Journal: Cell Reports

Article Title: Genomic landscape of drug response reveals mediators of anthelmintic resistance

doi: 10.1016/j.celrep.2022.111522

Figure Lengend Snippet: Outline of the genetic cross, X-QTL, and advanced intercross experiments (A) A genetic cross between the anthelmintic susceptible MHco3(ISE) and multi-drug-resistant MHco18(UGA) was used to map genetic loci associated with fenbendazole, levamisole, and ivermectin drug treatment. (B) An X-QTL experiment was performed on the F2 generation exposed to fenbendazole, levamisole, or ivermectin or not treated. (C) An advanced intercross experiment using the F3 generation was subjected to a half-dose followed by a double-standard dose of ivermectin. For both the X-QTL and advanced intercross experiments, pools of L 3 (n = 200) were collected pre- and post-treatment from drug-exposed and time-matched untreated controls, performed in triplicate ( Figure S1 ). Whole-genome sequencing was performed, and genetic diversity between pre- and post-treatment was compared.

Article Snippet: Ivermectin aglycone , Santa Cruz Biotechnology , CAS 123997-59-1.

Techniques: Sequencing

Journal: Cell Reports

Article Title: Genomic landscape of drug response reveals mediators of anthelmintic resistance

doi: 10.1016/j.celrep.2022.111522

Figure Lengend Snippet: A genetic cross followed by drug selection reveals discrete QTLs associated with each anthelmintic drug class (A) Genome-wide comparison of susceptible MHco3(ISE) and multidrug-resistant MHco18(UGA) parental strains revealed broad-scale genetic differentiation ( F ST ) on all chromosomes. In contrast, after the genetic cross, these signals of differentiation are lost in an untreated control (time-matched samples to the drug-treated groups). The dashed line represents the mean F ST + 3 standard deviations. (B) Comparison of genome-wide differentiation between F3 generation pooled infective-stage larvae (L 3 , n = 200) sampled pre- and post-treatment revealed distinct genomic regions or QTLs associated with fenbendazole, levamisole, and ivermectin drug treatment. In all plots, each point represents the -log 10 q value from the Z score distribution of mean genetic differentiation ( F ST ) from three biological replicates per 5 kb sliding window throughout the genome. The dashed line represents the Bonferroni genome-wide level of significance (α = 0.05, n = 56,476 windows). See Figure S1 for genome-wide replicate data of the drug selection experiments.

Article Snippet: Ivermectin aglycone , Santa Cruz Biotechnology , CAS 123997-59-1.

Techniques: Selection, Genome Wide

Journal: Cell Reports

Article Title: Genomic landscape of drug response reveals mediators of anthelmintic resistance

doi: 10.1016/j.celrep.2022.111522

Figure Lengend Snippet: Characterization of the major QTL associated with ivermectin resistance (A) QTL between pre- and post-ivermectin treatment on chromosome 5. Each data point represents the -log 10 q value from the Z score distribution of mean genetic differentiation ( F ST ) from three biological replicates per 5 kb sliding window throughout the genome; points are colored based on the concordance of individual replicates indicated by none (blue), 1 of 3 (yellow), 2 of 3 (orange), or all 3 (red) above the F ST genome-wide threshold. The horizontal dashed line represents the Bonferroni genome-wide level of significance (α = 0.05, n = 56,476 windows). A magnified aspect of the main chromosome 5 QTL, highlighting (B) -log 10 q value of F ST in the X-QTL cross, and (C) nucleotide diversity (Pi) on US farms, where each farm is colored by the degree of ivermectin resistance (EC 50 ) measured by larval development assays. In (A), (B), (C), and (D), the position of cky-1 is indicated by the vertical dashed line. (D) Reanalysis of RNA-seq data from ( Laing et al., 2022 ), highlighting the position of cky-1 in the QTL and overexpression after treatmenI(E) RT-qPCR analysis of cky-1 expression in H. contortus and T. circumcincta strains that differ in their ivermectin resistance phenotype. Data represent log 2 -transformed expression normalized to actin or GAPDH control genes for H. contortus and T. circumcincta, respectively, from three independent experiments. Downregulation of cky-1 expression in C. elegans by either (F) a balanced deletion or (G) RNAi-knockdown increases ivermectin sensitivity relative to the control N2 strain, based on developmental assays (n = 3 independent experiments) measuring the percentage of progeny surviving to adulthood relative to DMSO controls. In (F) and (G), each point represents an independent treatment condition, normalized to a DMSO control without ivermectin. A Kruskal-Wallis test was used to determine whether treatment condition differed from untreated control, where ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. Boxplots in (E), (F), and (G) show the median, 25th, and 75th percentiles of the data. The whiskers extend 1.5 times the inter-quartile range.

