IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.     

In vitro evaluation of co-exposure of arsenium and an organic nanomaterial (fullerene, C₆₀) in zebrafish hepatocytes

Taking into account the concept of the "Trojan Horse", where contaminants may have its entry into specific organs potentiated by its association with nanomaterials, the aim of this study was to analyze the joint toxic effects induced by an organic nanomaterial, fullerene (C(60)) with the metalloid arsenic (As(III)). Hepatocytes of zebrafish Danio rerio were exposed to As(III) (2.5 or 100 μM), C(60) or As+C(60) for 4h, not altering cells viability. Intracellular reactive oxygen species concentration was reduced in cells exposed only to the C(60) (1mg/L) and in the treatment of 100 μM As(III)+C(60). Co-exposure with C(60) abolished the peak of the antioxidant glutathione (GSH) registered in cells exposed to the lowest As(III) concentration (2.5 μM). A similar result was observed in terms of lipid damage (TBARS). Total antioxidant capacity was significantly higher at both As(III) concentrations co-exposed to C(60) when compared with the control group. Activity of glutathione-S-transferase omega, a limiting enzyme in the methylation pathway of As(III), was reduced in the 100 μM As(III)+C(60) treatment. Cells co-exposed to C(60) had a significantly higher accumulation of As(III), showing a "Trojan Horse" effect which did not result in higher cell toxicity. Instead, co-exposure of As(III) with C(60) showed to reduce cellular injury.     

Enhanced Passive Bat Rabies Surveillance in Indigenous Bat Species from Germany - A Retrospective Study

In Germany, rabies in bats is a notifiable zoonotic disease, which is caused by European bat lyssaviruses type 1 and 2 (EBLV-1 and 2), and the recently discovered new lyssavirus species Bokeloh bat lyssavirus (BBLV). As the understanding of bat rabies in insectivorous bat species is limited, in addition to routine bat rabies diagnosis, an enhanced passive surveillance study, i.e. the retrospective investigation of dead bats that had not been tested for rabies, was initiated in 1998 to study the distribution, abundance and epidemiology of lyssavirus infections in bats from Germany. A total number of 5478 individuals representing 21 bat species within two families were included in this study. The Noctule bat (Nyctalus noctula) and the Common pipistrelle (Pipistrellus pipistrellus) represented the most specimens submitted. Of all investigated bats, 1.17% tested positive for lyssaviruses using the fluorescent antibody test (FAT). The vast majority of positive cases was identified as EBLV-1, predominately associated with the Serotine bat (Eptesicus serotinus). However, rabies cases in other species, i.e. Nathusius'' pipistrelle bat (Pipistrellus nathusii), P. pipistrellus and Brown long-eared bat (Plecotus auritus) were also characterized as EBLV-1. In contrast, EBLV-2 was isolated from three Daubenton''s bats (Myotis daubentonii). These three cases contribute significantly to the understanding of EBLV-2 infections in Germany as only one case had been reported prior to this study. This enhanced passive surveillance indicated that besides known reservoir species, further bat species are affected by lyssavirus infections. Given the increasing diversity of lyssaviruses and bats as reservoir host species worldwide, lyssavirus positive specimens, i.e. both bat and virus need to be confirmed by molecular techniques.     

Macular Pigment Optical Density Measured by Heterochromatic Modulation Photometry

Purpose

To psychophysically determine macular pigment optical density (MPOD) employing the heterochromatic modulation photometry (HMP) paradigm by estimating 460 nm absorption at central and peripheral retinal locations.

Methods

For the HMP measurements, two lights (B: 460 nm and R: 660 nm) were presented in a test field and were modulated in counterphase at medium or high frequencies. The contrasts of the two lights were varied in tandem to determine flicker detection thresholds. Detection thresholds were measured for different R:B modulation ratios. The modulation ratio with minimal sensitivity (maximal threshold) is the point of equiluminance. Measurements were performed in 25 normal subjects (11 male, 14 female; age: 30±11 years, mean ± sd) using an eight channel LED stimulator with Maxwellian view optics. The results were compared with those from two published techniques – one based on heterochromatic flicker photometry (Macular Densitometer) and the other on fundus reflectometry (MPR).

Results

We were able to estimate MPOD with HMP using a modified theoretical model that was fitted to the HMP data. The resultant MPOD_HMP values correlated significantly with the MPOD_MPR values and with the MPOD_HFP values obtained at 0.25° and 0.5° retinal eccentricity.

