen des ÖLB entspricht. Das Projekt kann den Hauptaufgaben 2.1, 2.3, 2.4 und 2.14 des Forschungsplanes des BMELV zugeordnet werden und ist Grundlage für die Weiterentwicklung von Strategien zur Nutzung von Schwarzrostresistenzen sowie die Bewertung der Widerstandfähigkeit von Roggen gegenüber Schwarzrost