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Codeine is a natural opiate extracted from opium poppy (Papaver somniferum) and used to alleviate mild to moderate pain. The analgesic effect of this molecule results from its metabolism into morphine which is an agonist of the mu opioid receptor. Morphine's major metabolite morphine-3-glucuronide induces both thermal and mechanical hypersensitivies while codeine-6-glucuronide has been proposed to be antinociceptive. However, sex differences in codeine antinociceptive effect and pharmacokinetics were barely studied. To this purpose, we injected male and female mice with codeine (2.5, 5, 10, 20 and 40 mg/kg) and thermal hypersensitivity was assessed 30 min after injection using the Tail Immersion Test. Moreover, both peripheral and central metabolism of codeine were evaluated respectively in the blood or pain-related brain structures in the central nervous system. The amounts of codeine and its metabolites were quantified using the isotopic dilution method by liquid chromatography coupled to a mass spectrometer. Our results show that codeine induces a greater antinociceptive effect in males than females mice independently of the estrous cycle. Moreover, major sex differences were found in the peripheral metabolism of this molecule, with higher amounts of pronociceptive morphine-3-glucuronide and less antinociceptive codeine-6-glucuronide in females than in males. Concerning the central metabolism of codeine, we did not find significant sex differences in pain-related brain structures. Collectively, these findings support a greater codeine antinociceptive effect in males than females in mice. These sex differences could be influenced by a higher peripheral metabolism of this molecule in female mice rather than central metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The invasion of non-native amphipods often leads to severe changes in the composition of aquatic communities and may also result in the local replacement of native species. Particularly, a lower risk of being preyed upon resulting from high swimming velocities can be an advantage in interspecific competition. Furthermore, as swimming velocities are ecologically important, they are often used in ecotoxicological studies to estimate effects of different stressors. However, knowledge on swimming velocities of native and non-native amphipods is still rather limited. We experimentally investigated the maximum swimming velocities of three native and three non-native amphipods via video analyses in the laboratory. Results showed that non-native species reach higher maximum swimming velocities compared to natives probably leading to a higher predation success and reduced risk of being preyed upon. Additionally, body length correlates positively with swimming velocities, except for the invader Dikerogammarus villosus. As D. villosus can be cannibalistic, the high swimming velocities of the small individuals may reduce the intraspecific predation and may increase the survival rates of smaller specimen. Hence, knowledge about the swimming velocities of species contribute to the understanding of interspecific competition among species and might support explanation approaches for the success of invasive species. Furthermore, it provides baselines for ecotoxicological studies of stressor impacts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity., (© 2024. The Author(s).)

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/β-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/β-hydrolase fold formed by eight β-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The learning curve in minimally invasive surgery (MIS) is lengthened compared to open surgery. It has been reported that structured feedback and training in teams of two trainees improves MIS training and MIS performance. Annotation of surgical images and videos may prove beneficial for surgical training. This study investigated whether structured feedback and video debriefing, including annotation of critical view of safety (CVS), have beneficial learning effects in a predefined, multi-modal MIS training curriculum in teams of two trainees., Methods: This randomized-controlled single-center study included medical students without MIS experience (n = 80). The participants first completed a standardized and structured multi-modal MIS training curriculum. They were then randomly divided into two groups (n = 40 each), and four laparoscopic cholecystectomies (LCs) were performed on ex-vivo porcine livers each. Students in the intervention group received structured feedback after each LC, consisting of LC performance evaluations through tutor-trainee joint video debriefing and CVS video annotation. Performance was evaluated using global and LC-specific Objective Structured Assessments of Technical Skills (OSATS) and Global Operative Assessment of Laparoscopic Skills (GOALS) scores., Results: The participants in the intervention group had higher global and LC-specific OSATS as well as global and LC-specific GOALS scores than the participants in the control group (25.5 ± 7.3 vs. 23.4 ± 5.1, p = 0.003; 47.6 ± 12.9 vs. 36 ± 12.8, p < 0.001; 17.5 ± 4.4 vs. 16 ± 3.8, p < 0.001; 6.6 ± 2.3 vs. 5.9 ± 2.1, p = 0.005). The intervention group achieved CVS more often than the control group (1. LC: 20 vs. 10 participants, p = 0.037, 2. LC: 24 vs. 8, p = 0.001, 3. LC: 31 vs. 8, p < 0.001, 4. LC: 31 vs. 10, p < 0.001)., Conclusions: Structured feedback and video debriefing with CVS annotation improves CVS achievement and ex-vivo porcine LC training performance based on OSATS and GOALS scores., (© 2024. The Author(s).)

