Analysis of cytochrome P450 contribution to evolved plant toxin resistance in Drosophila sechellia

Drosophila sechellia is a dietary specialist species of fruit fly that has evolved resistance to the toxic secondary defence compounds produced by the fruit of its preferred host plant Morinda citrifolia. The genetic basis of adult toxin resistance is the result of evolution at five loci across the genome. Genetic mapping between D. sechellia and Drosophila simulans and subsequent functional studies in Drosophila melanogaster have identified candidate genes potentially underlying one locus involved in toxin resistance but the remainder of the genes involved are unknown. Genes in the mixed function oxidase or cytochrome P450 gene family are frequently utilized in evolved toxin resistance in insects, yet whether they play a role in D. sechellia's resistance to the toxins found in its host plant is unknown. Here we test the role of cytochrome P450 enzymatic activity in evolved resistance to the two primary toxins found in M. citrifolia fruit: octanoic acid and hexanoic acid. We found that although cytochrome P450 enzymatic activity is involved in basal resistance it is not involved in derived toxin resistance in D. sechellia.     

Exome‐wide association of deltamethrin resistance in Aedes aegypti from Mexico

Aedes aegypti is the major vector of a number of arboviruses that cause disease in humans. Without vaccines or pharmaceuticals, pyrethroid insecticides remain the major tool for public health protection. Pyrethroid resistance is now widespread. Replacement substitutions in the voltage‐gated sodium channel (vgsc) that reduce the stability of pyrethroid binding account for most of the resistance, but metabolic mechanisms also inactivate pyrethroids. High‐throughput sequencing and the A. aegypti L5 annotated physical map has allowed interrogation of the exome for genes and single‐nucleotide polymorphisms associated with pyrethroid resistance. We exposed females of A. aegypti from Mexico to a deltamethrin discriminating dose to designate them as resistant (active after 1 h) or susceptible (knocked down with no recovery after 4 h). The vgsc on chromosome 3 had the highest association, followed by genes proximal to vgsc. We identified potential detoxification genes located singly (eg HPX8C) or within clusters in chromosome 2 [three esterase clusters, two of cytochrome P450 monooxygenases (CYP)] and chromosome 3 (one cluster of 16 CYP325 and seven CYP9 genes). Deltamethrin resistance in A. aegypti is associated with mutations in the vgsc gene and a large assortment of genes.     

A case‐control study and meta‐analysis reveal the association between COX‐2 G‐765C polymorphism and primary end‐stage hip and knee osteoarthritis

Background

Osteoarthritis (OA) is a popular arthrosis featured as pain, limited joint activity, and deformity. Cyclooxygenase‐2 (COX‐2) has been reported to be up‐regulated in arthritic tissues and is integral to the progression of osteoarthritis (OA). Previous studies showed the COX‐2 promoter G‐765C polymorphism could influence COX‐2 expression. However, the relationship between the variant and OA risk is contrasting.

Methods

We conducted a case‐control study with 196 primary end‐stage hip and knee OA cases and 196 controls in a Chinese Han population. Subsequently, we integrated this case‐control study in a meta‐analysis to acquire greater statistical power. The results from our case‐control study using MassARRAY genotyping technology and binary logistic regression statistical methods.

Results

The variant carriers in the Chinese Han population had a lower primary end‐stage hip and knee OA susceptibility (C vs G: OR = 0.350, 95%CI: 0.154‐0.797, P = .012; GC vs GG: adjusted OR = 0.282, 95%CI: 0.118‐0.676, P = .005). Stratification studies indicated that a higher GC frequency in women decreased not only knee OA susceptibility but also unilateral knee OA risk. The meta‐analysis showed that the variant exhibited a significantly decreased OA risk through comparisons involving allelic, homozygous, heterozygous, and dominant models.

Conclusion

Our findings suggest that the COX‐2 G‐765C polymorphism exerts a protective effect against primary end‐stage knee osteoarthritis in a female Chinese Han population.

     

Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin‐resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene‐silencing experiments via double‐stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively.     

