Odorant-binding protein 19 in Monochamus alternatus involved in the recognition of a volatile strongly emitted from ovipositing host pines.

Monochamus alternatus is the primary carrier of pine wood nematodes, which pose a serious threat to Pinus spp. in many countries. Newly emerging M. alternatus adults feed on heathy host pines, while matured adults transfer to stressed host pines for mating and oviposition. Several odorant-binding proteins (OBPs) of M. alternatus have been proved to aid in the complex process of host location. To clarify the corresponding relations between OBPs and pine volatiles, more OBPs need to be studied. In this research, MaltOBP19 showed a specific expression in the antennae and mouthparts of M. alternatus, and it was marked in 4 types of antenna sensilla by immunolocalization. Fluorescence binding assays demonstrated the high binding affinity of MaltOBP19 with camphene and myrcene in vitro. In Y-tube olfactory experiments, M. alternatus adults were attracted by camphene and RNAi of OBP19 via microinjection significantly decreased their attraction index. Myrcene induced phobotaxis, but RNAi had no significant effect on this behavior. Further, we found that ingesting dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of MaltOBP19. These results suggest that MaltOBP19 may play a role in the process of host conversion via the recognition of camphene, which has been identified to be strongly released in stressed host pines. In addition, it is proved that knockdown of OBP can be achieved by oral administration of bacteria-expressed double-stranded RNA in M. alternatus adults, providing a new perspective in the control of M. alternatus.

Guar gum series affect nanostructured lipid carriers via electrostatic assembly or steric hindrance: Improving their oral delivery for phytosterols.

Surface modification of nanostructured lipid carriers (NLCs) can be an effective way to improve their oral delivery for active ingredients. In this study, four type of guar gum series modified NLCs for the delivery of phytosterols (PS) were constructed and the effects of the polysaccharides on their structure and physicochemical properties were studied. DLS and AFM results revealed that positively charged polysaccharides could bind to PS-NLCs through electrostatic attraction and made the complexes finally take positive charges, while negatively charged polysaccharides were more likely to fill in the gaps of NLC systems to achieve a balance between electrostatic repulsion and intermolecular forces. Although all four polysaccharides exhibited good storage stability and controlled release of PS in simulated intestinal digestion, PS-NLCs modified with partially hydrolyzed cationic guar gum (PHCG) at medium or high concentrations exhibited better gastric stability, mucoadhesion, and cellular uptake, which had considerable significance for improving the oral bioavailability of PS. This might be related to the coating structure of PHCG-PS-NLCs confirmed by AFM, FTIR, and Raman characterization. This study provide a reference value for designing suitable PS-NLC complexes without synthetic surfactants.

Effects of food formulation on bioavailability of phytosterols: phytosterol structures, delivery carriers, and food matrices.

Daily intake of phytosterols (PSs) as a diet supplement can lower blood-cholesterol levels and reduce the risks of cardiovascular diseases. However, the high crystallinity, low water solubility, easy oxidizability, and other characteristics of PSs restrict their application and bioavailability in food products. The formulation parameters including the structures of PSs, delivery carriers, and food matrices may play an important role in the release, dissolution, transport, and absorption of PSs in functional foods. In this paper, the effects of formulation parameters, including phytosterol structures, delivery carriers, and food matrices, on the bioavailability of phytosterols are summarized and suggestions are provided for the formulation design of functional foods. The side chain and hydroxyl esterification group of PSs may significantly affect their lipid or water solubilities and micellization capacities, which in turn affect the bioavailability of PSs. Selecting suitable delivery carriers based on the characteristics of the food system can reduce the crystallinity and oxidation of PSs and control the release of PSs, thereby improving the PS stability and delivery efficiency. Moreover, the ingredients of the carriers or food products would also influence the release, solubility, transport, and absorption of PSs in the gastrointestinal tract (GIT).

Impact of molecular interactions between hydrophilic phytosterol glycosyl derivatives and bile salts on the micellar solubility of cholesterol.

Hydrophilic phytosterol glycosyl derivatives are synthetic phytosterol analogues by coupling with the glycosyl moiety to improve the water solubility and bioaccessibility of free phytosterols. The aim of this study is to clarify the molecular interaction of phytosterol glycosyl derivatives with bile salts and the consequent impact on cholesterol solubilization. Sharp nonlinear decrease in the micellar solubility of cholesterol and accompanying changes in particle size, zeta potential and microtopography of mixed micelles were observed when phytosterol glycosyl derivatives were introduced in cholesterol-loaded bile salt micelles. These results suggested that ß-sitosterol glycosyl derivatives molecules indeed participated in the formation of mixed micelles. Further, nuclear magnetic resonance showed that the structural change of mixed micelles was caused by the insertion of ß-sitosterol glycosyl derivatives via hydrogen bonds with sodium taurocholate, which resulted in the low cholesterol solubilization. Moreover, the hydrogen-bond interactions were apparently influenced by the glycosyl moiety of ß-sitosterol glycosyl derivatives. These molecular mechanisms may contribute to the development of cholesterol-absorption inhibitors.

