Complex relationship between amino acids, fitness and food intake in Bombus terrestris.

The ratio of amino acids to carbohydrates (AA:C) that bumble bees consume has been reported to affect their survival. However, it is unknown how dietary AA:C ratio affects other bumble bee fitness traits (e.g., fecundity, condition) and possible trade-offs between them. Moreover, while individual AAs affect phenotype in many species, the effects of AA blend on bumble bee fitness and food intake are unclear. We test how the AA:C ratio that bumble bees (Bombus terrestris) consume affects their condition (abdomen lipid and dry mass), survival following food removal, and ovarian activation. We then compare ovarian activation and food intake in bees fed identical AA:C ratios, but where the blend of AAs in diets differ, i.e., diets contained the same 10 AAs in an equimolar ratio or in the same ratio as in bee collected pollen. We found that AA:C ratio did not significantly affect survival following food removal or ovarian activation; however, high AA intake increased body mass, which is positively correlated with multiple fitness traits in bumble bees. AA blend (i.e., equimolar versus pollen) did not significantly affect overall ovarian activation or consumption of each experimental diet. However, there was an interaction between AA mix and dietary AA:C ratio affecting survival during the feeding experiment, and signs that there may have been weak, interactive effects of AA mix and AA:C ratio on food consumption. These results suggest that the effect of total AA intake on bumble bee phenotype may depend on the blend of individual AAs in experimental diets. We suggest that research exploring how AA blend affects bumble bee performance and dietary intake is warranted, and highlight that comparing research on bee nutrition is complicated by even subtle variation in experimental diet composition.

Resistance to natural and synthetic gene drive systems.

Scientists are rapidly developing synthetic gene drive elements intended for release into natural populations. These are intended to control or eradicate disease vectors and pests, or to spread useful traits through wild populations for disease control or conservation purposes. However, a crucial problem for gene drives is the evolution of resistance against them, preventing their spread. Understanding the mechanisms by which populations might evolve resistance is essential for engineering effective gene drive systems. This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in general.

Genotoxicity Assessment of Drug Metabolites in the Context of MIST and Beyond.

While there are dedicated guidelines for industry regarding the assessment of the genotoxic potential of new pharmaceuticals and impurities, and the general safety assessment of major drug metabolites, only limited guidance exists on the assessment of potential genotoxic minor drug metabolites. In this Perspective, we discuss challenges associated with assessing the genotoxic potential of human metabolites and share five case studies within the context of an "aware-avoid-assess" paradigm. A special focus is on a class of potentially genotoxic carcinogens, aromatic amines (arylamines and anilines). This compound class is frequently used as building blocks and may show up as impurities, metabolites, or degradants in pharmaceuticals. We propose several recommendations that should help project teams at different stages of pharmaceutical development. In most cases, proactive interactions with the relevant health authority should be considered to endorse the proposed genotoxicity assessment strategy for minor drug metabolites.

Statistical analysis of in vivo alkaline comet assay data - Comparison of median and geometric mean as centrality measures.

The comet assay is one of the standard tests for evaluating the genotoxic potential of a test item able to detect DNA strand breaks in cells or isolated nuclei from various tissues. The in vivo alkaline comet assay is part of the standard test battery, given in option 2 of the ICH guidance S2 (R1) and a follow-up test in the EFSA framework on genotoxicity testing. The current OECD guideline for the testing of chemicals No. 489 directly affects the statistical analysis of comet data as it suggests using the median per slide and the mean of all medians per animal. However, alternative approaches can be used if scientifically justified. In this work, we demonstrated that the selection of different centrality measures to describe an average value per slide may lead to fundamentally different statistical test results and contradicting interpretations. Our focus was on geometric means and medians per slide for the primary endpoint "tail intensity". We compared both strategies using original and simulated data in different experimental settings incl. a varying number of animals, slides and cells per slide. In general, it turned out that the chosen centrality measure has an immense impact on the final statistical test result.

Effects of a male meiotic driver on male and female transcriptomes in the house mouse.