The environmental fate of plastic particles in water bodies is influenced by microbial biofilm formation. Invertebrate grazers may be affected when foraging biofilms on plastics compared to biofilms on natural substrata but the mechanistic basis for these effects is unknown. For analyzing these effects in ecotoxicological assays stable and reproducible biofilm communities are required that are related to the environmental site of interest. Here, a defined biofilm community was established and used to perform grazing experiments with a freshwater snail. For this, snippets of different plastic materials were incubated in the photic zone of three different freshwater sites. Amplicon sequencing of biofilms formed on these snippets showed that the site of incubation and not the plastic material dominated the microbial community composition. From these biofilms, individual microbial strains as well as photoautotrophic consortia were isolated; these consortia consisted of heterotrophic bacteria that were apparently nourished by microalga. While biofilms formed by defined dual cultures of a microalga and an Alphaproteobacterium were not accepted by the snail P. fontinalis , a photoautotrophic consortium (Co&#95;3) sustained growth and metabolism of this grazer. Amplicon sequencing revealed that consortium Co&#95;3, which could be stably maintained on solid medium under photoautotrophic conditions, reproducibly formed biofilms of a defined composition on three different plastic materials and on glass surfaces. In conclusion, our study shows that the generation of domesticated photoautotrophic microbial communities is a valid novel approach for establishing laboratory ecotoxicological assays with higher environmental relevance than those based on defined microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bakenhus, Jongsma, Michler-Kozma, Hölscher, Gabel, Holert and Philipp.)

Introduction: The learning curve in minimally invasive surgery (MIS) is steep compared to open surgery. One of the reasons is that training in the operating room in MIS is mainly limited to verbal instructions. The iSurgeon telestration device with augmented reality (AR) enables visual instructions, guidance, and feedback during MIS. This study aims to compare the effects of the iSurgeon on the training of novices performing repeated laparoscopic cholecystectomy (LC) on a porcine liver compared to traditional verbal instruction methods., Methods: Forty medical students were randomized into the iSurgeon and the control group. The iSurgeon group performed 10 LCs receiving interactive visual guidance. The control group performed 10 LCs receiving conventional verbal guidance. The performance assessment using Objective Structured Assessments of Technical Skills (OSATS) and Global Operative Assessment of Laparoscopic Skills (GOALS) scores, the total operating time, and complications were compared between the two groups., Results: The iSurgeon group performed LCs significantly better (global GOALS 17.3 ± 2.6 vs. 16 ± 2.6, p ≤ 0.001, LC specific GOALS 7 ± 2 vs. 5.9 ± 2.1, p ≤ 0.001, global OSATS 25.3 ± 4.3 vs. 23.5 ± 3.9, p ≤ 0.001, LC specific OSATS scores 50.8 ± 11.1 vs. 41.2 ± 9.4, p ≤ 0.001) compared to the control group. The iSurgeon group had significantly fewer intraoperative complications in total (2.7 ± 2.0 vs. 3.6 ± 2.0, p ≤ 0.001) than the control group. There was no difference in operating time (79.6 ± 25.7 vs. 84.5 ± 33.2 min, p = 0.087)., Conclusion: Visual guidance using the telestration device with AR, iSurgeon, improves performance and lowers the complication rates in LCs in novices compared to conventional verbal expert guidance., (© 2023. The Author(s).)

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil-based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C 2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C 2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN-20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Purpose: In rodents, morphine antinociception is influenced by sex. However, conflicting results have been reported regarding the interaction between sex and morphine antinociceptive tolerance. Morphine is metabolised in the liver and brain into morphine-3-glucuronide (M3G). Sex differences in morphine metabolism and differential metabolic adaptations during tolerance development might contribute to behavioural discrepancies. This article investigates the differences in peripheral and central morphine metabolism after acute and chronic morphine treatment in male and female mice., Experimental Approach: Sex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. After acute and chronic morphine treatment, morphine and M3G metabolic kinetics in the blood were evaluated using LC-MS/MS. They were also quantified in several CNS regions. Finally, the blood-brain barrier (BBB) permeability of M3G was assessed in male and female mice., Key Results: This study demonstrated that female mice showed weaker morphine antinociception and faster induction of tolerance than males. Additionally, female mice showed higher levels of M3G in the blood and in several pain-related CNS regions than male mice, whereas lower levels of morphine were observed in these regions. M3G brain/blood ratios after injection of M3G indicated no sex differences in M3G BBB permeability, and these ratios were lower than those obtained after injection of morphine., Conclusion: These differences are attributable mainly to morphine central metabolism, which differed between males and females in pain-related CNS regions, consistent with weaker morphine antinociceptive effects in females. However, the role of morphine metabolism in antinociceptive tolerance seemed limited., Linked Articles: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc., (© 2022 The British Pharmacological Society.)

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (MarR) family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites., Competing Interests: Declaration of interests The authors declare that they have no conflict of interest., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB 1 and AFG 2 ) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB 1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG 2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization., (© 2023. The Author(s).)

