Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.     

Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus‐growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species‐specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?

Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.

     

Systematics and evolutionary history of butterflies in the “Taygetis clade” (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography

The so-called “Taygetis clade” is a group of exclusively Neotropical butterflies classified within Euptychiina, one of the largest subtribes in the subfamily Satyrinae. Since the distribution of the ten genera belonging to this group ranges throughout the entire Neotropics, from lowlands to lower montane habitats, it offers a remarkable opportunity to study the region’s biogeographic history as well as different scenarios for speciation in upland areas. We inferred a robust and well-sampled phylogeny using DNA sequences from four genes (4035 bp in total) using maximum parsimony and Bayesian inference. We estimated divergence times using the Bayesian relaxed clock method calibrated with node ages from previous studies. Ancestral ranges of distribution were estimated using the dispersal–extinction–cladogenesis (DEC) model as implemented in the program Lagrange. We propose several taxonomic changes and recognize nine well-supported natural genera within the “Taygetis clade”: Forsterinaria (subsuming Guaianaza syn. nov.), Parataygetis, Posttaygetis, Harjesia (excluding Harjesia griseola and Harjesia oreba), Pseudodebis (including Taygetomorpha syn. nov.,), Taygetina (subsuming Coeruleotaygetis syn. nov., Harjesia oreba comb. nov., Taygetis weymeri comb. nov. and Taygetis kerea comb. nov.), Taygetis (excluding Taygetis ypthima, Taygetis rectifascia, Taygetis kerea and Taygetis weymeri), and two new genera, one containing Harjesia griseola, and the other Taygetis ypthima and Taygetis rectifascia. The group diversified mainly during late Miocene to Pliocene, coinciding with the period of drastic changes in landscape configuration in the Neotropics. Major dispersals inferred from the Amazon basin towards northwestern South America, the Atlantic forests and the eastern slope of the Andes have mostly shaped the evolution and diversification of the group. Furthermore, expansion of larval dietary repertoire might have aided net diversification in the two largest genera in the clade, Forsterinaria and Taygetis.     

Keep Your Opponents Close: Social Context Affects EEG and fEMG Linkage in a Turn-Based Computer Game

In daily life, we often copy the gestures and expressions of those we communicate with, but recent evidence shows that such mimicry has a physiological counterpart: interaction elicits linkage, which is a concordance between the biological signals of those involved. To find out how the type of social interaction affects linkage, pairs of participants played a turn-based computer game in which the level of competition was systematically varied between cooperation and competition. Linkage in the beta and gamma frequency bands was observed in the EEG, especially when the participants played directly against each other. Emotional expression, measured using facial EMG, reflected this pattern, with the most competitive condition showing enhanced linkage over the facial muscle-regions involved in smiling. These effects were found to be related to self-reported social presence: linkage in positive emotional expression was associated with self-reported shared negative feelings. The observed effects confirmed the hypothesis that the social context affected the degree to which participants had similar reactions to their environment and consequently showed similar patterns of brain activity. We discuss the functional resemblance between linkage, as an indicator of a shared physiology and affect, and the well-known mirror neuron system, and how they relate to social functions like empathy.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Dynamics and Management of Stage-Structured Fish Stocks

With increasing fishing pressures having brought several stocks to the brink of collapse, there is a need for developing efficient harvesting methods that account for factors beyond merely yield or profit. We consider the dynamics and management of a stage-structured fish stock. Our work is based on a consumer-resource model which De Roos et al. (in Theor. Popul. Biol. 73, 47–62, 2008) have derived as an approximation of a physiologically-structured counterpart. First, we rigorously prove the existence of steady states in both models, that the models share the same steady states, and that there exists at most one positive steady state. Furthermore, we carry out numerical investigations which suggest that a steady state is globally stable if it is locally stable. Second, we consider multiobjective harvesting strategies which account for yield, profit, and the recovery potential of the fish stock. The recovery potential is a measure of how quickly a fish stock can recover from a major disturbance and serves as an indication of the extinction risk associated with a harvesting strategy. Our analysis reveals that a small reduction in yield or profit allows for a disproportional increase in recovery potential. We also show that there exists a harvesting strategy with yield close to the maximum sustainable yield (MSY) and profit close to that associated with the maximum economic yield (MEY). In offering a good compromise between MSY and MEY, we believe that this harvesting strategy is preferable in most instances. Third, we consider the impact of harvesting on population size structure and analytically determine the most and least harmful harvesting strategies. We conclude that the most harmful harvesting strategy consists of harvesting both adults and juveniles, while harvesting only adults is the least harmful strategy. Finally, we find that a high percentage of juvenile biomass indicates elevated extinction risk and might therefore serve as an early-warning signal of impending stock collapse.