A Highly Expressed Antennae Odorant-Binding Protein Involved in Recognition of Herbivore-Induced Plant Volatiles in Dastarcus helophoroides.

Natural enemies such as parasitoids and parasites depend on sensitive olfactory to search for their specific hosts. Herbivore-induced plant volatiles (HIPVs) are vital components in providing host information for many natural enemies of herbivores. However, the olfactory-related proteins involved in the recognition of HIPVs are rarely reported. In this study, we established an exhaustive tissue and developmental expression profile of odorant-binding proteins (OBPs) from Dastarcus helophoroides, an essential natural enemy in the forestry ecosystem. Twenty DhelOBPs displayed various expression patterns in different organs and adult physiological states, suggesting a potential involvement in olfactory perception. In silico AlphaFold2-based modeling and molecular docking showed similar binding energies between six DhelOBPs (DhelOBP4, 5, 6, 14, 18, and 20) and HIPVs from Pinus massoniana. While in vitro fluorescence competitive binding assays showed only recombinant DhelOBP4, the most highly expressed in the antennae of emerging adults could bind to HIPVs with high binding affinities. RNAi-mediated behavioral assays indicated that DhelOBP4 was an essential functional protein for D. helophoroides adults recognizing two behaviorally attractive substances: p-cymene and γ-terpinene. Further binding conformation analyses revealed that Phe 54, Val 56, and Phe 71 might be the key binding sites for DhelOBP4 interacting with HIPVs. In conclusion, our results provide an essential molecular basis for the olfactory perception of D. helophoroides and reliable evidence for recognizing the HIPVs of natural enemies from insect OBPs' perspective.

Mass spectrometry in food authentication and origin traceability.

Food authentication and origin traceability are popular research topics, especially as concerns about food quality continue to increase. Mass spectrometry (MS) plays an indispensable role in food authentication and origin traceability. In this review, the applications of MS in food authentication and origin traceability by analyzing the main components and chemical fingerprints or profiles are summarized. In addition, the characteristic markers for food authentication are also reviewed, and the advantages and disadvantages of MS-based techniques for food authentication, as well as the current trends and challenges, are discussed. The fingerprinting and profiling methods, in combination with multivariate statistical analysis, are more suitable for the authentication of high-value foods, while characteristic marker-based methods are more suitable for adulteration detection. Several new techniques have been introduced to the field, such as proton transfer reaction mass spectrometry, ambient ionization mass spectrometry (AIMS), and ion mobility mass spectrometry, for the determination of food adulteration due to their fast and convenient analysis. As an important trend, the miniaturization of MS offers advantages, such as small and portable instrumentation and fast and nondestructive analysis. Moreover, many applications in food authentication are using AIMS, which can help food authentication in food inspection/field analysis. This review provides a reference and guide for food authentication and traceability based on MS.

NlugOBP8 in Nilaparvata lugens Involved in the Perception of Two Terpenoid Compounds from Rice Plant.

Odorant binding proteins (OBPs) play an important role in insect peripheral olfactory systems and exploring the physiological function of OBPs could facilitate the understanding of insects' chemical communication. Here, the functional analysis of an antenna-based NlugOBP8 from brown planthopper (BPH) Nilaparvata lugens (Stål) was performed both in vitro and in vivo. Recombinant NlugOBP8 exhibited strong binding affinity to 13 out of 26 rice plant volatiles and could form a stable complex with 9 of them according to the fluorescence binding and fluorescence quenching experiments. Circular dichroism spectra demonstrated that six volatiles could give rise to significant conformational change of recombinant NlugOBP8. H-tube olfactometer bioassay confirmed that BPHs were significantly attracted by nerolidol and significantly repelled by linalool, caryophyllene oxide, and terpinolene, respectively. Antennae of dsNlugOBP8-injected BPHs exhibited significantly lower electrophysiological response to linalool and caryophyllene oxide. Moreover, the repellent responses of BPHs to these two volatiles were also impaired upon silencing NlugOBP8. These data suggest that NlugOBP8 is involved in recognizing linalool and caryophyllene oxide and provide additional target for the sustainable control of BPHs.