An evaluation of gene–gene interaction between the CYP2C9 and VKORC1 genotypes affecting the anticoagulant effect of phenprocoumon and acenocoumarol

Background:  Previous studies have provided contradictory results regarding the interaction between the CYP2C9 and VKORC1 genotypes affecting various outcome measures. Objectives:  We aimed to provide a definite answer regarding the question whether there exists a gene–gene interaction between the CYP2C9 and VKORC1 genotypes affecting the anticoagulant effect of phenprocoumon and acenocoumarol. Patients/Methods:  The EU‐PACT cohort dataset, which contains data on 624 phenprocoumon and 471 acenocoumarol patients, was used. Patient characteristics, pharmacogenetic data, International Normalized Ratios (INRs) and dosages were available. We investigated whether there was an interaction between the CYP2C9 and VKORC1 genotypes affecting the maintenance dose, time to severe over‐anticoagulation and time to achieve stability during the first 180 days of phenprocoumon and acenocoumarol therapy, in addition to the effect of the separate genotypes. The interaction effect was investigated by adding the product term of the CYP2C9 and VKORC1 genotype classes for four different commonly used CYP2C9 classifications to the linear regression model – for the outcome measure maintenance dose – or to the Cox regression models – for the outcome measures time to severe over‐anticoagulation and time to achieve stability. Results:  No significant interactions – all P‐values above 0.23 for phenprocoumon and 0.30 for acenocoumarol – were observed for all outcome measures. Conclusions:  There are no interactions between the CYP2C9 and VKORC1 genotypes affecting the maintenance dose, time to severe over‐anticoagulation and time to achieve stability for phenprocoumon and acenocoumarol.     

Molecular characterization,expression pattern and metabolic activity of flavin‐dependent monooxygenases in Spodoptera exigua

Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin‐dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda‐cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over‐expressed in a metaflumizone‐resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over‐expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua.     

The molecular genetics of migraine

Within the past decade it has been possible to identify susceptibility gene loci that predispose to migraine using genetic markers distributed across the human genome. Five new loci with significant linkage to common types of migraine – migraine with or without aura – have been identified on four different chromosomes using a genome‐wide screen approach. So far, only the locus on 4q has been replicated but no specific, disease‐causing mutations have been described in these common forms of migraine. The best genetic evidence providing molecular insight into migraine still comes from the mutations detected in a rare Mendelian form of migraine with aura – familial hemiplegic migraine (FHM). In 50%–70% of FHM families, mutations in the calcium channel gene CACNA1A in chromosome 19p13 have been identified. In some families, mutations in the ATP1A2 gene encoding the α2 subunit of the Na ⁺ , K ⁺ ‐ATPase are associated with FHM, linked to 1q23. Here we discuss the current knowledge of the heritability of migraine and rare migraine variants as models for understanding the pathophysiology of common migraine and animal models that might contribute to understanding common forms of migraine.     

Alimentary microbes of winter‐form Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a damaging pest of fruit. Reproductively diapausing adults overwinter in woodlands and remain active on warmer winter days. It is unknown if this adult phase of the lifecycle feeds during the winter period, and what the food source may be. This study characterized the flora in the digestive tract of D. suzukii using a metagenomics approach. Live D. suzukii were trapped in four woodlands in the south of England and their guts dissected for DNA extraction and amplicon‐based metagenomics sequencing (internal transcribed spacer and 16S rRNA). Analysis at genus and family taxonomic levels showed high levels of diversity with no differences in digestive tract bacterial or fungal biota between woodland sites of winter‐form D. suzukii. Female D. suzukii at one site appeared to have higher bacterial diversity in the alimentary canal than males, but there was a site, sex interaction. Many of the biota were associated with cold, wet climatic conditions and decomposition. This study provides the first evidence that winter‐form D. suzukii may be opportunistic feeders during the winter period and are probably exploiting food sources associated with moisture on decomposing vegetation during this time. A core gut microbiome has been identified for winter‐form D. suzukii.     

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

P‐glycoprotein [P‐gp or the ATP‐binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P‐gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) gene‐editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P‐gp protein (SeP‐gp) predicted to display structural characteristics common to P‐gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP‐gp ORF was established using the CRISPR/Cas9 gene‐editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP‐gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta‐cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP‐gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP‐gp may contribute to abamectin and EB resistance in S. exigua.     