In the present book chapter we illustrate the state-of-the-art of time-resolved small-angle neutron scattering (TR-SANS) by a concrete example of a dynamic bio-macromolecular system, i.e., regulated protein degradation by the archaeal PAN-20S proteasome complex. We present the specific and unique structural information that can be obtained by this approach, in combination with bio-macromolecular deuteration and online spectrophotometric measurements of a fluorescent substrate (GFP). The complementarity with atomic-resolution structural biology techniques (SAXS, NMR, crystallography and cryo-EM) and with the advent of atomic structure prediction are discussed, as well as the respective limitations and future perspectives., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although self-imposed social isolation is an important way of reducing the risk of COVID-19 infection, previous research indicates that this behaviour varies substantially between different groups and individuals. Socially marginalized people are generally less involved in protective health behaviours, but there are few studies of their COVID-19 protective behaviours. The aims of the paper are therefore to: 1) compare self-imposed social isolation to avoid COVID-19 among socially marginalized groups, and to 2) examine factors influencing this, focusing especially on the role of social capital, risk awareness and sources of information about COVID-19. The study is based on survey data (N = 173) from people who are clients of social care organisations in Estonia, Norway, Hungary and Portugal. The sample involves clients living: a) in their homes, b) in facilities, and c) on the street or under temporary arrangements. Results indicate that the level of social isolation among the marginalized groups is comparable to that of the general population in previous studies. As hypothesized, we find that respondents living on the street or under temporary arrangements engage in less self-imposed social isolation than e.g. the respondents living in their homes. We also find lower levels of risk awareness, social capital and trust in authorities' information about COVID-19 among people living on the street or under temporary arrangements. Only linking social capital and trust in authorities' information was significantly related to respondents' social isolation, and not worry for COVID-19 infection. Thus, it seems that respondents largely self-isolated because of "duty" and not worry for infection., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tor-Olav Naevestad reports financial support was provided by European Union., (© 2022 The Authors.)

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å -1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å -1 , but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H 2 O and in D 2 O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å -1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set., (open access.)

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein-RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein-RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems., (open access.)

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impβ/Rev complex. We show that Impβ binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impβ cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impβ-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impβ cargos., (© 2022 Spittler et al.)

While social vulnerability in the face of disasters has received increasing academic attention, relatively little is known about the extent to which that knowledge is reflected in practice by institutions involved in disaster management. This study charts the practitioners' approaches to disaster vulnerability in eight European countries: Belgium; Estonia; Finland; Germany; Hungary; Italy; Norway; and Sweden. It draws on a comparative document analysis and 95 interviews with disaster managers and reveals significant differences across countries in terms of the ontology of vulnerability, its sources, reduction strategies, and the allocation of related duties. To advance the debate and provide conceptual clarity, we put forward a heuristic model to facilitate different understandings of vulnerability along the dimensions of human agency and technological structures as well as social support through private relations and state actors. This could guide risk analysis of and planning for major hazards and could be adapted further to particular types of disasters., (© 2021 The Authors Disasters © 2021 ODI.)

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Morphine remains the gold standard painkiller available to date to relieve severe pain. Morphine metabolism leads to the production of two predominant metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). This metabolism involves uridine 5'-diphospho-glucuronosyltransferases (UGTs), which catalyze the addition of a glucuronide moiety onto the C3 or C6 position of morphine. Interestingly, M3G and M6G have been shown to be biologically active. On the one hand, M6G produces potent analgesia in rodents and humans. On the other hand, M3G provokes a state of strong excitation in rodents, characterized by thermal hyperalgesia and tactile allodynia. Its coadministration with morphine or M6G also reduces the resulting analgesia. Although these behavioral effects show quite consistency in rodents, M3G effects are much more debated in humans and the identity of the receptor(s) on which M3G acts remains unclear. Indeed, M3G has little affinity for mu opioid receptor (MOR) (on which morphine binds) and its effects are retained in the presence of naloxone or naltrexone, two non-selective MOR antagonists. Paradoxically, MOR seems to be essential to M3G effects. In contrast, several studies proposed that TLR4 could mediate M3G effects since this receptor also appears to be essential to M3G-induced hyperalgesia. This review summarizes M3G's behavioral effects and potential targets in the central nervous system, as well as the mechanisms by which it might oppose analgesia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gabel, Hovhannisyan, Berkati and Goumon.)

