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Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development. [ABSTRACT FROM AUTHOR]

Xyleborus affinis, one of the most important pests of rubber trees, has caused severe damage to the natural rubber industry in Hainan province. The ability to detect host plants through a sensitive and specific olfactory system is crucial for Xyleborus affinis. Odorant binding proteins (OBPs) are believed to bind and carry hydrophobic active compounds from the environment to the surface of olfactory receptor neurons. To investigate the potential functional role of the highly expressed XaffOBP9 in binding with semiochemicals, we cloned and analyzed the cDNA sequence of XaffOBP9. The results showed that XaffOBP9 contains a 411bp open reading frame that encodes 136 amino acids. Then XaffOBP9 was expressed in Escherichia coli. The binding affinity of the recombinant OBP to 15 different ligands (14 host plant volatiles and 1 aggregation pheromone) was then examined using a fluorescence competitive binding approach. The results demonstrated that XaffOBP9 exhibited broad binding capabilities and strong affinities for 14 ligands. The structure of XaffOBP9 and its interactions with fourteen ligands were further analyzed by modeling and molecular docking, respectively. Based on the docking result, we found hydrophobic interactions are important between XaffOBP9 to these ligands and three amino acid residues (L71, Y106, and L114) were highly overlapped and contributed to the interaction with ligands. Mutation functional assays confirmed that the mutant L114A showed significantly reduced binding capacity to these ligands. This study suggested that XaffOBP9 may be involved in the chemoreception of semiochemicals and that it is helpful for the integrated management of X. affinis. [ABSTRACT FROM AUTHOR]

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

The bean bug Riptortus pedestris is a notorious insect pest that can damage various crops, especially soybean, in East Asia. In insects, the olfactory system plays a crucial role in host finding and feeding behaviour in which the odorant‐binding proteins (OBPs) are believed to be involved in initial step in this system. In this study, we produced the R. pedestris adult antennae‐expressed RpedOBP4 protein using a recombinant expression system in E. coli. Fluorescence competitive binding confirmed that RpedOBP4 has binding affinities to 7 of 20 soybean volatiles (ligands), and that a neutral condition is the best environment for it. The binding property of RpedOBP4 to these ligands was further revealed by integrating data from molecular docking, site‐directed mutagenesis and ligand binding assays. This demonstrated that five amino acid residues (I30, L33, Y47, I57 and Y121) are involved in the binding process of RpedOBP4 to corresponding ligands. These findings will not only help us to more thoroughly explore the olfactory mechanism of R. pedestris during feeding on soybean, but also lead to the identification of key candidate targets for developing environmental and efficient behaviour inhibitors to prevent population expansion of R. pedestris in the future. [ABSTRACT FROM AUTHOR]

Dioryctria abietella is a major pest of conifer cones that can initiate specific behaviours (locating hosts, finding mates and selecting oviposition sites) through sensitive olfactory systems. General odorant‐binding proteins are involved in the first step in odorant perception in insects, but their specific functional roles in D. abietella chemoreception have not been fully elucidated. In this study, quantitative real‐time polymerase chain reaction (PCR) examinations showed that DabiGOBP2 is predominantly expressed in the antennae of female adults. Western blot experiments further confirmed that these proteins show biased expression in the tissues. Binding property analysis demonstrated that DabiGOBP2 exhibited ligand specificity to terpenes, especially myrcene (Ki = 8.19 ± 0.05). Electroantennogram (EAG) and Y‐tube assays showed that three plant volatiles elicited high EAG responses, but only dodecanal showed a strong attraction to females. However, myrcene and isobutyl benzoate were significantly repellent to males at high doses in the behaviour test. In conclusion, exploring the functions of DabiGOBP2 can help in better understanding the mechanism of the olfactory system of D. abietella and developing novel strategies to control this pest. [ABSTRACT FROM AUTHOR]