Evolutionarily conserved odorant-binding proteins participate in establishing tritrophic interactions.

Attracting herbivores and their natural enemies is a standard method where plant volatiles mediate tritrophic interactions. However, it remains unknown whether the shared attraction has a shared chemosensory basis. Here we focus on the odorant-binding proteins (OBPs), a gene family integral to peripheral detection of odoriferous chemicals. Previous evidence suggests that the herbivorous beetle Monochamus alternatus and its parasitoid beetle Dastarcus helophoroides are attracted to stressed pines. In this study, (+)-fenchone, emitted by stressed pines, is found to be attracted to M. alternatus and D. helophoroides in behavioral assays. Meanwhile, two orthologous OBPs with a slower evolutionary rate, respectively, from the two insects are shown to bind with (+)-fenchone, and the attraction is abolished after RNAi. These results show the ability of evolutionarily conserved OBPs from herbivores and their enemies to detect the same plant volatiles, providing an olfactory mechanism of chemical signals-mediated tritrophic relationships.

BarH1 regulates odorant-binding proteins expression and olfactory perception of Monochamus alternatus Hope.

Insect odorant-binding proteins (OBPs) are a class of small soluble proteins that can be found in various tissues wherein binding and transport of small molecules are required. Thus, OBPs are not only involved in typical olfactory function by specific activities with odorants but also participate in other physiological processes in non-chemosensory tissues. To better understand the complex biological functions of OBPs, it is necessary to study the transcriptional regulation of their expression patterns. In this paper, an apparent gradient expression pattern of Obp19, that was highly and specifically expressed in antennae and played an essential role in the detection of camphene, was defined in the antennae of the Japanese pine sawyer. Further, the transcription factor BarH1, that also presented gradient expression pattern in antennae, was found to regulate expression of Obp19 directly through binding to its upstream DNA sequence. The condition of BarH1 gene silence, the gene expression levels of Obp19 significantly decreased. At the same time, additional olfactory genes also were regulated and thus influence camphene reception. These findings provide us an opportunity to incorporate Obps in the gene regulatory networks of insects, which contribute to a better understanding of the multiplicity and diversity of OBPs and the olfactory mediated behaviors.

Adulteration detection of essence in sesame oil based on headspace gas chromatography-ion mobility spectrometry.

Sesame oil is a traditional and delicious edible oil in China and Southeast Asia with a high price. However, sesame oil essence was often illegally added to cheaper edible oils to counterfeit sesame oil. In this study, a rapid and accurate headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) method was proposed to detect the counterfeit sesame oil where the other cheap oils were adulterated with essence. Combined with chemometric methods including principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and random forest (RF), authentic and counterfeit sesame oils adulterated with sesame essence (0.5%, w/w) were easily separated into two groups. More importantly, 2-methylbutanoic acid, 2-furfurylthiol, methylpyrazine, methional, and 2,5-dimethylpyrazine were found to be markers of sesame essence, which were used to directly identify the sesame essence. The determination of volatile compounds based on HS-GC-IMS was proven to be an effective method for adulteration detection of essence in sesame oil.

Functional Analysis of Two Odorant-Binding Proteins, MaltOBP9 and MaltOBP10, in Monochamus alternatus Hope.

Odorant-binding proteins (OBPs) are important for the perception of chemical signals by insects. Effective pest management strategies can be developed by understanding the host location mechanism and the physiological functions of OBPs in olfactory detection. In this study, we cloned two OBPs from Monochamus alternatus, where MaltOBP9 was highly expressed in multiple insect tissues and MaltOBP10 was highly expressed in the female antenna according to the results of qRT-PCR. The recombinant proteins were successfully purified in vitro. Immunocytochemistry indicated the high expression of MaltOBP9 and MaltOBP10 in the sensillum lymph of sensilla basiconica, sensilla trichodea, sensilla auricillica, and sensilla chaetica, thereby demonstrating their broad participation in semiochemical detection. Both proteins were localized in the inner cavity of mechanoreceptors and they exhibited broad binding abilities with volatiles from pine bark according to fluorescence competitive binding assays. Due to its broad binding ability and distribution, MaltOBP9 may be involved in various physiological processes as well as olfactory detection. MaltOBP10 appears to play a role in the fundamental olfactory recognition process of female adults according to its broad binding ability. These findings suggest that OBPs may have various physiological functions in insects, thereby providing novel insights into the olfactory receptive mechanism.

Identification and Validation of Metabolic Markers for Adulteration Detection of Edible Oils Using Metabolic Networks.

Food adulteration is a challenge faced by consumers and researchers. Due to DNA fragmentation during oil processing, it is necessary to discover metabolic markers alternative to DNA for adulteration detection of edible oils. However, the contents of metabolic markers vary in response to various factors, such as plant species, varieties, geographical origin, climate, and cultivation measures. Thus, it is difficult to identify a universal marker for all adulterants that may be present in some authentic samples. Currently, the specificity and selectivity of metabolic biomarkers are difficult to validate. Therefore, this study developed a screening strategy based on plant metabolic networks by developing a targeted analytical method for 56 metabolites in a metabolic network, using liquid/liquid extraction-liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified a chain of 11 metabolites that were related to isoflavonoid biosynthesis, which were detected in soybean oils but not rapeseed oils. Through multiple-marker mutual validation, these metabolites can be used as species-specific universal markers to differentiate soybean oil from rapeseed oil. Moreover, this method provides a model for screening characteristic markers of other edible vegetable oils and foods.

Extraction and Determination of Vitamin K1 in Foods by Ultrasound-Assisted Extraction, SPE, and LC-MS/MS.

Vitamin K1 is one of the important hydrophobic vitamins in fat-containing foods. Traditionally, lipase is employed in the determination of vitamin K1 to remove the lipids, which makes the detection complex, time-consuming, and insensitive. In this study, the determination of vitamin K1 in fat-containing foods was developed based on ultrasound-assisted extraction (UAE), solid-phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimal conditions for extraction of vitamin K1 were material-liquid ratio of 1:70 (g/mL), extraction temperature of 50 °C, extraction power of 700 W, extraction time of 50 min, material-wash fluid ratio of 1:60 (g/mL), and 8 mL of hexane/anhydrous ether (97:3, v/v) as the elution solvent. Then, vitamin K1 was analyzed on a ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 µm) by gradient elution with water (0.01% formic acid) and methanol (0.01 formic acid + 2.5 mmol/L ammonium formate) as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.16 µg/kg, respectively. Calibration curve was linear over the range of 10-500 ng/mL (R2 > 0.9988). The recoveries at three spiked levels were between 80.9% and 119.1%. The validation and application indicated that the proposed method was simple and sensitive in determination of vitamin K1 in fat-containing foods.

Phytosterol Contents of Edible Oils and Their Contributions to Estimated Phytosterol Intake in the Chinese Diet.

Phytosterols are important micronutrients in human diets. Evidence has shown that phytosterols play an essential role in the reduction of cholesterol in blood and therefore decrease cardiovascular morbidity. In this study, the content and composition of phytosterols in different kinds of vegetable oils were analyzed, and the total phytosterol intake and contribution of foods to intake were estimated based on consumption data. The results showed that the phytosterol contents of rice bran oil, corn oil, and rapeseed oil were higher than those of other vegetable oils and the intake of phytosterol in the Chinese diet was about 392.3 mg/day. The main sources of phytosterols were edible vegetable oils (46.3%), followed by cereals (38.9%), vegetables (9.2%), nuts (2.0%), fruits (1.5%), beans and bean products (1.4%), and tubers (0.8%). Among all vegetable oils, rapeseed oil was the main individual contributor to phytosterol intake (22.9%), especially for the southern residents of China.

Comparative Metabolomic Analysis of Rapeseeds from Three Countries.

Rapeseed is an important oilseed with proper fatty acid composition and abundant bioactive components. Canada and China are the two major rapeseed-producing countries all over the world. Meanwhile, Canada and Mongolia are major importers of rapeseed due to the great demand for rapeseed in China. To investigate the metabolites in rapeseeds from three countries, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based metabolomics was employed to analyze rapeseeds from China, Canada, and Mongolia. As results, 67, 53, and 68 metabolites showed significant differences between Chinese and Canadian, Chinese and Mongolian, and Canadian and Mongolian rapeseeds, respectively. Differential metabolites were mainly distributed in the metabolic pathways including phenylpropanoid biosynthesis, flavone and flavonol biosynthesis, and ubiquinone and other terpenoid-quinone biosynthesis. Among the differential metabolites, contents of sinapate and sinapine were higher in Chinese rapeseeds, while the contents of brassicasterol, stigmasterol, and campestanol were higher in Canadian rapeseeds. These findings might provide insight into the metabolic characteristics of rapeseeds from three countries to guide processing and consumption of the products of rapeseed.

Simultaneous determination of tocopherols, carotenoids and phytosterols in edible vegetable oil by ultrasound-assisted saponification, LLE and LC-MS/MS.