Microplastic debris affects marine as well as freshwater ecosystems and an increasing number of studies have documented the contamination in aquatic environments worldwide. However, while the research focuses on oceans and larger rivers, little is known about the situation in smaller rivers within rural catchments. Since microplastics pose various risks to ecosystems, wildlife and human health, it is important to identify potential sources, sinks and transport patterns, which are probably different for small rivers. In this study, we investigate the contamination with microplastic debris of the river Ems, representing a smaller river in Northwest Germany with an agricultural catchment. We hypothesised that with increasing river length the plastic concentration increases, especially downstream of towns, waste water treatment plant (WWTP) effluents and major tributaries as they may be important point sources of microplastics. We collected 36 surface water samples at 18 sampling sites within the first 70 km using manual driftnets. We sampled every 7 km and upstream and downstream of three larger towns, four major tributaries and four WWTP effluents. Overall, we found 1.54 ± 1.54 items m -3 , which corresponds to the plastic concentrations in larger streams. However, the shape of the detected items differed as we did not find potential primary microplastic. Furthermore, the pattern contradicts our assumption, that the contamination increased with distance to the river's source. Downstream of towns, we found significantly less floating microplastic indicating possible sinks due to sedimentation at sites with slowing flow velocity caused by weirs in towns. Hence, the non-linear distribution pattern of microplastics indicates potential sinks of microplastics due to flow alterations on the river course. This should be considered in future studies modelling microplastic distribution and transport. Furthermore, studies especially in smaller rivers are urgently needed to identify and quantify sources and sinks and to find applicable solutions to reduce microplastic loads., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

With an ever-increasing amount of plastic pollution in the various aquatic ecosystems around the world, the effects on organisms are still not fully understood. Most studies focus on direct effects posed by plastic intake or entanglement, but plastic debris can also affect primary production of biofilms and have an indirect impact on its consumers. This study investigates the primary production on three common plastic types in freshwater and its food quality for a benthic grazer. We hypothesized that different polymer types affect biofilm composition as well as the life parameters of its consumers. We incubated polyethylene (PE), polyethylene terephthalate (PET) and polystyrene (PS) as well as glass (control) in a productive freshwater creek for natural biofilm establishment. To account for changes in the environmental conditions, the experiment was conducted twice during winter and late spring, respectively. These biofilms were offered to the freshwater gastropod Physa fontinalis as sole food source. Growth and reproduction of the snails were measured to monitor sublethal effects. Additionally, biofilm composition was observed using confocal laser scanning microscopy (CLSM). In winter, snails feeding off PET and PE showed a significantly lower egg production and lower growth rates were observed on PET. No such effects occurred in spring. CLSM data revealed, that algal growth was significantly lower on PE and PET during the winter treatment compared to PS and glass. Since we could only find these effects during the colder and darker months (January-March), the microbial colonization on PE and PET was inhibited by the substrate under less favorable conditions of temperature and light. Hence, benign conditions may mask the adverse effects of microplastic on food webs. Our findings show that future studies on the plastisphere will need to consider such variations to further understand the influence of plastic pollution on primary production and higher trophic levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We present an overview of time-resolved small-angle neutron scattering (TR-SANS) applied to biological systems, with a focus on bio-macromolecules and assemblies they form, together with practical guidelines. After a brief introduction to the theory and practice of SANS, we present the general setup and specifics of time-resolved experiments, as well as an overview of diverse experimental results and applications from the past ≈25years. Subsequently, we provide guidelines and practical instructions for the design, planning and execution for TR-SANS experiments, as a function of the time- and length-scales of the biological processes of interest, the availability of sample amount and deuterium labeling, and the structural information sought. We conclude with a discussion of the most recent instrumental and sample environment developments and perspectives for the future., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Multiple studies in freshwater environments have verified that microplastic particles are present in water columns, sediment, and aquatic organisms. These studies indicated that certain freshwater ecosystems may act as temporary sinks of microplastic particles, leading to accumulation in the sediment and the ingestion by benthic organisms. Polyethylene terephthalate (PET) is one of the non-buoyant polymers that has been frequently found in aquatic sediments. This study aims to investigate a possible transfer of PET microfibers from aquatic to the terrestrial habitats and addressed selected effects (i.e. survival, general stress response, and growth) of PET microfibers using Chironomus riparius, a frequently applied model organism in ecotoxicological research. To assess the growth and development of C. riparius, a modified 28-day sediment chronic toxicity test was conducted, in which the main endpoint is time until emergence of the larvae. In this assay, C. riparius were exposed to artificial sediments spiked with PET microfibers. In addition, weight and head capsule lengths of the larvae were also measured. As a general stress response marker on the molecular level, Heat Shock Protein 70 (HSP70) levels were measured in two involved life stages, i.e. larvae and adults. Using staining method, ingestion of PET microfibers was verified in the adult sample. Our results clearly demonstrated that ingested microfibers by C. riparius larvae can be carried through subsequent life stages and end up in the adults. Accordingly, this is the first proof of aquatic-terrestrial transfer of PET microfibers for C. riparius. However, toxicity test results showed that there was no significant effect on the time until emergence, weight or head capsule lengths in the organisms exposed to PET microfibers compared to control organisms. HSP70 measurements showed no significant effects between control and exposure groups in the same life stage. The result suggests that PET microfibers in the applied concentration do not exert adverse effects both on organism and subcellular level in one generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice., Competing Interests: Declaration of Interests F.B. is an employee of the GSK group of companies. All the other authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The study of complex and dynamic biomolecular assemblies is a key challenge in structural biology and requires the use of multiple methodologies providing complementary spatial and temporal information. NMR spectroscopy is a powerful technique that allows high-resolution structure determination of biomolecules as well as investigating their dynamic properties in solution. However, for high-molecular-weight systems, such as biomolecular complexes or multi-domain proteins, it is often only possible to obtain sparse NMR data, posing significant challenges to structure determination. Combining NMR data with information obtained from other solution techniques is therefore an attractive approach. The combination of NMR with small-angle X-ray and/or neutron scattering has been shown to be particularly fruitful. These scattering approaches provide low-resolution information of biomolecules in solution and reflect ensemble-averaged contributions of dynamic conformations for scattering molecules up to megadalton molecular weight. Here, we review recent developments in the combination of nuclear magnetic resonance spectroscopy (NMR) and small-angle scattering (SAS) experiments. We briefly outline the different types of information that are provided by these techniques. We then discuss computational methods that have been developed to integrate NMR and SAS data, particularly considering the presence of dynamic structural ensembles and flexibility of the investigated biomolecules. Finally, recent examples of the successful combination of NMR and SAS are presented to illustrate the utility of their combination., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Equipping health systems with suitable incentives for efficient resource allocation remains a major health policy challenge. This study examines the impacts of 2015 regulatory changes in Danish dental care which aimed at effectuating a transition from six-to-twelve-monthly dental recall intervals, for every patient, towards a model where patients with higher need receive dental recalls systematically more frequently than patients with lower need. Exploiting administrative data from the years 2012-2016 from the Danish National Health Insurance database containing 72,155,539 treatment claims for 3,759,721 unique patients, we estimated a series of interrupted time-series regression models with patient-level fixed-effects. In comparison to the pre-reform period, the proportion of patients with recall intervals of up to 6 months was by 1.2%-points larger post-implementation; that of patients with 6-12-monthly recalls increased by 0.7%-points; that of patients with more than 12-monthly dental recalls decreased by 1.9%-points. The composition of care shifted more substantially: the proportion of treatment sessions including preventive care increased by 31.5%-points (95%-CI: 31.4;31.6); that of sessions including scaling increased by 24.1%-points (24.0;24.2); that of sessions including diagnostics decreased by 34.5%-points (34.4;34.6). These findings suggest that dental care providers may have responded differently to regulatory changes than intended by the health policy., (© 2020 The Authors. Health Economics published by John Wiley & Sons Ltd.)