BACKGROUND Athetis lepigone, a noctuid moth feeding on more than 30 different crops worldwide, has evolved a sophisticated, sensitive, and specific chemosensory system to detect and discriminate exogenous chemicals. Odorant‐binding proteins (OBPs) are the most important agent in insect chemosensory systems to be explored as an alternative target for environmentally friendly approaches to pest management. RESULTS: To investigate the olfactory function of A. lepigone OBPs (AlepOBPs), AlepOBP6 was identified and expressed in Escherichia coli. The binding affinity of the recombinant OBP to 20 different ligands was then examined using a competitive binding approach. The results revealed that AlepOBP6 can bind to two sex pheromones and ten maize volatiles, and its conformation stability is pH dependent. We also carried out a structure–function study using different molecular approaches, including structure modeling, molecular docking, and a mutation functional assay to identify amino acid residues (M39, V68, W106, Q107, and Y114) involved in the binding of AlepOBP6 to both sex pheromones and maize volatiles in A. lepigone. CONCLUSION: These results suggest that AlepOBP6 is likely involved in mediating the responses of A. lepigone to sex pheromones and maize volatiles, which may play a pivotal function in mating, feeding, and oviposition behaviors. This study not only provides new insight into the binding mechanism of OBPs to sex pheromones and host volatiles in moths, but also contributes to the discovery of novel target candidates for developing efficient behavior disruptors to control A. lepigone in the future. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Odorant-binding proteins (OBPs) capture and transport semiochemicals to olfactory receptors (OR) and function in the first step in insect olfaction. In the present study, we cloned a full-length cDNA sequence of BodoOBP5 from the insect pest Bradysia odoriphaga (Diptera: Sciaridae). Real-time PCR (qRT-PCR) analysis revealed that BodoOBP5 was expressed at higher levels in female adults than in other developmental stages. In the different tissues, BodoOBP5 was highly expressed in the female antennae, whereas low levels were expressed in the head and the male antennae, expression was negligible in other tissues. The recombinant protein of BodoOBP5 was successfully expressed with a bacterial system. Competitive binding assays with nine host plant volatiles and a putative sex pheromone revealed that purified BodoOBP5 strongly bound to two sulfur compounds (methyl allyl disulfide and diallyl disulfide); the corresponding dissolution constants (Ki) were 10.38 and 9.23 μM, respectively. Molecular docking indicated that Leu99, Leu103, Ala143, Tyr107, Phe142, and Trp144 in the hydrophobic cavity of BodoOBP5 are the key residues mediating the interaction of BodoOBP5 with methyl allyl disulfide and diallyl disulfide. RNAi-based Y-tube olfactometer assay indicated that there is no significant difference in methyl allyl disulfide and diallyl disulfide. The results of this study increase our understanding of the binding of BodoOBP5 with plant volatiles, facilitating the development of novel ways to control B. odoriphaga. [ABSTRACT FROM AUTHOR]

Peptide binding to major histocompatibility complexes (MHCs) is a central component of the immune system, and understanding the mechanism behind stable peptide-MHC binding will aid the development of immunotherapies. While MHC binding is mostly influenced by the identity of the so-called anchor positions of the peptide, secondary interactions from nonanchor positions are known to play a role in complex stability. However, current MHC-binding prediction methods lack an analysis of the major conformational states and might underestimate the impact of secondary interactions. In this work, we present an atomically detailed analysis of peptide-MHC binding that can reveal the contributions of any interaction toward stability. We propose a simulation framework that uses both umbrella sampling and adaptive sampling to generate a Markov state model (MSM) for a coronavirus-derived peptide (QFKDNVILL), bound to one of the most prevalent MHC receptors in humans (HLA-A24:02). While our model reaffirms the importance of the anchor positions of the peptide in establishing stable interactions, our model also reveals the underestimated importance of position 4 (p4), a nonanchor position. We confirmed our results by simulating the impact of specific peptide mutations and validated these predictions through competitive binding assays. By comparing the MSM of the wild-type system with those of the D4A and D4P mutations, our modeling reveals stark differences in unbinding pathways. The analysis presented here can be applied to any peptide-MHC complex of interest with a structural model as input, representing an important step toward comprehensive modeling of the MHC class I pathway. [ABSTRACT FROM AUTHOR]