A method was developed to simultaneously determine eight bioactive compounds in edible oil based on ultrasound-assisted saponification, liquid-liquid extraction and liquid chromatography coupled with tandem mass spectrometry. Central composite design was employed to optimize ultrasonic temperature and time of saponification. Sample treatment was conducted by ultrasound-assisted saponification at temperature of 75 °C for 40 min. Limits of detection and limits of quantification ranged from 2.0 to 3.2 and from 6.1 to 10.0 ng/mL, respectively. Linear correlations were obtained (R2 > 0.99) and the recoveries at three spiked levels were between 81.7% and 112.0%. This method was employed to determine eight compounds in camellia oils and olive oils. As results, the contents of stigmasterol, δ-tocopherol, γ-tocopherol, ß-carotene and lutein in camellia oils were significantly higher than those in olive oils (p < 0.05). The proposed method can be successfully used to determination of these eight active compounds in camellia oil and other edible oils.

Polymeric Micellar Delivery of Novel Microtubule Destabilizer and Hedgehog Signaling Inhibitor for Treating Chemoresistant Prostate Cancer.

Castration-resistant prostate cancer that has become resistant to docetaxel (DTX) represents one of the greatest clinical challenges in the management of this malignancy. There is an urgent need to develop novel therapeutic agents to overcome chemoresistance and improve the overall survival of patients. We have designed a novel microtubule destabilizer (2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (QW-296) and combined it with a newly synthesized hedgehog (Hh) signaling pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl)phenyl]-N 4,N 4- bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) to treat taxane-resistant (TXR) prostate cancer. The combination of QW-296 and MDB5 exhibited stronger anticancer activity toward DU145-TXR and PC3-TXR cells and suppressed tumor colony formation when compared with single-drug treatment. Because these drugs are hydrophobic, we synthesized the mPEG-p(TMC-MBC) [methoxy-poly(ethylene glycol)-block-poly(trimethylene carbonate-co-2-methyl-2-benzoxycarbonyl-propylene carbonate)] copolymer, which could self-assemble into micelles with loading capacities of 8.13% ± 0.75% and 9.12% ± 0.69% for QW-296 and MDB5, respectively. Further, these micelles provided controlled the respective drug release of 58% and 42% release of QW-296 and MDB5 within 24 hours when dialyzed against PBS (pH 7.4). We established an orthotopic prostate tumor in nude mice using stably luciferase expressing PC3-TXR cells. There was maximum tumor growth inhibition in the group treated with the combination therapy of QW-296 and MDB5 in micelles compared with their monotherapies or combination therapy formulated in cosolvent. The overall findings suggest that combination therapy with QW-296 and MDB5 has great clinical potential to treat TXR prostate cancer, and copolymer mPEG-p(TMC-MBC) could serve as an effective delivery vehicle to boost therapeutic efficacy in vivo.

Evaluation and comparison of in vitro antioxidant activities of unsaponifiable fraction of 11 kinds of edible vegetable oils.

The radical scavenging capabilities of the extracts from eleven edible vegetable oils were investigated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing ability of plasma (FRAP) assays. The results indicated that rapeseed oil and sesame oil showed higher radical scavenging abilities than other vegetable oils. When the radical scavenging capabilities of the extracts from virgin camellia oils and commercially available refined camellia oils were evaluated by FRAP assay, the results showed that the antioxidant capabilities of the former were higher than the latter. Therefore, it is recommended that moderate refining processes should be taken to minimize the loss of antioxidant components and people consume virgin oils or less processed edible vegetable oils for higher antioxidant activities.

Identification of Nutritional Components in Black Sesame Determined by Widely Targeted Metabolomics and Traditional Chinese Medicines.

Chemical composition of secondary metabolites is of great importance for quality control of agricultural products. Black sesame seeds are significantly more expensive than white sesame seeds, because it is thought that black sesame seeds are more beneficial to human health than white sesame seeds. However, the differences in nutrient composition between black sesame seeds and white sesame seeds are still unknown. The current study examined the levels of different metabolites in black and white sesame seeds via the use of a novel metabolomics strategy. Using widely targeted metabolomics data, we obtained the structure and content of 557 metabolites, out of which 217 metabolites were identified, and discovered 30 metabolic pathways activated by the secondary metabolites in both black and white sesame seeds. Our results demonstrated that the main pathways that were differentially activated included: phenylpropanoid biosynthesis, tyrosine metabolism, and riboflavin metabolism. More importantly, the biomarkers that were significantly different between black seeds and white sesame seeds are highly related to the functions recorded in traditional Chinese medicine. The results of this study may serve as a new theoretical reference for breeding experts to promote the genetic improvement of sesame seeds, and therefore the cultivation of higher quality sesame varieties.

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-ß-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (ß-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-ß-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, ß-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.