In 2012, The Netherlands established the so-called "free market experiment", which allowed providers of dental care to set the prices for their dental services themselves. The introduction of market mechanisms is intended to improve the quality of care and to contribute to cost containment, but increasing health expenditures for citizens have been observed in this context. Using large-volume health insurance claims data and exploiting the 2012 experiment in Dutch dental care, we identified the effects of a liberalization of service prices. Using pooled regression with individual fixed effects, we analyzed changes in utilization patterns of prevention-oriented dental services in response to the experiment as well as the elasticities in demand in response to variations in out-of-pocket (OOP) prices. We found substantial increases in prices and patients' OOP contributions for dental services following the liberalization with differences in increases between types of services. In response to the experiment, the proportion of treatment sessions containing preventive-oriented services decreased significantly by 3.4% among adults and by 5.3% for children and adolescents. Estimates of short-run price elasticities of demand for different services point towards differences in price sensitivity. One potential explanation for the observed variations in prices and utilization could be different extents of asymmetric information for first-stage and follow-on services. Price liberalization seems to have affected the composition of treatment sessions towards a decreasing use of preventive services, suggesting a shift in the reason for seeing a dental care provider from a regular-preventive perspective to a symptom-based restorative approach.

2'-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate-guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles., Competing Interests: AG, FG, JK, TC No competing interests declared, (© 2020, Graziadei et al.)

