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Gene drive technology has the potential to address major biological challenges. Well-studied homing suppression drives have been shown to be highly efficient in Anopheles mosquitoes, but for other organisms, lower rates of drive conversion prevent elimination of the target population. To tackle this issue, we propose a gene drive design that has two targets: a drive homing site where drive conversion takes place, and a distant site where cleavage induces population suppression. We model this design and find that the two-target system allows suppression to occur over a much wider range of drive conversion efficiency. Specifically, the cutting efficiency now determines the suppressive power of the drive, rather than the conversion efficiency as in standard suppression drives. We construct a two-target drive in Drosophila melanogaster and show that both components of the gene drive function successfully. However, cleavage in the embryo from maternal deposition as well as fitness costs in female drive heterozygotes both remain significant challenges for both two-target and standard suppression drives. Overall, our improved gene drive design has the potential to ease problems associated with homing suppression gene drives for many species where drive conversion is less efficient., (© 2024. The Author(s).)

Objective: This qualitative study explores when and why patients book video consultations through an online booking platform within the context of Danish general practice and how the technology affects patients' use of this consultation type., Methods: We conducted thirteen semi-structured interviews with patients from the same general practice who were experienced users of video consultations scheduled through the clinic's online booking platform. Interviews were analysed using thematic analysis and drawing on actor-network theory and Bruno Latour's concept of technical mediation as an analytical framework., Results: We introduce the concept of "hybrid patients," highlighting how values tied to video consultation that motivates patients to book them emerge through technical mediation in the network between patients, general practitioners (GPs) or practice staff, and the video technology. We identified three emerging values: efficiency, control and diminished presence. Video consultation affords efficient consultations that save patients' time. It mediates patients' sense of control when they experience certainty concerning their health issues. Video consultation mediates diminished presence that increases relational distance. However, it simultaneously allows for efficiency and emotional distance between patients and their GP, and between patients and their health issues., Conclusions: When initiating the use of video consultation, the patient plays an active and conscious role in adjusting to the mediated values (efficiency, control and diminished presence) linked to this form of consultation. These emerging values are context-specific, and patients employ them based on their individual requirements. Patients trust their GPs to prevent severe or vulnerable topics from being discussed in a video consultation., (© The Author(s) 2024.)

In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2,142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Developmental time is a key life-history trait with large effects on Darwinian fitness. In many insects, developmental time is currently under strong selection to minimize ecological mismatches in seasonal timing induced by climate change. The genetic basis of responses to such selection, however, is poorly understood. To address this problem, we set up a long-term evolve-and-resequence experiment in the beetle Tribolium castaneum and selected replicate, outbred populations for fast or slow embryonic development. The response to this selection was substantial and embryonic developmental timing of the selection lines started to diverge during dorsal closure. Pooled whole-genome resequencing, gene expression analysis and an RNAi screen pinpoint a 222 bp deletion containing binding sites for Broad and Tramtrack upstream of the ecdysone degrading enzyme Cyp18a1 as a main target of selection. Using CRISPR/Cas9 to reconstruct this allele in the homogenous genetic background of a laboratory strain, we unravel how this single deletion advances the embryonic ecdysone peak inducing dorsal closure and show that this allele accelerates larval development but causes a trade-off with fecundity. Our study uncovers a life-history allele of large effect and reveals the evolvability of developmental time in a natural insect population., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems., Competing Interests: Declaration of interests Kim Remans is a member of the STAR Protocols advisory board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The evolution of novel SARS-CoV-2 variants significantly affects vaccine effectiveness. While these effects can only be studied retrospectively, neutralizing antibody titers are most used as correlates of protection. However, studies assessing neutralizing antibody titers often show heterogeneous data., Methods: To address this, we investigated assay variance and identified virus infection time and dose as factors affecting assay robustness. We next measured neutralization against Omicron sub-variants in cohorts with hybrid or vaccine induced immunity, identifying a gradient of immune escape potential. To evaluate the effect of individual mutations on this immune escape potential of Omicron variants, we systematically assessed the effect of each individual mutation specific to Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5., Results: We cloned a library of pseudo-viruses expressing spikes with single point mutations, and subjected it to pooled sera from vaccinated hosts, thereby identifying multiple mutations that independently affect neutralization potency., Discussion: These data might help to predict antigenic features of novel viral variants carrying these mutations and support the development of broad monoclonal antibodies., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Katzmarzyk, Clesle, van den Heuvel, Hoffmann, Garritsen, Pöhlmann, Jacobsen and Čičin-Šain.)

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Auxier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genetic selection has been applied for many generations in animal, plant, and experimental populations. Selection changes the allelic architecture of traits to create genetic gain. It remains unknown whether the changes in allelic architecture are different for the recently introduced technique of genomic selection compared to traditional selection methods and whether they depend on the genetic architectures of traits. Here, we investigate the allele frequency changes of old and new causal loci under 50 generations of phenotypic, pedigree, and genomic selection, for a trait controlled by either additive, additive and dominance, or additive, dominance, and epistatic effects. Genomic selection resulted in slightly larger and faster changes in allele frequencies of causal loci than pedigree selection. For each locus, allele frequency change per generation was not only influenced by its statistical additive effect but also to a large extent by the linkage phase with other loci and its allele frequency. Selection fixed a large number of loci, and 5 times more unfavorable alleles became fixed with genomic and pedigree selection than with phenotypic selection. For pedigree selection, this was mainly a result of increased genetic drift, while genetic hitchhiking had a larger effect on genomic selection. When epistasis was present, the average allele frequency change was smaller (∼15% lower), and a lower number of loci became fixed for all selection methods. We conclude that for long-term genetic improvement using genomic selection, it is important to consider hitchhiking and to limit the loss of favorable alleles., Competing Interests: Conflicts of interest: The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1 . DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1 . Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction., Competing Interests: The authors declare no conflict of interest.

In the wake of the COVID-19 pandemic, video consultation was introduced in general practice in many countries around the world as a solution to provide remote health care to patients. It was assumed that video consultation would find widespread adoption in post-COVID-19 general practice. However, adoption rates remain low across countries in Northern Europe, suggesting that barriers to its use exist among general practitioners and other practice staff. In this viewpoint, we take a comparative approach, reflecting on similarities and differences in implementation conditions of video consultations in 5 Northern European countries' general practice settings that might have created barriers to its use within general practice. We convened at a cross-disciplinary seminar in May 2022 with researchers and clinicians from 5 Northern European countries with expertise in digital care in general practice, and this viewpoint emerged out of dialogues from that seminar. We have reflected on barriers across general practice settings in our countries, such as lacking technological and financial support for general practitioners, that we feel are critical for adoption of video consultation in the coming years. Furthermore, there is a need to further investigate the contribution of cultural elements, such as professional norms and values, to adoption. This viewpoint may inform policy work to ensure that a sustainable level of video consultation use can be reached in the future, one that reflects the reality of general practice settings rather than policy optimism., (©Elisabeth Assing Hvidt, Helen Atherton, Jelle Keuper, Eli Kristiansen, Elle Christine Lüchau, Børge Lønnebakke Norberg, Jost Steinhäuser, Johannes van den Heuvel, Lilian van Tuyl. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 22.05.2023.)

Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep ® tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.

Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.

Background: The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect., Methods: To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle., Results: Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi., Conclusions: The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate., (© 2023. The Author(s).)

Some COVID-19 survivors suffer from persistent pulmonary function impairment, but the extent and associated factors are unclear. This study aimed to characterize pulmonary function impairment three to five months after hospital discharge and the association with disease severity. Survivors of COVID-19 after hospitalization to the VieCuri Medical Centre between February and December 2020 were invited for follow-up, three to five months after discharge. Dynamic and static lung volumes, respiratory muscle strength and diffusion capacity were measured. The cohort comprised 257 patients after a moderate (n = 33), severe (n = 151) or critical (n = 73) COVID-19 infection with a median follow-up of 112 days (interquartile range 96-134 days). The main sequelae included reduced diffusion capacity (36%) and reduced maximal expiratory pressure (24%). Critically ill patients were more likely to have reduced diffusion capacity than moderate (OR 8.00, 95% CI 2.46-26.01) and severe cases (OR 3.74, 95% CI 1.88-7.44) and lower forced vital capacity (OR 3.29, 95% CI 1.20-9.06) compared to severe cases. Many COVID-19 survivors, especially after a critical disease course, showed pulmonary function sequelae, mainly DLCO impairments, three to five months after discharge. Monitoring is needed to investigate the persistence of these symptoms and the longer-term implications of the COVID-19 burden., (© 2023. The Author(s).)

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Our experimental work illustrates how microbial ecosystems can be shaped by selective pressures over long-term ecological time scales. Natural microbial ecosystems generally consist of various co-existing species, where community composition describes the frequency at which species or types are present. Overall functionality of the system is achieved by interacting species. Upon short-term selection, for instance by transfer to a novel environment, community composition and functionality may change in a process referred to as species sorting. Various factors, such as initial community composition and selective pressures from the environment, may influence this change. Mabisi is a traditional fermented food from Zambia that naturally contains a bacterial community of around twenty unique bacterial types. We used six comparable but different natural bacterial Mabisi communities, each split into five identical replicates, for 16 propagation cycles in a novel, common laboratory environment. Composition of the bacterial communities changed upon propagation. The influence of four main factors on community composition, i.e. initial composition (history), impact of the environment (adaptation), changes due to interaction between species and random processes (chance) in species dynamics, was tested using maximum likelihood ratios. Initial community composition seemed to determine the change in community composition, followed by random processes. Interestingly, we observed convergence at the level of ecosystem functionality, which was measured as profiles of metabolic output. This suggests different combinations of species or types can achieve similar eco-system functionality., Competing Interests: The authors declare no conflicts exist., (© 2022 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Little is known about the impact of age on the processes governing human intestinal drug absorption. The Ussing chamber is a system to study drug transport across tissue barriers, but it has not been used to study drug absorption processes in children. This study aimed to explore the feasibility of the Ussing chamber methodology to assess pediatric intestinal drug absorption. Furthermore, differences between intestinal drug transport processes of children and adults were explored as well as the possible impact of age. Fresh terminal ileal leftover tissues from both children and adults were collected during surgery and prepared for Ussing chamber experiments. Paracellular (enalaprilat), transcellular (propranolol), and carrier-mediated drug transport by MDR1 (talinolol) and BCRP (rosuvastatin) were determined with the Ussing chamber methodology. We calculated apparent permeability coefficients and efflux ratios and explored their relationship with postnatal age. The success rate for the Ussing chamber experiments, as determined by electrophysiological measurements, was similar between children (58%, N = 15, median age: 44 weeks; range 8 weeks to 17 years) and adults (67%, N = 13). Mean serosal to mucosal transport of talinolol by MDR1 and rosuvastatin by BCRP was higher in adult than in pediatric tissues (p = 0.0005 and p = 0.0091). In contrast, within our pediatric cohort, there was no clear correlation for efflux transport across different ages. In conclusion, the Ussing chamber is a suitable model to explore pediatric intestinal drug absorption and can be used to further elucidate ontogeny of individual intestinal pharmacokinetic processes like drug metabolism and transport., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical "Corona" aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

Objectives: Healthcare is required to be effectively organised to ensure that growing, aging and medically more complex populations have timely access to high-quality, affordable care. Cardiac surgery is no exception to this, especially due to the competition for and demand on hospital resources, such as operating rooms and intensive care capacity. This is challenged more since the COVID-19 pandemic led to postponed care and prolonged waiting lists. In other sectors, Quality Improvement Methodologies (QIM) derived from the manufacturing industry have proven effective in enabling more efficient utilisation of existing capacity and resources and in improving the quality of care. We performed a systematic review to evaluate the ability of such QIM to improve care in cardiac surgery., Methods: A literature search was performed in PubMed, Embase, Clarivate Analytics/Web of Science Core Collection and Wiley/the Cochrane Library according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis methodology., Results: Ten articles were identified. The following QIM were used: Lean, Toyota Production System, Six Sigma, Lean Six Sigma, Root Cause Analysis, Kaizen and Plan-Do-Study-Act. All reported one or more relevant improvements in patient-related (e.g., infection rates, ventilation time, mortality, adverse events, glycaemic control) and process-related outcomes (e.g., shorter waiting times, shorter transfer time and productivity). Elements to enhance the success included: multidisciplinary team engagement, a patient-oriented, data-driven approach, a sense of urgency and a focus on sustainability., Conclusions: In all ten papers describing the application of QIM initiatives to cardiac surgery, positive results, of varying magnitude, were reported. While the consistency of the available data is encouraging, the limited quantity and heterogenous quality of the evidence base highlights that more rigorous evaluation, including how best to employ manufacturing industry-derived QIM in cardiac surgery is warranted.

Since its outbreak in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been multiple times replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates. In order to find an explanation for why SARS-CoV-2 and its emerging mutated versions showed enhanced transmission efficiency compared with SARS-CoV (2002), an enhanced binding affinity of the spike protein to human angiotensin converting enzyme 2 (hACE2) has been proposed by crystal structure analysis and was identified in cell culture models. Kinetic analysis of the interaction of various spike protein constructs with hACE2 was considered to be best described by a Langmuir-based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with hACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of ~190 (primary versus secondary state) with an overall equilibrium dissociation constant ( K D ) of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wild type ("wt"; Wuhan strain) but also found in the B.1.1.7 variant, where its transition rate is 5-fold increased. IMPORTANCE The current SARS-CoV-2 pandemic is characterized by the high infectivity of SARS-CoV-2 and its derived variants of concern (VOCs). It has been widely assumed that the reason for its increased cell entry compared with SARS-CoV (2002) is due to alterations in the viral spike protein, where single amino acid residue substitutions can increase affinity for hACE2. So far, the interaction of a single unit of the CoV-2 spike protein has been described using the 1:1 Langmuir interaction kinetic. However, we demonstrate here that there is a secondary state binding step that may be essential for novel VOCs in order to further increase their infectivity. These findings are important for quantitatively understanding the infection process of SARS-CoV-2 and characterization of emerging SARS-CoV-2 variants of spike proteins. Thus, they provide a tool for predicting the potential infectivity of the respective viral variants based on secondary rate transition and secondary complex stability.

The sensitive detection of bacterial infections is a prerequisite for their successful treatment. The use of a chemiluminescent readout was so far hampered by an insufficient probe enrichment at the pathogens. We coupled siderophore moieties, that harness the unique iron transport system of bacteria, with enzyme-activatable dioxetanes and obtained seven trifunctional probes with high signal-to-background ratios (S/B=426-859). Conjugates with efficient iron transport capability into bacteria were identified through a growth recovery assay. All ESKAPE pathogens were labelled brightly by desferrioxamine conjugates, while catechols were weaker due to self-quenching. Bacteria could also be detected inside lung epithelial cells. The best probe 8 detected 9.1×10 3  CFU mL -1 of S. aureus and 5.0×10 4  CFU mL -1 of P. aeruginosa, while the analogous fluorescent probe 10 was 205-305fold less sensitive. This qualifies siderophore dioxetane probes for the selective and sensitive detection of bacteria., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell-endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl -deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair.

Background: Asthma and chronic obstructive pulmonary disease (COPD) affect millions of people worldwide. While medication can control and improve disease symptoms, incorrect use of medication is a common problem. The eHealth intervention SARA (Service Apothecary Respiratory Advice) aims to improve participants' correct use of inhalation medication by providing information and as-needed tailored follow-up support by a pharmacist., Objective: The primary aim of this study was to investigate the effect of SARA on exacerbation rates in participants with asthma and COPD. Secondary aims were to investigate its effects in terms of adherence to maintenance medication and antimycotic treatment., Methods: In this nonrandomized pre-post study, medication dispensing data from 382 Dutch community pharmacies were included. Exacerbation rates were assessed with dispensed short-course oral corticosteroids. Medication adherence between new and chronic users was assessed by calculating the proportion of days covered from dispensed inhalation maintenance medication. Antimycotic treatment was investigated from dispensed oral antimycotics in participants who were also dispensed inhaled corticosteroids (ICS). Outcomes were assessed 1 year before and 1 year after implementation of SARA and were compared between SARA participants and control participants. More specifically, for exacerbation rates and medication adherence, a difference score was calculated (ie, 1 year after SARA minus 1 year before SARA) and was subsequently compared between the study groups with independent-samples t tests. For antimycotics, the relative number of participants who were dispensed antimycotics was calculated and subsequently analyzed with a mixed-effects logistic regression., Results: The study population comprised 9452 participants, of whom 2400 (25.39%) were SARA participants. The mean age of the population was 60.8 (15.0) years, and approximately two-thirds (n=5677, 60.06%) were female. The results showed an increase in mean exacerbation rates over time for both study groups (SARA: 0.05; control: 0.15). However, this increase in exacerbation rates was significantly lower for SARA participants (t 9450 =3.10, 95% CI 0.04-0.16; P=.002; Cohen d=0.06). Chronic users of inhalation medication in both study groups showed an increase in mean medication adherence over time (SARA: 6.73; control: 4.48); however, this increase was significantly higher for SARA participants (t 5886 =-2.74, 95% CI -3.86 to -0.84; P=.01; Cohen d=-0.07). Among new users of inhalation medication, results showed no significant difference in medication adherence between SARA and control participants in the year after implementation of SARA (t 1434 =-1.85, 95% CI -5.60 to 0.16; P=.06; Cohen d=-0.10). Among ICS users, no significant differences between the study groups were found over time in terms of the proportion of participants who were dispensed antimycotics (t 5654 =0.29, 95% CI -0.40 to 0.54; P=.76; Cohen d=0)., Conclusions: This study provides preliminary evidence that the SARA eHealth intervention might have the potential to decrease exacerbation rates and improve medication adherence among patients with asthma and COPD., (©Kyma Schnoor, Anke Versluis, Robbert Bakema, Sanne van Luenen, Marcel J Kooij, J Maurik van den Heuvel, Martina Teichert, Persijn J Honkoop, Job F M van Boven, Niels H Chavannes, Jiska J Aardoom. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 08.06.2022.)

Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells. Our screen identified a group of 310 high confidence factors. These highlight the conservation of the process across eukaryotes and reveal the intricate connectivity of 60S subunit maturation with other key cellular processes, including splicing, translation, protein degradation, chromatin organization and transcription. Intriguingly, we also identified a cluster of hits comprising metabolic enzymes of the polyamine synthesis pathway. We demonstrate that polyamines, which have long been used as buffer additives to support ribosome assembly in vitro, are required for 60S maturation in living cells. Perturbation of polyamine metabolism results in early defects in 60S but not 40S subunit maturation. Collectively, our data reveal a novel function for polyamines in living cells and provide a rich source for future studies on ribosome synthesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Genomic selection has revolutionized genetic improvement in animals and plants, but little is known about its long-term effects. Here, we investigated the long-term effects of genomic selection on response to selection, genetic variance, and the genetic architecture of traits using stochastic simulations. We defined the genetic architecture as the set of causal loci underlying each trait, their allele frequencies, and their statistical additive effects. We simulated a livestock population under 50 generations of phenotypic, pedigree, or genomic selection for a single trait, controlled by either only additive, additive and dominance, or additive, dominance, and epistatic effects. The simulated epistasis was based on yeast data., Results: Short-term response was always greatest with genomic selection, while response after 50 generations was greater with phenotypic selection than with genomic selection when epistasis was present, and was always greater than with pedigree selection. This was mainly because loss of genetic variance and of segregating loci was much greater with genomic and pedigree selection than with phenotypic selection. Compared to pedigree selection, selection response was always greater with genomic selection. Pedigree and genomic selection lost a similar amount of genetic variance after 50 generations of selection, but genomic selection maintained more segregating loci, which on average had lower minor allele frequencies than with pedigree selection. Based on this result, genomic selection is expected to better maintain genetic gain after 50 generations than pedigree selection. The amount of change in the genetic architecture of traits was considerable across generations and was similar for genomic and pedigree selection, but slightly less for phenotypic selection. Presence of epistasis resulted in smaller changes in allele frequencies and less fixation of causal loci, but resulted in substantial changes in statistical additive effects across generations., Conclusions: Our results show that genomic selection outperforms pedigree selection in terms of long-term genetic gain, but results in a similar reduction of genetic variance. The genetic architecture of traits changed considerably across generations, especially under selection and when non-additive effects were present. In conclusion, non-additive effects had a substantial impact on the accuracy of selection and long-term response to selection, especially when selection was accurate., (© 2022. The Author(s).)

Severe respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious beta-class coronavirus. Although vaccinations have shown high efficacy, the emergence of novel variants of concern (VOCs) has already exhibited traits of immune evasion. Thus, the development of tailored antiviral medications for patients with incomplete, inefficient, or non-existent immunization, is essential. The attachment of viral surface proteins to the cell surface is the first crucial step in the viral replication cycle, which for SARS-CoV-2 is mediated by the high affinity interaction of the viral trimeric spike with the host cell surface-located human angiotensin converting enzyme-2 (hACE2). Here, we used a novel and efficient next generation sequencing (NGS) supported phage display strategy for the selection of a set of SARS-CoV-2 receptor binding domain (RBD)-targeting peptide ligands that bind to the target protein with low µM to nM dissociation constants. Compound CVRBDL-3 inhibits the SARS-CoV-2 spike protein association to hACE2 in a concentration-dependent manner for pre- as well as post-complex formation conditions. Further rational optimization yielded a CVRBDL-3 based divalent compound, which demonstrated inhibitory efficacy with an IC 50 value of 47 nM. The obtained compounds were not only efficient for the different spike constructs from the originally isolated "wt" SARS-CoV-2, but also for B.1.1.7 mutant trimeric spike protein. Our work demonstrates that phage display-derived peptide ligands are potential fusion inhibitors of viral cell entry. Moreover, we show that rational optimization of a combination of peptide sequences is a potential strategy in the further development of therapeutics for the treatment of acute COVID-19.

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload., Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf -/- ), and Mydgf bone marrow-chimeric or bone marrow-conditional transgenic mice with pressure overload-induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography-mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF., Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf -/- mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow-derived inflammatory cells attenuated pressure overload-induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein-coupled receptor agonist-induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf -/- mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf -/- mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload-induced hypertrophy and dysfunction. Pressure-overloaded Mydgf -/- mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival., Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.

Objective: To describe levels of perceived shared decision making (SDM), decisional conflict (DC), and decision regret (DR) in prenatal counseling by pregnant women, partners, neonatologists, and obstetricians regarding decision-making around imminent extreme premature birth in which a decision about palliative comfort care versus early intensive care had to be made., Study Design: Multicenter, cross-sectional study using surveys to determine perceived SDM at imminent extreme premature birth in parents and physicians, and to determine DC and DR in parents., Results: In total, 73 participants from 22 prenatal counseling sessions were included (21 pregnant women, 20 partners, 14 obstetricians, 18 neonatologists). High perceived levels of SDM were found (median 82,2), and low levels of DC (median 23,4) and DR at one month (median 12, 5)., Conclusions: Reported levels of self-perceived SDM in the setting of prenatal counseling in extreme prematurity were high, by both the parents and the physicians. Levels of DC and DR were low., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process., Competing Interests: Jv, CA, LG, KD, EW, IZ, UK No competing interests declared, (© 2021, van den Heuvel et al.)

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC 50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19., Competing Interests: Declaration of interests F.B., D.M., N.L., S. Steinke, P.A.H., R.B., M.R., K.-T.S., K.D.R.R., S.Z.-E., M.B., V.F., S.D., M. Schubert, and M.H. are inventors on a patent application on blocking antibodies against SARS-CoV-2. A.H., A.F., and T.S. are officers of CORAT Therapeutics GmbH, a company founded by YUMAB GmbH for the clinical and regulatory development of STE90-C11 (development name COR-101) (ClinicalTrials.gov ID: NCT04674566 for safety and efficacy of COR-101 in hospitalized patients with moderate to severe COVID-19). A.H. is a shareholder of CORAT Therapeutics GmbH. S.D. and M.H. are advisors to Corat Therapeutics GmbH. A.F., T.S., S.D., and M.H. are shareholders of YUMAB GmbH., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 ( 12 ), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Some multicellular organisms can fuse because mergers potentially provide mutual benefits. However, experimental evolution in the fungus Neurospora crassa has demonstrated that free fusion of mycelia favours cheater lineages, but the mechanism and evolutionary dynamics of this exploitation are unknown. Here we show, paradoxically, that all convergently evolved cheater lineages have similar fusion deficiencies. These mutants are unable to initiate fusion but retain access to wild-type mycelia that fuse with them. This asymmetry reduces cheater-mutant contributions to somatic substrate-bound hyphal networks, but increases representation of their nuclei in the aerial reproductive hyphae. Cheaters only benefit when relatively rare and likely impose genetic load reminiscent of germline senescence. We show that the consequences of somatic fusion can be unequally distributed among fusion partners, with the passive non-fusing partner profiting more. We discuss how our findings may relate to the extensive variation in fusion frequency of fungi found in nature.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.

The expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins. As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation. We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acute Aspergillus infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate. Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung., Competing Interests: Conflict of interest: T. Engel has nothing to disclose. Conflict of interest: P.E. Verweij reports grants from Gilead Sciences, MSD, Pfizer and F2G, and nonfinancial support from OLM and IMMY, outside the submitted work. Conflict of interest: J. van den Heuvel has nothing to disclose. Conflict of interest: D. Wangmo has nothing to disclose. Conflict of interest: J. Zhang has nothing to disclose. Conflict of interest: A.J.M. Debets has nothing to disclose. Conflict of interest: E. Snelders has nothing to disclose., (Copyright ©ERS 2020.)

Background: Cytostatic drugs are increasingly being prepared with a cytostatic robot, though it is not known whether the dose of the final product is more accurate after automated or manual preparation. This study is the first to compare accuracy and precision of automated preparations with manual preparations by measuring volumes and drug concentrations., Methods: The accuracy and precision of automated and manual preparations were compared by gravimetric and concentration measurements. During ten days 80 solutions were prepared; 40 robot preparations and 40 manual preparations. With both preparation methods, 20 methotrexate (MTX) and 20 cyclophosphamide (CP) bags were compounded. We simulated normal working conditions by performing the preparations on Monday till Friday. The MTX and CP concentrations were measured with validated ultra high performance liquid chromatography (UHPLC) methods on the last preparation day., Results: With UHPLC analysis, dose accuracy (mean dose error) of robotic or manual preparation of MTX were 1.70% and 0,96% respectively. With gravimetric analysis, these values were 0.50% and 1.96%. Precision (standard error) of the robotic preparation for MTX was significantly smaller than that of manual preparation (p < 0.001). Dose accuracy (mean dose error) of robotic or manual preparation of CP, with UHPLC analysis, were 6.10% and 5.20% respectively. With gravimetric analysis, these values were 0,67% and 0,18%., Conclusion: We conclude that both robotic and manual compounding produce accurate cytostatic products in which the mean percentage of active substance differs by less than 10% from the prescribed amount. Both preparation methods are compliant with the Dutch Medicines Act and the European Pharmacopoeia., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bracon brevicornis is an ectoparasitoid of a wide range of larval-stage Lepidopterans, including several pests of important crops, such as the corn borer, Ostrinia nubilalis . It is also one of the earliest documented cases of complementary sex determination in Hymenoptera. Here, we present the linked-read-based genome of B. brevicornis , complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloeum . We demonstrate the potential of linked-read assemblies in exploring regions of heterozygosity and search for structural and homology-derived evidence of the complementary sex determiner gene ( csd ).

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR 34 /L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR 34 3 /L98H evolved from a clinical TR 34 /L98H ancestor isolate, confirmed by whole genome sequencing. TR 34 3 /L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR 34 3 , thus supporting the possibility of in-host selection of TR 34 variants.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins 1,2 . Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1 3 . Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1 4 . The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae.

The preclinical multicomponent free-choice high-fat high-sucrose (fcHFHS) diet has strong validity to model diet-induced obesity (DIO) and associated maladaptive molecular changes in the central nervous system. fcHFHS-induced obese rats demonstrate increased sensitivity to intracerebroventricular infusion of the orexigenic Neuropeptide Y (NPY). The brain region-specific effects of NPY signaling on fcHFHS diet component selection are not completely understood. For example, fcHFHS-fed rats have increased intake of chow and fat following intracerebroventricular NPY infusion, whereas NPY administration in the nucleus accumbens, a key hub of the reward circuitry, specifically increases fat intake. Here, we investigated whether NPY infusion in the lateral hypothalamic area (LHA), which is crucially involved in the regulation of intake, regulates fcHFHS component selection, and if LHA NPY receptor subtypes 1 or 5 (NPYR1/5) are involved. Male Wistar rats were fed a chow or fcHFHS diet for at least seven days, and received intra-LHA vehicle or NPY infusions in a cross-over design. Diet component intake was measured two hours later. Separate experimental designs were used to test the efficacy of NPY1R- or NPY5R antagonism to prevent the orexigenic effects of intra-LHA NPY. Intra-LHA NPY increased caloric intake in chow- and fcHFHS-fed rats. This effect was mediated specifically by chow intake in fcHFHS-fed rats. The orexigenic effects of intra-LHA NPY were prevented by NPY1R and NPY5R antagonism in chow-fed rats, but only by NPY5R antagonism in fcHFHS-fed rats. Thus, NPY signaling has brain region-specific effects on fcHFHS component selection and LHA NPYR sensitivity is dysregulated during consumption of a fcHFHS diet., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many traditionally fermented milk products such as mabisi involve spontaneous fermentation, which can result in bacterial community composition variation due to selection pressure. The aim of this study was to determine the composition of bacterial communities in the different types of mabisi produced across Zambia and identify the factors that influence their composition. Samples of mabisi were collected across the country, and analyzed for pH and bacterial communities using 16S rRNA amplicon sequencing. We found that the bacterial community composition was dominated by members of two phyla, i.e., Firmicutes and Proteobacteria, from which the top 10 most abundant genera were Lactococcus, Lactobacillus, Streptococcus, Enterobacter, Citrobacter, Klebsiella, Kluyvera, Buttiauxella , Aeromonas , and Acinetobacter . The most dominant genus was Lactococcus , which was present in all types of mabisi produced from all regions. The mabisi products from traditional mabisi production regions (TMPRs) were dominated by lactic acid bacteria (LAB) whereas products from non-TMPRs were dominated by non-LAB species. Tonga mabisi, the most popular type of mabisi produced in non-TMPRs, had the most complex and diverse bacterial community composition compared to the other types, which included barotse, backslopping, creamy, and thick-tonga mabisi. Other factors that influenced bacterial community composition were geographical location, fermentation duration and pH while the type of fermentation container and producer did not. This study provides new insights that can be applied in starter culture development as well as microbial functionality studies., (Copyright © 2020 Moonga, Schoustra, van den Heuvel, Linnemann, Samad, Shindano and Smid.)

Background: Whilst adaptive facultative sex allocation has been widely studied at the phenotypic level across a broad range of organisms, we still know remarkably little about its genetic architecture. Here, we explore the genome-wide basis of sex ratio variation in the parasitoid wasp Nasonia vitripennis, perhaps the best studied organism in terms of sex allocation, and well known for its response to local mate competition., Results: We performed a genome-wide association study (GWAS) for single foundress sex ratios using iso-female lines derived from the recently developed outbred N. vitripennis laboratory strain HVRx. The iso-female lines capture a sample of the genetic variation in HVRx and we present them as the first iteration of the Nasonia vitripennis Genome Reference Panel (NVGRP 1.0). This panel provides an assessment of the standing genetic variation for sex ratio in the study population. Using the NVGRP, we discovered a cluster of 18 linked SNPs, encompassing 9 annotated loci associated with sex ratio variation. Furthermore, we found evidence that sex ratio has a shared genetic basis with clutch size on three different chromosomes., Conclusions: Our approach provides a thorough description of the quantitative genetic basis of sex ratio variation in Nasonia at the genome level and reveals a number of inter-related candidate loci underlying sex allocation regulation.

