Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids

Abstract Electrofusion between cells of yeast strains with different genetic markers in isotonic sorbitol solutions leads to high yields of hybrids when 0.1 mM Ca²⁺ and 0.5 mM Mg²⁺ salts are aded. On average, 1000–2000 hybrids are obtained when electrofusion is performed (in a helical chamber) compared to a yield of about 40–120 in the absence of these bivalent cations. A further increase in yield can be achieved by the addition of 1 mg/ml albumin, which results in up to 4000 hybrids per experimental run. The entire fusion process leads to very reproducible results in the presence of these substances.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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The Legal Regulation of Floats and Gliders—In Quest of a New Regime?

This article analyzes the legal status of unmanned instruments (particularly, floats and gliders) for observation purposes in the ocean environment. These new kinds of instruments are being deployed by the thousands into the oceans, not the least as part of the Argo Project of the International Oceanographic Commission. Their uncontrolled drifting has raised legal questions, especially when such instruments enter waters subject to the jurisdiction of foreign states. The authors argue that the current international legal framework is insufficient to address the pertinent issues, and that a new legal regime is needed.     

Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli

OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.     

Co‐occurrence patterns of trees along macro‐climatic gradients and their potential influence on the present and future distribution of Fagus sylvatica L.

Aim During recent and future climate change, shifts in large‐scale species ranges are expected due to the hypothesized major role of climatic factors in regulating species distributions. The stress‐gradient hypothesis suggests that biotic interactions may act as major constraints on species distributions under more favourable growing conditions, while climatic constraints may dominate under unfavourable conditions. We tested this hypothesis for one focal tree species having three major competitors using broad‐scale environmental data. We evaluated the variation of species co‐occurrence patterns in climate space and estimated the influence of these patterns on the distribution of the focal species for current and projected future climates. Location Europe. Methods We used ICP Forest Level 1 data as well as climatic, topographic and edaphic variables. First, correlations between the relative abundance of European beech (Fagus sylvatica) and three major competitor species (Picea abies, Pinus sylvestris and Quercus robur) were analysed in environmental space, and then projected to geographic space. Second, a sensitivity analysis was performed using generalized additive models (GAM) to evaluate where and how much the predicted F. sylvatica distribution varied under current and future climates if potential competitor species were included or excluded. We evaluated if these areas coincide with current species co‐occurrence patterns. Results Correlation analyses supported the stress‐gradient hypothesis: towards favourable growing conditions of F. sylvatica, its abundance was strongly linked to the abundance of its competitors, while this link weakened towards unfavourable growing conditions, with stronger correlations in the south and at low elevations than in the north and at high elevations. The sensitivity analysis showed a potential spatial segregation of species with changing climate and a pronounced shift of zones where co‐occurrence patterns may play a major role. Main conclusions Our results demonstrate the importance of species co‐occurrence patterns for calibrating improved species distribution models for use in projections of climate effects. The correlation approach is able to localize European areas where inclusion of biotic predictors is effective. The climate‐induced spatial segregation of the major tree species could have ecological and economic consequences.