Author Report for: Guo J

Imidacloprid (IMI) is a neonicotinoid insecticide used worldwide, either alone or in combination with other pesticides. The goal of this study was to assess the effects of IMI on the central nervous system of rats and its mechanism of oxidative stress-induced DNA damage by oxidant/antioxidant parameters. Fifteen male rats, divided into three groups, were used: the first group received 5 ml/kg body weight corn oil as a control, the second received a high oral dose of IMI (45 mg/kg body weight), while the third received a low dose (22 mg/kg body weight). After 28 days, acetylcholinesterase (AChE) activity, oxidative stress markers, histopathological alterations, and DNA damage were examined in the brains of these rats. The AChE activities decreased significantly after IMI exposure, reaching 2.45 and 2.75 nmol/min/mg protein in high dose and low dose, respectively, compared to the control group (3.75 nmol/g tissues), while the concentration of malondialdehyde MDA increased significantly (29.28 and 23.92 nmol/g tissues) vs. the control group (19.28 nmol/g tissues). The antioxidant status parameters such as reduced glutathione (GSH) content was 13.77 and 17.63 nmol/g, catalase (CAT) activity was 22.56 and 26.65 micromol/min/g, and superoxide dismutase (SOD) activity was 6.66 and 7.23 micromol/min/g in both doses against the control group (21.37 nmol/g, 30.67 micromol/min/g, 11.76 micromol/min/g), respectively, and histopathological changes in the brain tissues were observed. More in vivo research using epigenetic methods is needed to determine the ability of IMI and its metabolites to cause neurotoxicity and DNA lesions in mammalian brains.

Botanical molluscicides for controlling the invasive snail Pomacea canaliculata have attracted worldwide attention because of their cost and environmental friendliness. Aqueous extracts from discarded tobacco leaf (Nicotiana tobacum) were evaluated for molluscicidal activity against different-sized P. canaliculata under laboratory conditions. The results showed that over 90% of the snails died in 1 g/L tobacco extract within 4 days, and the survival of P. canaliculata was inversely proportional to the snail size, tobacco extract concentration and length of exposure time. Adult males were more susceptible to tobacco extract than females. The snails had few chances to feed or mate in 0.5 g/L tobacco extract, and reproduction was greatly limited in 0.2 g/L. The growth of juvenile snails was inhibited in 0.2 g/L tobacco extract, but adults were unaffected. The antioxidant capacity of P. canaliculata in response to tobacco extract can be size- and sex-dependent, and the activities of superoxide dismutase, catalase, and acetylcholinesterase and the contents of glutathione and malondialdehyde were increased in adult males. These results suggest that discarded tobacco leaves can be useful as a molluscicide for controlling the invasive snail P. canaliculata based on its effects on survival, behaviour, food intake, growth performance and antioxidant capacity.

Aroma is a crucial quality attribute of apple fruit, which significantly impacts its commercial value and consumer choice. Despite its importance the volatile aroma substances produced by the new variety 'Ruixue' after harvest remain unclear. In this study, we utilized headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) to investigate the changes in volatile substances, fruit hardness, crispness, and related aroma synthase activity of commercially mature 'Ruixue' apples during cold storage. Our findings revealed a gradual decline in fruit firmness and brittleness of 'Ruixue' apples during cold storage, with hexyl acetate, hexyl caproate, and hexyl thiocyanate being the main hexyl esters detected. To gain a better understanding of the metabolic pathway of esters, we identified 42 MdCXE gene members that are associated with ester degradation. Through RT-qPCR analysis, we discovered that carboxylesterase MdCXE20 exhibited higher expression levels compared to other MdCXE genes during cold storage. To confirm the role of MdCXE20, we conducted a transient injection of apple fruits and observed that overexpression of MdCXE20 led to the degradation of esters such as hexyl hexanoate, butyl hexanoate, butyl 2-methylbutyrate, hexyl butyrate, and hexyl 2-methylbutyrate. The results of the study showed that the virus-induced gene silencing of MdCXE20 found the opposite results. Additionally, the esters of OE-MdCXE20 callus showed a lower content of ester VOC than the control callus, according to the homologous stable transformation of 'Wanglin' callus. Overall, these findings suggest that the MdCXE20 gene plays a crucial role in the decrease of esters in 'Ruixue' apples, which ultimately affects their flavor.

Here, we have designed and synthesized a series of melatonin-alkylbenzylamine hybrids as multitarget agents for the treatment of Alzheimer's disease (AD). Most of them exhibited a potent multifunctional profile involving cholinesterase inhibition and antioxidant effects. Among these compounds, compound 5 was most the potent antioxidant (ORAC =5.13) and also an excellent selective inhibitor of BuChE (huBuChE IC(50)=1.20 microM, huAChE IC(50) = 177.49 microM, SIs= 147.91). Moreover, kinetic study indicated compound 5 was a mixed-type inhibitor for huBuChE. Furthermore, it could induce expression of the Nrf2 as well as its downstream markers at the protein level in cells. More importantly, compound 5 display no acute toxicity in mice at doses up to 2500 mg/kg. And we found compound 5 could improve memory function of scopolamine-induced amnesia mice. These results highlighted compound 5 as a possible hit molecule for further investigation of new anti-AD drugs.

Pesticide residues have serious environmental impacts on rice-based ecosystems. In rice fields, Chironomus kiiensis and Chironomus javanus provide alternative food sources to predatory natural enemies of rice insect pests, especially when pests are low. Chlorantraniliprole is a substitute for older classes of insecticides and has been used extensively to control rice pests. To determine the ecological risks of chlorantraniliprole in rice fields, we evaluated its toxic effects on certain growth, biochemical and molecular parameters in these two chironomids. The toxicity tests were performed by exposing third-instar larvae to a range of concentrations of chlorantraniliprole. LC(50) values at 24 h, 48 h, and 10 days showed that chlorantraniliprole was more toxic to C. javanus than to C. kiiensis. Chlorantraniliprole significantly prolonged the larval growth duration, inhibited pupation and emergence, and decreased egg numbers of C. kiiensis and C. javanus at sublethal dosages (LC(10) = 1.50 mg/L and LC(25) = 3.00 mg/L for C. kiiensis; LC(10) = 0.25 mg/L and LC(25) = 0.50 mg/L for C. javanus). Sublethal exposure to chlorantraniliprole significantly decreased the activity of the detoxification enzymes carboxylesterase (CarE) and glutathione S-transferases (GSTs) in both C. kiiensis and C. javanus. Sublethal exposure to chlorantraniliprole also markedly inhibited the activity of the antioxidant enzyme peroxidase (POD) in C. kiiensis and POD and catalase (CAT) in C. javanus. Expression levels of 12 genes revealed that detoxification and antioxidant abilities were affected by sublethal exposures to chlorantraniliprole. There were significant changes in the expression levels of seven genes (CarE6, CYP9AU1, CYP6FV2, GSTo1, GSTs1, GSTd2, and POD) in C. kiiensis and ten genes (CarE6, CYP9AU1, CYP6FV2, GSTo1, GSTs1, GSTd2, GSTu1, GSTu2, CAT, and POD) in C. javanus. These results provide a comprehensive overview of the differences in chlorantraniliprole toxicity to chironomids, indicating that C. javanus is more susceptible and suitable as an indicator for ecological risk assessment in rice ecosystems.

Honeybee (Apis mellifera) ingestion of toxic nectar plants can threaten their health and survival. However, little is known about how to help honeybees mitigate the effects of toxic nectar plant poisoning. We exposed honeybees to different concentrations of Bidens pilosa flower extracts and found that B. pilosa exposure significantly reduced honeybee survival in a dose-dependent manner. By measuring changes in detoxification and antioxidant enzymes and the gut microbiome, we found that superoxide dismutase, glutathione-S-transferase and carboxylesterase activities were significantly activated with increasing concentrations of B. pilosa and that different concentrations of B. pilosa exposure changed the structure of the honeybee gut microbiome, causing a significant reduction in the abundance of Bartonella (p<0.001) and an increase in Lactobacillus. Importantly, by using Germ-Free bees, we found that colonization by the gut microbes Bartonella apis and Apilactobacillus kunkeei (original classification as Lactobacillus kunkeei) significantly increased the resistance of honeybees to B. pilosa and significantly upregulated bee-associated immune genes. These results suggest that honeybee detoxification systems possess a level of resistance to the toxic nectar plant B. pilosa and that the gut microbes B. apis and A. kunkeei may augment resistance to B. pilosa stress by improving host immunity.

There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training.

4-Methylbenzylidene camphor (4-MBC), an emerging contaminant, is a widely-used ultraviolet (UV) filter incorporated into cosmetics because it protects the skin from UV rays and counters photo-oxidation. Despite the well-established estrogenic activity of 4-MBC, the link between this activity and its effects on neurobehavior and the liver remains unknown. Thus, we exposed zebrafish larvae to environmentally relevant concentrations of 4-MBC with 1.39, 4.17, 12.5 and 15.4 microg/mL from 3 to 5 days postfertilization. We found that 4-MBC produced an estrogenic effect by intensifying fluorescence in the transgenic zebrafish, which was counteracted by co-exposure with estrogen receptor antagonist. 4-MBC-upregulated estrogen receptor alpha (eralpha) mRNA, and an interaction between 4-MBC and ERalpha suggested ERalpha's involvement in the 4-MBC-induced estrogenic activity. RNA sequencing unearthed 4-MBC-triggered responses in estrogen stimulus and lipid metabolism. Additionally, 4-MBC-induced hypoactivity and behavioral phenotypes were dependent on the estrogen receptor (ER) pathway. This may have been associated with the disruption of acetylcholinesterase and acetylcholine activities. As a result, 4-MBC increased vitellogenin expression and caused lipid accumulation in the liver of zebrafish larvae. Collectively, this is the first study to report 4-MBC-caused estrogenic effects through the brain-liver-gonad axis. It provides novel insight into how 4-MBC perturbs the brain and liver development.

Phthalate esters (PAEs) are a ubiquitous kind of environmental endocrine that disrupt chemicals, causing environmental and health issues. EstJ6 is an effective phthalate-degrading hydrolase, and its mutant with a combination of three non-conservative distal mutations has an improved activity against PAEs with unknown molecular mechanisms. Herein, we attempt to fill the significant gap between distal mutations and the activity of this enzyme using computational approaches. We found that mutations resulted in a redistribution of the enzyme's preexisting conformational states and dynamic changes of key functional regions, especially the lid over the active site. The outward motion of the lid upon the mutations made it easier for substrates or products to enter or exit. Additionally, a stronger substrate binding affinity and conformational rearrangements of catalytic reaction-associated residues in the mutant, accompanied by the strengthened communication within the protein, could synergistically contribute to the elevated catalytic efficiency. Finally, an attempt was made to improve the thermostability of EstJ6 upon introducing a distal disulfide bond between residues A23 and A29, and the simulation results were as expected. Together, our work explored the allosteric effects caused by distal mutations, which could provide insights into the rational design of esterases for industrial applications in the future.

Based on the multitarget-directed ligands (MTDLs) strategy, a series of chromone-hydroxypyridinone hybrids were designed, synthesised, and evaluated as potential multimodal anti-AD ligands. Prospective iron-chelating effects and favourable monoamine oxidase B (MAO-B) inhibitory activities were observed for most of the compounds. Pharmacological assays led to the identification of compound 17d, which exhibited favourable iron-chelating potential (pFe(3+) = 18.52) and selective hMAO-B inhibitory activity (IC(50) = 67.02 +/- 4.3 nM, SI = 11). Docking simulation showed that 17d occupied both the substrate and the entrance cavity of MAO-B, and established several key interactions with the pocket residues. Moreover, 17d was determined to cross the blood-brain barrier (BBB), and can significantly ameliorate scopolamine-induced cognitive impairment in AD mice. Despite its undesired pharmacokinetic property, 17d remains a promising multifaceted agent that is worth further investigation.

A simple, sensitive method for pesticide distinguishment based on a colorimetric sensor array using diverse gold nanoparticles (AuNPs) at room temperature is presented in this study. Acetylcholinesterase (AChE) hydrolysis ability was influenced by different pesticides and produced different concentrations of thiocholine by hydrolyzing acetylthiocholine iodide (ATCh). Thiocholine could be easily linked to the AuNPs through an Aus-sS covalent bond, and AuNPs underwent aggregation, resulting in a visible color change due to alteration of surface plasmon resonance properties. Based on these results, we successfully distinguished eight pesticides (glyphosate, thiram, imidacloprid, tribenuron methyl, nicosulfuron, thifensulfuron methyl, dichlorprop, and fenoprop) utilizing five different AuNPs by colorimetric assay. The limit of detection (LOD) of this visual method for all pesticides was less than 1.5x 10(-7) M, which was more sensitive than the U.S. Environmental Protection Agency regulations specify (1.18s-s3.91x10(-6) M). This method was further improved by combining a portable smartphone device with a color picking application using (color name AR) and RGB (red, green, blue) values. The method was successfully applied to pesticide residue distinguishment in real samples by linear discriminant analysis (LDA).

BACKGROUND: Alzheimer's disease (AD) belongs to neurodegenerative disease, and the increasing number of AD patients has placed a heavy burden on society, which needs to be addressed urgently. ChEs/MAOs dual-target inhibitor has potential to treat AD according to reports. PURPOSE: To obtain effective multi-targeted agents for the treatment of AD, a novel series of hybrid compounds were designed and synthesized by fusing the pharmacophoric features of 3,4-dihydro-2 (1H)-quinolinone and dithiocarbamate. METHODS: All compounds were evaluated for their inhibitory abilities of ChEs and MAOs. Then, further biological activities of the most promising candidate 3e were determined, including the ability to cross the blood-brain barrier (BBB), kinetics and molecular model analysis, cytotoxicity in vitro and acute toxicity studies in vivo. RESULTS: Most compounds showed potent and clear inhibition to AChE and MAOs. Among them, compound 3e was considered to be the most effective and balanced inhibitor to both AChE and MAOs (IC(50)=0.28 microM to eeAChE; IC(50)=0.34 microM to hAChE; IC(50)=2.81 microM to hMAO-B; IC(50)=0.91 microM to hMAO-A). In addition, 3e showed mixed inhibition of hAChE and competitive inhibition of hMAO-B in the enzyme kinetic studies. Further studies indicated that 3e could penetrate the BBB and showed no toxicity on PC12 cells and HT-22 cells when the concentration of 3e was lower than 12.5 microM. More importantly, 3e lacked acute toxicity in mice even at high dose (2500 mg/kg, P.O.). CONCLUSION: This work indicated that compound 3e with a six-carbon atom linker and a piperidine moiety at terminal position was a promising candidate and was worthy of further study.

Alzheimer's disease (AD) possessed intricate pathogenesis. Currently, multi-targeted drugs were considered to have the potential to against AD by simultaneously triggering molecules in functionally complementary pathways. Hence, a series of molecules based on the pharmacophoric features of Dimethyl fumarate, Tranilast, and Dithiocarbate were designed and synthesized. These compounds showed significant AChE inhibitory activity in vitro. Among them, compound 4c(2) displayed the mighty inhibitory activity to hAChE (IC(50) = 0.053 microM) and held the ability to cross the BBB. Kinetic study and molecular docking pointed out that 4c(2) bound well into the active sites of hAChE, forming steady and sturdy interactions with key residues in hAChE. Additionally, 4c(2) as an Nrf2 activator could promote the nuclear translocation of Nrf2 protein and induce the expressions of Nrf2-dependent enzymes HO-1, NQO1, and GPX4. Moreover, 4c(2) rescued BV-2 cells from H(2)O(2)-induced injury and inhibited ROS accumulation. For the anti-neuroinflammatory potential of 4c(2), we observed that 4c(2) could lower the levels of pro-inflammatory cytokines (NO, IL-6 and TNF-alpha) and suppressed the expressions of iNOS and COX-2. In particular, 4c(2) was well tolerated in mice (2500 mg/kg, p.o.) and efficaciously recovered the memory impairment in a Scopolamine-induced mouse model. Overall, these results highlighted that 4c(2) was a promising multi-targeted agent for treating AD.

