The ESKAPE mobilome contributes to the spread of antimicrobial resistance and CRISPR-mediated conflict between mobile genetic elements

Mobile genetic elements (MGEs) mediate the shuffling of genes among organisms. They contribute to the spread of virulence and antibiotic resistance genes in human pathogens, including the particularly problematic group of ESKAPE pathogens, such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp. Here, we performed the first systematic analysis of MGEs, including plasmids, prophages, and integrative and conjugative/mobilizable elements (ICEs/IMEs), in the ESKAPE pathogens. We characterized over 1700 complete ESKAPE genomes and found that different MGE types are asymmetrically distributed across these pathogens. While some MGEs are capable of exchanging DNA beyond the genus (and phylum) barrier, horizontal gene transfer (HGT) is mainly restricted by phylum or genus. We further observed that most genes on MGEs have unknown functions and show intricate distribution patterns. Moreover, AMR genes and anti-CRISPRs are overrepresented in the ESKAPE mobilome. Our results also underscored species-specific trends shaping the number of MGEs, AMR, and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems. Finally, we found that CRISPR targets vary according to MGE type: while plasmid CRISPRs almost exclusively target other plasmids, ICEs/IME CRISPRs preferentially target ICEs/IMEs and prophages. Overall, our study highlights the general importance of the ESKAPE mobilome in contributing to the spread of AMR and mediating conflict among MGEs.

Decoding key transient inter-catalyst interactions in a metallaphotoredox-catalysed cross-electrophile coupling reaction

Metallaphotoredox chemistry has recently witnessed a renaissance through the use of abundant first-row transition metals combined with suitable photocatalysts. The intricate details arising from the combination of two (or more) catalytic components during the reaction and specially the inter-catalyst interactions remain poorly understood. As a representative example of a catalytic process featuring such intricacies, we here present a meticulous study of the mechanism of a cobalt-organophotoredox catalysed allylation of aldehydes. Importantly, the commonly proposed elementary steps in reductive metallaphotoredox chemistry are more complex than previously assumed. After initial reductive quenching, a transient charge-transfer complex forms that interacts with both the transition-metal catalyst, as well as the catalytic base. Surprisingly, the former interaction leads to deactivation due to induced charge recombination, while the latter promotes deprotonation of the electron donor, which is a crucial step in order to promote productive catalysis, but is often neglected. Due to the low efficiency of this process, the overall catalytic reaction is photon-limited and the cobalt catalyst remains in a dual resting state awaiting photoinduced reduction. These new insights are of general importance to the synthetic community, as photoredox chemistry has become a powerful tool used in the creation of elusive compounds through carbon-carbon bond formations. Understanding the underlying factors that determine the efficiency of such reactions provides a conceptually stronger reactivity paradigm to empower future approaches to synthetic challenges that rely on dual metallaphotoredox catalysis.

Unexpected arabinosylation after humanization of plant protein N-glycosylation

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

VISCOSITY IN SEAKEEPING

The paper addresses the issue of actuality in ship hydrodynamics: the estimation of ship’s linear and angular oscillations with respect to the state of equilibrium. The prediction of seakeeping properties raises a question about a relative importance of viscous and free-surface effects (Quérard et al. 2009), yet this question remains of more general importance in fluid mechanics, since it is related to the dynamic characteristics of objects/bodies immersed in a liquid. From a theoretical standpoint, the problem refers to flows with moving boundaries. It can also be considered in terms of fluid-structure interaction (FSI), however, not necessarily linked with the computation of the body deformation and stresses due to the flow. As the Author correctly notices, the computational solution to this problem in its full setup reveals to be extremely costly due to the 3D and unsteady nature of the fluid motion under turbulent flow conditions at nominally high Reynolds numbers (Re~109, as stated by the Author in Tab. 1) in presence of the free surface. For this reason, the full solution, or direct numerical simulation (DNS), of the governing Navier-Stokes (N-S) equations at these Re will remain unfeasible in the foreseeable future; see, e.g., Pozorski (2017) for an estimation of the DNS capability in simple wall-bounded turbulent flows. The situation gets even worse in ship hydrodynamics when a DNS of fluid flow would need to be coupled to the dynamics of the rigid body (of complex geometry, usually).

Open science practices and risks arising from misuse of biological research

The proliferation of open science may inadvertently increase the chance of deliberate or accidental misuse of research. Here, we examine the interaction between open science practices and biosecurity and biosafety to identify risks and opportunities for risk mitigation. We argue that open data, code, and materials may increase risks from research with misuse potential, despite their general importance. For instance, increased access to protocols, datasets, and computational methods for viral engineering may increase the risk of release of enhanced pathogens. For this dangerous subset of research, both open science and biosecurity goals may be achieved by using access-controlled repositories or application programming interfaces. The increased use of preprints could challenge any strategy for risk mitigation that relies on assessment at the publication stage, emphasising the need for earlier oversight in the research lifecycle. Preregistration of research, a practice promoted by the open science community, provides an opportunity for achieving biosecurity risk assessment at the conception of research. Open science and biosecurity experts have an important role to play in enabling responsible research with maximal societal benefit.