Article Snippet: Ivermectin aglycone , Santa Cruz Biotechnology , CAS 123997-59-1.

Techniques: Genome Wide, RNA Sequencing Assay, Over Expression, Quantitative RT-PCR, Expressing, Transformation Assay

Journal: Cell Reports

Article Title: Genomic landscape of drug response reveals mediators of anthelmintic resistance

doi: 10.1016/j.celrep.2022.111522

Figure Lengend Snippet:

Article Snippet: Ivermectin aglycone , Santa Cruz Biotechnology , CAS 123997-59-1.

Techniques: Recombinant, Staining, Expressing, Plasmid Preparation, Sequencing, Software

Journal: Journal of biochemical and molecular toxicology

Article Title: Combined in silico and in vitro approaches to identify P-glycoprotein-inhibiting pesticides.

doi: 10.1002/jbt.23588

Figure Lengend Snippet: FIGURE 1 Intracellular concentration of rhodamine 123 (A) and Hoechst 33342 (B) in MCF7R cells in the presence of either pesticide (white bars) or a reference inhibitor (black bars). The pesticides were tested at 100 μM (for cyflumetofen, dimethomorph, fenpicoxamid, oxathiapiprolin, pinoxaden, profoxydim, silaflulofen and tralkoxydim), or 10 μM (for ivermectin B1a, spinosad and emamectin B1 benzoate), while the reference inhibitors elacridar and verapamil were tested at 10 and 100 μM, respectively. Values are shown as mean ± SEM and expressed as percentages of the vehicle control. Dotted line: 150%. When the concentration of rhodamine 123 or Hoechst 33342 was higher than 150%, the tested compound was considered to be a P‐glycoprotein inhibitor. Three independent experiments were performed. *, **, ***: significantly different from the vehicle control (p < 0.05, p < 0.01 and p < 0.001, respectively).

Article Snippet: Dimethomorph, emamectin B1 benzoate, ivermectin B1a, profoxydim lithium salt (profoxydim), silafluofen, spinosad and tralkoxydim were purchased from Santa Cruz Biotechnology.

Techniques: Concentration Assay, Pesticides, Control

Journal: MethodsX

Article Title: A quick and simple method for the determination of ivermectin in dog plasma by LC–MS/MS

doi: 10.1016/j.mex.2018.11.011

Figure Lengend Snippet: Chromatograms of a blank sample (A), a blank sample spiked at the LLOQ (0.5 ng/mL) (B), and an actual dog sample collected 240 min after ivermectin oral administration (C), obtained by LC–MS/MS analysis. Early-eluting matrix contaminants and the last part of the LC run were diverted to waste through a Valco ® divert valve.

Article Snippet: Ivermectin chemical reference substance (CRS) was supplied by the European Directorate for the Quality of Medicines and HealthCare (EDQM).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: MethodsX

Article Title: A quick and simple method for the determination of ivermectin in dog plasma by LC–MS/MS

doi: 10.1016/j.mex.2018.11.011

Figure Lengend Snippet: Intraday and interday accuracy (D% = percentage difference between the experimental and theoretical values) and precision (CV%) for IVM (ivermectin), calculated on data obtained analyzing QC (quality control) samples prepared in three different days (total n = 54).

Article Snippet: Ivermectin chemical reference substance (CRS) was supplied by the European Directorate for the Quality of Medicines and HealthCare (EDQM).

Techniques: Control

Journal: MethodsX

Article Title: A quick and simple method for the determination of ivermectin in dog plasma by LC–MS/MS

doi: 10.1016/j.mex.2018.11.011

Figure Lengend Snippet:

Article Snippet: Ivermectin chemical reference substance (CRS) was supplied by the European Directorate for the Quality of Medicines and HealthCare (EDQM).

Techniques: Clinical Proteomics