BACKGROUND: Pentazocine produces a wide variety of actions in the treatment of perioperative analgesia. Neostigmine is a cholinesterase inhibitor used to antagonize the residual effects of muscle relaxants and also produces an analgesic effect. OBJECTIVES: To investigate the analgesic effects of intrathecally injected pentazocine and neostigmine and their interaction. METHODS: Sprague-Dawley rats were used to test the analgesic effect of pentazocine and neostigmine using the paw formalin pain model and the incision mechanical allodynia model. Pentazocine (3, 10, 30, and 100 microg), neostigmine (0.3, 1, 3, and 10 microg) or a pentazocine-neostigmine mixture were separately injected to evaluate their antinociceptive effects alone on the treatment groups. The corresponding control group received an intrathecal injection containing the same volume of saline. The formalin pain test, or the plantar incision pain behavior test were performed 30 minutes later. Isobolographic analysis was used to evaluate the interaction between pentazocine and neostigmine. Intrathecally administered selective mu-opioid receptor antagonist CTAP, selective kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), nonselective opioid receptor antagonist naloxone, and muscarinic acetylcholine receptor antagonist atropine were also used to test the possible interaction mechanism. These antagonists were used 30 minutes before the pentazocine and neostigmine mixtures which were intrathecally injected. RESULTS: Intrathecally administered pentazocine (3, 10, 30, and 100 microg) and neostigmine (0.3, 1, 3, and 10 microg) alone had a marked dose-related impact on suppressing the biphasic responses in the formalin test. Pentazocine (3, 10, 30, and 100 microg) and neostigmine (0.3, 1, 3, and 10 microg) alone attenuated the mechanical allodynia in a plantar incision model in a dose-dependent manner. Isobolographic analysis revealed that the mixture of intrathecal pentazocine and neostigmine synergistically decreased both phase I and II activity in the formalin test and mechanical allodynia in the plantar incision model. Pretreatment of intrathecally administered nor-BNI, naloxone, atropine, but not CTAP, antagonized the analgesic effect of the pentazocine-neostigmine mixture. CONCLUSIONS: All of these results suggest that the combined application of pentazocine and neostigmine is an effective way to relieve pain from formalin and acute incision mechanical allodynia. The synergistic effect between pentazocine and neostigmine is mostly attributed to the kappa-opioid receptor and the cholinergic receptor in the spinal cord.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase beta (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

Feruloyl esterase is a subclass of alpha/beta hydrolase, which could release ferulic acid from biomass residues for use as an efficient additive in food or pharmaceutical industries. In the present study, a feruloyl esterase with broad substrate specificity was characterised and secreted by Bacillus subtilis WB600. After codon usage optimisation and signal peptide library screening, the secretion amount of feruloyl esterase was enhanced by up to 10.2-fold in comparison with the base strain. The site-specific amino acid substitutions that facilitate protein folding further improved the secretion by about 1.5-fold. The purified rationally designed enzyme exhibited maximal activity against methyl ferulate at pH 6.5 and 65 degreesC. In the solid-state fermentation, the genetically engineered B. subtilis released about 37% of the total alkali-extractable ferulic acid in maize bran. This study provides a promising candidate for ferulic acid production and demonstrates that the secretion of a heterologous enzyme from B. subtilis can be cumulatively improved by changes in protein sequence features.

The sublethal effects of pesticide poisoning will have significant negative impacts on the foraging and learning of bees and bumblebees, so it has received widespread attention. However, little is known about the physiological effects of sublethal spinetoram and glyphosate exposure on bumblebees. We continuously exposed Bombus terrestris to sublethal (2.5 mg/L) spinetoram or glyphosate under controlled conditions for 10 days. The superoxide dismutase, glutathione-S-transferase, carboxylesterase, prophenoloxidase, alpha-amylase and protease activities, and changes in gut microbes were measured to understand the effects of sublethal pesticide exposure on the physiology and gut microbes of bumblebees. Sublethal pesticide exposure to significantly increased superoxide dismutase activity and significantly decreased gut alpha-amylase activity in bumblebees but had no significant effect on glutathione-S-transferase, carboxylesterase or gut protease activities. In addition, glyphosate increased the activity of prophenoloxidase. Interestingly, we observed that neither of the two pesticides had a significant effect on dominant gut bacteria, but glyphosate significantly altered the structure of the dominant gut fungal community, and reduced the relative abundance of Zygosaccharomyces associated with fat accumulation. These results suggest that sublethal spinetoram and glyphosate do not significantly affect the detoxification system of bumblebees, but may affect bumblebee health by inhibiting energy acquisition. Our results provide information on the sublethal effects of exposure to low concentrations of glyphosate and spinetoram on bumblebees in terms of physiology and gut microbes.

BACKGROUND: Acetylcholine (ACh), as a classical neurotransmitter, plays great roles in the nervous system. There is increasing evidence of its non-neuronal roles in regulating basic cell functions in vertebrates. However, knowledge about the non-neuronal cholinergic system in insects is scarce. RESULTS: A comparative transcriptome analysis was performed to investigate differences in the key molecular components of the cholinergic system between the head and ovary. The results showed that expression levels of most cholinergic system-related genes were higher in the head than in the ovary, and some cholinergic components were absent in the ovary. ACh contents ranged from 0.1 to 1.3 microg mg(-1) of wet weight during the development of the ovary, and weak acetylcholinesterase activity was also detected. Moreover, the ovary has a capacity for ACh synthesis. Bromoacetylcarnitine (BrACar), a specific carnitine acetyltransferase (CarAT) inhibitor, greatly inhibits ACh synthesis by 83.83% in ovary homogenates, but bromoacetylcholine (BrACh), a specific choline acetyltransferase (ChAT) inhibitor, has no effect on ACh synthesis in the ovary. These findings indicate that non-neuronal ACh in the ovary is only catalyzed by CarAT. CONCLUSION: This study reveals the existence of the non-neuronal cholinergic system in the ovary of M. separata, whose synthesis and release mechanisms are different from those of the head. These results provide novel insights into the non-neuronal cholinergic system in insects, and will be valuable in the discovery of new target genes and the future development of green pest control. 2022 Society of Chemical Industry.

Cholinesterase and monoamine oxidase are potential targets for the therapy of Alzheimer's disease. A series of novel AP2238-clorgiline hybrids as multi-target agents were designed, synthesized and investigated in vitro for their inhibition of cholinesterases and monoamine oxidases. Many compounds displayed balanced and good inhibitory activity against AChE, BuChE and MAO-B with an obvious selective inhibitory effect on MAO-B. Among them, Compound 5l showed the most balanced potency to inhibit ChEs (eeAChE: IC(50) = 4.03 +/- 0.03 microM, eqBuChE: IC(50) = 5.64 +/- 0.53 microM; hAChE: IC(50) = 8.30 +/- 0.04 microM, hBuChE: IC(50) = 1.91 +/- 0.06 microM) and hMAO-B (IC(50) = 3.29 +/- 0.09 microM). Molecular modeling and kinetic studies showed that 5l was a mixed inhibitor for both AChE and BuChE, and a competitive MAO-B inhibitor. Compound 5l exhibited no toxicity to PC12 and BV-2 cells at 12.5 microM and no acute toxicity at a dosage of 2500 mg/kg. Moreover, 5l can improve the memory function of mice with scopolamine-induced memory impairment and have an excellent ability to cross the blood-brain barrier. Overall, these findings suggested that compound 5l could be deemed as a promising, balanced multi-target drug candidate against Alzheimer's disease.

BACKGROUND: Phoxim is a widely used organophosphorus pesticide in agriculture. People are paying more and more attention to its toxicity. At present, there is no appropriate way to solve the phoxim poisoning of silkworm, which severely affected the development of sericulture. Fe(2+), Cu(2+), Rb(+) exerted their biological effects through various forms in vivo. METHODS: To evaluate the effect of Fe(2+)/Cu(2+)/Rb(+) on phoxim poisoning in silkworm, Bombyx mori were treated with fresh mulberry leaves soaked in 2.5 mg/L phoxim for 2 min with 50 mg/L FeCl(2), 150 mg/L CuCl(2), or 0.5 mg/L RbCl from 5 days of the fifth-instar silkworm. RESULTS: Fe(2+), Cu(2+), and Rb(+) pretreatments significantly inhibited the phoxim-induced reduction of survival rate and alleviated the phoxim-induced poisoning symptoms. The protective effects of Fe(2+), Cu(2+), and Rb(+) on phoxim poisoning might be due to their enhancement of superoxide dismutase (SOD), catalase (CAT), and carboxylesterase (CarE) in the hemolymph and fat body of silkworm. This enhancement might reduce reactive oxygen species (ROS) accumulation and oxidative stress (OS) caused by phoxim poisoning. Thereby it reduced the damage to silkworm tissues and cells. CONCLUSIONS: These results showed that Fe(2+), Cu(2+), and Rb(+) treatments protected the silkworm from phoxim poisoning by directly enhancing the activity of SOD, CAT, and CarE enzymes and reducing oxidative stress, but not dependent on the high expression of CYP genes. The use of Fe(2+), Cu(2+), and Rb(+) to enhance the activity of SOD, CAT, and CarE enzymes may be an underlying effective way to solve phoxim poisoning in the silkworm industry.

Liver failure has attracted attention in clinical work due to its high mortality, and the development of liver transplantation is restricted by various factors. Therefore, it is very important to carry out research on the mechanism of liver cell regeneration. This article has studied in depth the preparation of MED1 gene nanocarriers, collected human plasmids and cells through experimental materials and experimental instruments, and conducted comparative research on conventional culture. This question conducts a regeneration experiment on liver cells in chronic-onset acute liver failure, divides patients into an experimental group and a control group, and understands the recovery of liver function according to the screening of their plasma samples and separation of plasma. This article selects the commonly used clinical biological markers, such as Na+, AFP, Alb, CHE (serum cholinesterase) and other indicators to reflect the regeneration ability of liver function. The incidence of surgical complications in the control group, such as ascites, infection, bleeding, HE, hepatorenal syndrome, and hyponatremia were 71.3%, 87.4%, 16.1%, 41.4%, 19.5%, and 33.3%, respectively. Significantly higher than the experimental group, the difference was statistically significant (P < 0.05); while gender, age, PLT level and whether to use hormones, artificial liver or not there was no significant difference between the two groups (P > 0.05).

BACKGROUND AND AIMS: Myocardial infarction (MI) has been an important heart disease affecting human health. The aim of this study was to investigate the regulatory effect of abhydrolase domain containing 15 (ABHD15) on hypoxic cardiomyocytes. METHODS AND RESULTS: Hypoxic cardiomyocytes are commonly used as an vitro model for the study of MI. We found that cardiomyocyte viability was decreased under hypoxia, but cell glucose uptake, insulin receptor phosphorylation level and apoptosis were increased. Interestingly, ABHD15 expression was up-regulated in hypoxia-induced cardiomyocytes. Then, we identified the function of ABHD15 in hypoxic cardiomyocytes by using ABHD15 overexpression vector or short interfering RNA (siRNA) against ABHD15. The results showed that overexpression of ABHD15 promoted hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation (p-IR), and inhibited cell apoptosis. However, knockdown of ABHD15 attenuated hypoxic cardiomyocyte viability, glucose uptake and IR phosphorylation, and promoted apoptosis. Moreover, we found that ABHD15 promoted glucose transporter 4 (GLUT4) expression, translocation and enhance rate-limiting enzyme activation of glycolysis, thereby affecting glucose uptake. Furthermore, our study suggested that ABHD15 may affect the viability and apoptosis of hypoxic cardiomyocytes through IR/Ras/Raf/ERK/MEK and IR/PI3K/AKT/Bcl2/Bad/caspase9 signaling pathways, respectively. When the phosphorylation of IR, Raf or ERK was blocked by inhibitors, the protective effect of overexpressing ABHD15 on the viability of hypoxic cardiomyocytes was eliminated. Furthermore, inhibiting the phosphorylation of IR, AKT or Bcl2 abolished the inhibitory effect of overexpressing ABHD15 on hypoxic cardiomyocyte apoptosis. CONCLUSION: ABHD15 regulated myocardial cell viability, glycolysis, and apoptosis under hypoxia, providing a novel potential therapeutic strategy for MI.

The apoptosis of mature osteocytes is the main factor causing damage to the microstructure of cortical bone in glucocorticoid-induced osteoporosis (GIOP). Our previous research found damaged areas and empty osteocytes lacunae in the tibial cortical bone of GIOP mice. However, the specific mechanism has not been clarified. Recently, a study showed that the quality of the cortical bone significantly increased by knocking out Notum, a gene encoding alpha/beta hydrolase. However, it is not clear whether Notum affects cortical bone remodeling by participating in glucocorticoids (GCs)-induced apoptosis of osteocytes. The present study aimed to explore the correlation between Notum, osteocytes apoptosis, and cortical bone quality in GIOP. Prednisolone acetate was intragastrically administered to mice for two weeks. Histochemical staining was applied to evaluate changes in GIOP and Notum expression. Osteocytes were stimulated with prednisolone, and cell viability was assessed via CCK8. Hoechst 33342/PI staining, flow cytometry, RT-PCR, and western blot were used to detect osteocytes apoptosis, siRNA transfection efficiency, and expressions of pathway related factors. The results showed that the number of empty osteocytes lacunae increased in GIOP mice. TUNEL-stained apoptotic osteocytes and Notum immuno-positive osteocytes were also observed. Furthermore, prednisolone was found to promote Notum expression and osteocytes apoptosis in vitro. Knocking down Notum via siRNA partially restored osteocytes apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3beta (GSK3beta)/beta-catenin pathway. These findings showed GCs-induced osteocytes apoptosis by promoting Notum expression and inhibiting PI3K/AKT/GSK3beta/beta-catenin pathway. Thus, Notum might be a potential therapeutic target for the treatment of GIOP.

Chlorogenic acid (CGA) is a phenylpropanoid compound that is well known to improve the antioxidant capacity and other biological activities. However, the roles of CGA in the liver development of organisms are unclear. In the present study, we aimed to investigate the function of CGA in the hepatic development in thioacetamide (TAA)-induced zebrafish embryos. We found that CGA exerted certain beneficial effects on zebrafish larvae from TAA-exposed zebrafish embryos, such as increasing the liver size, body length, heart rate, acetylcholinesterase activity, and motor ability. In addition, CGA displayed an antioxidant effect on TAA-induced zebrafish embryos by enhancing the activities of superoxide dismutase (SOD), catalase (CAT), and glucose-6-phosphate dehydrogenase (G6PDH), and decreasing of the contents of malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO). The results of western blotting analysis showed that CGA inhibited cell apoptosis by increasing the levels of Bcl2 apoptosis regulator and decreasing the levels of Bcl2 associated X (Bax), apoptosis regulator and tumor protein P53. Moreover, CGA promoted cell proliferation in TAA-induced zebrafish larvae, as detected using proliferating cell nuclear antigen fluorescence immunostaining. In addition, CGA inhibited the expression of Wnt signaling pathway genes Dkk1 (encoding Dickkopf Wnt signaling pathway inhibitors), and promoted the expression of Lef1 (encoding lymphoid enhancer binding factor 1) and Wnt2bb (encoding wingless-type MMTV integration site family, member 2Bb). When the Wnt signal inhibitor IWR-1 was added, there was no significant change in liver development in the IWR-1 + TAA group compared with the IWR-1 + TAA + CGA group (p <0.05), which suggested that CGA regulates liver development via Wnt signaling pathway. Overall, our results suggested that CGA might alleviate TAA-induced toxicity in zebrafish and promote liver development through the Wnt signaling pathway, which provides a basis for the therapeutic effect of CGA on liver dysplasia.

Off-target drug release and insufficient drug delivery are the main obstacles for effective anticancer chemotherapy. Prodrug-based self-assembled nanoparticles bioactivated under tumor-specific conditions are one of the effective strategies to achieve on-demand drug release and effective tumor accumulation. Herein, stimuli-activable prodrugs are designed yielding smart tumor delivery by combination of the triglyceride-mimic (TG-mimetic) prodrug structure and disulfide bond. Surprisingly, these prodrugs can self-assemble into uniform nanoparticles (NPs) with a high drug loading (over 40%) and accumulate in tumor sites specifically. The super hydrophobic TG structure can act as a gate that senses lipase to selectively control over NP dissociation and affect the glutathione-triggered prodrug activation. In addition, the impacts of the double bonds in the prodrug NPs on parent drug release and the following cytotoxicity, pharmacokinetics, and antitumor efficiency are further demonstrated. Our findings highlight the promising potential of TG-mimetic structure-gated prodrug nanoparticles for tumor-specific drug delivery.