Conclusions

HMP is a flicker-based method with measurements taken at a constant mean chromaticity and luminance. The data can be well fit by a model that allows all data points to contribute to the photometric equality estimate. Therefore, we think that HMP may be a useful method for MPOD measurements, in basic and clinical vision experiments.     

Absence of co-phylogeny indicates repeated diatom capture in dinophytes hosting a tertiary endosymbiont

Tertiary endosymbiosis is proven through dinophytes, some of which (i.e. Kryptoperidiniaceae) have engulfed diatom algae containing a secondary plastid. Chloroplasts are usually inherited together permanently with the host cell, leading to co-phylogeny. We compiled a diatom sequence data matrix of two nuclear and two chloroplast loci. Almost all endosymbionts of Kryptoperidiniaceae found their closest relatives in free-living diatoms and not in other harboured algae, rejecting co-phylogeny and indicating that resident diatoms were taken up by dinophytes multiple times independently. Almost intact ultrastructure and insignificant genome reduction are supportive for young, if not recent events of diatom capture. With their selective specificity on the one hand and extraordinary degree of endosymbiotic flexibility on the other hand, dinophytes hosting diatoms share more traits with lichens or facultatively phototrophic ciliates than with green algae and land plants. Time estimates indicate the dinophyte lineages as consistently older than the hosted diatom lineages, thus also favouring a repeated uptake of endosymbionts. The complex ecological role of dinophytes employing a variety of organismic interactions may explain their high potential and plasticity in acquiring a great diversity of plastids.     

Membrane inlet—ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.     

iBench: A ground truth approach for advanced validation of mass spectrometry identification method

The discovery of many noncanonical peptides detectable with sensitive mass spectrometry inside, outside, and on cells shepherded the development of novel methods for their identification, often not supported by a systematic benchmarking with other methods. We here propose iBench, a bioinformatic tool that can construct ground truth proteomics datasets and cognate databases, thereby generating a training court wherein methods, search engines, and proteomics strategies can be tested, and their performances estimated by the same tool. iBench can be coupled to the main database search engines, allows the selection of customized features of mass spectrometry spectra and peptides, provides standard benchmarking outputs, and is open source. The proof-of-concept application to tryptic proteome digestions, immunopeptidomes, and synthetic peptide libraries dissected the impact that noncanonical peptides could have on the identification of canonical peptides by Mascot search with rescoring via Percolator (Mascot+Percolator).     

Rare Variants in PLXNA4 and Parkinson’s Disease

Approximately 20% of individuals with Parkinson’s disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.     

Zinc uptake and radial transport in roots of Arabidopsis thaliana: a modelling approach to understand accumulation

Background and Aims

Zinc uptake in roots is believed to be mediated by ZIP (ZRT-, IRT-like proteins) transporters. Once inside the symplast, zinc is transported to the pericycle, where it exits by means of HMA (heavy metal ATPase) transporters. The combination of symplastic transport and spatial separation of influx and efflux produces a pattern in which zinc accumulates in the pericycle. Here, mathematical modelling was employed to study the importance of ZIP regulation, HMA abundance and symplastic transport in creation of the radial pattern of zinc in primary roots of Arabidopsis thaliana.

Methods

A comprehensive one-dimensional dynamic model of radial zinc transport in roots was developed and used to conduct simulations. The model accounts for the structure of the root consisting of symplast and apoplast and includes effects of water flow, diffusion and cross-membrane transport via transporters. It also incorporates the radial geometry and varying porosity of root tissues, as well as regulation of ZIP transporters.

Key Results

Steady-state patterns were calculated for various zinc concentrations in the medium, water influx and HMA abundance. The experimentally observed zinc gradient was reproduced very well. An increase of HMA or decrease in water influx led to loss of the gradient. The dynamic behaviour for a change in medium concentration and water influx was also simulated showing short adaptation times in the range of seconds to minutes. Slowing down regulation led to oscillations in expression levels, suggesting the need for rapid regulation and existence of buffering agents.

Conclusions

The model captures the experimental findings very well and confirms the hypothesis that low abundance of HMA4 produces a radial gradient in zinc concentration. Surprisingly, transpiration was found also to be a key parameter. The model suggests that ZIP regulation takes place on a comparable timescale as symplastic transport.