Insects can exhibit flexible olfaction that is sensitive to complex natural chemical environments. Odorant-binding proteins (OBPs) in insects' antennal chemosensilla can act as transporters of plant volatiles and pheromones across the sensillar lymph. Although the physiological functions of OBPs have been widely reported, it is still unclear how OBP binds to ligands with various structures in detail. Here, we further investigated the ligand-binding modes and characteristics of AcerOBP2 from the Eastern honey bee (Apis cerana). The results showed that, as a specific protein distributed below the base of chemosensilla on the antennal surface, AcerOBP2 was strongly bound with the candidate floral volatiles and bee pheromones. By docking analysis and site-directed mutagenesis, four different binding modes were found in the five AcerOBP2 mutants between six ligands. Two key amino acids, Ser123 and Lys51, play a key role in AcerOBP2 binding to odors, depending on the presence or absence of hydrogen bonds. In addition, the binding modes depend on their chemical structures and the binding poses of the diverse ligands. These results not only further prompted the functional basis of the relationship between the chemical structures of odorants and bee OBPs, but also revealed the complexity of the flexible behavioral modes of odor binding in insect olfactory systems. • OBP2 is a specific protein distributed below the base of antennal chemosensilla. • OBP2 was strongly bound with the floral volatiles and bee pheromone components. • Four different binding modes were found in five OBP2 mutants between six ligands. • Ser123 and Lys51 play a key role in the binding of OBP2 to odors through H-bond. [ABSTRACT FROM AUTHOR]

Odorant binding proteins (OBPs) of insects play a critical role in chemical perceptions and choice of insect host plant. Bemisia tabaci is a notorious insect pest which can damage more than 600 plant species. In order to explore functions of OBPs in B. tabaci , here we investigated binding characteristics and function of odorant-binding protein 3 in B. tabaci (BtabOBP3). The results indicated that BtabOBP3 shows highly similar sequence with OBPs of other insects, including the typical signature motif of six cysteines. The recombinant BtabOBP3 protein was obtained, and the evaluation of binding affinities to tested volatiles of host plant was conducted, then the results indicated that β-ionone had significantly higher binding to BtabOBP3 among other tested plant volatiles. Furthermore, silencing of BtabOBP3 significantly altered choice behavior of B. tabaci to β-ionone. In conclusion, it has been demonstrated that BtabOBP3 exerts function as one carrier of β-ionone and the results could be contributed to reveal the mechanisms of choosing host plant in B. tabaci. [ABSTRACT FROM AUTHOR]

The belowground pest Bradysia odoriphaga (Diptera: Sciaridae) has a sophisticated and sensitive olfactory system to detect semiochemical signals from the surrounding environment. In particular, odorant-binding proteins (OBPs) are crucial in capturing and transporting these semiochemical signals across the sensilla lymph to the corresponding odorant receptors. In this study, we cloned a full-length cDNA sequence of BodoOBP8 from B. odoriphaga. Real-time PCR (qRT-PCR) analysis revealed that BodoOBP8 has the highest expression levels in males, with more pronounced expression in the male antennae than in other tissues. In this study, the recombinant protein BodoOBP8 was successfully expressed by a bacterial system to explore its function. Competitive binding assays with 33 host plant volatiles and one putative sex pheromone (n-heptadecane) revealed that purified BodoOBP8 strongly bound to two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and to n-heptadecane; the corresponding dissolution constants (Ki) were 4.04, 6.73, and 4.04 μM, respectively. Molecular docking indicated that Ile96, Ile103, Ala107, and Leu111, located in the hydrophobic cavity of BodoOBP8, are the key residues mediating the interaction of BodoOBP8 with two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and n-heptadecane. These results show that BodoOBP8 plays a role in the recognition of plant volatiles and sex pheromones, suggesting its application as a molecular target for the screening of B. odoriphaga attractants and repellents and facilitating a new mechanism of B. odoriphaga control.