Background: Tamoxifen is used to treat breast cancer and cancer recurrences. After administration, tamoxifen is converted into two more potent antitumor compounds, 4OH-tamoxifen and endoxifen by the CYP3A4/5 and 2D6 enzymes in human. These active compounds are inactivated by the same UDP-glucuronosyltransferase isoforms as those involved in the metabolism of morphine. Importantly, cancer-associated pain can be treated with morphine, and the common metabolic pathway of morphine and tamoxifen suggests potential clinically relevant interactions. Methods: Mouse liver microsomes were used to determine the impact of morphine on 4OH-tamoxifen metabolism in vitro . For in vivo experiments, female mice were first injected with tamoxifen alone and then with tamoxifen and morphine. Blood was collected, and LC-MS/MS was used to quantify tamoxifen, 4OH-tamoxifen, N-desmethyltamoxifen, endoxifen, 4OH-tamoxifen-glucuronide, and endoxifen-glucuronide. Results: In vitro , we found increased K m values for the production of 4OH-tamoxifen-glucuronide in the presence of morphine, suggesting an inhibitory effect on 4OH-tamoxifen glucuronidation. Conversely, in vivo morphine treatment decreased 4OH-tamoxifen levels in the blood while dramatically increasing the formation of inactive metabolites 4OH-tamoxifen-glucuronide and endoxifen-glucuronide. Conclusions: Our findings emphasize the need for caution when extrapolating results from in vitro metabolic assays to in vivo drug metabolism interactions. Importantly, morphine strongly impacts tamoxifen metabolism in mice. It suggests that tamoxifen efficiency could be reduced when both drugs are co-administered in a clinical setting, e.g., to relieve pain in breast cancer patients. Further studies are needed to assess the potential for tamoxifen-morphine metabolic interactions in humans., (Copyright © 2020 Gabel, Aubry, Hovhannisyan, Chavant, Weinsanto, Maduna, Darbon and Goumon.)

Small-angle neutron scattering (SANS) provides structural information on biomacromolecules and their complexes in dilute solutions at the nanometer length scale. The overall dimensions, shapes, and interactions can be probed and compared to information obtained by complementary structural biology techniques such as crystallography, NMR, and EM. SANS, in combination with solvent H 2 O/D 2 O exchange and/or deuteration, is particularly well suited to probe the internal structure of RNA-protein (RNP) complexes since neutrons are more sensitive than X-rays to the difference in scattering length densities of proteins and RNA, with respect to an aqueous solvent. In this book chapter we provide a practical guide on how to carry out SANS experiments on RNP complexes, as well as possibilities of data analysis and interpretation.

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. They could be considered as modular building blocks assembled in distinct fashion and connected by linkers of varying lengths and sequences. Due to their intrinsic flexibility, these linkers provide proteins a subtle way to modulate interactions and explore a wide range of conformational space. In the present study, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 UIM-SH3 dual domain protein with respect to molecular recognition. We have engineered several constructs of UIM-SH3 with different length linkers or domain deletion. By means of SAXS and NMR experiments, we have shown that the modification of the linker modifies the flexibility and the dynamics of UIM-SH3. Indeed, the global tumbling of both the UIM and SH3 domain is different but not independent from each other while the length of the linker has an impact on the ps-ns time scale dynamics of the respective domains. Finally, the modification of the flexibility and dynamics of the linker has a drastic effect on the interaction of UIM-SH3 with Lys63-linked diubiquitin with a roughly eight-time weaker dissociation constant.

Background: The evidence that large pay-for-performance schemes improve the health of populations is mixed-evidence regarding locally implemented schemes is limited., Objective: This study evaluates the effects in Stoke-on-Trent of a local, multifaceted Quality Improvement Framework including pay for performance in general practice introduced in 2009 in the context of the national Quality and Outcomes Framework that operated from 2004., Methods: We compared age-standardized mortality data from all 326 local authorities in England with the rates in Stoke-on-Trent using Difference-in-Differences, estimating a fixed-effects linear regression model with an interaction effect., Results: In addition to the existing downward trend in cardiovascular deaths, we find an additional annual reduction of 36 deaths compared with the national mean for coronary heart disease and 13 deaths per 100000 from stroke in Stoke-on-Trent. Compared with the national mean, there was an additional reduction of 9 deaths per 100000 people per annum for coronary heart disease and 14 deaths per 100000 people per annum for stroke following the introduction of the 2009 Stoke-on-Trent Quality Improvement Framework., Conclusion: There are concerns about the unintended consequences of large pay-for-performance schemes in health care, but in a population with a high prevalence of disease, they may at least initially be beneficial. This study also provides evidence that a local, additional scheme may further improve the health of populations. Such schemes, whether national or local, require periodic review to evaluate the balance of their benefits and risks., (© The Author(s) 2018. Published by Oxford University Press.)

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.