Not all genetic loci follow Mendel's rules, and the evolutionary consequences of this are not yet fully known. Genomic conflict involving multiple loci is a likely outcome, as restoration of Mendelian inheritance patterns will be selected for, and sexual conflict may also arise when sexes are differentially affected. Here, we investigate effects of the t haplotype, an autosomal male meiotic driver in house mice, on genome-wide gene expression patterns in males and females. We analysed gonads, liver and brain in adult same-sex sibling pairs differing in genotype, allowing us to identify t-associated differences in gene regulation. In testes, only 40% of differentially expressed genes mapped to the approximately 708 annotated genes comprising the t haplotype. Thus, much of the activity of the t haplotype occurs in trans, and as upregulation. Sperm maturation functions were enriched among both cis and trans acting t haplotype genes. Within the t haplotype, we observed more downregulation and differential exon usage. In ovaries, liver and brain, the majority of expression differences mapped to the t haplotype, and were largely independent of the differences seen in the testis. Overall, we found widespread transcriptional effects of this male meiotic driver in the house mouse genome.

Controlling invasive rodents via synthetic gene drive and the role of polyandry.

House mice are a major ecosystem pest, particularly threatening island ecosystems as a non-native invasive species. Rapid advances in synthetic biology offer new avenues to control pest species for biodiversity conservation. Recently, a synthetic sperm-killing gene drive construct called t-Sry has been proposed as a means to eradicate target mouse populations owing to a lack of females. A factor that has received little attention in the discussion surrounding such drive applications is polyandry. Previous research has demonstrated that sperm-killing drivers are extremely damaging to a male's sperm competitive ability. Here, we examine the importance of this effect on the t-Sry system using a theoretical model. We find that polyandry substantially hampers the spread of t-Sry such that release efforts have to be increased three- to sixfold for successful eradication. We discuss the implications of our finding for potential pest control programmes, the risk of drive spread beyond the target population, and the emergence of drive resistance. Our work highlights that a solid understanding of the forces that determine drive dynamics in a natural setting is key for successful drive application, and that exploring the natural diversity of gene drives may inform effective gene drive design.

No selection for change in polyandry under experimental evolution.

What drives mating system variation is a major question in evolutionary biology. Female multiple mating (polyandry) has diverse evolutionary consequences, and there are many potential benefits and costs of polyandry. However, our understanding of its evolution is biased towards studies enforcing monandry in polyandrous species. What drives and maintains variation in polyandry between individuals, genotypes, populations and species remains poorly understood. Genetic variation in polyandry may be actively maintained by selection, or arise by chance if polyandry is selectively neutral. In Drosophila pseudoobscura, there is genetic variation in polyandry between and within populations. We used isofemale lines to found replicate populations with high or low initial levels of polyandry and tracked polyandry under experimental evolution over seven generations. Polyandry remained relatively stable, reflecting the starting frequencies of the experimental populations. There were no clear fitness differences between high versus low polyandry genotypes, and there was no signature of balancing selection. We confirmed these patterns in direct comparisons between evolved and ancestral females and found no consequences of polyandry for female fecundity. The absence of differential selection even when initiating populations with major differences in polyandry casts some doubt on the importance of polyandry for female fitness.

The rat bone marrow micronucleus test: Statistical considerations on historical negative control data.

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

Meiotic drive changes sperm precedence patterns in house mice: potential for male alternative mating tactics?

BACKGROUND: With female multiple mating (polyandry), male-male competition extends to after copulation (sperm competition). Males respond to this selective pressure through physiological, morphological and behavioural adaptations. Sperm competitiveness is commonly decreased in heterozygote carriers of male meiotic drivers, selfish genetic elements that manipulate the production of gametes in males. This might give carriers an evolutionary incentive to reduce the risk of sperm competition. Here, we explore this possibility in house mice. Natural populations frequently harbour a well-characterised male driver (t haplotype), which is transmitted to 90 % of heterozygous (+/t) males' offspring. Previous research demonstrated strong detrimental effects on sperm competitiveness, and suggested that +/t males are particularly disadvantaged against wild type males when first-to-mate. Low paternity success in the first-to-mate role is expected to favour male adaptations that decrease the risk of sperm competition by preventing female remating. Genotype-specific paternity patterns (sperm precedence) could lead to genetically determined alternative reproductive tactics that can spread through gene level selection. Here, we seek confirmation that +/t males are generally disadvantaged when first-to-mate and address whether males of different genotypes differ in reproductive tactics (copulatory and morphological) to maximise individual or driver fitness. Finally, we attempt to explain the mechanistic basis for alternative sperm precedence patterns in this species. RESULTS: We confirmed that +/t males are weak sperm competitors when first to mate. When two +/t males competed, the second-to-mate was more successful, which contrasts with first male sperm precedence when wild type males competed. However, we found no differences between male genotypes in reproductive behaviour or morphology that were consistent with alternative reproductive tactics. Sperm of +/+ and +/t males differed with respect to in vitro sperm features. Premature hypermotility in +/t males' sperm can potentially explain why +/t males are very weak sperm competitors when first-to-mate. CONCLUSIONS: Our results demonstrate that meiotic drivers can have strong effects on sperm precedence patterns, and may provide a heritable basis for alternative reproductive tactics motivated by reduced sperm competitiveness. We discuss how experimental and evolutionary constraints may help explain why male genotypes did not show the predicted differences.