Xyleborus affinis, one of the most important pests of rubber trees, has caused severe damage to the natural rubber industry in Hainan province. The ability to detect host plants through a sensitive and specific olfactory system is crucial for Xyleborus affinis . Odorant binding proteins (OBPs) are believed to bind and carry hydrophobic active compounds from the environment to the surface of olfactory receptor neurons. To investigate the potential functional role of the highly expressed XaffOBP9 in binding with semiochemicals, we cloned and analyzed the cDNA sequence of XaffOBP9. The results showed that XaffOBP9 contains a 411bp open reading frame that encodes 136 amino acids. Then XaffOBP9 was expressed in Escherichia coli. The binding affinity of the recombinant OBP to 15 different ligands (14 host plant volatiles and 1 aggregation pheromone) was then examined using a fluorescence competitive binding approach. The results demonstrated that XaffOBP9 exhibited broad binding capabilities and strong affinities for 14 ligands. The structure of XaffOBP9 and its interactions with fourteen ligands were further analyzed by modeling and molecular docking, respectively. Based on the docking result, we found hydrophobic interactions are important between XaffOBP9 to these ligands and three amino acid residues (L71, Y106, and L114) were highly overlapped and contributed to the interaction with ligands. Mutation functional assays confirmed that the mutant L114A showed significantly reduced binding capacity to these ligands. This study suggested that XaffOBP9 may be involved in the chemoreception of semiochemicals and that it is helpful for the integrated management of X. affinis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Zhou, Zhang, Qin, Wu, Deng, Xu and Guo.)

The reference standards halo-GSK1482160 (F-, Br-, and I-) and their corresponding precursors desmethyl-halo-GSK1482160 (F-, Br-, and I-) were synthesized from (S)-1-methyl-5-oxopyrrolidine-2-carboxylic acid or (S)-5-oxopyrrolidine-2-carboxylic acid and 2-halo-3-(trifluoromethyl)benzylamine (F-, Br-, and I-) in one step with 45–93% yields. The target tracers [11C]halo-GSK1482160 (F-, Br-, and I-) were prepared from desmethyl-halo-GSK1482160 (F-, Br-, and I-) with [11C]CH 3 OTf under basic conditions (NaOH-Na 2 CO 3 , solid, w/w 1:2) through N -[11C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity (A M) at end of bombardment (EOB) was 370–740 GBq/μmol. The potency of halo-GSK1482160 (F-, Br-, and I-) in comparison with GSK1482160 (Cl-) was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity K i values for halo-GSK1482160 (F-, Br-, and I-) and GSK1482160 (Cl-) are 54.2, 2.5, 1.9 and 3.1 nM, respectively. [ABSTRACT FROM AUTHOR]

Abstract The overexpression of P2X7R is associated with neuroinflammation and plays an important role in various neurodegenerative diseases. The [18F]fluoropropyl derivative of GSK1482160, [18F]IUR-1602, has been first prepared and examined as a new potential P2X7R radioligand. The reference standard IUR-1602 was synthesized from tert -butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoropropylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 13% in three steps. The target tracer [18F]IUR-1602 was synthesized from desmethyl-GSK1482160 with 3-[18F]fluoropropyl tosylate, prepared from propane-1,3-diyl bis(4-methylbenzenesulfonate) and K[18F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 2–7% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1602 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity K i values for IUR-1602 and GSK1482160 are 23.6 and 3.07 nM, respectively. The initial in vitro evaluation results, 8-fold less potency of [18F]IUR-1602 compared to [11C]GSK1482160, prevent further in vivo evaluation of [18F]IUR-1602 in animals and human. Highlights • A new P2X7R radioligand [18F]IUR-1602 was synthesized. • A 2-step 2-pot fully automated [18F]-radiosynthesis for [18F]IUR-1602 was developed. • A semi-preparative RP HPLC-SPE technique was employed in radiosynthesis. • K i value of IUR-1602 was 23.6 ± 0.96 nM. [ABSTRACT FROM AUTHOR]