Small-angle X-ray scattering (SAXS) is increasingly used to extract structural information from a multitude of soft-matter and biological systems in aqueous solution, including polymers, detergents, lipids, colloids, proteins and RNA/DNA. When SAXS data are recorded at multiple contrasts, i.e. at different electron densities of the solvent, the internal electron-density profile of solubilized molecular systems can be probed. However, contrast-variation SAXS has been limited by the range of electron densities available by conventional agents such as sugars, glycerol and salt, and by the fact that many soft-matter and biological systems are modified in their presence. Here we present a pioneering SAXS contrast-variation study on DDM ( n -do-decyl-β-d-malto-pyran-oside) micelles by using two highly electron-rich contrast agents from biomedical imaging which belong to the families of gadolinium-based and iodinated molecules. The two agents, Gd-HPDO3A and iohexol, were allowed to attain modifications of the solvent electron density that are 50 to 100% higher than those obtained for sucrose, and are located between the electron densities of proteins and RNA/DNA. In the case of Gd-HPDO3A, an analysis of the internal micellar structure was possible and compared with results obtained with sucrose. In conclusion, medical contrast agents represent a promising class of molecules for SAXS contrast-variation experiments with potential appli-cations for numerous soft-matter and biological systems, including membrane proteins and protein-RNA/DNA complexes.

Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I 2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a μM affinity for morphine and binds to morphine in vitro . The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B 1-75 and CK-B 184-258 were identified as two specific morphine binding-peptides. In vitro , morphine (1-100 μM) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo .

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.

High amounts of plastic debris enter and accumulate in freshwater systems across the globe. The plastic contamination of benthic habitats in lakes and running waters poses a potential threat to freshwater ecosystems. This study investigates the effects of plastic on two trophic levels of the aquatic food web: primary production, that is, epiplastic biofilm, and primary consumption, that is, a benthic invertebrate grazer. Two plastic types, polymethyl methacrylate (PMMA) and polycarbonate (PC), and glass (control) were used as substrata for natural biofilm establishment. PMMA and PC are, for example, intensively used in the automobile, construction, and electronical industries and in cosmetics (PMMA), CDs, and DVDs (PC). These biofilms were fed to the freshwater gastropod  Radix balthica (Linnaeus 1758) in a laboratory-grazing experiment. Biofilm structure and composition were observed using confocal laser scanning microscopy before the grazing experiment. Sublethal effects on R. balthica were observed measuring consumption of biofilm and growth rates. The biofilm composition on PMMA significantly differed compared to PC and glass. The grazing experiments showed limited biofilm consumption and lower growth rates of R. balthica in both plastic treatments. Concluding, plastic in freshwaters has a direct effect on the primary production and an indirect effect on higher trophic levels.

Background and Purpose: Chronic administration of medication can significantly affect metabolic enzymes leading to physiological adaptations. Morphine metabolism in the liver has been extensively studied following acute morphine treatment, but such metabolic processes in the CNS are poorly characterized. Long-term morphine treatment is limited by the development of tolerance, resulting in a decrease of its analgesic effect. Whether or not morphine analgesic tolerance affects in vivo brain morphine metabolism and blood-brain barrier (BBB) permeability remains a major question. Here, we have attempted to characterize the in vivo metabolism and BBB permeability of morphine after long-term treatment, at both central and peripheral levels., Experimental Approach: Male C57BL/6 mice were injected with morphine or saline solution for eight consecutive days in order to induce morphine analgesic tolerance. On the ninth day, both groups received a final injection of morphine (85%) and d3-morphine (morphine bearing three 2 H; 15%, w/w). Mice were then killed and blood, urine, brain and liver samples were collected. LC-MS/MS was used to quantify morphine, its metabolite morphine-3-glucuronide (M3G) and their respective d3-labelled forms., Key Results: We found no significant differences in morphine CNS uptake and metabolism between control and tolerant mice. Interestingly, d3-morphine metabolism was decreased compared to morphine without any interference with our study., Conclusions and Implications: Our data suggests that tolerance to the analgesic effects of morphine is not linked to increased glucuronidation to M3G or to altered global BBB permeability of morphine., (© 2018 The British Pharmacological Society.)

Small-angle neutron scattering (SANS) has increasingly been used by the structural biology community in recent years to obtain low-resolution information on solubilized biomacromolecular complexes in solution. In combination with deuterium labelling and solvent-contrast variation (H 2 O/D 2 O exchange), SANS provides unique information on individual components in large heterogeneous complexes that is perfectly complementary to the structural restraints provided by crystallography, nuclear magnetic resonance and electron microscopy. Typical systems studied include multi-protein or protein-DNA/RNA complexes and solubilized membrane proteins. The internal features of these systems are less accessible to the more broadly used small-angle X-ray scattering (SAXS) technique owing to a limited range of intra-complex and solvent electron-density variation. Here, the progress and developments of biological applications of SANS in the past decade are reviewed. The review covers scientific results from selected biological systems, including protein-protein complexes, protein-RNA/DNA complexes and membrane proteins. Moreover, an overview of recent developments in instruments, sample environment, deuterium labelling and software is presented. Finally, the perspectives for biological SANS in the context of integrated structural biology approaches are discussed.

