A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

Aedes mosquitoes transmit many human viral pathogens including dengue, yellow fever and chikungunya. Most of these pathogens have no specific treatment or vaccine and hence their control is reliant on controlling the mosquito vectors, which usually involves the use of insecticides. In order to prevent the alarming prospect of mosquito control failure due to the rapid selection and spread of insecticide resistance in several mosquito populations worldwide, it is essential that effective resistance management strategies are implemented and adhered to. The development of simple diagnostic tests for the early identification and monitoring of resistance is an important prerequisite for this task. Here, we describe the development of a simple colorimetric test for the detection of GSTE2-2/DDTase-based resistance in individual mosquitoes. The novel assay combines the most desirable features of specificity and sensitivity with the low cost and ease of use required for a routine test in endemic countries. It can have direct application in routine vector monitoring as a resistance indicator and help improve the sustainability of insecticide based control strategies

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.The work of Hellenic Agricultural Organisation - 'Demeter' was partially supported by an ARIMnet2 StomP grand to A.T and E.R. This work was also partially funded from a fellowship granted to H.A.A.S. (CNPq - PQ - Proc 308461/2013-4). The Universidad Politécnica de Cartagena group would like to thank for partial financial support the Ministerio de Economía y Competitividad of Spain and FEDER (AGL2011-25164). Lidia García-Vidal holds a grant from the MECD (FPU13/01528). The Tuta absoluta strain from Gela, Sicily was collected under the frame a resistance monitoring program established among the Hellenic Agricultural Organisation - 'Demeter' and DuPont De Nemurs (data published in 2015). Finally, the Hellenic Agricultural Organisation - 'Demeter' would like to thank Fytochem S.A., Neo Mirtos, Ierapetra for supplies of plant material

Use of insecticide quantification kits to investigate the quality of spraying and decay rate of bendiocarb on different wall surfaces in Kagera region, Tanzania

Background Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 2012 as part of the interim national insecticide resistance management plan. This move followed reports of increasingly alarming levels of pyrethroid resistance across the country. This study used the insecticide quantification kit (IQK) to investigate the intra-operational IRS coverage and quality of spraying, and decay rate of bendiocarb on different wall surfaces in Kagera region. Methods To assess intra-operational IRS coverage and quality of spraying, 104 houses were randomly selected out of 161,414 sprayed houses. A total of 509 samples (218 in Muleba and 291 in Karagwe) were obtained by scraping the insecticide samples from wall surfaces. To investigate decay rate, 66 houses (36 in Muleba and 30 in Karagwe) were selected and samples were collected monthly for a period of five months. Laboratory testing of insecticide concentration was done using IQKTM [Innovative Vector Control Consortium]. Results Of the 509 samples, 89.5% met the World Health Organization (WHO) recommended concentration (between 100–400 mg/m2) for IRS target dosage. The proportion of samples meeting WHO standards varied between Karagwe (84.3%) and Muleba (96.3%) (p < 0.001). Assessment of quality of spraying at house level revealed that Muleba (84.8%) had a significantly higher proportion of households that met the expected target dosage (100–400 mg/m2) compared to Karagwe (68.9%) (p < 0.001). The quality of spraying varied across different wall substrates in both districts. Evaluation of bendiocarb decay showed that the proportion of houses with recommended concentration declined from 96.9%, 93.5% and 76.2% at months one, two, and three post IRS, respectively (p-trend = 0.03). The rate of decay increased in the fourth and fifth month post spraying with only 55.9% and 26.3% houses meeting the WHO recommendations, respectively. Conclusion IQK is an important tool for assessing IRS coverage and quality of spraying. The study found adequate coverage of IRS; however, residual life of bendiocarb was observed to be three months. Results suggest that in order to maintain the recommended concentrations with bendiocarb, a second spray cycle should be carried out after three months

Failed immune responses across multiple pathologies share pan-tumor and circulating lymphocytic targets

Tumor-infiltrating lymphocytes (TILs) are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes while preserving tumor reactivity and neoantigen specificity shared with circulating immune cells. We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and TILs in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Through comprehensive bioinformatics analyses, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, 3 genes (LEF1, FASLG, and MMP9) could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology, failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible noninvasive approach to pan-pathology diagnoses. The differentially expressed genes we have identified warrant future investigation into the development of their potential in noninvasive precision diagnostics and precision pan-disease immunotherapeutics. - 2019, American Society for Clinical Investigation.We thank all study participants and patients; The Cancer Genome Atlas; Mathieu Latour and Roula Albadine and supporting staff of the CHUM pathology department; Manon de Ladurantaye and Anne-Marie Mes-Masson from the CRCHUM for RNA quality profiling, Geneviève Cormier and Fred Saad from the CRCHUM for drawing blood from control donors; Gilles Corbeil of the CRCHUM genomics department for RNA quality testing and microarray profiling; Francois Harvey of the CRCHUM bioinformatics department; Peter Graf and Patrick Sabourin from Affymetrix for providing reagents and technical assistance; Zeeshan Farroq and Ofir Goldberger from Fluidigm; Erika Diaz from StemCell; Andrew Mouland from McGill University; Simon Turcotte from University of Montreal; and Sascha Ring from Biostars for their advice. This work was partially performed at the Institut du Cancer de Montréal CRCHUM and University of Montreal, in Montreal, Quebec, Canada. This work was supported by a Canadian Cancer Society Research Institute grant (CCSRI) (702036, to RL and IJ) and a Biomedical Research Grant from the Kidney Foundation of Canada (KFOC130019 to RL). RL is supported by the Quebec Cell, Tissue and Gene Therapy Network—ThéCell (a thematic network supported by the Fonds de recherche du Québec–Santé [FRQS]), the FRQS, and the Immunotherapy Network (iTNT) from the Terry Fox Research Institute (TFRI), A. Monette is supported by Mitacs, Merck, l’Institut du cancer de Montréal (ICM), the Society for Immunotherapy of Cancer, and the Lady Davis Institute for Medical Research. NAB is supported by the FRQS post-doctoral award and Qatar University. JBL is supported by l’Institut du Cancer de Montréal. JPR holds the Louis Lowenstein Chair, McGill University. DEK is supported by an FRQS Research Scholar Award (grant 31035), CIHR 377124, NHLBI RO1-HL-092565, and the Canada Foundation for Innovation (CFI) (grant 31756). IJ and computational analysis were supported by the Canada Research Chair Program (CRC) (grant 225404), Ontario Research Fund (grant 34876), the Natural Sciences Research Council (NSERC) (grant 203475), the CFI (grants 203373 and 30865), the Krembil Foundation, and IBM.Scopu

Comparative Genomics of the Anopheline Glutathione S-Transferase Epsilon Cluster

Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated, gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (ψAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, ψAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera, though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site within GSTE5 may represent adaptations to new habitats

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

Distinctive features of CD4+ T cell dysfunction in chronic viral infections

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