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a major pest of pines and it is also the key vector of the exotic pinewood nematode in China. In the present study, we cloned, expressed, and purified a chemosensory protein (CSP) in M. alternatus. We surveyed its expression in various developmental stages of male and female adult tissues and determined its binding affinities for different pine volatiles using a competitive binding fluorescence assay. A CSP known as CSP5 in M. alternatus was obtained from an antennal cDNA library and expressed in Escherichia coli. Quantitative reverse transcription polymerase chain reaction results indicated that the CSP5 gene was mainly expressed in male and female antennae. Competitive binding assays were performed to test the binding affinity of recombinant CSP5 to 13 odour molecules of pine volatiles. The results showed that CSP5 showed very strong binding abilities to myrcene, (+)-ß-pinene, and (-)-isolongifolene, whereas the volatiles 2-methoxy-4-vinylphenol, p-cymene, and (+)-limonene oxide have relatively weak binding affinity at pH 5.0. Three volatiles myrcene, (+)-ß-pinene, and (-)-isolongifolene may play crucial roles in CSP5 binding with ligands but this needs further study for confirmation. The sensitivity of insect to host plant volatiles can effectively be used to control and monitor the population through mass trapping as part of integrated pest management programs. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) play imperative functions in chemical and biochemical signaling of insects, as they distinguish and transfer ecological chemical indications to a sensory system in order to initiate behavioral responses. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), has emerged as the most destructive pest, causing serious damage to rice in extensive areas throughout Asia. Biotic characteristics like monophagy, dual wing forms, and annual long-distance migration imply a critical role of chemoreception in N. lugens. In this study, we cloned the full-length CSP8 gene from N. lugens. Protein sequence analysis indicated that NlugCSP8 shared high sequence resemblance with the CSPs of other insect family members and had the typical four-cysteine signature. Analysis of gene expression indicated that NlugCSP8 mRNA was specifically expressed in the wings of mated 3-day brachypterous females with a 175-fold difference compare to unmated 3-day brachypterous females. The NlugCSP8 mRNA was also highly expressed in the abdomen of unmated 5-day brachypterous males and correlated to the age, gender, adult wing form, and mating status. A competitive ligand-binding assay demonstrated that ligands with long chain carbon atoms, nerolidol, hexanal, and trans-2-hexenal were able to bind to NlugCSP8 in declining order of affinity. By using bioinformatics techniques, three-dimensional protein structure modeling and molecular docking, the binding sites of NlugCSP8 to the volatiles which had high binding affinity were predicted. In addition, behavioral experiments using the compounds displaying the high binding affinity for the NlugCSP8, revealed four compounds able to elicit significant behavioral responses from N. lugens. The in vivo functions of NlugCSP8 were further confirmed through the testing of RNAi and post-RNAi behavioral experiments. The results revealed that reduction in NlugCSP8 transcript abundance caused a decrease in behavioral response to representative attractants. An enhanced understanding of the NlugCSP8 is expected to contribute in the improvement of more effective and eco-friendly control strategies of BPH.

The reference standard IUR-1601 (( S )- N -(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from tert -butyl ( S )-5-oxopyrrolidine-2-carboxylate, fluoroethylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 12% in three steps. The target tracer [ 18 F]IUR-1601 (( S )- N -(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-[ 18 F]fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from desmethyl-GSK1482160 with 2-[ 18 F]fluoroethyl tosylate, prepared from 1,2-ethylene glycol-bis-tosylate and K[ 18 F ]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 1–3% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1601 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [ 11 C]GSK1482160, and the binding affinity K i values for IUR-1601 and GSK1482160 are 4.31 and 5.14 nM, respectively. [ABSTRACT FROM AUTHOR]

Brominated phenolic compounds (BPCs) are found in the environment, and in human and wildlife tissues, and some are considered to have endocrine disrupting activities. The goal of this study was to determine how structural differences of 3 BPC classes impact binding affinities for the thyroid receptor beta (TRß) in humans and zebrafish. BPC classes included halogenated bisphenol A derivatives, halogenated oxidative transformation products of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and brominated phenols. Affinities were assessed using recombinant TRb protein in competitive binding assays with 125I-triiodothyronine (125I-T3) as the radioligand. Zebrafish and human TRß displayed similar binding affinities for T3 (Ki=0.40 and 0.49 nM) and thyroxine (T4, Ki=6.7 and 6.8 nM). TRß affinity increased with increasing halogen mass and atomic radius for both species, with the iodinated compounds having the highest affinity within their compound classes. Increasing halogen mass and radius increases the molecular weight, volume, and hydrophobicity of a compound, which are all highly correlated with increasing affinity. TRb affinity also increased with the degree of halogenation for both species. Human TRb displayed higher binding affinities for the halogenate bisphenol A compounds, whereas zebrafish TRb displayed higher affinities for 2,4,6-trichlorophenol and 2,4,6-trifluorophenol. Observed species differences may be related to amino acid differences within the ligand binding domains. Overall, structural variations impact TRb affinities in a similar manner, supporting the use of zebrafish as a model for TRb disruption. Further studies are necessary to investigate how the identified structural modifications impact downstreamreceptor activities and potential in vivo effects. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) play imperative functions in chemical and biochemical signaling of insects, as they distinguish and transfer ecological chemical indications to a sensory system in order to initiate behavioral responses. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), has emerged as the most destructive pest, causing serious damage to rice in extensive areas throughout Asia. Biotic characteristics like monophagy, dual wing forms, and annual long-distance migration imply a critical role of chemoreception in N. lugens. In this study, we cloned the full-length CSP8 gene from N. lugens. Protein sequence analysis indicated that NlugCSP8 shared high sequence resemblance with the CSPs of other insect family members and had the typical four-cysteine signature. Analysis of gene expression indicated that NlugCSP8 mRNA was specifically expressed in the wings of mated 3-day brachypterous females with a 175-fold difference compare to unmated 3-day brachypterous females. The NlugCSP8mRNA was also highly expressed in the abdomen of unmated 5-day brachypterous males and correlated to the age, gender, adult wing form, and mating status. A competitive ligand-binding assay demonstrated that ligands with long chain carbon atoms, nerolidol, hexanal, and trans-2-hexenal were able to bind to NlugCSP8 in declining order of affinity. By using bioinformatics techniques, three-dimensional protein structure modeling and molecular docking, the binding sites of NlugCSP8 to the volatiles which had high binding affinity were predicted. In addition, behavioral experiments using the compounds displaying the high binding affinity for the NlugCSP8, revealed four compounds able to elicit significant behavioral responses from N. lugens. The in vivo functions of NlugCSP8 were further confirmed through the testing of RNAi and post-RNAi behavioral experiments. The results revealed that reduction in NlugCSP8 transcript abundance caused a decrease in behavioral response to representative attractants. An enhanced understanding of the NlugCSP8 is expected to contribute in the improvement of more effective and eco-friendly control strategies of BPH. [ABSTRACT FROM AUTHOR]

Aims: To confirm the different characteristics of genotype-specific and common neutralizing epitopes of hepatitis E virus (HEV). Methods: A competitive binding assay was established with known genotype-common neutralizing monoclonal antibodies (mAbs) 3G1 and 5G5 as well as genotype-specific neutralizing mAbs 2B1 and 4C5. HEV ORF2 recombinant p166W01 derived from genotype 1 and p166Chn derived from genotype 4 were used as coated antigens, to determine whether the mAbs recognize independent, similar, or overlapping epitopes. mAbs were produced, purified, and conjugated with horseradish peroxidase (HRP). HRP-conjugated 2B1 could react only with p166W01 but not p166Chn, HRP-conjugated 4C5 could react only with p166Chn but not p166W01, while HRP-conjugated 3G1 and 5G5 could react both with p166W01 and p166Chn. Thus, competitive binding assays were performed successively using p166W01 and p166Chn antigen. Results and Conclusion: The results of competitive binding assays revealed that the binding of HRP-conjugated 2B1 to p166W01 could not be inhibited by 5G5 or 3G1. Similarly, the binding of HRP-conjugated 4C5 to p166Chn could not be inhibited by 5G5 or 3G1. However, the mAbs 5G5 and 3G1 blocked each other’s binding to p166W01 and p166Chn, suggesting that common and genotype-specific neutralizing mAbs recognize independent epitopes. [ABSTRACT FROM AUTHOR]

Novel, molecularly imprinted polymers (MIPs) were developed for the highly sensitive and selective recognition of the stress marker cortisol. Oriented, homogeneous cavities with two binding sites for cortisol were fabricated by surface-initiated atom transfer radical polymerization, using a cortisol motif template molecule (TM1) which consists of a polymerizable moiety attached at the 3-carbonyl group of cortisol via an oxime linkage and an adamantane carboxylate moiety coupled with the 21-hydroxyl group. TM1 was orientationally immobilized on a β-cyclodextrin (β-CD)-grafted gold-coated sensor chip by inclusion of the adamantane moiety of TM1, followed by copolymerization of a hydrophilic comonomer, 2-methacryloyloxyethyl phosphorylcholine, with or without a cross-linker, N,N′-methylenebisacrylamide. Subsequent cleavage of the oxime linkage leaves the imprinted cavities that contain dual binding sites—namely, the aminooxy group and β-CD—capable of oxime formation and hydrophobic interaction, respectively. As an application, MIP-based picomolar level detection of cortisol was demonstrated by a competitive binding assay using a fluorescent competitor. Cross-linking of the MIP imparts rigidity to the binding cavities, and improves the selectivity and sensitivity significantly, reducing the limit of detection to 4.8 pM. In addition, detection of cortisol in saliva samples was demonstrated as a feasibility study.

Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9 h, and 40 nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E 2 ) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks. [ABSTRACT FROM AUTHOR]

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer’s disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[ 11 C]methyl-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ( N- [ 11 C]Me-5-BDBD analog, [ 11 C] 9 ) and 5-(3-Bromophenyl)-1-[ 11 C]methyl-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ( N- [ 11 C]Me-5-BDBD, [ 11 C] 8c ) were prepared from their corresponding desmethylated precursors with [ 11 C]CH 3 OTf through N -[ 11 C]methylation and isolated by HPLC combined with SPE in 30–50% decay corrected radiochemical yields with 370–1110 GBq/µmol specific activity at EOB. 5-(3-[ 18 F]Fluorophenyl)-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ([ 18 F]F-5-BDBD, [ 18 F] 5a ) and 5-(3-(2-[ 18 F]fluoroethoxy)phenyl)-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ([ 18 F]FE-5-BDBD, [ 18 F] 11 ) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[ 18 F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5–25% decay corrected radiochemical yields with 111–740 GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD. [ABSTRACT FROM AUTHOR]

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucinate ( 5 ) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucine ( 6 ) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [ 11 C]methyl (2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucinate ([ 11 C] 5 ) was prepared from the acid precursor with [ 11 C]CH 3 OTf through O -[ 11 C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [ 11 C]CO 2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ∼40-min from EOB. The radioligand depletion experiment of [ 11 C] 5 did not display specific binding to CX 3 CR1, and the competitive binding assay of ligand 5 found much lower CX 3 CR1 binding affinity. [ABSTRACT FROM AUTHOR]

Thyroid hormone (TH) disrupting compounds are potentially important environmental contaminants due to their possible adverse neurological and developmental effects on both humans and wildlife. Currently, the most successful bio-analytical method to detect and evaluate TH disruptors, which target the plasma transport of TH in environmental samples, is the radio-ligand thyroxine-transthyretin (T 4 -TTR) binding assay. Yet, costly materials and tedious handling procedures prevent the use of this assay in high throughput analysis that is nowadays urgently demanded in environmental quality assessment. For the first time a miniaturized fluorescence T 4 -TTR binding assay was developed in a 96 well microplate and tested with eight TH disrupting compounds. For most of the compounds, the sensitivity of the newly developed assay was slightly lower than the radio-ligand binding assay, however, throughput was enhanced at least 100-fold, while using much cheaper materials. The TH disrupting potency of 22 herring gull ( Larus argentatus ) egg extracts, collected from two different locations (Musvær and Reiaren) in Norway, was evaluated to demonstrate the applicability of the assay for environmental samples. [ABSTRACT FROM AUTHOR]