Human osteoarthritic chondrons outnumber patient‐ and joint‐matched chondrocytes in hydrogel culture—Future application in autologous cell‐based OA cartilage repair?

Autologous chondrocyte implantation (ACI) is used in 34–60% for osteoarthritic (OA) cartilage defects, although ACI is neither recommended nor designed for OA. Envisioning a hydrogel‐based ACI for OA that uses chondrons instead of classically used chondrocytes, we hypothesized that human OA chondrons may outperform OA chondrocytes. We compared patient‐ and joint surface‐matched human OA chondrons with OA chondrocytes cultured for the first time in a hydrogel, using a self‐assembling peptide system. We determined yield, viability, cell numbers, mRNA expression, GAPDH mRNA enzyme activity, Collagen II synthesis (CPII) and degradation (C2C), and sulfated glycosaminoglycan. Ex vivo, mRNA expression was comparable. Over time, significant differences in survival led to 3.4‐fold higher OA chondron numbers in hydrogels after 2 weeks (p = .002). Significantly, more enzymatically active GAPDH protein indicated higher metabolic activity. The number of cultures that expressed mRNA for Collagen Types I and VI, COMP, aggrecan, VEGF, TGF‐β1, and FGF‐2 (but not Collagen Types II and X) was different, resulting in a 3.5‐fold higher number of expression‐positive OA chondron cultures (p < .05). Measuring CPII and C2C per hydrogel, OA chondron hydrogels synthesized more than they degraded Collagen Type II, the opposite was true for OA chondrocytes. Per cell, OA chondrons but not OA chondrocytes displayed more synthesis than degradation. Thus, OA chondrons displayed superior biosynthesis and mRNA expression of tissue engineering and phenotype‐relevant genes. Moreover, human OA chondrons displayed a significant survival advantage in hydrogel culture, whose presence, drastic extent, and timescale was novel and is clinically significant. Collectively, these data highlight the high potential of human OA chondrons for OA ACI, as they would outnumber and, thus, surpass OA chondrocytes.     

Measures of Functioning in Patients With Episodic Migraine: Findings From a Double‐Blind,Randomized, Placebo‐Controlled Phase 2b Trial With Galcanezumab

Objective – To evaluate 12‐week changes from baseline of 2 disease‐specific patient‐reported outcome (PRO) measures in adults with migraine treated with galcanezumab, an investigational humanized antibody binding calcitonin gene‐related peptide (CGRP), or placebo. Background – Preventing headache‐related functional impairment is an important goal of migraine preventive treatment and a measurement target for PROs. Understanding which drugs have the potential to improve patient functioning in addition to preventing migraine headaches is vital to lessening patient burden. Design/Methods – This Phase 2b double‐blind, randomized, placebo‐controlled study enrolled adults with episodic migraine. Galcanezumab (120 mg subcutaneous injection; n = 60) or placebo (n = 127) was administered every 28 days for 12 weeks. Post hoc secondary analyses were conducted for those who completed 12 weeks of treatment on 2 PROs: The Migraine‐Specific Quality of Life Questionnaire (MSQ) v2.1 and the Headache Impact Test? (HIT‐6). Results – Analysis of covariance revealed significant differences in least square mean changes from baseline between galcanezumab and placebo for all MSQ domains including total mean change placebo of 18.63, galcanezumab of 27.36 (95% CI 2.449, 15.008; P‐value of .0067); Role Function‐Restrictive mean change placebo of 22.40, galcanezumab of 31.92 (95% CI 2.636, 16.518; P‐value of .0071); Role Function‐Preventive mean change placebo of 13.43, galcanezumab of 19.76 (95% CI 0.476, 12.185; P‐value of .0342); and Emotional Function mean change placebo of 16.88, galcanezumab of 26.61 (95% CI 2.789, 16.674; P‐value of .0063). At baseline, mean number of migraine headache days (MHDs) did not correlate with MSQ total scores or HIT‐6. At 12 weeks post‐treatment, MHD correlated with MSQ and HIT‐6 scores (all P < .0001). Change in MHD was associated with change in MSQ domains and change in HIT‐6 scores (all P < .0001). Conclusions – In comparison with placebo, treatment with galcanezumab was associated with significant functional improvements as reflected by changes in MSQ scores. Change in MHD was associated with improvements in MSQ and reductions in HIT‐6 scores, indicating the clinical importance of these changes in relation to PROs that measure function.     

Application of a dense genetic map for assessment of genomic responses to selection and inbreeding in Heliothis virescens

Adaptation of pest species to laboratory conditions and selection for resistance to toxins in the laboratory are expected to cause inbreeding and genetic bottlenecks that reduce genetic variation. Heliothis virescens, a major cotton pest, has been colonized in the laboratory many times, and a few laboratory colonies have been selected for Bacillus thuringiensis (Bt) resistance. We developed 350‐bp double‐digest restriction‐site associated DNA‐sequencing (ddRAD‐seq) molecular markers to examine and compare changes in genetic variation associated with laboratory adaptation, artificial selection and inbreeding in this nonmodel insect species. We found that allelic and nucleotide diversity declined dramatically in laboratory‐reared H. virescens as compared with field‐collected populations. The declines were primarily a result of the loss of low frequency alleles present in field‐collected H. virescens. A further, albeit modest decline in genetic diversity was observed in a Bt‐selected population. The greatest decline was seen in H. virescens that were sib‐mated for 10 generations, in which more than 80% of loci were fixed for a single allele. To determine which regions of the genome were resistant to fixation in our sib‐mated line, we generated a dense intraspecific linkage map containing three PCR‐based and 659 ddRAD‐seq markers. Markers that retained polymorphism were observed in small clusters spread over multiple linkage groups, but this clustering was not statistically significant. Overall, we have confirmed and extended the general expectations for reduced genetic diversity in laboratory colonies, provided tools for further genomic analyses and produced highly homozygous genomic DNA for future whole genome sequencing of H. virescens.     

Role of a gamma‐aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

The western corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was controlled by applications of cyclodiene insecticides from the late 1940s until resistance evolved ∼10 years later. Range expansion from the western plains into eastern USA coincides with resistance development. An alanine to serine amino acid substitution within the Rdl subunit of the gamma‐aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. We found that the non‐synonymous single nucleotide polymorphism (SNP) G/T at the GABA receptor cDNA position 838 (G/T⁸³⁸) of D. v. virgifera resulted in the alanine to serine change, and the codominant SNP allele T⁸³⁸ was genetically linked to survival of beetles in aldrin bioassays. A phenotypic gradient of decreasing susceptibility from west to east was correlated with higher frequencies of the resistance‐conferring T⁸³⁸ allele in the eastern‐most populations. This pattern exists in opposition to perceived selective pressures since the more eastern and most resistant populations probably experienced reduced exposure. The reasons for the observed distribution are uncertain, but historical records of the range expansion combined with the distribution of susceptible and resistant phenotypes and genotypes provide an opportunity to better understand factors affecting the species' range expansion.     

Attenuation of osteoarthritis progression in mice following intra‐articular administration of simvastatin‐conjugated gelatin hydrogel

Simvastatin is clinically used for the treatment of hypercholesterolaemia. This study investigated the effects of the intra‐articular administration of a simvastatin‐conjugated gelatin hydrogel on osteoarthritis (OA) progression in a murine model. The medial meniscus of the knee joint was de‐stabilized to induce OA in 10‐week‐old mice. Mice were then intra‐articularly injected with dimethyl sulfoxide (control), drug‐free gelatin hydrogel, simvastatin, or simvastatin‐conjugated gelatin hydrogel. At 8 weeks postsurgery, OA progression was significantly attenuated in the simvastatin‐conjugated gelatin hydrogel‐treated mice compared with the other three groups. Attenuation of OA in mice treated with simvastatin‐conjugated gelatin hydrogel was associated with decreased expression of cartilage‐degrading enzymes and interleukin (IL)‐1β and increased expression of type II collagen and an autophagic marker, LC3, in the articular cartilage. The effects of simvastatin on gene expression and autophagy in the mouse primary chondrocytes were also examined in the presence or absence of IL‐1β. Treatment with simvastatin down‐regulated Mmp‐13 and Il‐1β and up‐regulated Col2a1 and autophagic activity. Our findings suggest that the intra‐articular administration of simvastatin‐conjugated gelatin hydrogel could be used as a new potential therapy for OA.