PARma: identification of microRNA target sites in AGO-PAR-CLIP data

PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probabilities, cluster confidence scores and to assign the most probable microRNA. Based on differential PAR-CLIP analysis and comparison to RIP-Chip data, we show that PARma is more accurate than existing approaches. PARma is available from http://www.bio.ifi.lmu.de/PARma     

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.     

Complementing the Eukaryotic Protein Interactome

Protein interaction networks are important for the understanding of regulatory mechanisms, for the explanation of experimental data and for the prediction of protein functions. Unfortunately, most interaction data is available only for model organisms. As a possible remedy, the transfer of interactions to organisms of interest is common practice, but it is not clear when interactions can be transferred from one organism to another and, thus, the confidence in the derived interactions is low. Here, we propose to use a rich set of features to train Random Forests in order to score transferred interactions. We evaluated the transfer from a range of eukaryotic organisms to S. cerevisiae using orthologs. Directly transferred interactions to S. cerevisiae are on average only 24% consistent with the current S. cerevisiae interaction network. By using commonly applied filter approaches the transfer precision can be improved, but at the cost of a large decrease in the number of transferred interactions. Our Random Forest approach uses various features derived from both the target and the source network as well as the ortholog annotations to assign confidence values to transferred interactions. Thereby, we could increase the average transfer consistency to 85%, while still transferring almost 70% of all correctly transferable interactions. We tested our approach for the transfer of interactions to other species and showed that our approach outperforms competing methods for the transfer of interactions to species where no experimental knowledge is available. Finally, we applied our predictor to score transferred interactions to 83 targets species and we were able to extend the available interactome of B. taurus, M. musculus and G. gallus with over 40,000 interactions each. Our transferred interaction networks are publicly available via our web interface, which allows to inspect and download transferred interaction sets of different sizes, for various species, and at specified expected precision levels. Availability: http://services.bio.ifi.lmu.de/coin-db/.     

Are Bogs Reservoirs for Emerging Disease Vectors? Evaluation of Culicoides Populations in the Hautes Fagnes Nature Reserve (Belgium)

Several species of Culicoides (Diptera: Ceratopogonidae) biting midges serve as biological vectors for the bluetongue virus (BTV) and the recently described Schmallenberg virus (SBV) in northern Europe. Since their recent emergence in this part of the continent, these diseases have caused considerable economic losses to the sheep and cattle industries. Much data is now available that describe the distribution, population dynamics, and feeding habits of these insects. However, little is known regarding the presence of Culicoides in unusual habitats such as peaty marshes, nor their potential vector capacity. This study evaluated Culicoides biting midges present in the bogs of a Belgian nature reserve compared to those residing at a nearby cattle farm. Culicoides were trapped in 2011 at four different sites (broadleaved and coniferous forested areas, open environments, and at a scientific station) located in the Hautes Fagnes Nature Reserve (Belgium). An additional light trap was operated on a nearby cattle farm. Very high numbers of biting midges were captured in the marshy area and most of them (70 to 95%) were Culicoides impunctatus, a potential vector of BTV and other pathogens. In addition, fewer numbers of C. obsoletus/C. scoticus species, C. chiopterus, and C. dewulfi were observed in the bogs compared to the farm. The wet environment and oligotrophic nature of the soil were probably responsible for these changes in the respective populations. A total of 297,808 Culicoides midges belonging to 27 species were identified during this study and 3 of these species (C. sphagnumensis, C. clintoni and C. comosioculatus) were described in Belgium for the first time.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Molecular Mechanics Evaluation of the Proposed Mechanisms for the Degradation of Urea by Urease

Abstract

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the N_His272-Ni1-O-C_urea and Ni1-O-Curea-N_urea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional “His320 acts as a base” mechanism is unlikely, while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.     

CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene

The CRISPR/Cas technology has been successfully used to stimulate the integration of small DNA sequences in a target locus to produce gene mutations. However, many applications require homologous recombination using large gene-targeting constructs. Here we address the potential of CRISPR/Cas-mediated double-strand breaks to enhance the genetic engineering of large target sequences using a construct for “humanizing” the mouse Cnr2 gene locus. We designed a small-guide RNA that directs the induction of double strand breaks by Cas9 in the Cnr2 coding exon. By co-transfection of the CRISPR/Cas system with the 10 kb targeting construct we were able to boost the recombination frequency more than 200-fold from 0.27% to 67%. This simple technology can thus be used for the homologous integration of large gene fragments and should greatly enhance our ability to generate any kind of genetically altered mouse models.     

