The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Stimulation by 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and glucocorticoids of phosphoenolpyruvate carboxykinase in the isolated perfused rat liver (Short Communication)

Dibutyryl cyclic AMP stimulated the activity of phosphoenolpyruvate carboxykinase in perfused livers of rats, fed on a low-protein diet, linearly over a 6h period. The enzyme activity was also significantly elevated by dexamethasone, the effect being considerably lower than that of the cyclic nucleotide. Since the time-course of phosphoenolpyruvate carboxykinase activity in response to dibutyryl cyclic AMP resembled that observed after dibutyryl cyclic AMP injection into intact animals, it is suggested that induction of the enzyme in vivo is due to a direct action of the cyclic nucleotide on the liver. Combined administration of dibutyryl cyclic AMP and glucocorticoids did not lead to an additive increase of liver phosphoenolpyruvate carboxykinase activity, either in vivo or in the perfused organ.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Although there is no Physical Short‐Term Scarcity of Phosphorus,its Resource Efficiency Should be Improved

The German government has adopted a law that requires sewage plants to go beyond the recovery of phosphorus from wastewater and to promote recycling. We argue that there is no physical global short‐ or mid‐term phosphorus scarcity. However, we also argue that there are legitimate reasons for policies such as those of Germany, including: precaution as a way to ensure future generations’ long‐term supply security, promotion of technologies for closed‐loop economics in a promising stage of technology development, and decrease in the current supply risk with a new resource pool.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

<Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> sp. nov., A Wound-infesting Fungus of Hardwood Trees in Africa

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, β-tubulin and translation elongation factor 1-α gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.     

Survey of electromagnetic field exposure in bedrooms of residences in lower Austria

Previous investigations of exposure to electric, magnetic, or electromagnetic fields (EMF) in households were either about electricity supply EMFs or radio frequency EMFs (RF‐EMFs). We report results from spot measurements at the bedside that comprise electrostatic fields, extremely low‐frequency electric fields (ELF‐EFs), extremely low‐frequency magnetic fields (ELF‐MFs), and RF‐EMFs. Measurements were taken in 226 households throughout Lower Austria. In addition, effects of simple reduction measures (e.g., removal of clock radios or increasing their distance from the bed, turning off Digital Enhanced Cordless Telecommunication (DECT) telephone base stations) were assessed. All measurements were well below International Commission on Non‐Ionizing Radiation Protection (ICNIRP) guideline levels. Average night‐time ELF‐MFs (long‐term measurement from 10 pm to 6 am, geometric mean over households) above 100 nT were obtained in 2.3%, and RF‐EMFs above 1000 µW/m² in 7.1% of households. Highest ELF‐EFs were primarily due to lamps beside the bed (max = 166 V/m), and highest ELF‐MFs because of transformers of devices (max = 1030 nT) or high current of power lines (max = 380 nT). The highest values of RF‐EMFs were caused by DECT telephone base stations (max = 28979 µW/m²) and mobile phone base stations (max = 4872 µW/m²). Simple reduction measures resulted in an average decrease of 23 nT for ELF‐MFs, 23 V/m for ELF‐EFs, and 246 µW/m² for RF‐EMFs. A small but statistically significant correlation between ELF‐MF exposure and overall RF‐EMF levels of R = 0.16 (P = 0.008) was computed that was independent of type (flat, single family) and location (urban, rural) of houses. Bioelectromagnetics 31:200–208, 2010. © 2009 Wiley‐Liss, Inc.     

Forschungsschwerpunkte der paläobotanik und palynologie der Universität Wien

An initial accent on the investigation of megafossils by some well-known Austrian paleobotanists (like Ettinghausen, Stur, Unger) changed with time to a higher degree of research on the histology and anatomy of fossil plants, especially of wood and cuticles by the late E. Hofmann. Due to the large quantity of spores and pollen grains in peat, brown coal, salt rocks, phosphorites, clay and shales in Austria, the major topic of investigation at the present time is clearly devoted to palynology. Quaternary, Neogene and Permo-Triassic microfloras occupy the first place in research. Concerning the determination of pollen and morphologic studies of a selection of modern comparison species by all kinds of light and electron microscopy have been initiated to find out whether biospecific determination could be carried out in the Quaternary and Neogene. An example of pollen-morphological features within the genus Pinus is presented.The microflora of Upper Permian and Triassic sediments of the east Alpine salt rocks, their environment and inclusions receive comprehensive microphotographic documentation and will be backed from a stratigraphic point of view by sulphur-isotope investigations, which promise to be of great value in dating the Permo-Triassic boundary.