Protein 4.1B contributes to the organization of peripheral myelinated axons

Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber.     

Elevated [11C]-D-deprenyl uptake in chronic Whiplash Associated Disorder suggests persistent musculoskeletal inflammation

There are few diagnostic tools for chronic musculoskeletal pain as structural imaging methods seldom reveal pathological alterations. This is especially true for Whiplash Associated Disorder, for which physical signs of persistent injuries to the neck have yet to be established. Here, we sought to visualize inflammatory processes in the neck region by means Positron Emission Tomography using the tracer (11)C-D-deprenyl, a potential marker for inflammation. Twenty-two patients with enduring pain after a rear impact car accident (Whiplash Associated Disorder grade II) and 14 healthy controls were investigated. Patients displayed significantly elevated tracer uptake in the neck, particularly in regions around the spineous process of the second cervical vertebra. This suggests that whiplash patients have signs of local persistent peripheral tissue inflammation, which may potentially serve as a diagnostic biomarker. The present investigation demonstrates that painful processes in the periphery can be objectively visualized and quantified with PET and that (11)C-D-deprenyl is a promising tracer for these purposes.     

Tissue tropism and target cells of NSs-deleted rift valley fever virus in live immunodeficient mice

Background

Rift Valley fever virus (RVFV) causes disease in livestock and humans. It can be transmitted by mosquitoes, inhalation or physical contact with the body fluids of infected animals. Severe clinical cases are characterized by acute hepatitis with hemorrhage, meningoencephalitis and/or retinitis. The dynamics of RVFV infection and the cell types infected in vivo are poorly understood.

Methodology/Principal Findings

RVFV strains expressing humanized Renilla luciferase (hRLuc) or green fluorescent protein (GFP) were generated and inoculated to susceptible Ifnar1-deficient mice. We investigated the tissue tropism in these mice and the nature of the target cells in vivo using whole-organ imaging and flow cytometry. After intraperitoneal inoculation, hRLuc signal was observed primarily in the thymus, spleen and liver. Macrophages infiltrating various tissues, in particular the adipose tissue surrounding the pancreas also expressed the virus. The liver rapidly turned into the major luminescent organ and the mice succumbed to severe hepatitis. The brain remained weakly luminescent throughout infection. FACS analysis in RVFV-GFP-infected mice showed that the macrophages, dendritic cells and granulocytes were main target cells for RVFV. The crucial role of cells of the monocyte/macrophage/dendritic lineage during RVFV infection was confirmed by the slower viral dissemination, decrease in RVFV titers in blood, and prolonged survival of macrophage- and dendritic cell-depleted mice following treatment with clodronate liposomes. Upon dermal and nasal inoculations, the viral dissemination was primarily observed in the lymph node draining the injected ear and in the lungs respectively, with a significant increase in survival time.

Conclusions/Significance

These findings reveal the high levels of phagocytic cells harboring RVFV during viral infection in Ifnar1-deficient mice. They demonstrate that bioluminescent and fluorescent viruses can shed new light into the pathogenesis of RVFV infection.     

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals¹. Though being a crucial part of the visual processing by shaping the retinal output², most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue^3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage⁵. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments⁶.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.     

Phytophthora gemini sp. nov., a new species isolated from the halophilic plant Zostera marina in the Netherlands

Eight strains belonging to the Oomycete genus Phytophthora were isolated from Zostera marina (seagrass) in The Netherlands over the past 25 y. Based on morphology, isozymes, temperature-growth relationships and ITS sequences, these strains were found to belong to two different Phytophthora species. Five strains, four of them isolated from rotting seeds and one isolated from decaying plants, could not be assigned to a known species and hence belong to a new species for which we propose the name Phytophthora gemini sp. nov. Three strains were isolated from decaying plants and were identified as Phytophthora inundata, thereby expanding the known habitat range of this species from fresh to brackish-saline areas. The possible role of both Phytophthora species in the decline of Z. marina in The Netherlands and the evolutionary significance of the presence of Phytophthora species in marine environments are discussed.     

Real-time ichthyoplankton drift in Northeast Arctic cod and Norwegian spring-spawning herring

Background

Individual-based biophysical larval models, initialized and parameterized by observations, enable numerical investigations of various factors regulating survival of young fish until they recruit into the adult population. Exponentially decreasing numbers in Northeast Arctic cod and Norwegian Spring Spawning herring early changes emphasizes the importance of early life history, when ichthyoplankton exhibit pelagic free drift. However, while most studies are concerned with past recruitment variability it is also important to establish real-time predictions of ichthyoplankton distributions due to the increasing human activity in fish habitats and the need for distribution predictions that could potentially improve field coverage of ichthyoplankton.

Methodology/Principal Findings

A system has been developed for operational simulation of ichthyoplankton distributions. We have coupled a two-day ocean forecasts from the Norwegian Meteorological Institute with an individual-based ichthyoplankton model for Northeast Arctic cod and Norwegian Spring Spawning herring producing daily updated maps of ichthyoplankton distributions. Recent years observed spawning distribution and intensity have been used as input to the model system. The system has been running in an operational mode since 2008. Surveys are expensive and distributions of early stages are therefore only covered once or twice a year. Comparison between model and observations are therefore limited in time. However, the observed and simulated distributions of juvenile fish tend to agree well during early fall. Area-overlap between modeled and observed juveniles September 1^st range from 61 to 73%, and 61 to 71% when weighted by concentrations.

Conclusions/Significance

The model system may be used to evaluate the design of ongoing surveys, to quantify the overlap with harmful substances in the ocean after accidental spills, as well as management planning of particular risky operations at sea. The modeled distributions are already utilized during research surveys to estimate coverage success of sampled biota and immediately after spills from ships at sea.     

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.     

Translational database selection and multiplexed sequence capture for up front filtering of reliable breast cancer biomarker candidates

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.     

Increased expression of cannabinoid CB₁ receptors in Achilles tendinosis

Background

The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB₁) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology

Cannabinoid CB₁ receptor immunoreactivity (CB₁IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings

CB₁IR was seen as a granular pattern in the tenocytes. CB₁IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB₁ receptor expression in tendinosis tissue compared to control tissue.

Conclusion

Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.     

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.