A chemosensory protein named HarmCSP5 in cotton bollworm Helicoverpa armigera (Hübner) was obtained from antennal cDNA libraries and expressed in Escherichia coli. The real time quantitative PCR (RT-qPCR) results indicated that HarmCSP5 gene was mainly expressed in male and female antennae but also expressed in female legs and wings. Competitive binding assays were performed to test the binding affinity of recombinant HarmCSP5 to 60 odor molecules including some cotton volatiles. The resules showed that HarmCSP5 showed strong binding abilities to 4-ehtylbenzaldehyde and 3,4-dimethlbenz aldehyde, whereas methyl phenylacetate, 2-decanone, 1-pentanol, carvenol, isoborneol, nerolidol, 2-nonanone and ethyl heptanoate have relatively weak binding affinity. Moreover, the predicted 3D model of HarmCSP5 consists of six α-helices located among residues 33–38 (α1), 40–48 (α2), 62–72 (α3), 80–96 (α4), 98–108 (α5), and 116–119 (α6), two pairs of disulfide bridges Cys49-Cys55, Cys75-Cys78. The two amino acid residues, Ile94 and Trp101, may play crucial roles in HarmCSP5 binding with ligands and need further study for confirmation.

Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid's optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic "target" and magnetic "probe" nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1 × 5 array of 50 μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ~25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology's potential to translate magnetic nanoparticle assays into a user-free optical analysis platform. [ABSTRACT FROM AUTHOR]

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. [ABSTRACT FROM AUTHOR]

Two chromium(III) complexes containing derivatives of 1,10-phenanthroline were synthesized and characterized using different spectroscopic methods. The interaction of the synthesized complexes with DNA was performed. From the absorption titration data, the binding constant for the complexes [Cr(imiphen) 2 Cl 2 ] + ( 1 ) and [Cr(furphen) 2 Cl 2 ] + ( 2 ) with DNA were found to be 5.69 ± 0.3 × 10 4 M − 1 for 1 and 3.7 ± 0.2 × 10 4 M − 1 for 2 , respectively. The viscosity studies show that both the complexes bind to DNA in a partial intercalative mode. The results of competitive binding assay reveal that both complexes displace EB from DNA marginally. Gel electrophoresis data show that both the complexes 1 and 2 requires co-reagent to induce DNA cleavage but complex 2 exhibit better nuclease activity compared to complex 1 . The results from this study describe the role of ligand structure in the binding and cleavage of DNA as furan based ligand exhibit better cleaving ability compared to imidazole moiety. [ABSTRACT FROM AUTHOR]

Extracellular signal-regulated kinase 2 (ERK2) is a drug target for type 2 diabetes mellitus. A peptide-type ERK2 inhibitor (PEP) was discovered in the previous study through the knowledge-based method and showed physiological effects on the db / db mice model of type 2 diabetes. Here, the crystal structure showed that PEP bound to the allosteric site without the interruption of the ATP competitive inhibitor binding to ERK2. An in silico biased-screening using the focused library rendered three compounds with inhibitory activity of IC 50 <100 μM. Among them, two compounds revealed the concentration-dependent competition with PEP and could be lead compounds for antidiabetic medicine. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate receptors (S1PRs) are important regulators of vascular permeability, inflammation, angiogenesis and vascular maturation. Identifying a specific S1PR PET radioligand is imperative, but it is hindered by the complexity and variability of current for binding affinity measurement procedures. Herein, we report a streamlined protocol for radiosynthesis of [ 32 P]S1P with good radiochemical yield (36–50%) and high radiochemical purity (>99%). We also report a reproducible procedure for determining the binding affinity for compounds targeting S1PRs in vitro . [ABSTRACT FROM AUTHOR]