Genotoxicity assessment of peptide/protein-related biotherapeutics: points to consider before testing.

The ICH S6(R1) recommendations on safety evaluation of biotherapeutics have led to uncertainty in determining what would constitute a cause for concern that would require genotoxicity testing. A Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee Workgroup was formed to review the current practice of genotoxicity assessment of peptide/protein-related biotherapeutics. There are a number of properties of peptide/protein-related biotherapeutics that distinguish such products from traditional 'small molecule' drugs and need to be taken into consideration when assessing whether genotoxicity testing may be warranted and if so, how to do it appropriately. Case examples were provided by participating companies and decision trees were elaborated to determine whether and when genotoxicity evaluation is needed for peptides containing natural amino acids, non-natural amino acids and other chemical entities and for unconjugated and conjugated proteins. From a scientific point of view, there is no reason for testing peptides containing exclusively natural amino acids irrespective of the manufacturing process. If non-natural amino acids, organic linkers and other non-linker chemical components have already been tested for genotoxicity, there is no need to re-evaluate them when used in different peptide/protein-related biotherapeutics. Unless the peptides have been modified to be able to enter the cells, it is generally more appropriate to evaluate the peptides containing the non-natural amino acids and other non-linker chemical moieties in vivo where the cleavage products can be formed. For linkers, it is important to determine if exposure to reactive forms are likely to occur and from which origin. When the linkers are anticipated to be potential mutagenic impurities they should be evaluated according to ICH M7. If linkers are expected to be catabolic products, it is recommended to test the entire conjugate in vivo, as this would ensure that the relevant 'free' linker forms stemming from in vivo catabolism are tested.

Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice.

Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males.We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype inhouse mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge,the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry.

A practical application of two in silico systems for identification of potentially mutagenic impurities.

The International Conference on Harmonization (ICH) M7 guidance for the assessment and control of DNA reactive impurities in pharmaceutical products includes the use of in silico prediction systems as part of the hazard identification and risk assessment strategy. This is the first internationally agreed guidance document to include the use of these types of approaches. The guideline requires the use of two complementary approaches, an expert rule-based method and a statistical algorithm. In addition, the guidance states that the output from these computer-based assessments can be reviewed using expert knowledge to provide additional support or resolve conflicting predictions. This approach is designed to maximize the sensitivity for correctly identifying DNA reactive compounds while providing a framework to reduce the number of compounds that need to be synthesized, purified and subsequently tested in an Ames assay. Using a data set of 801 chemicals and pharmaceutical intermediates, we have examined the relative predictive performances of some popular commercial in silico systems that are in common use across the pharmaceutical industry. The overall accuracy of each of these systems was fairly comparable ranging from 68% to 73%; however, the sensitivity of each system (i.e. how many Ames positive compounds are correctly identified) varied much more dramatically from 48% to 68%. We have explored how these systems can be combined under the ICH M7 guidance to enhance the detection of DNA reactive molecules. Finally, using four smaller sets of molecules, we have explored the value of expert knowledge in the review process, especially in cases where the two systems disagreed on their predictions, and the need for care when evaluating the predictions for large data sets.

The condition-dependence of male genital size and shape.

The male genitals of internal fertilisers evolve rapidly and divergently, and sexual selection is generally responsible for this. Many sexually selected traits are condition-dependent-with their expression dependent upon the resources available to be allocated to them-as revealed by genetic or environmental manipulations of condition. However, it is not clear whether male genitals are also condition-dependent. Here we manipulate condition in two ways (via inbreeding and diet) to test the condition-dependence of the genital arch of Drosophila simulans. We found that genital size but not genital shape suffered from inbreeding depression, whereas genital size and shape were affected by dietary manipulation of condition. The differential effects of these treatments likely reflect underlying genetic architecture that has been shaped by past selection: inbreeding depression is only expected when traits have a history of directional selection, while diet impacts traits regardless of historical selection. Nonetheless, our results suggest genitals can be condition-dependent like other sexually selected traits.

Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

Use of in silico systems and expert knowledge for structure-based assessment of potentially mutagenic impurities.