Traditional fermented foods and beverages are common in many countries, including Zambia. While the general (nutritional) benefits of fermented foods are widely recognised, the nutritional composition of most traditional fermented foods is unknown. Furthermore, fermentation is known to add nutritional value to raw materials, mainly by adding B-vitamins and removing anti-nutritional factors. In the case of traditional fermentation, the composition of microbial communities responsible for fermentation varies from producer to producer and this may also be true for the nutritional composition. Here, we characterized the nutrient profile and microbial community composition of two traditional fermented foods: milk-based Mabisi and cereal-based Munkoyo. We found that the two products are different with respect to their nutritional parameters and their microbial compositions. Mabisi was found to have higher nutritional values for crude protein, fat, and carbohydrates than Munkoyo . The microbial community composition was also different for the two products, while both communities were dominated by lactic acid bacteria. Our analyses showed that variations in nutritional composition, defined as the amount of consumption that would contribute to the estimated average requirement (EAR), might be explained by variations in microbial community composition. Consumption of Mabisi appeared to contribute more than Munkoyo to the EAR and its inclusion in food-based recommendations is warranted. Our results show the potential of traditional fermented foods such as Mabisi and Munkoyo to add value to current diets and suggests that variations in microbial composition between specific product samples can result in variations in nutritional composition.

A model and data toolbox is presented to assess risks from combined exposure to multiple chemicals using probabilistic methods. The Monte Carlo Risk Assessment (MCRA) toolbox, also known as the EuroMix toolbox, has more than 40 modules addressing all areas of risk assessment, and includes a data repository with data collected in the EuroMix project. This paper gives an introduction to the toolbox and illustrates its use with examples from the EuroMix project. The toolbox can be used for hazard identification, hazard characterisation, exposure assessment and risk characterisation. Examples for hazard identification are selection of substances relevant for a specific adverse outcome based on adverse outcome pathways and QSAR models. Examples for hazard characterisation are calculation of benchmark doses and relative potency factors with uncertainty from dose response data, and use of kinetic models to perform in vitro to in vivo extrapolation. Examples for exposure assessment are assessing cumulative exposure at external or internal level, where the latter option is needed when dietary and non-dietary routes have to be aggregated. Finally, risk characterisation is illustrated by calculation and display of the margin of exposure for single substances and for the cumulation, including uncertainties derived from exposure and hazard characterisation estimates., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis., Competing Interests: CM, Jv, CA, KD, AM, SJ, IZ, PP, LG, UK No competing interests declared, (© 2020, Montellese et al.)

Background: Hospital admission during pregnancy complications is considered to be an event of significant impact. Besides conventional in-clinic maternal and fetal monitoring, recent technologies enable home-based telemonitoring with self-measurements in high risk pregnancy. This study is part of a feasibility pilot to explore the usability and acceptability of telemonitoring and aims to gain insight in the experiences and preferences of high risk pregnant women concerning the novel strategy of telemonitoring, opposed to women who were hospitalized in pregnancy., Methods: Using secured Facebook Groups, we conducted four online focus groups: two focus groups with women who were admitted during pregnancy (n = 11) and two with women who received home telemonitoring in the pilot phase (n = 11). The qualitative data were analyzed thematically., Results: Four major themes emerged from both participant groups: [1] care experience, [2] emotions regarding pregnancy, [3] privacy and [4] impact on daily life. Different views were reported on all four themes, resulting in a direct comparison of experiences during hospitalization and telemonitoring. Most admitted patients reported a growing sense of boredom and anxiety during their clinical admission. Lack of privacy on ward was a great concern, as it affected their contact with hospital staff and family. This issue was not reported amongst telemonitored women. These participants still felt like a patient at times but responded that the comfort of their own home and bed was pleasant. Only a minority of telemonitored participants reported being anxious at times at home, while not having a physician or nurse nearby. Being at home resulted in less travel time for partners or family for hospital visits, which had its positive effects on family life., Conclusions: Telemonitoring of a high-risk pregnancy provides an innovative manner to monitor fetal and maternal condition from home. Compared to the experiences of hospital admission in high risk pregnancy, it allows women to be in a comforting and private environment during an anxious time in their lives. As future studies should further investigate the safety and cost effectiveness of this novel strategy, women's views on the preference of telemonitoring need to be taken into consideration.

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para -aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL -1 , and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL -1 ). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens., Competing Interests: GT, MB, CG, RM, JH, HP, MH, WAME, RWH and AR are co-inventors on patent applications on natural and synthetic cystobactamids., (This journal is © The Royal Society of Chemistry.)