A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.

This is the final version of the article. Available from the publisher via the DOI in this record.Open Access funded by Biotechnology and Biological Sciences Research CouncilSpinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.This work was, in part, funded by a research grant (BB/G023352/1) from the Biotechnology and Biological Sciences Research Council of the UK to CB

Directing the use of DDR kinase inhibitors in cancer treatment.

Introduction Defects in the DNA damage response (DDR) drive the development of cancer by fostering DNA mutation but also provide cancer-specific vulnerabilities that can be exploited therapeutically. The recent approval of three different PARP inhibitors for the treatment of ovarian cancer provides the impetus for further developing targeted inhibitors of many of the kinases involved in the DDR, including inhibitors of ATR, ATM, CHEK1, CHEK2, DNAPK and WEE1. Areas covered: We summarise the current stage of development of these novel DDR kinase inhibitors, and describe which predictive biomarkers might be exploited to direct their clinical use. Expert opinion: Novel DDR inhibitors present promising candidates in cancer treatment and have the potential to elicit synthetic lethal effects. In order to fully exploit their potential and maximize their utility, identifying highly penetrant predictive biomarkers of single agent and combinatorial DDR inhibitor sensitivity are critical. Identifying the optimal drug combination regimens that could used with DDR inhibitors is also a key objective

Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis

This is the final version. Available from the publisher via the DOI in this record.The Osmia bicornis whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MPJT00000000. The RNAseq data generated in this study has been deposited in the Sequence Read Archive (SRA) under accession SRP065762. Accession numbers of the bee P450 genes manually curated in this study are shown in S5 Table. All other relevant data are within the paper and its Supporting Information files.The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.Biotechnology and Biological Science Research Council (BBSRC)Bayer AGEuropean Research Council (ERC

Can public spaces effectively be used as cleaner indoor air shelters during extreme smoke events?

During extreme air pollution events, such as bushfires, public health agencies oftenrecommend that vulnerable individuals visit a nearby public building with central air conditioningto reduce their exposure to smoke. However, there is limited evidence that these “cleaner indoorair shelters” reduce exposure or health risks. We quantified the impact of a “cleaner indoor airshelter” in a public library in Port Macquarie, NSW, Australia when concentrations of fine particulatematter (PM2.5) were elevated during a local peat fire and nearby bushfires. Specifically, we evaluatedthe air quality improvements with central air conditioning only and with the use of portable highefficiency particulate air (HEPA) filter air cleaners. We measured PM2.5 from August 2019 untilFebruary 2020 by deploying pairs of low-cost PM2.5 sensors (i) inside the main library, (ii) in asmaller media room inside the library, (iii) outside the library, and (iv) co-located with regulatorymonitors located in the town. We operated two HEPA cleaners in the media room from August untilOctober 2019. We quantified the infiltration efficiency of outdoor PM2.5concentrations, defined asthe fraction of the outdoor PM2.5 concentration that penetrates indoors and remains suspended, aswell as the additional effect of HEPA cleaners on PM2.5 concentrations. The infiltration efficiency ofoutdoor PM2.5 into the air-conditioned main library was 30%, meaning that compared to the PM2.5concentration outdoors, the concentrations of outdoor-generated PM2.5 indoors were reduced by 70%.In the media room, when the HEPA cleaners were operating, PM2.5 concentrations were reducedfurther with a PM2.5 infiltration efficiency of 17%. A carefully selected air-conditioned public buildingcould be used as a cleaner indoor air shelter during episodes of elevated smoke emissions. Furtherimprovements in indoor air quality within the building can be achieved by operating appropriatelysized HEPA cleaners

A global optimisation approach to range-restricted survey calibration

Survey calibration methods modify minimally unit-level sample weights to fit domain-level benchmark constraints (BC). This allows exploitation of auxiliary information, e.g. census totals, to improve the representativeness of sample data (addressing coverage limitations, non-response) and the quality of estimates of population parameters. Calibration methods may fail with samples presenting small/zero counts for some benchmark groups or when range restrictions (RR), such as positivity, are imposed to avoid unrealistic or extreme weights. User-defined modifications of BC/RR performed after encountering non-convergence allow little control on the solution, and penalization approaches modelling infeasibility may not guarantee convergence. Paradoxically, this has led to underuse in calibration of highly disaggregated information, when available. We present an always-convergent flexible two-step Global Optimisation (GO) survey calibration approach. The feasibility of the calibration problem is assessed, and automatically controlled minimum errors in BC or changes in RR are allowed to guarantee convergence in advance, while preserving the good properties of calibration estimators. Modelling alternatives under different scenarios, using various error/change and distance measures are formulated and discussed. The GO approach is validated by calibrating the weights of the 2012 Health Survey for England to a fine age-gender-region cross-tabulation (378 counts) from the 2011 Census in England and Wales

A global optimisation approach to range-restricted survey calibration

Survey calibration methods modify minimally unit-level sample weights to fit domain-level benchmark constraints (BC). This allows exploitation of auxiliary information, e.g. census totals, to improve the representativeness of sample data (addressing coverage limitations, non-response) and the quality of estimates of population parameters. Calibration methods may fail with samples presenting small/zero counts for some benchmark groups or when range restrictions (RR), such as positivity, are imposed to avoid unrealistic or extreme weights. User-defined modifications of BC/RR performed after encountering non-convergence allow little control on the solution, and penalization approaches modelling infeasibility may not guarantee convergence. Paradoxically, this has led to underuse in calibration of highly disaggregated information, when available. We present an always-convergent flexible two-step Global Optimisation (GO) survey calibration approach. The feasibility of the calibration problem is assessed, and automatically controlled minimum errors in BC or changes in RR are allowed to guarantee convergence in advance, while preserving the good properties of calibration estimators. Modelling alternatives under different scenarios, using various error/change and distance measures are formulated and discussed. The GO approach is validated by calibrating the weights of the 2012 Health Survey for England to a fine age-gender-region cross-tabulation (378 counts) from the 2011 Census in England and Wales