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Robin, C.

Publications and source records attributed to Robin, C..

At least 19 recordsLinked to original sources

Variation in aggressiveness plasticity between northern and southern European populations of Hymenoscyphus fraxineus, an invasive fungal pathogen of ash

Hymenoscyphus fraxineus is an invasive fungal pathogen responsible for the ash dieback epidemic, which continues to cause severe mortality of common ash (Fraxinus excelsior L.) across Europe. Following its likely introduction in northeastern Europe, the pathogen rapidly colonized most regions where common ash is present. As it spread southward, H. fraxineus encountered warmer climates and a higher occurrence of Fraxinus ornus L.), a species resistant to the disease, which may have imposed novel selective pressures on H. fraxineus at the epidemic front. Virulence is a key trait that is expected to evolve during epidemics and to exhibit plasticity in response to environmental variation. We investigated whether the plasticity of virulence in response to temperature and ash species has evolved in H. fraxineus. Using leaf inoculations, we characterized individual reaction norms for virulence in a long-established Lithuanian population and a recently established Italian population of H. fraxineus. At moderate temperature, the two H. fraxineus populations showed similar levels of virulence on F. excelsior. Virulence decreased to a similar extent on F. ornus in both pathogen populations, indicating similar plasticity of their virulence in relation to ash species. Interestingly, increasing temperature reduced virulence in the Lithuanian population but did not significantly change the virulence expressed by the Italian population, which remained stable across temperatures. Although these findings warrant further investigation, they suggest that ash populations in southern Europe may be more vulnerable to ash dieback under warm conditions than previously anticipated.

evolutionary biology↗

Introducing furanocoumarin biosynthetic genes in tomato results in coumarins accumulation and impacted growth

Over the past three decades, eeorts to decipher plant metabolism have shed light on key enzymes driving specialized metabolite biosynthesis. Although only few pathways have been completely investigated to date, their characterization paves the way for exploring the potential eeects of specialized metabolites on plant physiology. Among them is the linear furanocoumarin pathway, which was recently completed to produce up to psoralen. In this study, we report the first metabolic engineering of the linear furanocoumarin pathway to enable artificial psoralen production in tomato, through the integration of four genes coding for the enzymes: Umbelliferone Synthase, Demethylsuberosin Synthase, Marmesin Synthase and Psoralen Synthase. Interestingly, coumarins were produced instead of furanocoumarins. Using morphophysiological, metabolomic, and transcriptomic analyses, we suggest how coumarins, particularly scopoletin, can impact growth and aeect plant physiology, even at low concentrations. As coumarins have increasingly attracted interest for agricultural applications due to their minimal environmental impact, this work both expands and challenges their potential by highlighting the physiological costs and benefits they may impose on tomato. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/663522v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1df0bdeorg.highwire.dtl.DTLVardef@843d9borg.highwire.dtl.DTLVardef@1e898b6org.highwire.dtl.DTLVardef@18205eb_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

plant biology↗

A tardigrade cytochrome P450 perturbs Drosophila melanogaster development

An analysis (Nelson, 2018) of Hypsibius exemplaris cytochrome P450 genes has revealed the existence of a gene (dubbed Cyp18k1) showing moderate sequence similarity with the ecdysteroid-inactivating arthropod Cyp18a group. Using a transgenic H. exemplaris Cyp18k1 construct, we have demonstrated that ubiquitous CYP18K1 expression results in lethal failure of abdominal histoblast proliferation in Drosophila melanogaster.

evolutionary biology↗

Before the Black Box

Ecdysteroid hormones are responsible for regulating arthropod moulting, but unlike other taxa which used sterol-derived hormones (e.g. vertebrate sex-determining hormones), arthropods cannot synthesis sterols de novo. Ecdysteroid synthesis is thus dependent on funnelling diverse dietary sterols into the conserved reactions of ecdysteroidogenesis. While the later steps of ecdysteroid synthesis are dominated by cytochrome P450s, earlier stages of this pathway (i.e. those acting in or before the Black Box in which 7-dehydrocholesterol is converted to 5,22,25-trideoxyecdysone) involve more diverse enzymes. While numerous comparative analyses of arthropod cytochrome P450s have been conducted, little comparable work has been done on the pre-Black Box FAD-dependent oxidoreductase DHCR24 and the Rieske domain oxygenase Neverland and the Black Box short-chain dehydrogenase/reductase Shroud. However, there is some evidence for evolutionary shifts among these enzymes across arthropods, e.g. the absence of DHCR24 from Drosophila melanogaster. We have sought to systematically identify such changes in copy number in DHCR24, Neverland and Shroud across arthropods, as well as to identify correlations between these changes and dietary factors with reference to the literature. Among other results, we find that Neverland is absent from all beetles (providing support for mycophagy as an important element of the beetle ancestral diet) and that losses of DHCR24 correlate with production of alkylated ecdysteroids.

genomics↗

Genetic characterisation of candidate ecdysteroid kinases in Drosophila melanogaster

Ecdysteroids are major hormones in insects and control moulting, growth, reproduction, physiology, and behaviour. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterised, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date only two EcK-encoding genes have been identified, in the silkworm Bombyx mori and the mosquito Anopheles gambiae. Previously, we identified two ecdysteroid kinase-like (EcKL) genes--Wallflower (Wall) and Pinkman (pkm)--in the model fruit fly Drosophila melanogaster that are orthologs of the ecdysteroid 22-kinase gene BmEc22K. Here, using gene knockdown, knockout and misexpression, we explore Wall and pkms possible functions and genetically test the hypothesis that they encode EcKs. Wall and pkm null mutants are viable and fertile, suggesting they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artefacts. However, misexpression of Wall results in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle and pigmentation. Wall misexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression of Cyp18a1, suggesting Wall does not directly inactivate 20E. Additionally, Wall misexpression phenotypes are not attenuated in Cyp18a1 mutants, strongly suggesting Wall is not an ecdysteroid 26-kinase. We hypothesise that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles in Drosophila development, and may highlight aspects of insect endocrinology that are as-yet uncharacterised. We also provide preliminary evidence that CG5644 encodes an ecdysteroid 22-kinase conserved across Diptera.

genetics↗

Designing Bt constructs for Brassicas, with minimal IP issues: A case study

As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Cry-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM). The two main motivations for modifying the DNA sequences of these genes were to minimise any licencing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might affect gene activity in plants. To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) Subterranean Clover Stunt Virus promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 0.81 to 17.69 g Cry1CM/g fresh weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. Under laboratory conditions, even low-level expression of Cry1BM and Cry1CM was sufficient to cause insect mortality, suggesting that these modified CryM genes are suitable for the development of insect resistant GM crops. Except for the Cry1B/Cry1C genes themselves, which remain under patent until 2027 and the PAT gene in the USA, our assessment of the intellectual property landscape of the constructs described here suggest that they can be used without the need for further licencing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

plant biology↗

Acetylcholine esterase of Drosophila melanogaster: a laboratory model to explore applications of insecticide susceptibility gene drives

BACKGROUNDOne of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses. RESULTSWe have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygosed, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of Gene Drive Resistance (GDR): rather than being mediated by the conventional Non-Homologous End-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas. CONCLUSIONSInsecticide susceptibility gene drives could be useful tools to control pest insects however problems associated with particularities of the target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as a safety-switch to prevent the unwanted spread of gene drives.

genetics↗

Phylogenomics of the ecdysteroid kinase-like (EcKL) gene family in insects highlights roles in both steroid hormone metabolism and detoxification

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid moulting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We also provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. This work provides a robust framework for future functional studies into the EcKL gene family and opens promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied co-evolution of butterflies with their host plants. Significance statementThe ecdysteroid kinase-like (EcKL) gene family has been linked to both steroid inactivation and detoxification in insects, but the functions of most of its members are unknown. Here, we study the evolutionary history of the EcKLs and showcase how phylogenetics can inform the functional characterisation of enzyme families. EcKL family size varies over an order of magnitude and is associated with chemically complex diets, implicating numerous genes in detoxification. We also find a surprising link between a steroid-metabolising clade and host plant diversity in butterflies and moths, suggesting new detoxification functions may evolve through an insect-plant chemical arms-race. This work advances numerous functional hypotheses for multiple EcKL clades and proposes a new classification system for this poorly characterised gene family.

genomics↗

The haplolethal gene wupA of Drosophila exhibits potential as a target for an X-poisoning gene drive

A synthetic gene drive that targets haplolethal genes on the X-chromosome can skew the sex ratio towards males. Like an X-shredder it does not involve homing and that has advantages including the reduction of gene drive resistance allele formation. We examine this X-poisoning strategy by targeting four of the 11 known X-linked haplolethal/haplosterile genes of Drosophila melanogaster with CRISPR/Cas9. We find that targeting the wupA gene during spermatogenesis skews the sex ratio so fewer than 14% of progeny are daughters. That is unless we cross the mutagenic males to X^XY female flies that bear attached-X chromosomes, which reverses the inheritance of the poisoned X chromosome so that sons inherit it from their father; in which case only 2% of the progeny are sons. These sex ratio biases suggests that most of the CRISPR/Cas9 mutants we induced in the wupA gene are haplolethal but some are recessive lethal. The males generating wupA mutants do not suffer from reduced fertility rather the haplolethal mutants arrest development in the late stages of embryogenesis well after fertilized eggs have been laid. This provides a distinct advantage over genetic manipulation strategies involving sterility which can be countered by the remating of females. We also find that wupA mutants that destroy the nuclear localization signal of shorter isoforms are not haplolethal as long as the open reading frame remains intact. Like D. melanogaster wupA orthologs of D. suzukii and Anopheles mosquitos are found on X chromosomes making wupA a viable X-poisoning target in multiple species.

genetics↗

Discovery of a druggable copper-signaling pathway that drives cell plasticity and inflammation

Inflammation is a complex physiological process triggered in response to harmful stimuli. It involves specialized cells of the immune system able to clear sources of cell injury and damaged tissues to promote repair. Excessive inflammation can occur as a result of infections and is a hallmark of several diseases. The molecular basis underlying inflammatory responses are not fully understood. Here, we show that the cell surface marker CD44, which characterizes activated immune cells, acts as a metal transporter that promotes copper uptake. We identified a chemically reactive pool of copper(II) in mitochondria of inflammatory macrophages that catalyzes NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with a rationally-designed dimer of metformin triggers distinct metabolic and epigenetic states that oppose macrophage activation. This drug reduces inflammation in mouse models of bacterial and viral (SARS-CoV-2) infections, improves well-being and increases survival. Identifying mechanisms that regulate the plasticity of immune cells provides the means to develop next-generation medicine. Our work illuminates the central role of copper as a regulator of cell plasticity and unveils a new therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

cell biology↗

Ecdysteroid kinase-like (EcKL) paralogs confer developmental tolerance to caffeine in Drosophila melanogaster

A unique aspect of metabolic detoxification in insects compared to other animals is the presence of xenobiotic phosphorylation, about which little is currently understood. Our previous work raised the hypothesis that members of the taxonomically restricted ecdysteroid kinase-like (EcKL) gene family encode the enzymes responsible for xenobiotic phosphorylation in the model insect Drosophila melanogaster (Diptera: Ephydroidea)--however, candidate detoxification genes identified in the EcKL family have yet to be functionally validated. Here, we test the hypothesis that EcKL genes in the rapidly evolving Dro5 clade are involved in the detoxification of plant and fungal toxins in D. melanogaster. The mining and reanalysis of existing data indicated multiple Dro5 genes are transcriptionally induced by the plant alkaloid caffeine and that adult caffeine susceptibility is associated with a novel naturally occurring indel in CG31370 (Dro5-8) in the Drosophila Genetic Reference Panel (DGRP). CRISPR-Cas9 mutagenesis of five Dro5 EcKLs substantially decreased developmental tolerance of caffeine, while individual overexpression of two of these genes--CG31300 (Dro5-1) and CG13659 (Dro5-7)--in detoxification-related tissues increased developmental tolerance. In addition, we found Dro5 loss-of-function animals also have decreased developmental tolerance of the fungal secondary metabolite kojic acid. Taken together, this work provides the first compelling functional evidence that EcKLs encode detoxification enzymes and suggests that EcKLs in the Dro5 clade are involved in the metabolism of multiple ecologically relevant toxins in D. melanogaster. We also propose a biochemical hypothesis for EcKL involvement in caffeine detoxification and highlight the many unknown aspects of caffeine metabolism in D. melanogaster and other insects. HighlightsO_LIPhosphorylation is an under-characterised detoxification reaction in insects C_LIO_LIDro5 EcKL genes are good detoxification candidate genes in Drosophila melanogaster C_LIO_LIKnockout and misexpression of some Dro5 genes modulated tolerance to caffeine C_LIO_LIDro5 genes may also confer tolerance to the fungal toxin kojic acid C_LIO_LICaffeine tolerance could be adaptive for Drosophila associating with Citrus fruits C_LI

genetics↗

Generation of a new Tbx6-inducible reporter mouse line to trace presomitic mesoderm derivatives throughout development and in adults

The presomitic mesoderm (PSM) is initially an unsegmented structure localized on each side of the neural tube of the developing embryo, which progressively segments to form the somites. The somites will segregate and partition to generate the dorsal dermomyotome and the ventral sclerotome. Endothelial and myogenic cells of both the trunk and limbs are derived from the somites. There is a lack of efficient reporter mouse models to label and trace the PSM derivatives, despite their crucial contribution to many developmental processes. In this study, we generated a tamoxifen inducible transgenic Tbx6 mouse line, Tg(Tbx6_Cre/ERT2)/ROSA-eYFP, to tag and follow PSM-derivatives from early embryonic stages until adulthood. After induction, endothelial and myogenic cells can be easily identified within the trunk and limbs with proper expression patterns. Since our Tg(Tbx6_Cre/ERT2)/ROSA-eYFP model allows to permanently label the PSM-derived cells, their progeny can be studied at long-term, opening the possibility to perform lineage tracing of stem cells upon aging.

developmental biology↗

BMP signalling directs a fibroblast-to-myoblast conversion at the connective tissue/muscle interface to pattern limb muscles

Positional information driving limb muscle patterning is contained in lateral plate mesoderm-derived tissues, such as tendon or muscle connective tissue but not in myogenic cells themselves. The long-standing consensus is that myogenic cells originate from the somitic mesoderm, while connective tissue fibroblasts originate from the lateral plate mesoderm. We challenged this model using cell and genetic lineage tracing experiments in birds and mice, respectively, and identified a subpopulation of myogenic cells at the muscle tips close to tendons originating from the lateral plate mesoderm and derived from connective tissue gene lineages. Analysis of single-cell RNA-sequencing data obtained from limb cells at successive developmental stages revealed a subpopulation of cells displaying a dual muscle and connective tissue signature, in addition to independent muscle and connective tissue populations. Active BMP signalling was detected in this junctional cell sub-population and at the tendon/muscle interface in developing limbs. BMP gain- and loss-of-function experiments performed in vivo and in vitro showed that this signalling pathway regulated a fibroblast-to-myoblast conversion. We propose that localised BMP signalling converts a subset of lateral plate mesoderm-derived fibroblasts to a myogenic fate and establishes a boundary of fibroblast-derived myonuclei at the muscle/tendon interface to control the muscle pattern during limb development.

developmental biology↗

Identifying candidate detoxification genes in the ecdysteroid kinase-like (EcKL) and cytochrome P450 gene families in Drosophila melanogaster by integrating evolutionary and transcriptomic data