The high cost and easy denaturation of natural enzymes under environmental conditions hinder their practical usefulness in sensing devices. In this study, peroxidase (POD)-like metal-organic frameworks (MOFs) were in situ grown in the nanochannels of an anodized TiO(2) membrane (TiO(2)NM) as an electrochemical platform for multitarget sensing. By directly using a nanochannel wall as the precursor of metal nodes, Ti-MOFs were in situ derived on the nanochannel wall. Benefitting from the presence of bipyridine groups on the ligands, the MOFs in the nanochannels provide plenty of sites for Fe(3+) anchoring, thus endowing the resulting membrane (named as Fe(3+):MOFs/TiO(2)NM) with remarkable POD-like activity. Such Fe(3+)-induced POD-like activity is very sensitive to thiol-containing molecules owing to the strong coordination effect of thiols on Fe(3+). Most importantly, the POD-like activity of nanochannels can be in situ characterized by the current-potential (I-V) properties via catalyzing the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) substrate to the corresponding positively charged product ABTS(+). As a proof-of-concept application, the free-standing POD-like membranes were applied as a label-free assay in sensing cysteine, as well as monitoring acetylcholinesterase (AChE) activity through the generated thiol-containing product. Furthermore, based on the toxicity effect of organophosphorus (OP) compounds on AChE, the robust membranes were successfully utilized to evaluate the toxicity of diverse OP compounds. The POD-like nanochannels open up an innovative way to expand the application of nanochannel-based electrochemical sensing platforms in drug inspection, food safety, and environmental pollution.

In this study, a series of multifunctional hybrids against Alzheimer's disease were designed and obtained by conjugating the pharmacophores of xanthone and alkylbenzylamine through the alkyl linker. Biological activity results demonstrated that compound 4j was the most potent and balanced dual ChEs inhibitor with IC(50) values 0.85 microM and 0.59 microM for eeAChE and eqBuChE, respectively. Kinetic analysis and docking study indicated that compound 4j was a mixed-type inhibitor for both AChE and BuChE. Additionally, it exhibited good abilities to penetrate BBB, scavenge free radicals (4.6 trolox equivalent) and selectively chelate with Cu(2+) and Al(3+) at a 1:1.4 ligand/metal molar ratio. Importantly, after assessments of cytotoxic and acute toxicity, we found compound 4j could improve memory function of scopolamine-induced amnesia mice. Hence, the compound 4j can be considered as a promising lead compound for further investigation in the treatment of AD.

Dimethyl terephthalate (DMT) is a primary ingredient widely used in the manufacture of polyesters and industrial plastics; its environmental fate is of concern due to its global use. Microorganisms play key roles in the dissipation of DMT from the environment; however, the enzymes responsible for the initial transformation of DMT and the possible altered toxicity due to this biotransformation have not been extensively studied. To reduce DMT toxicity, we identified the esterase gene dmtH involved in the initial transformation of DMT from the AOPP herbicide-transforming strain Sphingobium sp. C3. DmtH shows 24-41% identity with alpha/beta-hydrolases and belongs to subfamily V of bacterial esterases. The purified recombinant DmtH was capable of transforming DMT to mono-methyl terephthalate (MMT) and potentially transforming other p-phthalic acid esters, including diallyl terephthalate (DAT) and diethyl terephthalate (DET). Using C. elegans as an assay model, we observed the severe toxicity of DMT in inducing reactive oxygen species (ROS) production, decreasing locomotion behavior, reducing lifespan, altering molecular basis for oxidative stress, and inducing mitochondrial stress. In contrast, exposure to MMT did not cause obvious toxicity, induce oxidative stress, and activate mitochondrial stress in nematodes. Our study highlights the usefulness of Sphingobium sp. C3 and its esterase DmtH in transforming p-phthalic acid esters and reducing the toxicity of DMT to organisms.

As a new protective and therapeutic fungicide, studies on famoxadone-cymoxanil are rare, and its toxicity to aquatic organisms has not been reported. In the present study, zabrafish embryos were exposed to several concentrations of famoxadone-cymoxanil at 10 hpf. Then, the changes of their shape, heart rate, development and function of innate and adaptive immune cells, oxidative stress, apoptosis, the expression of apoptosis-related genes and immune-related genes, the locomotor behavior were observed and detected in acute toxicity of famoxadone-cymoxanil. Our studies showed that, after exposure to famoxadone-cymoxanil, zebrafish embryos had decreased heart rate, shortened body length, swollen yolk sac. Secondly, the number of innate and adaptive immune cells was significantly reduced; and neutrophil migration and retention at the injury area were inhibited, indicating the developmental toxicity and immunotoxicity of famoxadone-cymoxanil on the zebrafish. We also found that the oxidative stress related indicators of embryos were changed significantly, and apoptosis were substantially increased. Further investigation of changes of some key genes in TLR signaling including TLR4, MYD88 and NF-kappaB p65 revealed that the mRNA expression of these genes was up-regulated. Meanwhile, the mRNA expression of some proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL6 and IL-1beta was also up-regulated. In addition, the activity, the total distance, time and average speed were decreased along with the increase of exposure concentration. The absolute turn angle, sinuosity and the enzymatic activity of acetylcholinesterase (AChE) were also increased. These results suggested that famoxadone-cymoxanil can induce developmental toxicity, immunotoxicity and neurobehavioral toxicity in zebrafish larvae.

Alpinia oxyphylla Miq. (i.e., A. oxyphylla), a traditional Chinese medicine, can exert neuroprotective effects in ameliorating mild cognitive impairment and improving the pathological hallmarks of Alzheimer's disease (AD). Here, 50 active compounds and 164 putative targets were collected and identified with 251 clinically tested AD-associated target proteins using network pharmacology approaches. Based on the Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway enrichments, the compound-target-pathway-disease/protein-protein interaction network constructions, and the network topological analysis, we concluded that A. oxyphylla may have neuroprotective effects by regulating neurotransmitter function, as well as brain plasticity in neuronal networks. Moreover, closely-related AD proteins, including the amyloid-beta precursor protein, the estrogen receptor 1, acetylcholinesterase, and nitric oxide synthase 2, were selected as the bottleneck nodes of network for further verification by molecular docking. Our analytical results demonstrated that terpene, as the main compound of A. oxyphylla extract, exerts neuroprotective effects, providing new insights into the development of a natural therapy for the prevention and treatment of AD.

Background: Neuropathic pain not only affects individual life quality but also increases economic burden for the society. Treatment to alleviate neuropathic pain is required. Methodology: Fifty rats were randomly assigned into sham, spinal nerve ligation, and three treatment groups with different doses of Tempol (100, 200, and 300 mg/kg, respectively), with 10 rats in each group. A neuropathic pain model was created with spinal nerve L5 and L6 ligation. Mechanical allodynia and thermal hyperalgesia were tested preoperatively (day 0) and postoperatively (days 1, 3, 5, and 7). Spinal cord levels of nitric oxide, as well as activities of nitric oxide synthase and acetylcholinesterase, were tested in postoperative day 7. Results: Compared with rats in the spinal nerve ligation group, rats in Tempol treatment groups had decreased responses to mechanical pain and cold plate stimulations. A high dose of Tempol produced more attenuating effects. The level of nitric oxide and activity of nitric oxide synthase were also decreased with Tempol treatments, whereas no significant changes were observed in the activity of acetylcholinesterase. Conclusions: Tempol attenuated an experimental rat model with neuropathic pain by inhibiting nitric oxide production.

A cosmid metagenomic library containing 1.3 x 10(5) clones was created from a soil sample. A novel gene (fae-xuan) encoding a feruloyl esterase was identified through functional screening. Primary sequence analysis showed that the gene consisted of 759 base pairs and encoded a protein of 252 amino acids. The gene was expressed in Escherichia coli BL21 (DE3) and the corresponding purified recombinant enzyme exhibited a molecular weight of 29 kDa. The FAE-Xuan showed high activity (40.0 U/mg) toward methyl ferulate with an optimal temperature and pH of 30 degreesC and 5.0, respectively. Besides methyl ferulate, FAE-Xuan can also hydrolyze methyl sinapate and methyl p-coumarate. The substrate utilization preferences and phylogenetic analysis indicated that FAE-Xuan belongs to type A FAE. FAE-Xuan was quite stable over a broad pH range from 3.0 to 10.0. The activity reduced remarkably in presence of Cu(2+). FAE-Xuan can enhance the quantity of ferulic acid from de-starched wheat bran in presence of xylanase. The work presented here highlighted the effectiveness of metagenomic strategy in identifying novel FAEs with diverse properties for potential use in industrial production.

To understand how ambient temperature affect the gypsy moth larvae, and provide a theoretical basis for pest control in different environments. Fourth instar gypsy moth larvae were incubating for 3 hr at 15, 20, 25, 30, 35, and 40, respectively. Afterward, digestive and antioxidant enzyme activities, total antioxidant capacity, and intestinal microflora community were analyzed to reveal how the caterpillars respond to ambient temperature stress. Results showed that both digestive and antioxidant enzymes were regulated by the ambient temperature. The optimum incubation temperatures of protease, amylase, trehalase, and lipase in gypsy moth larvae were 30, 25, and 20, respectively. When the incubation temperature was deviated optimum temperatures, digestive enzyme activities would be downregulated depending on the extent of temperature stress. In addition, glutathione S-transferase, peroxidase, catalase, and polyphenol oxidase would be activated under a sufferable temperature stress, but superoxide dismutase and carboxylesterase (CarE) would be inhibited. In addition, results showed that the top two abundant phyla were Proteobacteria and Firmicutes. The phylum Firmicutes abundance was decreased and phylum Proteobacteria abundance was increased by ambient temperature stress. Moreover, it suggested that gypsy moth caterpillars at different ambient temperature mainly differed from each other by Escherichia-Shigella and Bifidobacterium in control, Acinetobacter in T15, and Lactobacillus in T40, respectively.

Triclosan (TCS), a commonly used antimicrobial compound, has recently been detected in the eggs of wild avian species. Exposure to TCS in rodents is known to interfere with thyroid hormone (TH), disrupt immune responses and cause liver disease. However, no attempt has been made to clarify the effects of TCS in avian species. The aim of this study is therefore to evaluate the toxic effects of in ovo exposure to TCS and explore the molecular mechanism by transcriptome analysis in the embryonic liver of a model avian species, chicken (Gallus gallus). Embryos were treated with graded concentration of TCS (0.1, 1 and 10mug/g egg) at Hamburger Hamilton Stage (HHS) 1 (1st day), followed by 20days of incubation to HHS 46. At the administration of 10mug TCS/g egg, embryo mortality increased from 20% in control to 37% accompanied with 8% attenuation in tarsus length. While liver somatic index (LSI) in TCS treatments was enhanced, statistical difference was only observed at the treatment of 0.1mug TCS/g egg in females. The up-regulation of several crucial differentially expressed genes (DEGs) in transcriptome analysis suggested that TCS induced xenobiotic metabolism (e.g. CYP2C23a, CYP2C45 and CYP3A37 in males; CYP2C45 in females) and activated the thyroid hormone receptor (THR) - mediated downstream signaling (e.g. THRSPB and DIO2 in males; THRSPB in females). In females, TCS may further activate the lipogenesis signaling (e.g. ACSL5, ELOVL2) and repress the lipolysis signaling (e.g. ABHD5, ACAT2). A battery of enriched transcription factors in relation to these TCS-induced signaling and phenotypes were found, including activated SREBF1, PPARa, LXRa, and LXRb in males and activated GLI2 in females; COUP-TFII was predicted to be suppressed in both genders. Finally, we developed adverse outcome pathways (AOPs) that provide insights into the molecular mechanisms underlying the alteration of phenotypes.

This study mainly investigated the ameliorative effect of lotus seedpod proanthocyanidins (LSPC) and the mechanism underlying such effect on cognitive impairment and brain aging induced by d-galactose. Aging mice induced by d-galactose (150mg/kg, sc injection daily for 6weeks) were chosen for the experiment. LSPCs (30, 60, and 90mg/kg, ig) were provided after d-galactose injection. Learning and memory functions were detected by Y-maze and step-down avoidance tests. Then, some biochemical indexes related to cognitive ability and aging were measured. Histopathological feature and P53 protein expression in the hippocampus were observed. Results showed that the three different doses of LSPC could significantly ameliorate the learning and memory abilities impaired by d-galactose. LSPC significantly reduced the levels of malondialdehyde and nitric oxide (i.e. 90mg/kg LSPC group vs. model group, P=0.008), reduced the content of beta-amyloid peptide 1-42 (i.e. 90mg/kg LSPC group vs. model group, P=0.009), decreased the activities of acetylcholinesterase, monoamine oxidase B, total nitric oxide synthase (i.e. 90mg/kg LSPC group vs. model group, P=0.006), and neuronal nitric oxide synthase and synchronously increased the activities of superoxide dismutase and glutathione peroxidase in the brain. Furthermore, LSPC could prevent neuron damage and could lessen the expression of P53 protein in the hippocampus. These findings demonstrated that LSPC effectively attenuated cognitive damage and improved parameters related to brain aging in senescent mice induced by d-galactose, and may be used to treat Alzheimer's disease.

Hyperactivity is a symptom found in several neurological and psychiatric disorders, including Fragile X syndrome (FXS). The animal model of FXS, fragile X mental retardation gene (Fmr1) knockout (KO) mouse, exhibits robust locomotor hyperactivity. Alpha (alpha)-asarone, a major bioactive component isolated from Acorus gramineus, has been shown in previous studies to improve various disease conditions including central nervous system disorders. In this study, we show that treatment with alpha-asarone alleviates locomotor hyperactivity in Fmr1 KO mice. To elucidate the mechanism underlying this improvement, we evaluated the expressions of various cholinergic markers, as well as acetylcholinesterase (AChE) activity and acetylcholine (ACh) levels, in the striatum of Fmr1 KO mice. We also analyzed the AChE-inhibitory activity of alpha-asarone. Striatal samples from Fmr1 KO mice showed decreased m1 muscarinic acetylcholine receptor (m1 mAChR) expression, increased AChE activity, and reduced ACh levels. Treatment with alpha-asarone improved m1 mAChR expression and ACh levels, and attenuated the increased AChE activity. In addition, alpha-asarone dose-dependently inhibited AChE activity in vitro. These results indicate that direct inhibition of AChE activity and up-regulation of m1 mAChR expression in the striatum might contribute to the beneficial effects of alpha-asarone on locomotor hyperactivity in Fmr1 KO mice. These findings might improve understanding of the neurobiological mechanisms responsible for locomotor hyperactivity.

Long noncoding RNAs (lncRNAs) play vital roles in tumorigenesis. However, the diagnostic values of most lncRNAs are largely unknown. To investigate whether gastric juice lncRNA-ABHD11-AS1 can be a potential biomarker in the screening of gastric cancer, 173 tissue samples and 130 gastric juice from benign lesion, gastric dysplasia, gastric premalignant lesions, and gastric cancer were collected. ABHD11-AS1 levels were detected by reverse transcription-polymerase chain reaction. Then, the relationships between ABHD11-AS1 levels and clinicopathological factors of patients with gastric cancer were investigated. The results showed that ABHD11-AS1 levels in gastric cancer tissues were significantly higher than those in other tissues. Its levels in gastric juice from gastric cancer patients were not only significantly higher than those from cases of normal mucosa or minimal gastritis, atrophic gastritis, and gastric ulcers but also associated with gender, tumor size, tumor stage, Lauren type, and blood carcinoembryonic antigen (CEA) levels. More importantly, when using gastric juice ABHD11-AS1 as a marker, the positive detection rate of early gastric cancer patients was reached to 71.4 %. Thanks to the special origin of gastric juice, these results indicate that gastric juice ABHD11-AS1 may be a potential biomarker in the screening of gastric cancer.

A lot of attention has been given to novel diabetes treatment, which is used to replace injectable insulin. A novel dipeptidyl peptidase IV inhibitor (HWH-10d) for treating type 2 diabetes was previously determined to have comparable efficacy to the marketed drug (alogliptin) in ICR male mice. Therefore, a sensitive and rapid liquid chromatography-tandem mass spectrometric method was developed and validated for the further evaluation of HWH-10d in monkey. The analytes were extracted through a liquid-liquid extraction with ethyl acetate. The linear detection range for HWH-10d in monkey plasma was from 0.5 to 2000ng/mL with the lower limit of quantification of 0.5ng/mL. The relative standard deviation was measured to be less than 10.4% for determination of inter- and intra-day precisions, and the relative error was determined to be within +/-7.2% for all accuracy measurements. The simple and rapid LC-MS/MS method for HWH-10d in monkey plasma could be used for the pharmacokinetics studies of HWH-10d in monkeys. The oral bioavailability of HWH-10d in monkeys is 57.8%.

In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method.

Long noncoding RNAs (lncRNAs) play an important role in basic physiological processes, also affect tumor occurrence and development. However, there are still many unknown relationships between the lncRNA expression levels and gastric tumor process. In our study, we selected ABHD11 Antisense RNA 1 (ABHD11-AS1) as a representative lncRNAs to study the different expression levels between gastric tumor and adjacent non-tumor tissues. At the same time, we analyzed the relationship between the expression levels of ABHD11-AS1 in gastric cancer tissues and the clinicopathological features of patients with gastric cancer and evaluated the diagnostic value through the receiver operation characteristic (ROC) curve. Results show that compared with adjacent non-tumor tissues the expression level of ABHD11-AS1 in gastric cancer tissues was significantly increased (P = 0.027). The expression level was also significantly related with the differentiation (P = 0.022), Lauren histologic classification (P = 0.004) and carbohydrate antigen 19-9 (CA19-9) (P = 0.007), and the area under ROC curve was up to 0.613. Thus, ABHD11-AS1 might be a potential biomarker for diagnosis of gastric cancer.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD-hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4-RBD binding interface were important on hDPP4-RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection.

In order to observe the toxic effects of butyl benzyl phthalate (BBP) on zebrafish, the AChE and SOD activity of zebrafish exposed to different concentrations of BBP (0, 0.332, 0.665, 1.33 mg L(-1)) in a short-term (7d) test were determined. Semi-quantitative PCR was used to determine the mRNA transcript levels of the AChE and SOD gene in zebrafish brain and muscle. The results showed: AChE activity decreased with increased exposure concentration, and was significantly inhibited (p < 0.01) compared with the control group at 0.665 mg L(-1) concentration. Low BBP concentrations stimulated and high concentrations inhibited SOD activity with a concentration of 0.332 mg L(-1) resulting in a significant induction (p < 0.05) compared with the control, and 0.665 and 1.33 mg L(-1) concentrations resulting in significant inhibition (p < 0.05, p < 0.01) relative to the control group. The RT-PCR data showed a decrease in brain and muscle mRNA transcription of AChE gene with an increase in exposure concentration. The mRNA transcription of SOD in the brain was not different between the exposed groups and control group; in muscle, the mRNA transcription inhibition decreased and then increased: all differences from the control were statistically significant.

Six Gram-stain-positive, motile, filamentous and/or rod-shaped, spherical spore-forming bacteria (strains GY32(T), L31, F01, F03, F06 and F07) showing polybrominated diphenyl ether transformation were investigated to determine their taxonomic status. After spore germination, these organisms could grow more than one hundred microns long as intact single cells and then divide into rod cells and form endospores in 33 h. The cell-wall peptidoglycan of these strains was type A4alpha, the predominant menaquinone was MK-7 and the major fatty acids were iso-C(16:0), iso-C(15:0) and C(16:1)omega7C. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were detected in the polar lipid profile. Analysis of the 16S rRNA gene sequences indicated that these strains should be placed in the genus Lysinibacillus and they were most closely related to Lysinibacillus sphaericus DSM 28(T) (99% 16S rRNA gene sequence similarity). The gyrB sequence similarity and DNA-DNA relatedness between strain GY32(T) and L. sphaericus JCM 2502(T) were 81% and 52%, respectively. The G+C content of the genomic DNA of strain GY32(T) was 43.2 mol%. In addition, strain GY32(T) showed differences in nitrate reduction, starch and gelatin hydrolysis, carbon resource utilization and cell morphology. The phylogenetic distance from its closest relative measured by DNA-DNA relatedness and DNA G+C content, and its phenotypic properties demonstrated that strain GY32(T) represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus varians sp. nov. is proposed. The type strain is GY32(T) ( = NBRC 109424(T) = CGMCC 1.12212(T) = CCTCC M 2011307(T)).

Ralstonia solanacearum is an important etiological agent that can cause serious bacterial wilt in a very wide range of potential host plants, including ginger. Here, we report the complete genome sequence of R. solanacearum SD54, a race 4 biovar 4 (R4B4) strain from a diseased ginger plant in China.

Shewanella decolorationis is a valuable microorganism for degrading diverse synthetic textile dyes. Here, we present an annotated draft genome sequence of S. decolorationis S12, which contains 4,219 protein-coding genes and 86 structural RNAs. This information regarding the genetic basis of this bacterium can greatly advance our understanding of the physiology of this species.

Digestive and detoxification enzyme activity and nutrient composition were examined in the body of fourth instar beet armyworms, Spodoptera exigua (Hubner), fed on transgenic Bacillus thuringiensis (Bt) and non-Bt cotton for different time periods. Nutrient composition and specific enzyme activities differed significantly between the S. exigua fed Bt vs. non-Bt cotton. At 1, 6 and 24 h, free fatty acid and glucose levels were significantly lower in S. exigua fed on Bt cotton than those fed on non-Bt cotton. S. exigua fed on Bt cotton had significantly higher trypsin and total superoxide dismutase (T-SOD) activities and significantly lower lipase, carboxylesterase and acetylcholinesterase activities than non-Bt fed worms for all feeding time periods. Differences were also observed among feeding times within each cotton variety group. Significantly lower free fatty acid and total amino acid were observed in S. exigua fed on Bt cotton for 24 h than in those fed for 1 h. Significantly lower activities of lipase and trypsin were detected in S. exigua fed on Bt cotton for 24 h than those for 1 and 4 h. However, carboxylesterase and acetylcholinesterase activities in S. exigua fed on Bt cotton for 24 h were significantly higher than those for 1, 4 and 6 h. The interaction between cotton variety and feeding time significantly affected the activities of lipase, trypsin, acetylcholinesterase and T-SOD enzymes in S. exigua. Measuring the temporal allocation of protection and detoxification enzyme activities in the body of S. exigua in response to B. thuringiensis can provide a meaningful evaluation on the metabolic tolerance of herbivorous insects under the continuous selection pressure of a toxic protein.

With citrate-coated Au nanoparticles as colorimetric probe, a novel visual method for rapid assay of organophosphorus pesticides has been developed. The assay principle is based on catalytic hydrolysis of acetylthiocholine into thiocholine by acetylcholinesterase, which induces the aggregation of Au nanoparticles and the color change from claret-red to purple or even grey. The original plasmon absorption of Au nanoparticles at 522 nm decreases, and simultaneously, a new absorption band appears at 675 nm. The irreversible inhibition of organophosphorus pesticides on acetylcholinesterase prevents aggregation of Au nanoparticles. Under optimum conditions, the absorbance at 522 nm of Au nanoparticles is related linearly to the concentration of mathamidophos in the range of 0.02-1.42 mug/mL with a detection limit of 1.40 ng/mL. This colorimetric method has been successfully utilized to detect mathamidophos in vegetables with satisfactory results. The proposed colorimetric assay exhibits good reproducibility and accuracy, providing a simple and rapid method for the analysis of organophosphorus pesticides.

The complete gene (PG37 lipI) encoding an alkaline lipase (PG37 LipI) was cloned from the genomic DNA of Penicillium cyclopium PG37. The cloned PG37 lipI is 2020 bp in length, consisting of 632 bp of the 5' flanking promoter region and 1388 bp of the downstream fragment that contains 6 exons and 5 short introns. The promoter region harbors putative TATA box, CAAT box and several transcription factor binding sites. The open reading frame (ORF) encodes a PG37 LipI of 285 amino acid residues, which was predicted to contain a 20-aa signal peptide, a 7-aa propeptide and a 258-aa mature peptide with a conserved motif Gly-X-Ser-X-Gly. However, PG37 LipI shows only 32%, 30%, 28% and 26% identity with lipases of Aspergillus parasiticus, Penicillium camembertii, Thermomyces lanuginosus and Rhizomucor miehei, respectively. It was predicted that the main secondary structures of PG37 LipI are alpha-helix and random coil. Three amino acid residues, Ser132-Asp188-His241, compose the enzymatic active center in the tertiary structure.

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of alpha/beta hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the alpha/beta fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis.

BACKGROUND: It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined. METHODOLOGY/PRINCIPAL FINDINGS: We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant beta-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0-10.0 and at temperatures <= 40 degrees C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C(2)-C(8)), and its suitable substrate was p-nitrophenyl caproate (C(6)) with optimal catalytic conditions of 39 degrees C and pH 8.0. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.

The Rv0045c protein is predicted to be an esterase that is involved in lipid metabolism in Mycobacterium tuberculosis. The protein was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The Rv0045c protein crystals diffracted to a resolution of 2.7A using a synchrotron-radiation source and belonged to space group P3(1) or P3(2), with unit-cell parameters a=b=73.465, c=48.064A, alpha=beta=90, =120deg. Purified SeMet-labelled Rv0045c protein was also crystallized and formed crystals that diffracted to a resolution of 3.0A using an in-house X-ray radiation source.

Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology. Here we report the draft genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next-generation Illumina GA sequencing technologies to obtain 72.2-fold genome coverage. The absence of recent whole-genome duplication, along with the presence of few tandem duplications, explains the small number of genes in the cucumber. Our study establishes that five of the cucumber's seven chromosomes arose from fusions of ten ancestral chromosomes after divergence from Cucumis melo. The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and 'fresh green' odor. We also identify 686 gene clusters related to phloem function. The cucumber genome provides a valuable resource for developing elite cultivars and for studying the evolution and function of the plant vascular system.

The nutrient composition and enzyme activities in larvae of the beet armyworm, Spodoptera exigua (Hubner), fed on high, medium or low gossypol cotton cultivars were examined at different time intervals. Significantly lower free fatty acid was observed in larvae fed for 6 h on high gossypol 'M9101' compared to larvae fed on the low (ZMS13) and intermediate (HZ401) gossypol cultivars. Significantly higher trypsin activity was observed in larvae fed on high gossypol 'M9101' for 24 h compared to those fed for 1, 4 and 6 h. Significantly higher catalase and total superoxide dismutase enzyme activities were observed in larvae of S. exigua fed on high gossypol 'M9101' compared with low gossypol cultivars 'ZMS13' and 'HZ401' for 1, 4, 6 and 24 h. However, significantly lower carboxylesterase and acetylcholinesterase enzyme activities were found in larvae fed on high gossypol 'M9101' compared with the other cultivars for 1, 4, 6 and 24 h. The interaction between cotton variety and beet armyworm infestation time significantly affected the carboxylesterase enzyme activity in S. exigua. The characterization of the effects of plant allelochemicals on herbivorous larvae is important for aiding understanding of plant-insect interaction as well as in devising solutions to pest problems by breeding plant resistance, identifying metabolic targets for insecticide development, etc.

Adipose triglyceride lipase (ATGL) was recently identified and described as a major novel triglyceride lipase in animals. In this study, we aimed to study the tissue-specific and developmental expression pattern of porcine ATGL (pATGL) and the effect of resveratrol (RES) on expression of pATGL in vitro. The full-length cDNA sequence of pATGL was 1,958 bp (accession no. EF583921), with a 1,458-bp open reading frame encoding a 486-AA protein (the predicted molecular mass of 53.2 kDa, accession no. ABS58651). Comparison of the deduced AA sequence with the bovine, mouse, rat, dog, and human adipose triglyceride lipase showed 87, 84, 83, 81, and 80% similarity, respectively. Furthermore, the pATGL was highly expressed in porcine adipose tissue, to a lesser degree in kidney, heart, and muscle, and least but detectable in brain. In s.c. adipose tissue, pATGL mRNA was low at birth (1 kg of BW) and then increased, reaching a maximal value at 20 kg of BW (approximately 8 wk old; P < 0.01). In peritoneal and omental adipose tissue, the greatest expression of pATGL was observed at 40 kg of BW (approximately 12 wk old). In vitro, exposure of cultured adipocytes to 40 and 80 muM RES for 24 h increased the mRNA levels of pATGL by 95.3% (P < 0.05) and 146.8% (P < 0.01), respectively. Accordingly, lipid accumulation was decreased by 25.7% (P < 0.05) and 60.8% (P < 0.01), respectively. When treated with RES for 48 h, the mRNA levels of pATGL were increased by 104.1% (P < 0.05) and 163.1% (P < 0.01), respectively. As expected, lipid accumulation was decreased by 9.7% (P > 0.05) and 29.0% (P < 0.05), respectively. These results add to our understanding of the role of pATGL in adipose tissue development and as a potential target for regulating fat deposition and meat quality.

Severe hypertriglyceridemia (HTG) is a metabolic disturbance often seen in clinical practice. It is known to induce life-threatening acute pancreatitis, but its role in atherogenesis remains elusive. Hemorheological abnormality was thought to play an important role in pathogenesis of both pancreatitis and atherosclerosis. However, hemorheology in severe HTG was not well investigated. Recently, we established a severe HTG mouse model deficient in lipoprotein lipase (LPL) in which severe HTG was observed to cause a significant increase in plasma viscosity. Disturbances of erythrocytes were also documented, including decreased deformability, electrophoresis rate, and membrane fluidity, and increased osmotic fragility. Scanning electron microscopy demonstrated that most erythrocytes of LPL deficient mice deformed with protrusions, irregular appearances or indistinct concaves. Analysis of erythrocyte membrane lipids showed decreased cholesterol (Ch) and phospholipid (PL) contents but unaltered Ch/PL ratio. The changes of membrane lipids may be partially responsible for the hemorheological and morphologic abnormalities of erythrocytes. This study indicated that severe HTG could lead to significant impairment of hemorheology and this model may be useful in delineating the role of severe HTG in the pathogenesis of hyperlipidemic pancreatitis and atherosclerosis.

A new analytical method for gas chromatography (GC) or GC-mass spectrometry (MS) using the direct sampling technique is described. This direct sampling technique, which bypasses the conventional complicated sample pretreatment process, is applicable to cases of fast detection of pesticide residues in foods and large-scale screening of samples by portable GC in field detection. By a direct sampling technique, the vegetable sample is ground into paste, and 30 mg is placed directly into the evaporating chamber for GC-MS identification and quantitation (by full-scan mode). The GC column used is an HP-5 (30.0-m x 250-microm x 0.25-microm, 5% phenyl methyl siloxane). Chlorpyrifos, bromophos, fenpropathrin, gamma-666, and pp'-DDT are chosen to represent organophosphorus, pyrethrins, and organochlorine pesticides because they are chief objects of the detection of pesticide residues in vegetables. Rape, a common and mass-consumed vegetable in China, is chosen as the sample in this study. The detection limits for these pesticides by the full-scan mode are all below the maximum pesticide residue limit of vegetables set by the Ministry of Agriculture of China, and the reproducibility of this method is acceptable. This analysis method is proven to be simple, quick, and reliable and is suitable for multipesticide residues analysis of vegetables. It can also be used in the analysis of vegetable components and signal chemicals.

Acetylcholinesterase (AChE) is an interesting research target not only because of its high enzyme catalytic rate but also because of the wide range of health effects resulting from its inhibition. This paper discusses results of a theoretical study of acetylcholinesterase inhibition using several simulation techniques. In the first technique, a novel method was developed and used for predicting the binding affinity of human AChE (huAChE) inhibitors. Results are also presented for classical molecular dynamics and quantum mechanical simulations. Theoretical proton NMR shift results are obtained and compared to experiment, and the importance of the Glu199 residue is discussed in the context of the model.

Mechanisms that coordinate cell growth with division are thought to determine the timing of initiation of cell division and to limit overall cell proliferation. To identify genes involved in this process in Saccharomyces cerevisiae, we describe a method that does not rely on cell size alterations or resistance to pheromone. Instead, our approach was based on the cell surface deposition of the Flo1p protein in cells having passed START. We found that over-expression of HXT11 (which encodes a plasma membrane transporter), PPE1 (coding for a protein methyl esterase), or SIK1 (which encodes a protein involved in rRNA processing) shortened the duration of the G1 phase of the cell cycle, prior to the initiation of DNA replication. In addition, we found that, although SIK1 was not part of a mitotic checkpoint, SIK1 over-expression caused spindle orientation defects and sensitized G2/M checkpoint mutant cells. Thus, unlike HXT11 and PPE1, SIK1 over-expression is also associated with mitotic functions. Overall, we used a novel enrichment approach and identified genes that were not previously associated with cell cycle progression. This approach can be extended to other organisms.

Acetylcholinesterase (AChE) inhibition is an important research topic because of its wide range of associated health implications. A receptor-specific scoring function was developed herein for predicting binding affinities for human AChE (huAChE) inhibitors. This method entails a statistically trained weighted sum of electrostatic and van der Waals (VDW) interactions between ligands and the receptor residues. Within the 53 ligand training set, a strong correlation was found (R2 = 0.89) between computed and experimental inhibition constants. Leave-one-out cross-validation indicated high predictive power (Q2 = 0.72), and analysis of a separate 16-compound test set also produced very good correlation with experiment (R2 = 0.69). Scoring function analysis has permitted identification and characterization of important ligand-receptor interactions, producing a list of those residues making the most important electrostatic and VDW contributions within the main active site, gorge area, acyl binding pocket, and periferal site. These analyses are consistent with X-ray crystallographic and site-directed mutagenesis studies.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.