Mineral stability, brine development and rock-fluid reaction at repository-relevant temperatures (<i>T</i> < 200 °C) in the potential host rock rock salt

For a repository of heat generating radioactive waste, the thermal behaviour of the host rock and the impact of temperature increase on rock properties is of general importance. In the German Site Selection Act (2017), the maximum temperature of the container surface is preliminarily limited to 100 ∘C but this limit might change in the future based on scientific and technological findings. Rock salt, as one of the possible host rocks, consists predominantly of halite with varying amounts of accessory minerals (e.g., Hudec and Jackson, 2007); however, some lithological units within a salt deposit, e.g. potash seams, show a different mineralogical composition with high amounts of potash minerals. Most of them are not very stable regarding temperature resistance and stress, contain water in the crystal lattice, and therefore react sensitively to changes in the environment. The melting point of most evaporated minerals is higher than the expected temperatures in a repository but dehydration and partial melting might occur at relevant temperatures, depending on the confining pressure. For example, the temperature of dehydration of carnallite is ca. 80 ∘C at 0.1 MPa confining pressure but increases to ca. 145 ∘C at 10 MPa confining pressure (Kern and Franke, 1986). The melting point of carnallite increases from ca. 145∘C/8MPa to ca. 167∘C/24MPa, which corresponds to a depth of ca. 1000 m. Depending on the mineral paragenesis and composition of saline solutions, different minerals develop with increasing temperature. For instance, a salt rock with an initial composition of kieserite + kainite + carnallite + solution R (25 ∘C) reacts solely to kieserite and solution R, when the temperature increases to 78 ∘C. A rock with a composition of kieserite + carnallite + bischofite + solution Z (25 ∘C) reacts to kieserite + carnallite from 25 to 50 ∘C, from 50 to 73 ∘C only kieserite is stable, and at temperatures >73 ∘C kieserite and bischofite develop (Usdowski and Dietzel, 1998). For the construction of an underground repository, the mineralogical composition of the host rocks and fluids have to be evaluated carefully and play an important role for the site selection and design of the underground facility.

Non-native plant drives the spatial dynamics of its herbivores: the case of black locust (Robinia pseudoacacia) in Europe

Non-native plants typically benefit from enemy release following their naturalization in non-native habitats. However, over time, herbivorous insects specializing on such plants may invade from the native range and thereby diminish the benefits of enemy release that these plants may experience. In this study, we compare rates of invasion spread across Europe of three North American insect folivores: the Lepidoptera leaf miners Macrosaccus robiniella and Parectopa robiniella, and the gall midge Obolodiplosis robiniae, that specialize on Robinia pseudoacacia. This tree species is one of the most widespread non-native trees in Europe. We find that spread rates vary among the three species and that some of this variation can be explained by differences in their life history traits. We also report that geographical variation in spread rates are influenced by distribution of Robinia pseudoacacia, human population and temperature, though Robinia pseudoacacia occurrence had the greatest influence. The importance of host tree occurrence on invasion speed can be explained by the general importance of hosts on the population growth and spread of invading species.

The Molecular Basis for Erns Dimerization in Classical Swine Fever Virus

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein Erns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant Erns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form Erns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of Erns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

Functional (un-)Coupling: Impairment, Compensation, and Future Progression in Alzheimer's Disease

Background: Functional (un-)coupling (task-related change of functional connectivity) between different sites of the brain is a mechanism of general importance for cognitive processes. In Alzheimer's disease (AD), prior research identified diminished cortical connectivity as a hallmark of the disease. However, little is known about the relation between the amount of functional (un-)coupling and cognitive performance and decline in AD. Method: Cognitive performance (based on CERAD-Plus scores) and electroencephalogram (EEG)-based functional (un-)coupling measures (connectivity changes from rest to a Face-Name-Encoding task) were assessed in 135 AD patients (age: M = 73.8 years; SD = 9.0). Of these, 68 patients ( M = 73.9 years; SD = 8.9) participated in a follow-up assessment of their cognitive performance 1.5 years later. Results: The amounts of functional (un-)coupling in left anterior-posterior and homotopic interhemispheric connections in beta1-band were related to cognitive performance at baseline (β = .340; p < .001; β = .274; P = .001, respectively). For both markers, a higher amount of functional coupling was associated with better cognitive performance. Both markers also were significant predictors for cognitive decline. However, while patients with greater functional coupling in left anterior-posterior connections declined less in cognitive performance (β = .329; P = .035) those with greater functional coupling in interhemispheric connections declined more (β = −.402; P = .010). Conclusion: These findings suggest an important role of functional coupling mechanisms in left anterior–posterior and interhemispheric connections in AD. Especially the complex relationship with cognitive decline in AD patients might be an interesting aspect for future studies.

Structural and functional consequences of NEDD8 phosphorylation

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins.