Protocol: An improved and universal procedure for whole-mount immunolocalization in plants

Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment

Rational design of a peptide capture agent for CXCL8 based on a model of the CXCL8:CXCR1 complex

Protein-capture agents are widely used for the detection, immobilization and isolation of proteins and are the foundation for the development of in vitro diagnostic chips. The chemokine CXCL8 is an interesting protein target due to its involvement in the human inflammatory response. We constructed a novel structural model of CXCL8 interaction with its G-protein coupled receptor CXCR1, taking into account previously reported experimental data. From this CXCL8:CXCR1 model complex, the interaction of CXCL8 with residues near the extracellular domains 3 and 4 of CXCR1 were used as a scaffold for the rational design of a peptide capture agent called 'IL8RPLoops'. A molecular dynamics simulation of IL8RPLoops indicates a stable helical conformation consistent with the CXCR1 structure from which it was derived. CXCL8 capture in fluorescence-based assays on beads and on glass demonstrates that IL8RPLoops is an effective capture agent for CXCL8. Additionally, we found IL8RPLoops to be a potent inhibitor of CXCL8-induced neutrophil migration and CXCL8:CXCR1 association. A theoretical binding model for IL8RPLoops:CXCL8 is proposed, which shows the peptide predominantly interacting with CXCL8 via electrostatic contacts with the ELR motif at the CXCL8 N-terminus

Long-Term Summertime Investigations of Pelagic and Benthic Realms with Continuous Observations of Vertical Particle Flux in the Fram Strait and the Central Arctic Ocean

Sea ice volume and extent currently experience massive reduction in the Arctic Ocean due to climate change. Our long-term study aims at tracing effects of environmental changes in pelagic and benthic systems and investigate accompanying impacts on the fate of organic matter produced in the upper water column on its way down to the seafloor. Since the start of our observations in 1999, we have already seen some effects and will present selected data sets from the upper water column and benthic data during summer expeditions as well as results from vertical particle flux measurements that were obtained from annually deployed sediment traps at the LTER (Long-Term Ecological Research) observatory HAUSGARTEN in the eastern Fram Strait (79°/4°E) and on fewer occasions in the central Arctic Ocean (CAO). Highest biomass was found in the eastern Fram Strait and lowest in the heavily ice-covered regions in the CAO. Flux rates of POC where at least one order of magnitude lower in the CAO than in the eastern Fram Strait. While in the CAO ice algae dominate the recognizable flux fraction, faecal material prevailed in eastern Fram Strait traps. This points towards different systems of organic matter production and modification and, thus, different mechanisms determine the efficiency of the biological carbon pump. These differences are also reflected in the benthic communities in the CAO and in the eastern Fram Strait. These first results have shown the importance of long-term observations and encouraged the continuation of the Arctic Ocean Observing System FRAM (FRontiers in Arctic marine Monitoring) to record environmental and biological data at high temporal and spatial resolution

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

The gut of most invertebrates is lined by a protective layer of chitin and glycoproteins, often designated as a peritrophic matrix. Previous research suggests that it forms a barrier that may protect the midgut epithelium from abrasive food particles and pathogens. Parasitic invertebrates ingesting vertebrate plasma have evolved additional strategies to protect themselves from hazardous host molecules consumed during feeding. An important part of the immediate defense in vertebrate plasma is complement-mediated killing. The Complement system is a complex network of more than 35 proteins present in human plasma that results in killing of foreign cells including the gut epithelial cells of a feeding parasite. Recently we found that scabies mites, who feed on skin containing plasma, produce several proteins that inhibit human complement within the mite gut. The mites excrete these molecules into the upper epidermis where they presumably also inhibit complement activity. Mite gut antigens that initially trigger the complement cascade have not been identified previously. Obvious possible targets of complement attack within the mite gut could be peritrophins. Our study describes the first peritrophin identified in scabies mites and indicates a possible role in complement activation

LCC – Ausgangspunkt für Kostensenkungen in der Eisenbahnsignaltechnik

Für die Eisenbahninfrastrukturbetreiber stellt sich immer häufiger die Frage, welche Signaltechnik bei Erst- oder Ersatzinvestitionen aus wirtschaftlichen Gesichtspunkten zu wählen ist. Wesentlicher Indikator für die Wirtschaftlichkeit der Anlagen der Schieneninfrastruktur sind deren Lebenszykluskosten (LCC). Sie umfassen alle mit einer Anlage verbundenen Kosten über deren gesamten Lebenszyklus, von der Entwicklung über den Anlagenbetrieb bis zur Entsorgung. Um das Bewusstsein für das zeitliche Kostenverhalten der Anlagen sowohl bei Anlagenhersteller als auch -betreiber zu schärfen, ist eine strukturierte Erfassung und Auswertung der Kostenpositionen notwendig. Die Möglichkeit hierzu bieten Lebenszykluskostenmodelle. Diese sind ein wesentlicher Bestandteil des Life Cycle Costing, dessen Ziel die Ermittlung und Analyse der Lebenszykluskosten ist. Aufgrund der praktischen Bedeutung des Life Cycle Costing für die Eisenbahnsignaltechnik sollen im vorliegenden Artikel die notwendigen Arbeitsschritte an einem Beispiel erläutert und die Vorteile und Grenzen dieses Konzepts in der praktischen Anwendung herausgestellt werden

Capnocytophaga canimorsus. interaction with the innate immune system

We show that Capnocytophaga canimorsus strain 5 (Cc5) is even more resistant to phagocytosis and killing by murine macrophages (J774.1) and human polymorphonuclear neutrophils (PMNs) than Yersinia enterocolitica, which is known as a model bacterium for resistance against phagocytosis due to its type 3 secretion system (Grosdent et al., 2002). We observed that Cc5 even becomes completely resistant to phagocytosis at high multiplicity of infection (moi of 50). In addition, we demonstrate that the Cc5 transposon mutant Y1C12, identified during a serum sensitivity screen, has an increased sensitivity to phagocytosis and killing by either murine macrophages or human PMNs even in the unopsonized state. This indicated that not an increased susceptibility for antibody binding or complement deposition led to an increased phagocytosis of the mutant, but that rather the outer surface was more readily recognized by the phagocytes. Furthermore, we demonstrate that Cc5 induces the formation of neutrophil extracellular traps upon infection of human PMNs in vitro and that Cc5 is trapped and killed within neutrophil extracellular traps, indicating sensitivity of Cc5 towards antimicrobial peptides present in PMN granules. Analysis of serum resistance in Cc5 revealed that serum resistance is probably linked to its lipopolysaccharide, which prevents deposition of the membrane attack complex on the bacterial surface. Moreover, we have observed that upon growth in the presence of cells, Cc5 releases or modifies factor(s) in the medium, which interfere with the killing ability of macrophages. Investigating the underlying mechanism, we could show that Cc5 does not affect phagosome maturation, but blocks the oxidative burst. This capacity was shown to depend on the release of the zinc metallopeptidase pitrilysin by Cc5. First analyses on the prevalence of the hypothetical virulence factors serum resistance and interference with the oxidative burst indicated that C. canimorsus strains might display strain variability. While 59% of the strains (50% of case strains, 61% of dog isolates) were able to block the killing ability of macrophages, 60% of the strains were highly serum resistant (100% of case strains, 54% of dog isolates). However, serum resistance could not be directly linked to a specific polysaccharide structure in C. canimorsus. November 2009, Salome Casutt-Meye

Neural correlates of metonymy resolution

Metonymies are exemplary models for complex semantic association processes at the sentence level. We investigated processing of metonymies using event-related functional magnetic resonance imaging (fMRI). During an 1.5 Tesla fMRI scan, 14 healthy subjects (12 female) read 124 short German sentences with either literal (like “Africa is arid”), metonymic (“Africa is hungry”), or nonsense (“Africa is woollen”) content. Sentences were constructed so that they obey certain grammatical, semantic, and plausibility conditions and were matched for word frequency, semantic association, length and syntactic structure. We concentrated on metonymies that were not yet fossilised; we also examined a wide variety of metonymic readings. Reading metonymies relative to literal sentences revealed signal changes in a predominantly left-lateralised fronto-temporal network with maxima in the left and right inferior frontal as well as left middle temporal gyri. Left inferior frontal activation may reflect both inference processes and access to world knowledge during metonymy resolution

Inhibition of Midkine Augments Osteoporotic Fracture Healing.

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications