Discovery selective acetylcholinesterase inhibitors to control Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite, Tetranychus urticae Koch, has a broad host plant range and presents an extreme capacity for developing pesticide resistance, becoming a major economic pest in agriculture. Anticholinesterase insecticides still account for a big part of global insecticide sales. However, there is a growing concern about the serious resistance problems of anticholinesterase insecticides and their nontarget toxicity. In this study, structure-based virtual screening was performed to discover selective AChE inhibitors from the ChemBridge database, and 39 potential species-specific AChE inhibitor were obtained targeting T. urticae AChE, but not human AChE. Among them, compound No. 8 inhibited AChE from T. urticae, but not from human, and had an inhibitory activity comparable to that of eserine. Compound No. 8 had dose-dependent toxicity to T. urticae in glass slide-dipping assay and had significant mite control effects in a pot experiment, but required a high concentration to achieve similar control effects to spirodiclofen. The toxicity evaluation suggested that compound No. 8 had no acute toxicity on pollinator honey bees and natural predator N. californicus and did not affect strawberry growth in our assay. Compound No. 8 is a potential lead compound for developing novel acaricides with reduced nontarget toxicity.

Exposure of chlorothalonil and acetamiprid reduce the survival and cause multiple internal disturbances in Apis mellifera larvae reared in vitro.

Background: Chlorothalonil and acetamiprid are chemical pesticides commonly used in agricultural production and have been shown to have negative effects on bee's fitness. Despite many studies have revealed that honey bee (Apis mellifera L.) larvae are posting a high risk on exposure to pesticides, but the toxicology information of chlorothalonil and acetamiprid on bee larvae remain limited. Results: The no observed adverse effect concentration (NOAEC) of chlorothalonil and acetamiprid for honey bee larvae were 4 µg/mL and 2 µg/mL, respectively. Except for CarE, the enzymic activities of GST and P450 were not influenced by chlorothalonil at NOAEC, while chronic exposure to acetamiprid slightly increased the activities of the three tested enzymes at NOAEC. Further, the exposed larvae showed significantly higher expression of genes involved in a series of different toxicologically relevant process following, including caste development (Tor (GB44905), InR-2 (GB55425), Hr4 (GB47037), Ac3 (GB11637) and ILP-2 (GB10174)), immune system response (abaecin (GB18323), defensin-1 (GB19392), toll-X4 (GB50418)), and oxidative stress response (P450, GSH, GST, CarE). Conclusion: Our results suggest that the exposure to chlorothalonil and acetamiprid, even at concentrations below the NOAEC, showed potentially effects on bee larvae's fitness, and more important synergistic and behavioral effects that can affect larvae fitness should be explored in the further.

Chitin deacetylase 2 is essential for molting and survival of Tetranychus urticae.

Chitin metabolism has long been considered promising targets for development of biorational pesticides. Considering the increasing challenges of controlling the twospotted spider mite, Tetranychus urticae Koch, the roles of chitin deacetylases (CDAs) during molting process and mite development are explored. TuCDA1 and TuCDA2 differ in expression patterns during the development process. Feeding of double-strand RNA (dsRNA) against TuCDA1 or TuCDA2 has lethal effects on the mites. Especially TuCDA2 displays a much stronger phenotype than TuCDA1 (p = 0.0003). The treated mites fail to shed the old cuticle and are trapped within exuviate until they die. The aberrant cuticle structure observed by scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM) may be responsible for the lethal phenotype of TuCDA1 and TuCDA2 knocked down mites. However, treatment with both dsRNA-CDA1 and dsRNA-CDA2 cannot significantly enhance the lethal effects of dsRNA-CDA2, which indicates partially redundant function of TuCDA1 and TuCDA2. TuCDA2 may play a key role during the molting and development process. Chitin-modifying enzyme such as TuCDA2 is potential target of RNA interference through feeding.

Acetylcholinesterase target sites for developing environmentally friendly insecticides against Tetranychus urticae (Acari: Tetranychidae).

The non-target toxicity and resistance problems of acetylcholinesterase (AChE) insecticides, such as organophosphates and carbamates, are of growing concern. To explore the potential targets for achieving inhibitor selectivity, the AChE structures at or near the catalytic pocket of Tetranychus urticae (TuAChE), honey bees, and humans were compared. The entrances to the AChE catalytic pocket differ significantly because of their different peripheral sites. The role of these potential mite-specific sites in AChE function was further elucidated by site-directed mutagenesis of these sites and then examining the catalytic activities of TuAChE mutants. The spider mite E316, H369, and V105 active sites are important for AChE function. By further analyzing their physostigmine inhibitory properties and the detailed interaction between physostigmine and TuAChE, the peripheral site H369 locating near the gorge entrance, and S154 at the oxyanion hole, affects substrate and inhibitor trafficking. The discovery of conserved mite-specific residues in Tetranychus will enable the development of safer, effective pesticides that target residues present only in mite AChEs, potentially offering effective control against this important agricultural pest.

LigMate: A Multifeature Integration Algorithm for Ligand-Similarity-Based Virtual Screening.

Ligand-similarity-based virtual screening is one of the most applicable computer-aided drug design techniques. The current methodology relies heavily on several descriptors of molecular features, including atoms (zero-dimensional, 0D), the presence or absence of structural features (one-dimensional, 1D), topological descriptors (two-dimensional, 2D), geometry and volume (three-dimensional, 3D), or stereoelectronic and stereodynamic properties (four-dimensional, 4D). These descriptors have been frequently used in virtual screening; however, they are usually used independently without integration, which may hinder effective and precise virtual screening. In this study, we developed a multifeature integration algorithm named LigMate, which employs a Hungarian algorithm-based matching and a machine learning-based nonlinear combination of various descriptors, including the new relevant descriptors focusing on the maximum common substructures (maximum common substructure score, MCSS), the relative distance of atoms from the ligand mass center (intraligand distance score, ILDS), as well as the ring differences (ring score, RS). In the benchmark tests, LigMate achieved an overall enrichment factor of the first percent (EF1) of 36.14 and an area under the curve (AUC) value of 0.81 on the DUD-E data set, as well as an EF1 of 15.44 and an AUC of 0.69 on the maximum unbiased validation (MUV) data set, outperforming the control methods that are based on single descriptors. Thus, our study provides a new framework for multiple feature integration, which can benefit ligand-similarity-based virtual screening. LigMate is freely available for noncommercial users at http://cao.labshare.cn/ligmate/.

Comparative characterization of putative chitin deacetylases from Tetranychus cinnabarinus.

Considering the challenges of controlling carmine spider mite and protecting pollinators, chitin deacetylase genes (TecCDA1 and TecCDA2) of spider mite were characterized as group I, and were expressed in stage-specific pattern, which imply their role during development. The differences in sequences and structures between T. cinnabarinus and honeybee CDAs offer possibilities to design new selective pesticides protecting pollinator honeybees.

A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses.

Fungal plant pathogens, like rust-causing biotrophic fungi, secrete hundreds of effectors into plant cells to subvert host immunity and promote pathogenicity on their host plants by manipulating specific physiological processes or signal pathways, but the actual function has been demonstrated for very few of these proteins. Here, we show that the PgtSR1 effector proteins, encoded by two allelic genes (PgtSR1-a and PgtSR1-b), from the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt), suppress RNA silencing in plants and impede plant defenses by altering the abundance of small RNAs that serve as defense regulators. Expression of the PgtSR1s in plants revealed that the PgtSR1s promote susceptibility to multiple pathogens and partially suppress cell death triggered by multiple R proteins. Overall, our study provides the first evidence that the filamentous fungus P. graminis has evolved to produce fungal suppressors of RNA silencing and indicates that PgtSR1s suppress both basal defenses and effector triggered immunity.

Quantitative Proteomics Analysis of Developmental Reprogramming in Protoplasts of the Moss Physcomitrella patens.

The moss Physcomitrella patens is a model system for studying Plant developmental processes. To better understand the biochemical and physiological changes involved in developmental reprogramming, we conducted a quantitative proteomics analysis for protonemata, protoplasts made therefrom and protoplasts regenerated for 2 d. Using an iTRAQ peptide labeling strategy and liquid chromatography-tandem mass spectrometry (LC-MS/MS), >3,000 peptides and 1,000 proteins were quantified. Of these, 162 proteins were identified as having differential abundances during developmental reprogramming. These proteins were involved in various biological functions, such as defense, energy production, translation, metabolism, protein destination and storage, transcription, transport, cell growth/division, cell structure and signal transduction. Of these, the proteins involved in energy production and translation increased in abundance, while many of the metabolism and defense proteins decreased in abundance. In addition, most of the cell growth/division, protein stability and cell structure proteins were also down-regulated. This is the first report on the metabolic changes involved in developmental reprogramming in protoplasts. The significance of metabolic networks in developmental programming is beginning to emerge. Our study suggested that stress signals, energy metabolism and ribosomal proteins are pivotal components during developmental programming.

Novel and selective acetylcholinesterase inhibitors for Tetranychus cinnabarinus (Acari: Tetranychidae).

The carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychidae), is an economically important and extremely polyphagous herbivorous pest, with the title of "resistance champion" among arthropods. Anticholinesterase insecticides such as organophosphate and carbamate account for more than one-third of global insecticide sales. The non-target toxicity and resistance problem of organophosphate and carbamate have become of growing concern, which may be due to the fact that they target the ubiquitous catalytic serine residue of acetylcholinesterase (AChE) in mammals, birds, and beneficial insects. In this study, the structural differences between T. cinnabarinus AChE and human AChE, at or near the catalytic pocket, were illustrated. From the SPECS chemical lead-compound database, 55 AChE inhibitor candidates were screened for high affinity for T. cinnabarinus AChE, but low affinity for human AChE, using the DOCK 6 and AutoDock Vina software. Three of the fifty-five candidates had inhibitory activity greater than that of the reversible AChE inhibitor eserine, with no observed inhibitory activities against human AChE. Two of the three had toxicity to T. cinnabarinus comparable to that of natural insecticidal pyrethrins. However, their potency is low compared with that of etoxazole, and further work is needed to optimize their potency. The selectivity of the three compounds over human and mite AChE may be due to their interaction with the mite-specific residues, as analyzed by Cyscore. The three compounds are potential lead compounds for development of novel acaricides against T. cinnabarinus with reduced toxicity to non-target species and a low propensity for resistance.

Cloning and Characterization of the Acetylcholinesterase1 Gene of Tetranychus cinnabarinus (Acari: Tetranychidae).

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a major agriculture pest. It can be found worldwide, has an extensive host plant range, and has shown resistance to pesticides. Organophosphate and carbamate insecticides account for more than one-third of all insecticide sales. Insecticide resistance and the toxicity of organophosphate and carbamate insecticides to mammals have become a growing concern. Acetylcholinesterase (AChE) is the major targeted enzyme of organophosphate and carbamate insecticides. In this study, we fully cloned, sequenced and characterized the ace1 gene of T. cinnabarinus, and identified the differences between T. cinnabarinus AChE1, Tetranychus urticae Koch AChE1, and human AChE1. Resistance-associated target-site mutations were displayed by comparing the AChE amino acid sequences and their AChE three-dimensional (3D) structures of the insecticide-susceptible strains of T. cinnabarinus and T. urticae to that of a T. urticae-resistant strain. We identified variation in the active-site gorge and the sites interacting with gorge residues by comparing AChE1 3D structures of T. cinnabarinus, T. urticae, and humans, though their 3D structures were similar. Furthermore, the expression profile of T. cinnabarinus AChE, at the different developmental stages, was determined by quantitative real-time polymerase chain reaction; the transcript levels of AChE were higher in the larvae stage than in other stages. The changes in AChE expression between different developmental stages may be related to their growth habits and metabolism characteristics. This study may offer new insights into the problems of insecticide resistance and insecticide toxicity of nontarget species.

Transcriptome Analysis of the Carmine Spider Mite, Tetranychus cinnabarinus (Boisduval, 1867) (Acari: Tetranychidae), and Its Response to ß-Sitosterol.

Tetranychus cinnabarinus (Acari: Tetranychidae) is a worldwide polyphagous agricultural pest that has the title of resistance champion among arthropods. We reported previously the identification of the acaricidal compound ß-sitosterol from Mentha piperita and Inula japonica. However, the acaricidal mechanism of ß-sitosterol is unclear. Due to the limited genetic research carried out, we de novo assembled the transcriptome of T. cinnabarinus using Illumina sequencing and conducted a differential expression analysis of control and ß-sitosterol-treated mites. In total, we obtained >5.4 G high-quality bases for each sample with unprecedented sequencing depth and assembled them into 22,941 unigenes. We identified 617 xenobiotic metabolism-related genes involved in detoxification, binding, and transporting of xenobiotics. A highly expanded xenobiotic metabolic system was found in mites. T. cinnabarinus detoxification genes-including carboxyl/cholinesterase and ABC transporter class C-were upregulated after ß-sitosterol treatment. Defense-related proteins, such as Toll-like receptor, legumain, and serine proteases, were also activated. Furthermore, other important genes-such as the chloride channel protein, cytochrome b, carboxypeptidase, peritrophic membrane chitin binding protein, and calphostin-may also play important roles in mites' response to ß-sitosterol. Our results demonstrate that high-throughput-omics tool facilitates identification of xenobiotic metabolism-related genes and illustration of the acaricidal mechanisms of ß-sitosterol.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

beta2-glycoprotein i is a cofactor for tissue plasminogen activator-mediated plasminogen activation.

OBJECTIVE: Regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. The purpose of this study was to investigate the effects of beta(2)-glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation. METHODS: The effect of beta(2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta(2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta(2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured. RESULTS: Beta(2)-glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta(2)GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta(2)GPI was inhibited by monoclonal anti-beta(2)GPI antibodies as well as by anti-beta(2)GPI antibodies from patients with APS. CONCLUSION: These findings suggest that beta(2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta(2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.

IgG antibodies to plasminogen and their relationship to IgG anti-beta(2)-glycoprotein 1 antibodies and thrombosis.

Reduced fibrinolytic activity has been described in primary antiphospholipid syndrome (PAPS) and may be responsible for thrombotic events. Some evidence supports a relationship between anti-plasminogen (PLG) antibodies, anti-beta(2)-glycoprotein 1 (beta(2)GP1) antibodies, and fibrinolysis, but their relationship is still unclear. The aim of study is to evaluate the association between IgG anti-beta(2)GP1 and IgG anti-PLG antibodies and thrombosis. Two groups of consecutive patients with PAPS and systemic lupus erythematosus (SLE): 32 patients with lupus anticoagulant (LAC), 32 patients without LAC, and 40 healthy controls were included. IgG against beta(2)GP1 and PLG antibodies were measured by enzyme-linked immunosorbent assay, and a value above the 99th percentile of the normal healthy control was considered as positive, and their interrelationship with thrombosis was evaluated by Pearson Chi-squared test. Cross-reactive antibodies binding to PLG and beta(2)GP1 were determined in a competitive and cross-inhibition assay. Levels of fibrinolytic activity in the presence of IgG fractions from patients and healthy controls were examined using a plasmin fluorogenic substrate assay. A high frequency of IgG anti-PLG antibodies (35.9%) was found in 64 patients, and its presence was associated with thrombosis (p = 0.001), which may be due to its ability to inhibit exogenous fibrinolysis. Coexistence of IgG anti-PLG and IgG anti-beta(2)GP1 antibodies was found in 11 of 64 patients and was related with thrombosis (p = 0.001). Cross-reactive antibody binding to PLG and beta(2)GP1 was found in IgG fractions from three patients and a monoclonal anti-beta(2)GP1 antibody BD4, and one of these three patients had thrombotic history. However, no significant association was found between IgG anti-PLG and IgG anti-beta(2)GP1 antibodies in patients. In conclusion, the prevalence of IgG anti-PLG was high in patients with PAPS and SLE and might relate with thrombosis. Cross-reactivity of IgG anti-beta(2)GP1 antibodies with PLG may occur in the sera of patients.

Isolation, identification, and comparison of four isolates of avian paramyxovirus serotype 2 in China.

Four Yucaipa-like viruses of avian paramyxovirus serotype 2 (APMV-2) were isolated in China from the imported Gouldian Finch (Chloebia gouldiae) and broilers in 1998-2002, and were named F4, F6, F8, and NK, respectively. Examined under electron microscope, the isolates were found to be round in shape and varying in size. The results of the hemagglutination inhibition test and indirect enzyme-linked immunosorbent assay (using monoclonal antibodies) showed some differences between the isolates and the reference strain Yucaipa. The isolates derived from chickens had a closer relationship to Yucaipa virus than did those of finches. Sequence comparison of the fusion gene and the haemagglutinin-neuraminidase gene showed similar results, although the variations were lesser among APMV-2 viruses in nucleotide and amino acid sequence. By sequence comparison, it was also revealed that at the molecular level the four virus strains belong to APMV-2, and that two of the strains were isolated from the same group of imported Gouldian Finches.