Functional characterization of CYP6G4 from the house fly in propoxur metabolism and resistance.

The house fly (Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides. The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica, in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b5) were functionally expressed in Escherichia coli. Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min-1. Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation, O-depropylation and N-demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance.

PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

Ultra-highly sensitive organophosphorus biosensor based on chitosan/tin disulfide and British housefly acetylcholinesterase.

Pesticides have been extensively applied worldwide to protect crops from worms and insects; however, the continuous use of pesticides affects ecosystems, agricultural product safety, nontarget organisms, and human health. In this paper, we report a highly sensitive biosensor for the determination of pesticides based on tin sulfide (SnS2) and chitosan (CHIT) nanocomposites decorated with a unique British housefly acetylcholinesterase (AChE). The hydrothermally synthesized nano-SnS2 mixed with chitosan solution (CHIT-SnS2) was drop-casted onto a glassy carbon electrode (GCE). Subsequently, the British housefly AChE was immobilized on the CHIT/SnS2-coated GCE that was then employed for pesticide detection. The developed biosensor showed an ultra-high sensitivity and wide linear detection range from 0.02 nM to 20000 nM with a detection limit of 0.02 nM for the detection of chlorpyrifos as the model pesticide. Furthermore, the AChE/CHIT-SnS2/GCE exhibited acceptable storage stability, good reproducibility, and selectivity.

Activities of Select Enzymes Involved in Insecticide Resistance in Spinosad-Resistant and -Susceptible Strains of Musca domestica (Diptera: Muscidae).

A Musca domestica L. strain collected from Pakistan has recently been shown to be resistant to spinosad; however, there is scarce information about the mechanism of resistance. For this reason, we explored whether a metabolic-based mechanism was responsible by analyzing the activities of the metabolic detoxifying enzymes, carboxylesterases, glutathione S-transferases, and mixed-function oxidases, in both a spinosad-selected (Spin-SEL) strain of M. domestica and a susceptible counterpart (Lab-susceptible). The results revealed that both strains were statistically at par in terms of enzyme activities. The activity of carboxylesterases in the Lab-susceptible strain was 78.17 ± 3.06 in comparison to 79.16 ± 3.31 nmol min-1 mg-1 in the Spin-SEL strain. The activity of mixed-function oxidases was 51.58 ± 4.20 in the Lab-susceptible strain, whereas 54.33 ± 4.08 pmol min-1 mg-1 was recorded in the Spin-SEL strain. The activity of glutathione S-transferases was 86.50 ± 4.59 (Lab-susceptible) and 90.33 ± 2.81 nmol min-1 mg-1 (Spin-SEL). These results revealed that the studied enzymes might not be responsible for spinosad resistance in the studied strain of M. domestica. Therefore, studies should be extended to find out other possible mechanisms of spinosad resistance.

Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach.

Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

Design, Synthesis and Insecticide Activity of Novel Acetylcholinesterase Inhibitors: Triazolinone and Phthalimide Heterodimers.

Based on the "cluster effect" and the structure characters of acetylcholinesterase (AChE; EC 3.1.1.7), a new series of 1,2,4-triazolin-3-one and phthalimide heterodimers were designed, synthesized, and evaluated as potent dual acetylcholinesterase inhibitors (AChEIs). Most of the synthesized compounds showed good in vitro inhibitory activities towards both Drosophila melanogaster acetylcholinesterase (DmAChE) and Musca domestica acetylcholinesterase (MdAChE). Among them, 5g was found to be the most potent anti-AChE derivate (5g, IC50 = 8.07 µM to DmAChE, IC50 = 32.24 µM to MdAChE). It was 2.31- and 1.35-fold more active than the positive control ethion (CP, IC50 = 18.62 µM to DmAChE, IC50 = 43.56 µM to MdAChE). The docking model study revealed that 5g possessed the fitted spatial structure and bound to the central pocket and peripheral site of DmAChE. Moreover, most compounds demonstrated high insecticidal activity to Lipaphis erysimi and Tetranychus cinnabarinus at the concentration of 300 mg/L.

MdRDH1, a HSP67B2-like rhodanese homologue plays a positive role in maintaining redox balance in Musca domestica.

Rhodanese homology domains (RHODs) are the structural modules of ubiquitous tertiary that occur in three major evolutionary phyla. Despite the versatile and important physiological functions of RHODs containing proteins, little is known about their invertebrate counterparts. A novel HSP67B2-like single-domain rhodanese homologue, MdRDH1 from Musca domestica, whose expression can be induced by bacterial infection or oxidative stress. Silencing MdRDH1 through RNAi causes important accumulations of reactive oxygen species (ROS) and malondialdehyde (MDA), and increases mortality in the larvae treated with bacterial invasion. The E. coli with MdRDH1 and the mutant MdRDH1C135A are transformed, with significant rhodanese activity of the recombinant protein of MdRDH1 in vitro found, without no detection of enzyme activity of the mutant MdRDH1C135A, revealing that catalytic Cys135 in the active-site loop is essential in the sulfurtransferase activity of MdRDH1. When oxidative stress is insulted by phenazine methosulfate (PMS), the MdRDH1 transformed E. coli shows enhanced survival rates compared with those bacteria transformed with MdRDH1C135A. Our research indicates that MdRDH1 confers oxidative stress tolerance, thus rendering evidence for the idea that rhodanese family genes play a critical role in antioxidant defenses. This paper yields novel insights into the potential antioxidative and immune functions of HSP67B2-like rhodanese homologues in invertebrate.

In silico approaches to evaluate the molecular properties of organophosphate compounds to inhibit acetylcholinesterase activity in housefly.

Organophosphate compounds (OPC) have become the primary choice as insecticides and are widely used across the world. Additionally, OPCs were also commonly used as a chemical warfare agent that triggers a great challenge to public safety. Exposure of OPCs to human causes immediate excitation of cholinergic neurotransmission through transient elevation of synaptic acetylcholine (ACh) levels and accumulations. Likewise, prolonged exposure of OPCs can affect the processes in immune response, carbohydrate metabolism, cardiovascular toxicity, and several others. Studies revealed that the toxicity of OPCs was provoked by inhibition of acetylcholinesterase (AChE). Therefore, combined in silico approaches - pharmacophore-based 3D-QSAR model; docking and Molecular Dynamics (MD) - were used to assess the precise and comprehensive effects of series of known OP-derived compounds together with its -log LD50 values. The selected five-featured pharmacophore model - AAHHR.61 - displayed the highest correlation (R2 = .9166), cross-validated coefficient (Q2 = .8221), F = 63.2, Pearson-R = .9615 with low RMSE = .2621 values obtained using five component PLS factors. Subsequently, the well-validated model was then used as a 3D query to search novel OPCs using a high-throughput virtual screening technique. Simultaneously, the docking studies predicted the binding pose of the most active OPC in the MdAChE binding pocket. Additionally, the stability of docking was verified using MD simulation. The results revealed that OP22 and predicted lead compounds bound tightly to S315 of MdAChE through potential hydrogen bond interaction over time. Overall, this study might provide valuable insight into binding mode of OPCs and hit compounds to inhibit AChE in housefly.

Crystal structure of the delta-class glutathione transferase in Musca domestica.

Among the various glutathione transferase (GST) isozymes in insects, the delta- and epsilon-class GSTs fulfill critical functions during the detoxification of insecticides. We crystalized MdGSTD1, the major delta-class GST isozyme in the housefly (Musca domestica), in complex with glutathione (GSH) and solved its structure at a resolution of 1.4 Å. The overall folding of MdGSTD1 resembled other known delta-class GSTs. Its substrate binding pocket was exposed to solvent and considerably more open than in the epsilon-class GST from M. domestica (MdGSTE2). However, their C-terminal structures differed the most because of the different lengths of the C-terminal regions. Although this region does not seem to directly interact with substrates, its deletion reduced the enzymatic activity by more than 70%, indicating a function in maintaining the proper conformation of the binding pocket. Binding of GSH to the GSH-binding region of MdGSTD1 results in a rigid conformation of this region. Although MdGSTD1 has a higher affinity for GSH than the epsilon class enzymes, the thiol group of the GSH molecule was not close enough to serine residue 9 to form a hydrogen-bond with this residue, which is predicted to act as the catalytic center for thiol group deprotonation in GSH.

Characterization of a new multifunctional beta-glucosidase from Musca domestica.

OBJECTIVE: To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications. RESULTS: A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZαA and expressed in P. pastoris with a yield of 500 mg l-1. The enzyme has the maximum activity with 27.6 U mg-1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 °C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 ± 1.8 U mg-1), cellobiose (40.1 ± 2.3 U mg-1) and cellulose (27.6 ± 3.5 U mg-1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Ι and possesses O-glycosylation sites. CONCLUSIONS: Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.

Binary combinations of organophosphorus and synthetic pyrethroids are more potent acetylcholinesterase inhibitors than organophosphorus and carbamate mixtures: An in vitro assessment.

Anticholinesterase insecticides such as organophosphorous (OP) and carbamates pesticides (CB); and synthetic pyrethroids (SP) pesticides commonly co-occur in the environment. This raises the possibility of antagonistic, additive, or synergistic neurotoxicity in exposed organisms. Acetylcholinesterase (AChE) inhibition has been demonstrated to be useful as a biomarker for exposure to OP and CBs in many environments. This study investigated the response of housefly (Musca domestica) head AChE (HF-AChE) exposed to five OPs; chlorpyrifos (CPF), malathion (MLT), triazophos (TRZ), monocrotophos (MCP) and profenofos (PRF) and two CBs; carbaryl (CRB) and carbofuran (CBF) as individual compounds and as binary mixtures of OPs and CBs under in vitro conditions. In addition, the selected OPs and CBs were evaluated for their toxicity in binary combinations with two SPs; deltamethrin (DLT) and cypermethrin (CYP) at fixed concentrations of 0.1 and 10µg/L. The toxicological interaction of five OPs with two CBs pesticides was evaluated under oxidised and un-oxidised conditions using a toxic unit (TU) approach and a concentration addition (CA) model. Pyrethroid combinations were assessed only under oxidised conditions. Since OPs and CBs act by a similar mechanism of inhibition of AChE, a dose additive effect was expected, but not conclusively found. TRZ with either CBF or CRB exhibited synergism under oxidised and un-oxidised conditions but the degree of synergism was stronger under un-oxidised conditions. Additivity was exhibited by CBF+MCP, CRB+MCP, CRB+MLT and CBF+MCP under un-oxidised conditions and CRB+MCP and CRB+CPF under oxidised conditions. Pyrethorids in combination with OPs (TRZ, MLT and CPF) were highly synergistic. In the present study, we used pure housefly head AChE without any interference of monooxygenase and/or esterase enzyme activities. Therefore these other enzymes were not producing the observed deviations from concentration-addition in the binary combinations between OPs, CBs and SPs. The mechanisms of OP, CB and SP interactions in pesticide mixtures requires further investigation.

Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 µM/min and 23.48 µM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

Cyromazine resistance in a field strain of house flies, Musca domestica L.: Resistance risk assessment and bio-chemical mechanism.