Role of Tachykinin 1 and 4 Gene-Derived Neuropeptides and the Neurokinin 1 Receptor in Adjuvant-Induced Chronic Arthritis of the Mouse

Objective

Substance P, encoded by the Tac1 gene, is involved in neurogenic inflammation and hyperalgesia via neurokinin 1 (NK1) receptor activation. Its non-neuronal counterpart, hemokinin-1, which is derived from the Tac4 gene, is also a potent NK1 agonist. Although hemokinin-1 has been described as a tachykinin of distinct origin and function compared to SP, its role in inflammatory and pain processes has not yet been elucidated in such detail. In this study, we analysed the involvement of tachykinins derived from the Tac1 and Tac4 genes, as well as the NK1 receptor in chronic arthritis of the mouse.

Methods

Complete Freund’s Adjuvant was injected intraplantarly and into the tail of Tac1^−/−, Tac4^−/−, Tacr1^−/− (NK1 receptor deficient) and Tac1^−/−/Tac4^−/− mice. Paw volume was measured by plethysmometry and mechanosensitivity using dynamic plantar aesthesiometry over a time period of 21 days. Semiquantitative histopathological scoring and ELISA measurement of IL-1β concentrations of the tibiotarsal joints were performed.

Results

Mechanical hyperalgesia was significantly reduced from day 11 in Tac4^−/− and Tacr1^−/− animals, while paw swelling was not altered in any strain. Inflammatory histopathological alterations (synovial swelling, leukocyte infiltration, cartilage destruction, bone damage) and IL-1β concentration in the joint homogenates were significantly smaller in Tac4^−/− and Tac1^−/−/Tac4^−/− mice.

Conclusions

Hemokinin-1, but not substance P increases inflammation and hyperalgesia in the late phase of adjuvant-induced arthritis. While NK1 receptors mediate its antihyperalgesic actions, the involvement of another receptor in histopathological changes and IL-1β production is suggested.     

Closed-Cell Stent-Assisted Coiling of Intracranial Aneurysms: Evaluation of Changes in Vascular Geometry Using Digital Subtraction Angiography

BackgroundStent-assisted coil embolization (SACE) plays an important role in the treatment of intracranial aneurysms. The purpose of this study was to investigate geometrical changes caused by closed-cell design stents in bifurcation and sidewall aneurysms.Methods31 patients with 34 aneurysms underwent SACE with closed-cell design stents. Inflow angle α, determined by aneurysm neck and afferent vessel, and angle between afferent and efferent vessel close to (δ₁), respectively, more remote from the aneurysm neck (δ₂) were graphically determined in 2D angiography projections.ResultsStent assisted coiling resulted in a significant increase of all three angles from a mean value (±SEM) of α = 119° (±6.5°) pretreatment to 130° (±6.6°) posttreatment (P ≤ .001), δ₁ = 129° (±6.4°) to 139° (±6.1°), (P ≤ .001) and δ₂ = 115° (±8.4°) to 126° (±7.5°), (P ≤ .01). Angular change of δ₁ in AcomA aneurysms was significant greater compared to sidewall aneurysms (26°±4.9° versus 8°± 2.3°, P ≤ .05). The initial angle of δ₁ and δ₂ revealed a significantly inverse relationship to the angle increase (δ₁: r = -0.41, P ≤ .05 and δ₂: r = -0.47, P ≤ .01). Moreover, angle δ₁ was significantly higher in unruptured compared to ruptured aneurysms (135°±7.1° versus 103°±10.8°, P ≤ .05).ConclusionStent deployment modulates the geometry of the aneurysm-vessel complex, which may lead to favorable hemodynamic changes more similar to unruptured than to ruptured aneurysms. Our findings also suggest that the more acute-angled aneurysm-vessel anatomy, the larger the angular change. Further studies are needed to investigate whether these changes improve the clinical outcome.     

Effects of heat and drought stress on post‐illumination bursts of volatile organic compounds in isoprene‐emitting and non‐emitting poplar

Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO₂ concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO₂ and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.