The capacity to detoxify toxic compounds is essential for adaptation to the ecological niches of many organisms, especially insects. However, detoxification in insects is often viewed through the lens of mammalian detoxification research, even though the organ and enzyme systems involved have diverged for over half a billion years. Phosphorylation is a non-canonical phase II detoxification reaction that, among animals, occurs near exclusively in insects, but the enzymes responsible have never been cloned or otherwise identified. We propose the hypothesis that members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family encode detoxicative kinases. To test this hypothesis, we annotated the EcKL gene family in 12 species of Drosophila and explored their evolution within the genus. Many ancestral EcKL clades are evolutionarily unstable and have experienced repeated gene gain and loss events, while others are conserved as single copy orthologs. Leveraging multiple published gene expression datasets from D. melanogaster, and using the cytochrome P450s--a canonical detoxification family--as a test case, we demonstrate relationships between xenobiotic induction, detoxification tissue-enriched expression and evolutionary instability in the EcKLs and the P450s. We also found previously unreported genomic and transcriptomic variation in a number of EcKLs and P450s associated with toxic stress phenotypes using a targeted phenome-wide association study (PheWAS) approach. Lastly, we devised a systematic method for identifying candidate detoxification genes in large gene families that is concordant with experimentally determined functions of P450 genes in D. melanogaster. Applying this method to the EcKLs suggested a significant proportion of these genes play roles in detoxification, and that the EcKLs may constitute a detoxification gene family in insects. Additionally, we estimate that between 11-16 uncharacterised D. melanogaster P450s are strong detoxification candidates. HighlightsO_LIThe poorly characterised ecdysteroid kinase-like (EcKL) gene family is hypothesised to encode enzymes responsible for detoxification by phosphorylation in insects. C_LIO_LIAn integrative detoxification score method accurately categorises the known functions of a canonical detoxification family, the cytochrome P450s, and suggests many EcKLs are also involved in detoxification. C_LIO_LIA targeted phenome-wide association study finds novel associations between EcKL/P450 variation and a number of toxic stress phenotypes, such as two unlinked EcKL paralogs that are both associated with developmental methylmercury resistance. C_LI

genetics↗

The spread of resistance to imidacloprid is restricted by thermotolerance in natural populations of Drosophila melanogaster

Imidacloprid, the worlds most utilised insecticide1, has raised considerable controversy due to its harmful effects on non-pest species2-6 and there is increasing evidence showing that insecticides have become the primary selective force in many insect species7-14. The genetic response to insecticides is heterogeneous across population and environment15-17, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, demonstrated a polygenic basis to resistance and a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicate enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.

evolutionary biology↗

Cis and trans-acting variants contribute to survivorship in a naive Drosophila melanogaster population exposed to ryanoid insecticides

Insecticide resistance is a paradigm of microevolution and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster. Here we use a naive population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome wide association studies of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliproles mode of action. Genome-wide association studies also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homologue of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naive to this new insecticide chemistry and it adumbrates a selective response among natural populations to these chemicals.\n\nSignificanceAround the world insecticides are being deregistered and banned, as their environmental costs are deemed too great or their efficacy against pest insects is reduced through the evolution of insecticide resistance. With the introduction of replacement insecticides comes the responsibility to assess the way new insecticides perturb various levels of biological systems; from insect physiology to ecosystems. We used a systems genetics approach to identify genetic variants affecting survivorship of Drosophila melanogaster exposed to chlorantraniliprole. The study population was completely naive to this insecticide chemistry and yet we find associations with variants in neuromuscular genes and co-regulated detoxification genes. We predict that these variants will increase in populations of this sentinel species as these insecticides are applied in the environment.

genetics↗

Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains

Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.

cell biology↗

High-quality SNPs from genic regions highlight introgression patterns among European white oaks (Quercus petraea and Q. robur).

The Src homology-2 domain containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key "allosteric switch" driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the "allosteric switch" model, we critically reviewed structural data of SHP2 and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution, and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central {beta}- sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation. Significance StatementSHP2 is a multi-domain protein, playing an important role in up-regulating cellular processes such as cell survival, proliferation, and programmed cell death. SHP2 mutations cause developmental disorders and were found in many cancer types, including neuroblastoma, breast cancer, and leukemia. In healthy cells, SHP2 mainly takes an autoinhibited, inactive form, and SHP2 is activated upon binding of phosphopeptides to the N-SH2 domain. For the past two decades, the widening of the binding cleft upon peptide binding has been considered as the key event driving SHP2 activation. Here, by analyzing crystallographic data and molecular simulations, we demonstrate that the binding cleft in N-SH2 is, instead, already open and accessible in solution, and its degree of opening does not influence SHP2 activation.

evolutionary biology↗