Developing resistance management strategies for eco-friendly insecticides is essential for the management of insect pests without harming the environment. Cyromazine is a biorational insecticide with very low mammalian toxicity. Resistance to cyromazine has recently been reported in house flies from Punjab, Pakistan. In order to propose a resistance management strategy for cyromazine, experiments were planned to study risk for resistance development, possibility of cross-resistance and bio-chemical mechanisms. A field strain of house flies with 8.78 fold resistance ratio (RR) to cyromazine was re-selected under laboratory conditions. After seven rounds of selection (G1-G7), the RR values rapidly increased from 8.8 to 211 fold. However, these values declined to 81fold when the cyromazine selected (CYR-SEL) strain was reared without selection pressure, suggesting an unstable nature of resistance. The CYR-SEL strain showed lack of cross-resistance to pyriproxyfen, diflubenzuron, and methoxyfenozide. Synergism bioassays using enzyme inhibitors: piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF), and metabolic enzyme analyses revealed increased activity of carboxylesterase (CarE) and mixed-function oxidase (MFO) in the CYR-SEL strain compared to the laboratory susceptible (Lab-susceptible) strain, suggesting the metabolic resistance mechanism responsible for cyromazine resistance in the CYR-SEL strain. In conclusion, risk of rapid development of cyromazine resistance under consistent selection pressure discourages the sole reliance on cyromazine for controlling house flies in the field. The unstable nature of cyromazine resistance provides window for restoring cyromazine susceptibility by uplifting selection pressure in the field. Moreover, lack of cross-resistance between cyromazine and pyriproxyfen, diflubenzuron, or methoxyfenozide in the CYR-SEL strain suggest that cyromazine could be rotated with these insecticides whenever resistance crisis occur in the field.

QSAR analyses of organophosphates for insecticidal activity and its in-silico validation using molecular docking study.

The present work was carried out to design and develop novel QSAR models using 2D-QSAR and 3D-QSAR with CoMFA methodology for prediction of insecticidal activity of organophosphate (OP) molecules. The models were validated on an entirely different external dataset of in-house generated combinatorial library of OPs, by completely different computational approach of molecular docking against the target AChE protein of Musca domestica. The dock scores were observed to be in good correlation with 2D-QSAR and 3D-QSAR with CoMFA predicted activities and had the correlation coefficients (r(2)) of -0.62 and -0.63, respectively. The activities predicted by 2D-QSAR and 3D-QSAR with CoMFA were also observed to be highly correlated with r(2)=0.82. Also, the combinatorial library molecules were screened for toxicity in non-target organisms and degradability using USEPA-EPI Suite. The work was first step towards computer aided design and development of novel OP pesticide candidates with good insecticidal property but lower toxicity in non-targeted organisms and having biodegradation potential.

Transcriptomic Analysis of Musca domestica to Reveal Key Genes of the Prophenoloxidase-Activating System.

The proPO system regulates melanization in arthropods. However, the genes that are involved in the proPO system in housefly Musca domestica remain unclear. Thus, this study analyzed the combined transcriptome obtained from M. domestica larvae, pupae, and adults that were either normal or bacteria-challenged by an Escherichia coli and Staphylococcus aureus mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were de novo assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in M. domestica.

Thiamethoxam Resistance in the House Fly, Musca domestica L.: Current Status, Resistance Selection, Cross-Resistance Potential and Possible Biochemical Mechanisms.

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC50 ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.

Binary combinations of organophosphorus pesticides exhibit differential toxicity under oxidised and un-oxidised conditions.

Acetylcholinesterase (AChE) inhibition has been demonstrated to be useful as a biomarker for exposure to organophosphorus (OP) insecticides in many environments. The objective of this study was to investigate the response of housefly (Musca domestica) head AChE (HF-AChE) exposed to five OPs as individual compounds and their binary mixtures under in vitro conditions. To examine the effects of oxidation on OP potency in the HF-AChE system, bromine water was used as an oxidisng agent. With oxidation, the sensitivity of HF-AChE to chlorpyrifos (CPF), malathion (MLT) and triazophos (TRZ) increased significantly. Monocrotophos (MCP) and profenofos (PRF) did not exhibit any significant differences in toxicity under oxidised and un-oxidised conditions. The toxicological interaction of five organophosphorus pesticides was evaluated using the concentration addition model, the combination index-isobologram equation and the toxic unit approach. All three models provided similar predictions for the 10 binary combinations of OPs under oxidised and un-oxidised conditions. In the present study, the antagonistic effects of the binary combination of OPs (CPF+PRF, CPF+MLT, MCP+MLT, PRF+MLT, MLT+TRZ and PRF+TRZ) were observed under oxidised conditions. This may be due to dispositional and/or receptor antagonism. Most of the binary combinations assayed under un-oxidised conditions exhibited synergistic responses. Triazophos showed very strong synergism in binary combinations with CPF, MCP and PRF un-oxidised conditions. In contrast, under oxidised conditions, only CPF+TRZ exhibited synergism. The results obtained indicate differential toxicity of binary combinations of OPs under oxidised and un-oxidised conditions. This information could be a valuable tool in understanding the mechanisms of OPs interactions and the interpretation of future in vivo studies with mixtures of OP insecticides.

Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica.

Glutathione transferases (GSTs) play an important role in the detoxification of insecticides, and as such, they are a key contributor to enhanced resistance to insecticides. In the housefly (Musca domestica), two epsilon-class GSTs (MdGST6A and MdGST6B) that share high sequence homology have been identified, which are believed to be involved in resistance against insecticides. The structural determinants controlling the substrate specificity and enzyme activity of MdGST6s are unknown. The aim of this study was to crystallize and perform structural analysis of the GST isozyme, MdGST6B. The crystal structure of MdGST6B complexed with reduced glutathione (GSH) was determined at a resolution of 1.8 Å. MdGST6B was found to have a typical GST folding comprised of N-terminal and C-terminal domains. Arg113 and Phe121 on helix 4 were shown to protrude into the substrate binding pocket, and as a result, the entrance of the substrate binding pocket was narrower compared to delta- and epsilon-class GSTs from Africa malaria vector Anopheles gambiae, agGSTd1-6 and agGSTe2, respectively. This substrate pocket narrowing is partly due to the presence of a π-helix in the middle of helix 4. Among the six residues that donate hydrogen bonds to GSH, only Arg113 was located in the C-terminal domain. Ala substitution of Arg113 did not have a significant effect on enzyme activity, suggesting that the Arg113 hydrogen bond does not play a crucial role in catalysis. On the other hand, mutation at Phe108, located just below Arg113 in the binding pocket, reduced the affinity and catalytic activity to both GSH and the electrophilic co-substrate, 1-chloro-2,4-dinitrobenzene.

Design, synthesis and biological evaluation of organophosphorous-homodimers as dual binding site acetylcholinesterase inhibitors.

The cluster effect is an effective strategy to explore new lead compounds, and has been successfully applied in rational drug design and screening. A series of novel organophosphorous-homodimers were designed and synthesized based on the dual-site structure characteristics of acetylcholinesterase (AChE). The compounds were evaluated in vitro for their inhibitory activity to AChE extracted from Drosophila melanogaster and Musca domestic. Compound 4H showed an excellent inhibitor activity to both Drosophila melanogaster and Musca domestic with the corresponding IC(50) values of 23 and 168 nM, respectively. Meanwhile, its activities against Drosophila melanogaster and Musca domestic AChE were more than 10,00,000 and 100,000-fold higher compared with the parent compound (MH), and was up to 245 and 107-fold higher than those of the positive control omethoate. The molecular docking study revealed that 4H possessed an optimal spacer length and can perfectly fit into the central pocket, active gorge, and peripheral site of DmAChE, and consequently exhibited highly improved inhibitor potency to DmAChE. The bioassay tests showed that 4 series compounds showed prominent insecticidal activities against both Lipaphser erysimi and Tetranychus cinnbarinus at a concentration of 200mg/L. The insecticide activity of compound 4H was particularly significant that can cause 96% mortality to Tetranychus cinnbarinus after 24h of treatment.