In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.

Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a. Here, by using an integrative structural biology approach, we show that hnRNP A1 forms a 1:1 complex with pri-mir-18a where both RNA recognition motifs (RRMs) bind to cognate RNA sequence motifs in the terminal loop of pri-mir-18a. Terminal loop binding induces an allosteric destabilization of base-pairing in the pri-mir-18a stem that promotes its downstream processing. Our results highlight terminal loop RNA recognition by RBPs as a potential general principle of miRNA biogenesis and regulation.

Background: Dental diseases are among the most frequent diseases globally and tooth loss imposes a substantial burden on peoples' quality of life. Non-experimental evidence suggests that individuals with more children have more missing teeth than individuals with fewer children, but until now there is no causal evidence for or against this., Methods: Using a Two-Stage Least Squares (2SLS) instrumental variables approach and large-scale cross-sectional data from the Survey of Health, Ageing, and Retirement in Europe (study sample: 34 843 non-institutionalised individuals aged 50+ from 14 European countries and Israel; data were collected in 2013), we investigated the causal relationship between the number of biological children and their parents' number of missing natural teeth. Thereby, we exploited random natural variation in family size resulting from (i) the birth of multiples vs singletons, and (ii) the sex composition of the two first-born children (increased likelihood of a third child if the two first-born children have the same sex)., Results: 2SLS regressions detected a strong causal relationship between the number of children and teeth for women but not for men when an additional birth occurred after the first two children had the same sex. Women then had an average of 4.27 (95% CI: 1.08 to 7.46) fewer teeth than women without an additional birth whose first two children had different sexes., Conclusions: This study provides novel evidence for causal links between the number of children and the number of missing teeth. An additional birth might be detrimental to the mother's but not the father's oral health., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

In order to predict which species can successfully cope with global warming and how other environmental stressors modulate their vulnerability to climate-related environmental factors, an understanding of the ecophysiology underpinning thermal limits is essential for both conservation biology and invasion biology.Heat tolerance and the extent to which heat tolerance differed with oxygen availability were examined for four native and four alien freshwater peracarid crustacean species, with differences in habitat use across species. Three hypotheses were tested: (1) Heat and lack of oxygen synergistically reduce survival of species; (2) patterns in heat tolerance and the modulation thereof by oxygen differ between alien and native species and between species with different habitat use; (3) small animals can better tolerate heat than large animals, and this difference is more pronounced under hypoxia.To assess heat tolerances under different oxygen levels, animal survival was monitored in experimental chambers in which the water temperature was ramped up (0.25°C min -1 ). Heat tolerance (CTmax) was scored as the cessation of all pleopod movement, and heating trials were performed under hypoxia (5 kPa oxygen), normoxia (20 kPa) and hyperoxia (60 kPa).Heat tolerance differed across species as did the extent by which heat tolerance was affected by oxygen conditions. Heat-tolerant species, for example, Asellus aquaticus and Crangonyx pseudogracilis , showed little response to oxygen conditions in their CTmax, whereas the CTmax of heat-sensitive species, for example, Dikerogammaru s villosus and Gammarus fossarum , was more plastic, being increased by hyperoxia and reduced by hypoxia.In contrast to other studies on crustaceans, alien species were not more heat-tolerant than native species. Instead, differences in heat tolerance were best explained by habitat use, with species from standing waters being heat tolerant and species from running waters being heat sensitive. In addition, larger animals displayed lower critical maximum temperature, but only under hypoxia. An analysis of data available in the literature on metabolic responses of the study species to temperature and oxygen conditions suggests that oxygen conformers and species whose oxygen demand rapidly increases with temperature (low activation energy) may be more heat sensitive.The alien species D. villosus appeared most susceptible to hypoxia and heat stress. This may explain why this species is very successful in colonizing new areas in littoral zones with rocky substrate which are well aerated due to continuous wave action generated by passing ships or prevailing winds. This species is less capable of spreading to other waters which are poorly oxygenated and where C. pseudogracilis is the more likely dominant alien species. A http://onlinelibrary.wiley.com/doi/10.1111/1365-2435.13050/suppinfo is available for this article.

Using claims data for research is well established. However, most claims data analyses are focused on single countries. Multi-national approaches are scarce. The application of different anonymization techniques before data are shared for research as well as differences in the reimbursement systems hamper the use of claims data from multiple countries. This paper analyses data conflicts that occur when international claims data sets are used for research and develops a generic process to detect and resolve these conflicts. The approach was successfully applied in the EU-funded ADVOCATE (Added Value for Oral Care) project that acquired data from health insurance providers, health funds or health authorities in six European countries.