Genotoxicity hazard identification is part of the impurity qualification process for drug substances and products, the first step of which being the prediction of their potential DNA reactivity using in silico (quantitative) structure-activity relationship (Q)SAR models/systems. This white paper provides information relevant to the development of the draft harmonized tripartite guideline ICH M7 on potentially DNA-reactive/mutagenic impurities in pharmaceuticals and their application in practice. It explains relevant (Q)SAR methodologies as well as the added value of expert knowledge. Moreover, the predictive value of the different methodologies analyzed in two surveys conveyed in the US and European pharmaceutical industry is compared: most pharmaceutical companies used a rule-based expert system as their primary methodology, yielding negative predictivity values of ⩾78% in all participating companies. A further increase (>90%) was often achieved by an additional expert review and/or a second QSAR methodology. Also in the latter case, an expert review was mandatory, especially when conflicting results were obtained. Based on the available data, we concluded that a rule-based expert system complemented by either expert knowledge or a second (Q)SAR model is appropriate. A maximal transparency of the assessment process (e.g. methods, results, arguments of weight-of-evidence approach) achieved by e.g. data sharing initiatives and the use of standards for reporting will enable regulators to fully understand the results of the analysis. Overall, the procedures presented here for structure-based assessment are considered appropriate for regulatory submissions in the scope of ICH M7.

Inroads to predict in vivo toxicology-an introduction to the eTOX Project.

There is a widespread awareness that the wealth of preclinical toxicity data that the pharmaceutical industry has generated in recent decades is not exploited as efficiently as it could be. Enhanced data availability for compound comparison ("read-across"), or for data mining to build predictive tools, should lead to a more efficient drug development process and contribute to the reduction of animal use (3Rs principle). In order to achieve these goals, a consortium approach, grouping numbers of relevant partners, is required. The eTOX ("electronic toxicity") consortium represents such a project and is a public-private partnership within the framework of the European Innovative Medicines Initiative (IMI). The project aims at the development of in silico prediction systems for organ and in vivo toxicity. The backbone of the project will be a database consisting of preclinical toxicity data for drug compounds or candidates extracted from previously unpublished, legacy reports from thirteen European and European operation-based pharmaceutical companies. The database will be enhanced by incorporation of publically available, high quality toxicology data. Seven academic institutes and five small-to-medium size enterprises (SMEs) contribute with their expertise in data gathering, database curation, data mining, chemoinformatics and predictive systems development. The outcome of the project will be a predictive system contributing to early potential hazard identification and risk assessment during the drug development process. The concept and strategy of the eTOX project is described here, together with current achievements and future deliverables.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

The impact of female mating strategies on the success of insect control technologies.

Attempts to control insect pests and disease vectors have a long history. Recently, new technology has opened a whole new range of possible methods to suppress or transform natural populations. But it has also become clear that a better understanding of the ecology of targeted populations is needed. One key parameter is mating behaviour. Often modified males are released which need to successfully reproduce with females while competing with wild males. Insect control techniques can be affected by target species' mating ecology, and conversely mating ecology is likely to evolve in response to manipulation attempts. A better understanding of (female) mating behaviour will help anticipate and overcome potential challenges, and thus make desirable outcomes more likely.

The baculum affects paternity success of first but not second males in house mouse sperm competition.

The vast variation observed in genital morphology is a longstanding puzzle in evolutionary biology. Studies showing that the morphology of the mammalian baculum (penis bone) can covary with a male's paternity success indicate a potential impact of baculum morphology on male fitness, likely through influencing sperm competition outcomes. We therefore measured the size (measurements of length and width) and shape (geometric morphometric measurements) of the bacula of male house mice used in previously published sperm competition experiments, in which two males mated successively with the same female in staged matings. This enabled us to correlate baculum morphology with sperm competition success, incorporating potential explanatory variables related to copulatory plugs, male mating behavior and a selfish genetic element that influences sperm motility. We found that a wider baculum shaft increased a male's paternity share when mating first, but not when mating second with a multiply-mating female. Geometric morphometric shape measurements were not clearly associated with fertilization success for either male. We found limited evidence that the effect of baculum morphology on male fertilization success was altered by experimental removal of the copulatory plug. Furthermore, neither genetic differences in sperm motility, nor covariation with male mating behavior mediated the effect of baculum morphology on male fertilization success. Taken together with previous findings, the mating-order effects we found here suggest that baculum-mediated stimulation by the first male might be particularly important for fertilization.

DNA repair inhibitors sensitize cells differently to high and low LET radiation.

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6-1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest.