Title: In-situ growth of SnO(2) nanoparticles on Nb(2)CT(x) nanosheets as highly sensitive electrochemical sensing platform for organophosphorus pesticide detection Guo W, Liang L, Zhao Y, Zhao C, Lu X, Cao Y, Gao F Ref: Colloids Surf B Biointerfaces, 224:113238, 2023 : PubMed
In this study, the SnO(2)/Nb(2)CT(x) MXene nanocomposite containing 0D/2D interfaces was prepared by situ growth strategy of one-step hydrothermal method. A SnO(2)/Nb(2)CT(x) MXene based acetylcholinesterase (AChE) biosensor was constructed for pesticide detection. Highly conductive Nb(2)CT(x) MXene, acting as substrate material, restrained the agglomeration of nanoparticles (NPs) and accelerated electron migration due to the confinement effect and well-known accordion-like layered structure. In addition, SnO(2) anchored on both sides of the Nb(2)CT(x) MXene nanosheets effectively provided a large surface area, abundant surface groups and active sites, which preserved numbers of electrons at the interface of the heterojunction. The SnO(2)/Nb(2)CT(x) MXene hybrids with outstanding conductivity, good biocompatibility and structural stability were beneficial for AChE immobilization. Under the optimized conditions, as-fabricated electrochemical biosensor demonstrated superior performance with linear detection range of 5.1 x 10(-14) - 5.1 x 10(-7) M for chlorpyrifos, along with the limit of detection (LOD) down to 5.1 x 10(-14) M (calculated for 10% inhibition). Furthermore, it is highly expected that this biosensor can be applied for the detection of other organophosphorus pesticides in the environment, providing an effective nanoplatform in biosensing field.
AIM: The objective of this study is to examine the correlation between patient serum cholinesterase (SCHE) concentration and weaning failure in the context of invasive mechanical ventilation (IMV), as well as to identify predictors of ventilator weaning failure. Additionally, this study investigates the potential relationship between SCHE and nutritional risk for developing more effective weaning strategies. METHOD: A retrospective observational study was conducted. The sample was collected from 227 patients with IMV over 48h who underwent SBT before weaning. Relevant experimental samples and data collection were analyzed at the time of patient admission and before the initiation of the SBT. The correlation between SCHE and weaning failure was determined by multifactorial logistic regression and propensity matching scores. RESULTS: Weaning was successful in 127 patients and failed in 100 patients. Depending on the difficulty of weaning, 55 of these patients had difficulty in weaning and 45 had long-term weaning. In the crude cohort, experimental data collected on the day of SBT showed that SCHE concentrations were higher in patients with successful weaning than in those with failed weaning (4,514 u/l vs. 3,190 u/l p<0.01). The critical value for predicting weaning failure was SCHE 3,228 u/l (p<0.01). Ventilator weaning failure was predicted by multifactorial logistic regression analysis of SCHE, heart rate, and PaO(2) before SBT, with SCHE predicting ventilator weaning failure (AUC 0.714; 95% CI 0.647-0.782) better than heart rate (AUC 0.618; 95% CI 0.545-0.690), PaO(2) (AUC 0.59; 95% CI 0.515-0.664). After propensity-matched scores, SCHE remained an independent predictor of weaning failure (p=0.05). And the SCHE concentration was strongly correlated with the patient's weaning difficulties (p<0.01). The Nutrition Risk in Critically Ill (NUTRIC) score was also significantly correlated with SCHE according to Spearman's correlation analysis (p<0.01). CONCLUSION: Our study revealed that the patients who experienced weaning failure exhibited lower SCHE values compared to those who successfully underwent weaning. Before spontaneous breathing trial (SBT), SCHE, heart rate, and PaO(2) were identified as independent predictors of weaning failure. Following propensity score matching (PSM), SCHE and heart rate remained independent predictors. Patients with SCHE levels below 3,228 u/l should undergo careful evaluation before weaning. Our findings suggest that malnutrition may be a contributing factor to weaning failure in patients.
Title: Synthesis, Bioactivity and Molecular Docking of Nereistoxin Derivatives Containing Phosphonate Yan Q, Lu X, Zhang Z, Jin Q, Gao R, Li L, Wang H Ref: Molecules, 28:, 2023 : PubMed
Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via (31)P, (1)H and (13)C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) against Mythimna separata Walker, Myzus persicae Sulzer and Rhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound 7f displayed good activity against all three insect species, showing LC(50) values of 136.86 microg/mL for M. separata, 138.37 microg/mL for M. persicae and 131.64 microg/mL for R. padi. Compound 7b had the highest activity against M. persicae and R. padi, with LC(50) values of 42.93 microg/mL and 58.19 microg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.
Title: Inside Out Computational Redesign of Cavities for Improving Thermostability and Catalytic Activity of Rhizomucor Miehei Lipase Zhang Z, Long M, Zheng N, Lu X, Zhu C, Osire T, Xia X Ref: Applied Environmental Microbiology, :e0217222, 2023 : PubMed
Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (T(m)) of 8 mutants showed an increase of more than 2 degreesC. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The T(m) of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0 degreesC, together with a 28.7-fold increase in the half-life at 65 degreesC and a specific activity increase of 9.9-fold (up to 5,828 U mg(-1)), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.
Title: Diketopyrrolopyrrole-based fluorescent probe for visualizing over-expressed carboxylesterase in fever via ratiometric imaging Zhang B, Qin S, Wang N, Lu X, Jiao J, Zhang J, Zhao W Ref: Talanta, 266:124971, 2023 : PubMed
Fever is the result of inflammation and the innate self-defense response of organisms, can cause abnormal changes in the activity of many enzymes in organisms, including the important carboxylesterase (CE). Monitoring the activity changes of CE in vivo during a fever will help to understand heat-related pathological mechanisms. In this paper, we designed diketopyrrolopyrrole-based ratiometric fluorescent probes DPP-FBC-P and DPP-FBO-P containing alkyl chain and diethylene glycol monomethyl ether chain respective for detection of CE. Both probes could realized fast response to CE and displayed good selectivity and high sensitivity. Compared with DPP-FBO-P, DPP-FBC-P had better biocompatibility, larger signal to noise ratio (225-fold vs 125-fold) and lower detection limit (1.6 x 10(-5) U/mL vs 4.2 x 10(-5) U/mL). Moreover, the probe DPP-FBC-P had been successfully applied to image the endogenous CE in HepG2 cells and solid tumors, and also visualized the over expressed CE in fever cells. Most importantly, the changes of CE level in the liver of fever mice model induced by LPS were monitored with the assistance of DPP-FBC-Pvia dual channel ratio imaging for the first time. In addition, fluorescence color signal in solution was captured by smart phone, and the linear relationship between RGB ratio (G/R) and CE concentration was established. This work will provide a potential approach for investigating the physiological and pathological processes of heat related diseases.
Title: The porous hollow cobalt-based oxides encapsulated with bimetallic PdAu Nanoparticles of electrochemical biosensor for highly sensitive pesticides detection Zhao Y, Liang L, Guo W, Lu X, Zhao C, Gao F Ref: Nanotechnology, :, 2023 : PubMed
Efficient and portable electrochemical biosensors are received to evaluation of pesticides in the environment, which can make great significance for food safety. In this study, the Co-based oxides with a kind of hierarchical porous hollow and nanocages were constructed, in which the materials (Co3O4-NC) were encapsulated with PdAu nanoparticles (NPs). Due to the unique porous structure, the changeable valence state of cobalt and the synergistic effect of bimetallic PdAuNPs, PdAu@Co3O4-NC possessed excellent electron pathways, and showed more exposed active sites. Accordingly, the porous Co-based oxides have been applied to construct an acetylcholinesterase (AChE) electrochemical biosensor, which showed good performance for organophosphorus pesticides (OPs) detection. The optimum biosensing platform based on nanocomposites was applied to exhibit highly sensitive determination of omethoate and chlorpyrifos, with the relative low detection limit of 6.125 x 10-15 M and 5.10 x 10-13 M, respectively. And a wide detection range of 6.125 x 10-15 ~ 6.125 x 10-6 M and 5.10 x 10-13 ~ 5.10 x 10-6 M for these two pesticides were achieved. Therefore, the PdAu@Co3O4-NC may represent a powerful tool for ultrasensitive sensing of OPs, and have great potential application.
Title: Promising candidates from drug clinical trials: Implications for clinical treatment of Alzheimer's disease in China Cao Y, Yu F, Lyu Y, Lu X Ref: Front Neurol, 13:1034243, 2022 : PubMed
Alzheimer's disease is the most common neurodegenerative disease. Prior to 2017, National Medical Products Administration approved only four drugs to treat Alzheimer's disease, including three cholinesterase inhibitors and one N-methyl-D-aspartate receptor antagonist. We queried ClinicalTrials.gov to better understand Alzheimer's drug development over the past 5 years and found 16 promising candidates that have entered late-stage trials and analyzed their impact on clinical treatment of Alzheimer's disease in China. The 16 compounds selected include disease-modifying therapies and symptomatic therapies. The research and development pipeline now focuses on disease-modifying therapies such as gantenerumab, aducanumab, ALZ-801, ALZT-OP1, donanemab, lecanemab, simufilam, NE3107, semaglutide, and GV-971, which could put an end to the situation where Alzheimer's patients in China have no effective treatment alternatives. The reuse of drugs or combinations currently under investigation for the psychiatric treatment of Alzheimer's disease, including AXS-05, AVP-786, nabilone, brexpiprazole, methylphenidate, and pimavanserin, could provide physicians with additional treatment options. Although most of these drugs have not been explored in China yet, due to the current development trend in this field in China, it is expected that China will be involved in research on these drugs in the future.
Herein, we report a series of selective sub-nanomolar inhibitors against butyrylcholinesterase (BChE). These compounds, bearing a novel N-benzyl benzamide scaffold, inhibited BChE with IC(50) from picomolar to nanomolar. The inhibitory activity was confirmed by the surface plasmon resonance assay, showing a sub-nanomolar K(D) value, which revealed that the compounds exert the inhibitory effect through directly binding to BChE. Several compounds showed neuroprotective effects verified by the oxidative damage model. Furthermore, the safety of S11-1014 and S11-1033 was demonstrated by the in vivo acute toxicity test. In the behavior study, 0.5 mg/kg S11-1014 or S11-1033 exhibited a marked therapeutic effect, which was almost equal to the treatment with 1 mg/kg rivastigmine, against the cognitive impairment induced by Abeta(1-42). The pharmacokinetics studies characterized the metabolic stability of S11-1014. Thus, N-benzyl benzamide inhibitors are promising compounds with drug-like properties for improving cognitive dysfunction, providing a potential strategy for the treatment of Alzheimer's disease.
Title: Design, synthesis, and biological evaluation of aromatic tertiary amine derivatives as selective butyrylcholinesterase inhibitors for the treatment of Alzheimer's disease Lu X, Qin N, Liu Y, Du C, Feng F, Liu W, Chen Y, Sun H Ref: Eur Journal of Medicinal Chemistry, 243:114729, 2022 : PubMed
Butyrylcholinesterase (BChE) is recently regarded as a biomarker in progressed Alzheimer's disease (AD), the development of selective BChE inhibitors has attracted a great deal of interest and may be a viable therapeutic strategy for AD. Previously, an aromatic tertiary amine derivative (S17-1001) was screened and validated as a selective BChE inhibitor. Structured-based molecular modification guided the synthesis of 43 analogs. Biological test of cholinesterase inhibition, in vitro blood brain barrier permeation assay, neurotoxicity assay and neuroprotective effects assay indicated two optimal compounds 17c and 19c. Both compounds showed selective BChE inhibitory (hBChE < 20 nM, eeAChE > 10 microM), good BBB permeation and primary cell safety. Besides, 17c can dose-response protect cell from Abeta(1-42) induced damage. It also demonstrated that 17c and 19c were able to restore cognitive impairment in vivo test. These data suggest that 17c and 19c represent promising candidate for follow-up in the drug-discovery process against AD.
BACKGROUND: Carboxylesterase (CXE) is a type of hydrolase with alpha/beta sheet hydrolase activity widely found in animals, plants and microorganisms, which plays an important role in plant growth, development and resistance to stress. RESULTS: A total of 72, 74, 39, 38 CXE genes were identified in Gossypium barbadense, Gossypium hirsutum, Gossypium raimondii and Gossypium arboreum, respectively. The gene structure and expression pattern were analyzed. The GBCXE genes were divided into 6 subgroups, and the chromosome distribution of members of the family were mapped. Analysis of promoter cis-acting elements showed that most GBCXE genes contain cis-elements related to plant hormones (GA, IAA) or abiotic stress. These 6 genes we screened out were expressed in the root, stem and leaf tissues. Combined with the heat map, GBCXE49 gene was selected for subcellular locate and confirmed that the protein was expressed in the cytoplasm. CONCLUSIONS: The collinearity analysis of the CXE genes of the four cotton species in this family indicated that tandem replication played an indispensable role in the evolution of the CXE gene family. The expression patterns of GBCXE gene under different stress treatments indicated that GBCXE gene may significantly participate in the response to salt and alkaline stress through different mechanisms. Through the virus-induced gene silencing technology (VIGS), it was speculated that GBCXE49 gene was involved in the response to alkaline stress in G. barbadense.
Title: Retrospective detection for V-type OPNAs exposure via phosphonylation and disulfide adducts in albumin Wang J, Sun F, Lu X, Gao R, Pei C, Wang H Ref: Sci Rep, 12:10979, 2022 : PubMed
Organophosphorus nerve agents (OPNAs) that damage the central nervous system by inhibiting acetylcholinesterase activity, pose severe threats to human health and life security. Reliable biomarkers that quickly and accurately detect OPNAs exposure are urgently needed to help diagnose quickly and treat in time. Albumins that covalently bind to OPNAs could serve as important targets for retrospective verification of OPNAs exposure. The goal of this study is to explore the potential biomarkers in albumins with high reactivity and good stability and expand the group of potential biomarkers in different species for detecting the exposure of V-type OPNAs including O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). Taking human serum albumin (HSA), bovine serum albumin (BSA) and rabbit serum albumin (RSA) as the research objectives, multiple active sites including phosphonylation and disulfide adduct sites were observed in albumins from different species. Numerous phosphonylation sites labeled by all agents in one type of albumin were found. Among the different species, four shared phosphonylation sites with high reactivity include K499, K549, K249, and Y108. In addition, Y108 on ETY*GEMADCCAK, Y287 on Y*ICENQDSISSK, Y377 on TY*ETTLEK and Y164 on YLY*EIAR in HSA were stably phosphonylated by all agents in gradient concentration, making them stable and suitable potential biomarkers for V-type OPNAs exposure. Notably, Y108 on ETY*GEMADCCAK in HSA, on DTY*GDVADCCEK in RSA, and on ETY*GDMADCCEK in BSA were highly reactive to all V-type agents, regardless of species. It was also successfully labeled in HSA exposed to class V agents in gradient concentration. Y108 is expected to be used to screen and identify the exposure of V-type agents in the retrospective research. Disulfide adducts sites, consisted of four sites in HSA and two sites in BSA were also successfully labeled by V-type agents, and characteristic ion fragments from these disulfide adducts were also identified by secondary mass spectrometry. Molecular simulation of the stably modified sites were conducted to discover the promoting factors of covalent adduct formation, which help further clarify formation mechanism of albumin adducts at active sites.
Title: A stable enzyme sensor via embedding enzymes into zeolitic imidazolate frameworks for pesticide determination Zhao Y, Lu X, Gao F Ref: Analytical Biochemistry, :114628, 2022 : PubMed
The stability of biosensors is of significant importance for practical applications, and the natural biomineralization processes in living organisms have inspired us from a new perspective. In this work, acetylcholinesterase (AChE) was embedded into zeolitic imidazolate framework-8 carriers (with negligible cytotoxicity) via biomimetic mineralization, being demonstrated to be a stable strategy for enzyme immobilization. When further coupled with the conductive and catalytic Au nanoparticles, the biocomposites were explored as electrochemical pesticide detection biosensor, which showed favorable analytical performance, and improved stability comparing with other biosensors. This work provides a new strategy for the reasonable design of stable biosensors for different analytes monitoring.
Endothelial lipase (LIPG/EL) performs fundamental and vital roles in the human body, including cell composition, cytokine expression, and energy provision. Since LIPG predominantly functions as a phospholipase as well as presents low levels of triglyceride lipase activity, it plays an essential role in lipoprotein metabolism, and involves in the metabolic syndromes such as inflammatory response and atherosclerosis. Cytokines significantly affect LIPG expression in endothelial cells in many diseases. Recently, it is suggested that LIPG contributes to cancer initiation and progression, and LIPG attached increasing importance to its potential for future targeted therapy.
Background: Alzheimer's disease is a multifactorial neurological disorder seen in elderly people. Loss of cholinergic transmission and unbalanced tryptophan metabolism kynurenine pathway have been demonstrated in neuropsychiatric diseases. Methods & results: Among the two series of synthesized compounds, compounds 5c and 5h were identified as effective dual BChE/IDO1 inhibitors, with well-balanced micromolar activity. Compounds 5c and 5h exhibited promising ability to ameliorate behavioral impairment by Morris water maze. The safety of miconazole analogs was also validated by PC12 and SH-SY5Y cell lines. Conclusion: These results highlight the ability of 5c and 5h to treat Alzheimer's disease.
Title: Nitrogen-Doped Graphdiyne as a Robust Electrochemical Biosensing Platform for Ultrasensitive Detection of Environmental Pollutants Niu K, Gao J, Wu L, Lu X, Chen J Ref: Analytical Chemistry, :, 2021 : PubMed
Owing to its unique chemical structure, natural pores, high structure defects, good surface hydrophilicity and biocompatibility, and favorable electrical conductivity, nitrogen-doped graphdiyne (NGDY) has been attracting attention in the application of electrochemical sensing. Taking advantage of these fascinating electrochemical properties, for the first time, two types of electrochemical enzymatic biosensors were fabricated for the respective detection of organophosphorus pesticides (OPs) and phenols based on the immobilization of acetylcholinesterase or tyrosinase with NGDY. Results revealed that the sensitivities of the NGDY-based enzymatic biosensors were almost twice higher than that of the matching biosensor in the absence of NGDY, proving that NGDY plays a vital role in immobilizing the enzymes and improving the performance of the fabricated biosensors. The effects of nitrogen doping on improving the biosensing performance were studied in depth. Graphitic N atoms can enhance the electrical conductivity, while imine N and pyridinic N can help to adsorb and accumulate the substance molecules to the electrode surface, all of which contribute to the significantly improved performance. Furthermore, these two types of biosensors also demonstrated excellent reproducibility, high stability, and good recovery rate in real environmental samples, which showed a valuable way for the rapid detection of OPs and phenols in the environment. With these excellent performances, it is strongly anticipated that NGDY has tremendous potential to be applied to many other biomedical and environmental fields.
Title: Identification of S419 on human serum albumin as a novel biomarker for sarin and cyclosarin exposure Fu F, Liu H, Lu X, Zhang R, Li L, Gao R, Xie J, Wang H, Pei C Ref: Rapid Commun Mass Spectrom, :e8721, 2020 : PubMed
RATIONALE: Organophosphorus nerve agents are highly toxic because they inhibit acetylcholinesterase activity, thereby causing a series of symptomatic poisoning. Upon entering the body, nerve agents bind active amino acid residues to form phosphonylated adducts. A potentially beneficial method for specific verification of exposure of nerve agents is based on albumin adducts, which have a half-life of 18 days. This appears to be more effective than the fluoride reactivation method, based on acetylcholinesterase. METHODS: After the exposure of human serum albumin to nine nerve agents, human serum albumin was denatured, reduced, alkylated and digested with trypsin according to standard mass spectrometry-based proteomics procedures. The phosphonylated peptides of human serum albumin were identified using positive ion electrospray ionization with a quadrupole orbitrap mass spectrometer. RESULTS: The peptide KVPQVSTPTLVESR showed a good mass spectrometric response to the nine nerve agents. The tendency of sarin and cyclosarin was to bind to S419 on the peptide, while the other nerve agents (tabun, soman, and V-type nerve agents) were shown to bind more readily to K414 on the peptide. CONCLUSIONS: This research revealed the new site, S419, of the tryptic peptide KVPQVSTPTLVEVSR on human albumin to be a valuable biomarker for sarin/cyclosarin exposure, helping to further distinguish sarin and cyclosarin poisoning from nerve agents and providing an important tool for identification of sarin or cyclosarin in terrorist attacks.
Title: Collaborative Biosynthesis of a Class of Bioactive Azaphilones by Two Separate Gene Clusters Containing Four PKS/NRPSs with Transcriptional Crosstalk in Fungi Huang X, Zhang W, Tang S, Wei S, Lu X Ref: Angew Chem Int Ed Engl, 59:4349, 2020 : PubMed
Azaphilones are a family of fungal polyketide metabolites with diverse chemical structures and biological activities with a highly oxygenated pyranoquinone bicyclic core. Here, a class of azaphilones possessing a 6/6/6/6 tetracyclic ring system was identified in Aspergillus terreus, and exhibited potential anticancer activities. The gene deletions and biochemical investigations demonstrated that these azaphilones were collaboratively synthesized by two separate clusters containing four core-enzymes, two nonreducing PKSs, one highly reducing PKS, and one NRPS-like. More interestingly, we found that the biosynthesis is coordinately regulated by a crosstalk mechanism between these two gene clusters based on three transcriptional factors. This is a meaningful mechanism of fungal secondary metabolism, which allows fungi to synthesize more complex compounds and gain new physiological functions. The results provide a new insight into fungal natural product biosynthesis.
Title: Structural insights into the catalytic mechanism of lovastatin hydrolase Liang Y, Lu X Ref: Journal of Biological Chemistry, 295:1047, 2020 : PubMed
The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure-function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 A), PcEST in complex with monacolin J (2.48 A), PcEST complexed with the substrate analog simvastatin (2.4 A), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 A). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser(57) (nucleophile)-Tyr(170) (general base)-Lys(60) (general acid) catalytic triad, the hydrogen-bond network (Trp(344) and Tyr(127)) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2',2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure-function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.
Title: Correction to Near-Infrared Fluorescence Probe for Evaluating Acetylcholinesterase Activity in PC12 Cells and In Situ Tracing AChE Distribution in Zebrafish Ma J, Si T, Yan C, Li Y, Li Q, Lu X, Guo Y Ref: ACS Sens, :, 2020 : PubMed
Title: Near-Infrared Fluorescence Probe for Evaluating Acetylcholinesterase Activity in PC12 Cells and In Situ Tracing AChE Distribution in Zebrafish Ma J, Si T, Yan C, Li Y, Li Q, Lu X, Guo Y Ref: ACS Sens, 5:83, 2020 : PubMed
Acetylcholinesterase (AChE) plays crucial roles in numerous physiological processes such as cell differentiation, cell apoptosis, and nerve tissue developments. Hence, it is highly necessary to design a fluorescent probe for monitoring AChE activity in complex living organisms. In this work, a near-infrared (NIR) off-on probe (CyN) was developed for AChE detection. CyN was exactly synthesized by introducing an N,N-dimethyl carbamyl moiety to hemicyanine (CyOH). AChE can "light up" strong NIR fluorescence through a cleavage special ester bond and transform CyN into CyOH. Moreover, CyN was qualified for imaging the dynamic change of AChE activity in PC12 cells with retinoic acid or hypoxia stimulation. In particular, the probe has been successfully applied for in situ tracing the intact distribution of AChE in living zebrafish. The observations indicate that major occurrence sites of endogenic AChE on zebrafish are the yolk sac and neuromasts. Overall, CyN shows great potential for use in AChE-related physiological studies.
Title: Rational design of a near-infrared fluorescence probe for highly selective sensing butyrylcholinesterase (BChE) and its bioimaging applications in living cell Ma J, Lu X, Zhai H, Li Q, Qiao L, Guo Y Ref: Talanta, 219:121278, 2020 : PubMed
In the current work, a near-infrared (NIR) fluorescent probe (CyClCP) was developed for fast (35 min), highly sensitive (LOD of 3.75 U/L) and selective response to BChE in vitro and in vivo. Upon the addition of BChE, CyClCP could be efficiently activated with remarkable NIR ((em) = 708 nm) fluorescence enhancement and obvious absorbance red shift (581 nm-687 nm). Specifically, according to the subtle differences structural features and substrate preference between BChE and its sister enzyme AChE, CyClCP was constructed by introducing chlorine atom at the ortho-position of the phenolic hydroxyl in the previous reported probe (CyCP). Fortunately, CyClCP exhibited better selectivity towards BChE over AChE compared with CyCP. This molecular design strategy was further rationalized by docking molecular of fluorescence probes (CyClCP and CyCP) and enzymes (BChE and AChE). Finally, CyClCP was membrane permeable and successfully applied to image endogenous BChE level in HepG2 and LO2 cells. Therefore, CyClCP could serve as a promising tool for BChE-related physiological function studies in complex biological systems.
Clonostachys rosea is a promising saprophytic filamentous fungus that belongs to phylum Ascomycota. Clonostachys rosea is widespread around the world and exists in many kinds of habitats, with the highest frequency in soil. As an excellent mycoparasite, C. rosea exhibits strong biological control ability against numerous fungal plant pathogens, nematodes and insects. These behaviours are based on the activation of multiple mechanisms such as secreted cell-wall-degrading enzymes, production of antifungal secondary metabolites and induction of plant defence systems. Besides having significant biocontrol activity, C. rosea also functions in the biodegradation of plastic waste, biotransformation of bioactive compounds, as a bioenergy sources and in fermentation. This mini review summarizes information about the biology and various applications of C. rosea and expands on its possible uses.
Title: Discovery and Characterization of a PKS-NRPS Hybrid in Aspergillus terreus by Genome Mining Tang S, Zhang W, Li Z, Li H, Geng C, Huang X, Lu X Ref: Journal of Natural Products, 83:473, 2020 : PubMed
Fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrids have been characterized to produce polyketide-amino acid compounds with striking structural features and biological activities. In this study, a PKS-NRPS hybrid enzyme was found in Aspergillus terreus by genome mining. By activating the cluster-specific transcriptional regulator, this cryptic PKS-NRPS gene cluster was successfully activated and ten products (1-10) were identified as pyranterreones. Using functional genetics, bioinformatics, and isotope-labeling feeding analysis, the biosynthetic pathway was revealed. This is the second PKS-NRPS hybrid identified in A. terreus.
BACKGROUND: Penehyclidine is a newly developed anticholinergic agent. We aimed to investigate the role of penehyclidine in acute organophosphorus pesticide poisoning (OP) patients. METHODS: We searched the Pubmed, Cochrane library, EMBASE, Chinese National Knowledge Infrastructure (CNKI), Chinese Biomedical literature (CBM) and Wanfang databases. Randomized controlled trials (RCTs) recruiting acute OP patients were identified for meta-analysis. Main outcomes included cure rate, mortality rate, time to atropinization, time to 60% normal acetylcholinesterase (AchE) level, rate of intermediate syndrome (IMS) and rate of adverse drug reactions (ADR). RESULTS: Sixteen RCTs involving 1,334 patients were identified. Compared with the atropine- or penehyclidine-alone groups, atropine combined with penehyclidine significantly increased the cure rate (penehyclidine+atropine vs. atropine, 0.97 vs. 0.86, RR 1.13, 95% CI [1.07-1.19]; penehyclidine+atropine vs. penehyclidine, 0.93 vs. 0.80, RR 1.08, 95% CI [1.01-1.15]) and reduced the mortality rate (penehyclidine+atropine vs. atropine, 0.015 vs. 0.11, RR 0.17, 95% CI [0.06-0.49]; penehyclidine+atropine vs. penehyclidine, 0.13 vs. 0.08, RR 0.23, 95% CI [0.04-1.28]). Atropine combined with penehyclidine in OP patients also helped reduce the time to atropinization and AchE recovery, the rate of IMS and the rate of ADR. Compared with a single dose of atropine, a single dose of penehyclidine also significantly elevated the cure rate, reduced times to atropinization, AchE recovery, and rate of IMS. CONCLUSION: Atropine combined with penehyclidine benefits OP patients by enhancing the cure rate, mortality rate, time to atropinization, AchE recovery, IMS rate, total ADR and duration of hospitalization. Penehyclidine combined with atropine is likely a better initial therapy for OP patients than atropine alone.
Title: Overexpressed CES2 has prognostic value in CRC and knockdown CES2 reverses L-OHP-resistance in CRC cells by inhibition of the PI3K signaling pathway Zhang Y, Sun L, Sun Y, Chen Y, Wang X, Xu M, Chi P, Xu Z, Lu X Ref: Experimental Cell Research, :111856, 2020 : PubMed
CES-2 (carboxylesterase-2) belongs to the carboxylesterase gene family, which plays crucial roles in lipid mobilization and chemosensitivity to irinotecan. However, its role in chemosensitivity to oxaliplatin (L-OHP) remains unclear. Herein, L-OHP-resistant cells (HCT-116L and RKOL) were established by increasing the concentration of L-OHP. The results showed that CES2 expression was upregulated in L-OHP-resistant tissues and cells lines (P<0.01). Low expression of CES2 correlated with a better survival, and the results were further confirmed in the R2 platform: a biologist friendly web-based genomics analysis and visualization application. Downregulation of CES2 suppressed cell proliferation, induced apoptosis and reversed L-OHP resistance by medicating the PI3K signaling pathway in L-OHP-resistant cells. However, both PI3K inhibitor (LY294002) and activator (IGF-1) could not medicate CES2 expression. These findings indicated that CES2 may be utilized as a novel biomarker and therapeutic target for L-OHP resistance in CRC treatment.
Title: Discovery of a Selective 6-Hydroxy-1, 4-Diazepan-2-one Containing Butyrylcholinesterase Inhibitor by Virtual Screening and MM-GBSA Rescoring Zhou Y, Hu Y, Lu X, Yang H, Li Q, Du C, Chen Y, Hong KH, Sun H Ref: Dose Response, 18:1559325820938526, 2020 : PubMed
Alzheimer disease (AD) is the most common form of dementia characterized by the loss of cognitive abilities through the death of central neuronal cells. In this study, structure-based virtual screens of 2 central nervous system-targeted libraries followed by molecular mechanics/generalized born surface area rescoring were performed to discover novel, selective butyrylcholinesterase (BChE) inhibitors, which are one of the most effective therapeutic strategies for the treatments in late-stage AD. Satisfyingly, compound 5 was identified as a highly selective low micromolar inhibitor of BChE (BChE IC50 = 1.4 muM). The binding mode prediction and kinetic analysis were performed to obtain detailed information about compound 5. Besides, a preliminary structure-activity relationship investigation of compound 5 was carried out for further development of the series. The present results provided a valuable chemical template with a novel scaffold for the development of selective BChE inhibitors.
Title: Novel BuChE-IDO1 inhibitors from sertaconazole: Virtual screening, chemical optimization and molecular modeling studies Zhou Y, Lu X, Du C, Liu Y, Wang Y, Ho Hong K, Chen Y, Sun H Ref: Bioorganic & Medicinal Chemistry Lett, :127756, 2020 : PubMed
In our effort towards the identification of novel BuChE-IDO1 dual-targeted inhibitor for the treatment of Alzheimer's disease (AD), sertaconazole was identified through a combination of structure-based virtual screening followed by MM-GBSA rescoring. Preliminary chemical optimization was performed to develop more potent and selective sertaconazole analogues. In consideration of the selectivity and the inhibitory activity against target proteins, compounds 5c and 5d were selected for the next study. Further modification of compound 5c led to the generation of compound 10g with notably improved selectivity towards BuChE versus AChE. The present study provided us with a good starting point to further design potent and selective BuChE-IDO1 inhibitors, which may benefit the treatment of late stage AD.
A series of novel chalcone-O-alkylamine derivatives were designed, synthesized and evaluated as multifunctional anti-Alzheimer's disease agents. Based on the experimental results, compound 23c exhibited good inhibitory potency on both acetylcholinesterase (IC50=1.3+/-0.01muM) and butyrylcholinesterase (IC50=1.2+/-0.09muM). Besides, 23c exhibited selective MAO-B inhibitory activity with IC50 value of 0.57+/-0.01muM. Compound 23c was also a potential antioxidant and neuroprotectant. In addition, compound 23c could inhibit self-induced Abeta1-42 aggregation. Moreover, compound 23c was a selective metal chelator, and could inhibit and disaggregate Cu(2+)-induced Abeta1-42 aggregation, which was supported by the further transmission electron microscopy images. Furthermore, 23c could cross the blood-brain barrier in vitro, and improved scopolamine-induced memory impairment in vivo assay. Molecular modeling studies showed that 23c could bind to the active site of AChE, BuChE, Abeta1-42 and MAO-B. Taken together, these results suggested that compound 23c might be a potential multifunctional agent for the treatment of AD.
Title: Tracing and attribution of V-type nerve agents in human exposure by strategy of assessing the phosphonylated and disulfide adducts on ceruloplasmin Fu F, Chen J, Zhao P, Lu X, Gao R, Chen D, Liu H, Wang H, Pei C Ref: Toxicology, 430:152346, 2019 : PubMed
V-type agents are highly toxic organophosphorus nerve agents that inhibit acetylcholinesterase in the nervous system, causing a series of poison symptoms. Trace analytical methods are essential for the specific verification of exposure to these agents, especially for human exposure. This paper investigates the phosphonylated and disulfide adducts between human ceruloplasmin and O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2-(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). After being digested by trypsin, the mixture of peptides was separated by a nano-liquid chromatography (nano-LC) and analyzed using quadrupole-orbitrap mass spectrometry (Q-Orbitrap-MS). The sensitive LC-MS/MS-assisted proteomics approach was developed to achieve the identification of human exposure to V-type agents based on these modified sites; results revealed that potential biomarkers could be derived from adducts based on the sulfur- and phosphorus-containing groups of V-type agents. This work offered a novel insight into the mechanism of disulfide-containing adducts resulting from the replacement of disulfide bridges by the thiolate groups from the V-type agents. Moreover, four disulfide adducts on human ceruloplasmin were also discovered during this research, specifically confirming exposure to the V-type agents. Furthermore, molecular simulation testified to the reactivity of the modified sites. Collectively, our findings suggest that the eleven binding sites on human ceruloplasmin have the potential use as a selective marker for prediction the V-type agent exposure in humans.
Title: Expansion of the scaffold diversity for the development of highly selective butyrylcholinesterase (BChE) inhibitors: Discovery of new hits through the pharmacophore model generation, virtual screening and molecular dynamics simulation Lu X, Yang H, Li Q, Chen Y, Zhou Y, Feng F, Liu W, Guo Q, Sun H Ref: Bioorg Chem, 85:117, 2018 : PubMed
Butyrylcholinesterase (BChE) is recently considered as a new target for the treatment of Alzheimer's disease (AD). There is an increasing interest in the development of BChE inhibitors. In the present study, a set of pharmacophore models for BChE was developed and validated. Based on the models, virtual screening was performed on five compound collections, from which seventeen potential hits were retained for biological investigation. In total, eight of these seventeen potential hits showed selective BChE inhibitory activity. Moreover, four compounds displayed IC50 values in sub-micromolar range on eqBChE and three displayed IC50 values <2muM on huBChE. The diverse scaffolds of the active compounds provided good starting point further development of selective BChE inhibitors. As far as we concerned, here we disclose the first selective pharmacophore model targeting BChE. The high rate of the model in the identification of active hits indicates it is a valuable tool for the development of selective BChE inhibitors, which may benefit the treatment of AD.
Title: Epigenetic mechanisms underlying the effects of triptolide and tripchlorolide on the expression of neuroligin-1 in the hippocampus of APP/PS1 transgenic mice Lu X, Yang B, Yu H, Hu X, Nie J, Wan B, Zhang M, Lu C Ref: Pharm Biol, 57:453, 2019 : PubMed
Context: Neuroligin-1 (NLGN1) is a cell adhesion protein located on the excitatory postsynaptic membrane. beta-Amyloid (Abeta)-induced neuroinflammation decreases NLGN1 expression through epigenetic mechanisms. Triptolide (T10) and tripchlorolide (T4) exert protective effects on synapses in Alzheimer's disease (AD) mice, but the mechanisms remain unclear. Objective: The effects of T10 and T4 on hippocampal NLGN1 expression in AD mice and the epigenetic mechanisms were assessed using chromatin immunoprecipitation and methylated DNA immunoprecipitation. Materials and methods: Sixty APP/PS1 transgenic mice were randomly divided into an AD model group, a T10-treated group and a T4-treated group (n = 20); 20 wild-type littermates served as the control group. APP/PS1 transgenic mice were intraperitoneally injected with T10 (0.1 mg/kg) and T4 (25 mug/kg) once per day for 60 days. NLGN1 expression was examined using western blotting and quantitative PCR. Results: T10 and T4 increased the levels of the NLGN1 protein and mRNA in hippocampus of AD mice. T10 and T4 inhibited the binding of HDAC2 (p< 0.01) and MeCP2 (p< 0.01 and p< 0.05, respectively) to the NLGN1 promoter, and cytosine methylation (1.2305 +/- 0.1482/1.2554 +/- 0.3570 vs. 1.6578 +/- 0.1818, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice. T10 and T4 increased the level of acetylated histone H3 (0.7733 +/- 0.1611/0.8241 +/- 0.0964 vs. 0.5587 +/- 0.0925, p< 0.01) at the NLGN1 promoter in the hippocampus of AD mice. Conclusions: T10 and T4 may increase hippocampal NLGN1 expression in AD mice through epigenetic mechanisms, providing a new explanation for the mechanism underlying the protective effects of T10 and T4 on synapses.
Title: Discovery, molecular dynamic simulation and biological evaluation of structurally diverse cholinesterase inhibitors with new scaffold through shape-based pharmacophore virtual screening Yang H, Du C, Li Q, Chen T, Lu X, Feng F, Chen Y, Liu W, Sun H Ref: Bioorg Chem, 92:103294, 2019 : PubMed
Designing small molecule inhibitors targeting cholinesterases (ChEs) is considered as an efficient strategy for the treatment of Alzheimer's disease (AD). In the present study, based on a shaped-based pharmacophore (SBP) model that we reported previously, virtual screening was performed on four commercial compound databases, from which eight small molecules containing new structurally scaffolds were retained and evaluated. In general, six of these potential hits were identified to be selective ChEs inhibitors. Three compounds exhibited IC50 values and Ki values in micromolar range on acetylcholinesterase (AChE), the most active compound 4 showed IC50 value of 6.31+/-2.68muM and Ki value of 4.76muM. Other three compounds displayed IC50 values and Ki values in micromolar range on butyrylcholinesterase (BChE) with high target selectivity, the most active compound 1 showed IC50 value of 3.87+/-2.48muM and Ki value of 1.52muM. Multiple biological evaluations were performed to determine their cytotoxicity, cyto-protective effects, antioxidant effect as well as druglike properties. These compounds provide new cores for the further design and optimization, with the aim to discover new ChEs inhibitors for the treatment of AD.
Title: Efficacy and outcomes of lipid resuscitation on organophosphate poisoning patients: A systematic review and meta-analysis Yu S, Zhang L, Gao Y, Walline J, Lu X, Ma Y, Zhu H, Yu X, Li Y Ref: Am J Emerg Med, 37:1611, 2019 : PubMed
OBJECTIVE: Organophosphate (OP) pesticides are still widely available in developing countries, leading to numerous accidental or suicidal poisonings every year. Lipid emulsion treatments are commonly used in resuscitating OP poisoning patients but few studies regarding their use have been reported. Our meta-analysis aimed to analyze the efficacy and outcomes of lipid resuscitation on OP poisoning patients. METHODS: A systematic search for associated studies was conducted in Pubmed, EMBASE, MEDLINE, the Cochrane Library and the Chinese National Knowledge Infrastructure. Collected data was pooled using Revman v5.3. Outcomes included prognosis (cured vs. mortality rates), hepatic function (serum ALT, AST, Total Bilirubin (TBIL) level), serum acetylcholinesterase (AchE) level and respiratory function (rate of respiratory muscular paralysis). RESULTS: Seven randomized controlled studies consisting of 630 patients meeting inclusion criteria were identified. Lipid emulsion helped to improve the cure rate [OR=2.54, 95% CI (1.33, 4.86), p=0.005] and lower the mortality rate [OR=0.31, 95% CI (0.13, 0.74), p=0.009]. Serum ALT, AST and TBIL in patients undergoing lipid resuscitation were lower than those in the control groups [ALT, SMD=-1.52, 95% CI (-2.64, 0.40), p=0.008; AST, SMD=-1.66, 95% CI (-3.15, 0.16), p=0.03; TBIL, SMD=-1.26, 95% CI (-2.32, 0.20), p=0.02]. Serum AchE level were increased in patients treated with lipid emulsion [SMD=2.15, 95% CI (1.60, 2.71), p<0.00001]. Rate of respiratory muscular paralysis was lower in patients undergoing lipid resuscitation than those in the control groups [OR=0.19, 95% CI (0.05, 0.71), p=0.01]. CONCLUSION: Based on our meta-analysis of included RCT reports, lipid resuscitation seems likely to help improve prognosis and liver function of OP poisoning patients. However, larger multi-center RCTs are still recommended.
As there are increased levels and activity of butyrylcholiesterase (BChE) in the late stage of Alzheimer's disease (AD), development of selective BChE inhibitors is of vital importance. In this study, a workflow combining computational technologies and biological assays were implemented to identify selective BChE inhibitors with new chemical scaffolds. In particular, a pharmacophore model served as a 3D search query to screen three compound collections containing 3.0 million compounds. Molecular docking and cluster analysis were performed to increase the efficiency and accuracy of virtual screening. Finally, 15 compounds were retained for biological investigation. Results revealed that compounds 8 and 18 could potently and highly selectively inhibit BChE activities (IC50 values < 10 muM on human BChE, selectivity index BChE > 30). These active compounds with novel scaffolds provided us with a good starting point to further design potent and selective BChE inhibitors, which may be beneficial for the treatment of AD.
Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC(50)) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC(50) The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs.IMPORTANCE Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group beta-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.
In our endeavor towards the development of potent multi-target ligands for the treatment of Alzheimer's disease, miconazole was identified to show BuChE-IDO1 dual-target inhibitory effects. Morris water maze test indicated that miconazole obviously ameliorated the cognitive function impaired by scopolamine. Furthermore, it showed good safety in primary hepatotoxicity evaluation. Based on these results, we designed, synthesized, and evaluated a series of miconazole derivatives as BuChE-IDO1 dual-target inhibitors. Out of the 12 compounds, 5i and 5j exhibited the best potency in enzymatic evaluation, thus were selected for subsequent behavioral study, in which the two compounds exerted much improved effect than tacrine. Meanwhile, 5i and 5j displayed no apparent hepatotoxicity. The results suggest that miconazole analogue offers an attractive starting point for further development of new BuChE-IDO1 dual-target inhibitors against Alzheimer's disease.
Title: Amorphous metal boride as a novel platform for acetylcholinesterase biosensor development and detection of organophosphate pesticides Lu X, Li Y, Tao L, Song D, Wang Y, Gao F Ref: Nanotechnology, 30:055501, 2018 : PubMed
The exploration of new materials for modifying electrodes is important to advance electrochemical biosensors. Herein, we demonstrated that amorphous bimetallic boride material (Co-2Ni-B) prepared by a simple and facile aqueous reaction is an efficient matrix to immobilize acetylcholinesterase (AChE) to construct a biosensor for the determination of organophosphate pesticides. The effects of different composition and crystallinity on its electrochemical performance are investigated, and the optimization studies of the biological transducer were also discussed. Under optimal conditions, the fabricated sensor showed good analytical performance for the determination of chlorpyrifos with a low limit of detection (2.83 pM) and a wide linear range (3 pM-300 nM). The proposed biosensor also demonstrated high reproducibility, stability and accuracy. The impressive performance was due to the excellent conductivity and the unique amorphous bimetal-metalloid complex nanostructure. These results introduce a new class of promising materials as a robust platform for biosensor applications.
Flexible epidermal tattoo and textile-based electrochemical biosensors have been developed for vapor-phase detection of organophosphorus (OP) nerve agents. These new wearable sensors, based on stretchable organophosphorus hydrolase (OPH) enzyme electrodes, are coupled with a fully integrated conformal flexible electronic interface that offers rapid and selective square-wave voltammetric detection of OP vapor threats and wireless data transmission to a mobile device. The epidermal tattoo and textile sensors display a good reproducibility (with RSD of 2.5% and 4.2%, respectively), along with good discrimination against potential interferences and linearity over the 90-300mg/L range, with a sensitivity of 10.7microAcm3mg-1 (R2 = 0.983) and detection limit of 12mg/L in terms of OP air density. Stress-enduring inks, used for printing the electrode transducers, ensure resilience against mechanical deformations associated with textile and skin-based on-body sensing operations. Theoretical simulations are used to estimate the OP air density over the sensor surface. These fully integrated wearable wireless tattoo and textile-based nerve-agent vapor biosensor systems offer considerable promise for rapid warning regarding personal exposure to OP nerve-agent vapors in variety of decentralized security applications.
Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.
Title: Ultra-thin bimetallic alloy nanowires with porous architecture/monolayer MoS2 nanosheet as a highly sensitive platform for the electrochemical assay of hazardous omethoate pollutant Song D, Li Q, Lu X, Li Y, Wang Y, Gao F Ref: J Hazard Mater, 357:466, 2018 : PubMed
A novel electrochemical biosensor was designed for sensitive detection of organophosphate pesticides based on three-dimensional porous bimetallic alloy architecture with ultrathin nanowires (PdCo NWs, PdCu NWs, PdNi NWs) and monolayer MoS2 nanosheet (m-MoS2). The bimetallic alloy NWs/m-MoS2 nanomaterials were used as a sensing platform for electrochemical analysis of omethoate, a representative organophosphate pesticide, via acetylcholinesterase inhibition pathway. We demonstrated that all three bimetallic alloy NWs enhanced electrochemical responses of enzymatic biosensor, benefited from bimetallic synergistic action and porous structure. In particular, PdNi NWs outperformed other two bimetallic alloy. Moreover, PdNi NWs/m-MoS2 as an electronic transducer is superior to the corresponding biosensor in the absence of monolayer MoS2 nanosheet, which arise from synergistic signal amplification effect between different components. Under optimized conditions, the developed biosensor on the basis of PdNi NWs/m-MoS2 shows outstanding performance for the electrochemical assay of omethoate, such as a wide linear range (10(-13) M approximately 10(-7) M), a low detection limit of 0.05pM at a signal-to-noise ratio of 3, high sensitivity and long-time stability. The results demonstrate that bimetallic alloy NWs/m-MoS2 nanocomposites could be excellent transducers to promote electron transfer for the electrochemical reactions, holding great potentials in the construction of current and future biosensing devices.
Title: Isosteroidal alkaloids as potent dual-binding site inhibitors of both acetylcholinesterase and butyrylcholinesterase from the bulbs of Fritillaria walujewii Liu YM, Feng YD, Lu X, Nie JB, Li W, Wang LN, Tian LJ, Liu QH Ref: Eur Journal of Medicinal Chemistry, 137:280, 2017 : PubMed
Five new isosteroidal alkaloids, walujewine A (1), walujewine B (4), walujewine C (5), walujewine D (6), walujewine E (10) were isolated from the bulbs of Fritillaria walujewii together with seven known isosteroidal alkaloids (2, 3, 7-9, 11, 12). Their structures were elucidated on the basis of IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR spectroscopic data analyses and single-crystal X-ray diffraction. All the isolates were tested for ChE inhibiting activity by the Ellman's method. Compounds 3-5 and 8-10 were potent dual AChE-BChE inhibitors, and compound 1 showed highly selective AChE inhibition. The structure-activity relationship of compounds 1-12 was discussed in details. And kinetic analysis showed that compounds 1, 3-5, and 8-10 were mixed-type reversible inhibitors of AChE, simultaneously binding to the catalytic and peripheral anionic sites, which was verified by in silico docking studies. The docking simulation also showed that active compound 3 and 8 created many interactions with the CAS and PAS gorges of BChE, revealing their mixed-type inhibition. ADMET analysis further confirmed the therapeutic potential of some isosteroidal alkaloids based on their high BBB-penetration.
Title: Palladium-copper nanowires-based biosensor for the ultrasensitive detection of organophosphate pesticides Song D, Li Y, Lu X, Sun M, Liu H, Yu G, Gao F Ref: Anal Chim Acta, 982:168, 2017 : PubMed
A highly sensitive acetylcholinesterase (AChE) electrochemical biosensor for the quantitative determination of organophosphate pesticides (OPs) in vegetables and fruits based on palladium-copper nanowires (Pd-Cu NWs) was reported. AChE immobilized on the modified electrode could catalyze hydrolysis of acetylthiocholine chloride (ATCl), generating an irreversible oxidation peak. When exposed to the OPs, the activity of the AChE was inhibited and the current significantly decreased. The detection mechanism is based on the inhibition of AChE. The Pd-Cu NWs not only provide a large active surface area (0.268 +/- 0.01) cm2 for the immobilization of AChE, which was approximately 3.8 times higher than the bare glass carbon electrode, but also exhibit excellent electro-catalytic activity and remarkable electron mobility. The biosensor modified with Pd-Cu NWs displayed a good affinity to ATCl and catalyzed hydrolysis of ATCl, with a low Michaelis-Menten constant (KM) of 50.56 muM. Under optimized conditions, the AChE-Cs/Pd-Cu NWs/GCE biosensor detected malathion with wide linear ranges of 5-1000 ppt and 500-3000 ppb, and the low detection limit was 1.5 ppt (4.5 pM). In addition, the OPs biosensor has been applied to the analysis of malathion in commercial vegetable and fruit samples, with excellent recoveries in the range of 98.5%-113.5%. This work provides a simple, sensitive and effective platform for biosensors and exhibits future potential in practical application for the OPs assay.
Title: Dietary phosphatidylcholine impacts on growth performance and lipid metabolism in adult Genetically Improved Farmed Tilapia (GIFT) strain of Nile tilapia Oreochromis niloticus Tian J, Wen H, Lu X, Liu W, Wu F, Yang CG, Jiang M, Yu LJ Ref: British Journal of Nutrition, :1, 2017 : PubMed
This study aimed to determine the effects of supplementing the diet of adult Nile tilapia Oreochromis niloticus with phosphatidylcholine (PC) on growth performance, body composition, fatty acid composition and gene expression. Genetically Improved Farmed Tilapia fish with an initial body weight of 83.1 (sd 2.9) g were divided into six groups. Each group was hand-fed a semi-purified diet containing 1.7 (control diet), 4.0, 6.5, 11.5, 21.3 or 41.0 g PC/kg diet for 68 d. Supplemental PC improved the feed efficiency rate, which was highest in the 11.5 g PC/kg diet. Weight gain and specific growth rate were unaffected. Dietary PC increased PC content in the liver and decreased crude fat content in the liver, viscera and body. SFA and MUFA increased and PUFA decreased in muscle with increasing dietary PC. Cytoplasmic phospholipase A 2 and secreted phospholipase A 2 mRNA expression were up-regulated in the brain and heart in PC-supplemented fish. PC reduced fatty acid synthase mRNA expression in the liver and visceral tissue but increased expression in muscle. Hormone-sensitive lipase and lipoprotein lipase expression increased in the liver with increasing dietary PC. Growth hormone mRNA expression was reduced in the brain and insulin-like growth factor-1 mRNA expression in liver reduced with PC above 6.5 g/kg. Our results demonstrate that dietary supplementation with PC improves feed efficiency and reduces liver fat in adult Nile tilapia, without increasing weight gain, representing a novel dietary approach to reduce feed requirements and improve the health of Nile tilapia.
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.
BACKGROUND: Parasitism is a major ecological niche for a variety of nematodes. Multiple nematode lineages have specialized as pathogens, including deadly parasites of insects that are used in biological control. We have sequenced and analyzed the draft genomes and transcriptomes of the entomopathogenic nematode Steinernema carpocapsae and four congeners (S. scapterisci, S. monticolum, S. feltiae, and S. glaseri). RESULTS: We used these genomes to establish phylogenetic relationships, explore gene conservation across species, and identify genes uniquely expanded in insect parasites. Protein domain analysis in Steinernema revealed a striking expansion of numerous putative parasitism genes, including certain protease and protease inhibitor families, as well as fatty acid- and retinol-binding proteins. Stage-specific gene expression of some of these expanded families further supports the notion that they are involved in insect parasitism by Steinernema. We show that sets of novel conserved non-coding regulatory motifs are associated with orthologous genes in Steinernema and Caenorhabditis. CONCLUSIONS: We have identified a set of expanded gene families that are likely to be involved in parasitism. We have also identified a set of non-coding motifs associated with groups of orthologous genes in Steinernema and Caenorhabditis involved in neurogenesis and embryonic development that are likely part of conserved protein-DNA relationships shared between these two genera.
Title: Nonsynonymous polymorphisms in PLA2G7 gene are associated with the risk of coronary heart disease in a southern Chinese population Hong M, Zhang M, Lu X Ref: Mamm Genome, 26:191, 2015 : PubMed
Lipoprotein-associated phospholipase A2 (Lp-PLA2) plays an important role in coronary heart disease (CHD). This study was aimed to investigate the associations of polymorphisms (R92H, V279F, I198T, and A379V) in PLA2G7 with CHD. A total of 322 patients with CHD and 414 CHD-free controls were included in the study. Polymorphisms in PLA2G7 were sequenced by DNA Sequencer and statistical analyses were performed to study the associations between polymorphisms and CHD. RH + HH genotype, RH genotype, and H allele of R92H were significantly associated with an increased risk of CHD (P = 0.005, P = 0.009, and P = 0.003, respectively), while no associations were observed between V279F and I198T and CHD (A379V was not analyzed because of deviation from Hardy-Weinberg equilibrium). Correlations between R92H and CHD still existed after adjustment for confounding risk factors of CHD (P = 0.001). Furthermore, stratified analyses showed subgroups of the senior, hypertension, non-smoking, non-diabetics, and male subjects brought a higher risk for CHD (P = 0.015, P = 0.001, P = 0.001, P = 0.002, and P = 0.004, respectively). We also observed a lower level of protective factor HDL-C in CHD patients carrying genotype RH + HH than patients with RR (P = 0.047). Furthermore, we conducted haplotype analysis and detected more harmful effects of haplotypes HVI and RVT as compared with other haplotypes (P = 2.538 x 10(-3) and P = 0.031). These findings indicated that R92H variant in PLA2G7 gene might contribute to CHD susceptibility in a southern Chinese population.
Title: Whole Genome Sequence of the Probiotic Strain Lactobacillus paracasei N1115, Isolated from Traditional Chinese Fermented Milk Wang S, Zhu H, He F, Luo Y, Kang Z, Lu C, Feng L, Lu X, Xue Y, Wang H Ref: Genome Announc, 2:, 2014 : PubMed
Lactobacillus paracasei N1115 is a new strain with probiotic properties isolated from traditional homemade dairy products in Inner Mongolia, China. Here, we report the complete genome sequence of L. paracasei N1115, which shows high similarity to the well-studied probiotic Lactobacillus rhamnosus GG, and 3 structures turned out to be inversions, according to the colinearity analysis of the BLAST alignment.
Mycoplasma columbinum is a member of nonglycolytic Mycoplasma species which can hydrolyze arginine. Increasingly research has revealed that M. columbinum is associated with respiratory disease of pigeons and that the respiratory disease symptoms could be eliminated via the use of mycoplasma treatment medicine. Here we report the genome sequence of M. columbinum strain SF7, which is the first genome report for M. columbinum.
Many single-nucleotide polymorphisms (SNPs) have been reported to be associated with lipid concentrations in recent genome-wide association studies. The aim of this study was to validate the associations of rs2197089 in the lipoprotein lipase (LPL) gene with serum lipid concentrations and gene expression levels in the Chinese Han population and examine the potential interactions. A total of 9339 participants were recruited and genotyped for rs2197089. Gene expression levels of LPL in blood cells of 309 participants were evaluated by real-time PCR. We observed significant associations between rs2197089 and decreased triglycerides (TG) (P=0.0006), but not high-density lipoprotein cholesterol (HDL-C) concentration (P=0.0881). However, weak evidence of interaction between cigarette smoking and rs2197089 was detected (P=0.0362). In smokers, significant association between rs2197089 and increased HDL-C concentration was found (P=0.0068). Participants with the minor allele A had higher expression levels of LPL (P=0.0243). The results of our study indicated that rs2197089 was significantly associated with TG but it was associated with HDL-C only in smokers. This SNP seemed to have influence on the expression level of LPL.
Title: 3-Hydroxybutyrate methyl ester as a potential drug against Alzheimer's disease via mitochondria protection mechanism Zhang J, Cao Q, Li S, Lu X, Zhao Y, Guan JS, Chen JC, Wu Q, Chen GQ Ref: Biomaterials, 34:7552, 2013 : PubMed
Alzheimer's disease (AD) is induced by many reasons, including decreased cellular utilization of glucose and brain cell mitochondrial damages. Degradation product of microbially synthesized polyhydroxybutyrate (PHB), namely, 3-hydroxybutyrate (3HB), can be an alternative to glucose during sustained hypoglycemia. In this study, the derivative of 3HB, 3-hydroxybutyrate methyl ester (HBME), was used by cells as an alternative to glucose. HBME inhibited cell apoptosis under glucose deprivation, rescued activities of mitochondrial respiratory chain complexes that were impaired in AD patients and decreased the generation of ROS. Meanwhile, HBME stabilized the mitochondrial membrane potential. In vivo studies showed that HBME crossed the blood brain barrier easier compared with charged 3HB, resulting in a better bioavailability. AD mice treated with HBME performed significantly better (p < 0.05) in the Morris water maze compared with other groups, demonstrating that HBME has a positive in vivo pharmaceutical effect to improve the spatial learning and working memory of mice. A reduced amyloid-beta deposition in mouse brains after intragastric administration of HBME was also observed. Combined with the in vitro and in vivo results, HBME was proposed to be a drug candidate against AD, its working mechanism appeared to be mediated by various effects of protecting mitochondrial damages.
Despite the importance of PEGylation in achieving long nanoparticle circulation times, many nanoparticles are coated with PEGylating agents susceptible to enzymatic degradation. In this study, solid lipid nanoparticles (SLNs) prepared with ester-containing compounds were evaluated for their stability in the presence of carboxylesterase. SLN suspensions became turbid within 30 min of enzymatic exposure, indicating possible disassociation of a portion of the nanoparticles. The particle size of SLNs incubated with the enzyme was smaller than the size of controls, although their morphologies appeared similar in transmission electron microscopy images. Although SLNs offered some protection over micelles, PEG6000 monostearate was rapidly degraded within 15 min. Hydrolysis of polysorbate 60 was much slower, reaching only 36% in 2 h. These studies reveal the importance of confirming the stability of PEG surface coatings prior to undertaking in vivo experiments in small animal models, which can have considerably higher plasma esterase activity than humans.
Domestic yaks (Bos grunniens) provide meat and other necessities for Tibetans living at high altitude on the Qinghai-Tibetan Plateau and in adjacent regions. Comparison between yak and the closely related low-altitude cattle (Bos taurus) is informative in studying animal adaptation to high altitude. Here, we present the draft genome sequence of a female domestic yak generated using Illumina-based technology at 65-fold coverage. Genomic comparisons between yak and cattle identify an expansion in yak of gene families related to sensory perception and energy metabolism, as well as an enrichment of protein domains involved in sensing the extracellular environment and hypoxic stress. Positively selected and rapidly evolving genes in the yak lineage are also found to be significantly enriched in functional categories and pathways related to hypoxia and nutrition metabolism. These findings may have important implications for understanding adaptation to high altitude in other animal species and for hypoxia-related diseases in humans.
Title: Synthesis and evaluation of in vitro bioactivity for vesicular acetylcholine transporter inhibitors containing two carbonyl groups Tu Z, Wang W, Cui J, Zhang X, Lu X, Xu J, Parsons SM Ref: Bioorganic & Medicinal Chemistry, 20:4422, 2012 : PubMed
To identify selective high-affinity ligands for the vesicular acetylcholine transporter (VAChT), we have incorporated a carbonyl group into the structures of trozamicol and prezamicol scaffolds, and also converted the secondary amines of the piperidines of trozamicols and prezamicols into amides. Of 18 new racemic compounds, 4 compounds displayed high affinity for VAChT (K(i)=10-20 nM) and greater than 300-fold selectivity for VAChT over sigma(1) and sigma(2) receptors, namely (4-(4-fluorobenzoyl)-4'-hydroxy-[1,3'-bipiperidin]-1'-yl)(3-methylthiophen-2-yl)m ethanone oxalate (9g) (K(i-VAChT)=11.4 nM, VAChT/sigma(1)=1063, VAChT/sigma(2)=370), (1'-benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10c) (K(i-VAChT)=15.4 nM, VAChT/sigma(1)=374, VAChT/sigma(2)=315), (4'-hydroxy-1'-(thiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)me thanone oxalate (10e) (K(i-VAChT)=19.0 nM, VAChT/sigma(1)=1787, VAChT/sigma(2)=335), and (4'-hydroxy-1'-(3-methylthiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyp henyl)methanone oxalate (10g) (K(i-VAChT)=10.2 nM, VAChT/sigma(1)=1500, VAChT/sigma(2)=2030). These four compounds can be radiosynthesized with C-11 or F-18 to validate their possibilities of serving as PET probes for quantifying the levels of VAChT in vivo.
We previously reported a highly potent DPP-IV inhibitor 6 with low in vivo efficacy. While trying to maintain consistent in vitro and in vivo biological activity, we initiated a pharmacokinetic property-driven optimization to improve the metabolic stability and permeability of inhibitor 6. A simple scaffold replacement of thienopyrimidine with pyrrolopyrimidine (21a) led to significantly improved metabolic stability (4% vs. 65% remaining). Further modification of the pyrrolopyrimidine scaffold to produce compound 21j resulted in much better oral bioavailability than 6. Importantly, compound 21j exhibits greater in vivo efficacy than does 6 and Alogliptin and is worthy of further development.
Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.
Title: Correlation between carboxylesterase alleles and insecticide resistance in Culex pipiens complex from China Liu Y, Zhang H, Qiao C, Lu X, Cui F Ref: Parasit Vectors, 4:236, 2011 : PubMed
BACKGROUND: In China, large amounts of chemical insecticides are applied in fields or indoors every year, directly or indirectly bringing selection pressure on vector mosquitoes. Culex pipiens complex has evolved to be resistant to all types of chemical insecticides, especially organophosphates, through carboxylesterases. Six resistant carboxylesterase alleles (Ester) were recorded previously and sometimes co-existed in one field population, representing a complex situation for the evolution of Ester genes. RESULTS: In order to explore the evolutionary scenario, we analyzed the data from an historical record in 2003 and a recent investigation on five Culex pipiens pallens populations sampled from north China in 2010. Insecticide bioassays showed that these five populations had high resistance to pyrethroids, medium resistance to organophosphates, and low resistance to carbamates. Six types of Ester alleles, EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11 were identified, and the overall pattern of their frequencies in geographic distribution was consistent with the report seven years prior to this study. Statistical correlation analysis indicated that Ester8 and Ester9 positively correlated with resistance to four insecticides, and EsterB10 to one insecticide. The occurrences of these three alleles were positively correlated, while the occurrence of EsterB1 was negatively correlated with Ester8, indicating an allelic competition. CONCLUSION: Our analysis suggests that one insecticide can select multiple Ester alleles and one Ester allele can work on multiple insecticides. The evolutionary scenario of carboxylesterases under insecticide selection is possibly "one to many".
To identify the ligands for sigma(1) receptors that are potent and selective, analogues of prezamicol and trozamicol scaffolds of carbonyl-containing vesicular acetylcholine transporter (VAChT) inhibitors were explored. Of the 23 analogues synthesized and tested, 5 displayed very high affinity for sigma(1) (K(i) = 0.48-4.05 nM) and high selectivity for sigma(1) relative to sigma(2) receptors (sigma(1)/sigma(2) selectivity of >749-fold). Four of the five compounds (14a, 14b, 14c, and 14e) showed very low affinity for VAChT (K(i) > 290 nM), and the fifth compound (14g) showed moderate affinity for VAChT (K(i) = 44.2 nM). The compound [1'-(4-fluorobenzyl)-3'-hydroxy[1,4']bipiperidinyl-4-yl]-(4-fluorophenyl)methanon e (14a) displayed very high affinity and selectivity for sigma(1) receptor (K(i) = 0.48 nM, sigma(1)/sigma(2) > 3600). All four of these most promising compounds (14a, 14b, 14c, and 14e) can be radiosynthesized with fluorine-18 or carbon-11, which will allow further evaluation of their properties as PET probes for imaging sigma(1) receptor in vivo.
Title: Differential control of ATGL-mediated lipid droplet degradation by CGI-58 and G0S2 Lu X, Yang X, Liu J Ref: Cell Cycle, 9:2719, 2010 : PubMed
Lipid droplets (LDs) are intracellular storage sites for triacylglyerols (TAGs)and steryl esters, and play essential roles in energy metabolism and membrane biosynthesis. Adipose triglyceride lipase (ATGL) is the key enzyme for TAG hydrolysis (lipolysis) in adipocytes and LD degradation in nonadipocyte cells. Lipase activity of ATGL in vivo largely depends on its C-terminal sequence as well as coactivation by CGI-58. Here we demonstrate that the C-terminal hydrophobic domain in ATGL is required for LD targeting and CGI-58-independent LD degradation. Overexpression of wild type ATGL causes a dramatic decrease in LD size and number, whereas a mutant lacking the hydrophobic domain fails to localize to LDs and to affect their morphology. Interestingly, coexpression of CGI-58 is able to promote LD turnover mediated by this ATGL mutant. Recently we have discovered that G0S2 acts as an inhibitor of ATGL activity and ATGL-mediated lipolysis. Here we show that G0S2 binds to ATGL irrelevantly of its activity state or the presence of CGI-58. In G0S2-expressing cells, the combined expression of CGI-58 and ATGL is incapable of stimulating LD turnover. We propose that CGI-58 and G0S2 regulate ATGL via non-competing mechanisms.
Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.
INTRODUCTION: Variants of PLA2G7 gene have been reported to be associated with coronary heart disease (CHD) since ten years ago, but the available data on this relationship are inconsistent. A meta-analysis was conducted to assess the effect of PLA2G7 gene on CHD. MATERIALS AND METHODS: Association studies were identified from the databases of PubMed, EMbase, Chinese National Knowledge Infrastructure (CNKI) and Wanfang by two investigators and pooled effects (odds ratio (OR), together with 95% confidence interval (CI)) were calculated. RESULTS: 14 association studies focusing on three polymorphisms (A379V, V279F and R92H) in PLA2G7 gene and risk of CHD were included in meta-analysis, covering a total of 8,280 cases and 5,656 controls. Concerning R92H, a significantly increased CHD risk was observed in recessive model, with an OR of 1.31(1.02, 1.68). Nevertheless, combined analyses of studies of the A379V and V279F variants showed no significant overall association with CHD, yielding ORs of 0.99(0.85, 1.15) and 1.09(0.88, 1.35) in allelic analysis, with strong evidence of heterogeneity. Similar results were also obtained in dominant and recessive models. CONCLUSIONS: The results indicate 92H allele had probably increased the risk of CHD, while the hypothesized effects of A379V and V279F polymorphisms on CHD cannot be confirmed in present data. However, given the limited number of studies and the potential biases, the influence of these polymorphisms on CHD risk needs further investigation.
Title: Identification of a novel splicing isoform of murine CGI-58 Yang X, Lu X, Liu J Ref: FEBS Letters, 584:903, 2010 : PubMed
The comparative gene identification-58 (CGI-58) gene, mutations of which are linked to Chanarin-Dorfman syndrome, encodes a protein of the alpha/beta hydrolase domain subfamily. We report here a new alternative splicing isoform of the murine CGI-58 gene, termed mCGI-58S. Sequence comparison indicates the lack of second and third exons in this cDNA variant. While the full-length protein displayed perilipin-dependent localization to lipid droplets, mCGI-58S showed a predominant cytoplasmic staining when expressed in cells. mCGI-58S was incapable of activating adipose triglyceride lipase but retained the capacity to acylate lysophosphatidic acid. Overexpression of mCGI-58S failed to promote lipid droplet turnover and loss of intracellular triacylglycerols. These results suggest that this splicing event may be involved in the regulation of lipid homeostasis.
Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.
Title: Associations of PLA2G7 gene polymorphisms with plasma lipoprotein-associated phospholipase A2 activity and coronary heart disease in a Chinese Han population: the Beijing atherosclerosis study Hou L, Chen S, Yu H, Lu X, Chen J, Wang L, Huang J, Fan Z, Gu D Ref: Hum Genet, 125:11, 2009 : PubMed
The human PLA2G7 gene encodes lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an emerging risk factor for cardiovascular diseases. In the present study, seven single nucleotide polymorphisms (SNPs) in the PLA2G7 gene were genotyped in 827 patients with coronary heart disease (CHD), of which 512 were patients with myocardial infarction (MI), and 947 age- and gender-matched controls in a Chinese Han population. Plasma Lp-PLA(2) activity was measured in 416 randomly selected controls and 689 randomly selected CHD patients, including 423 MI patients. Lp-PLA(2) activity in CHD and MI cases was significantly higher (233.42+/-57.66 and 234.27+/-59.51 nmol ml(-1) min(-1), respectively) than in controls (211.47+/-58.61 nmol ml(-1) min(-1)). After adjusting for traditional risk factors by logistic regression, the odds ratios for CHD and MI per 1 standard deviation increment of Lp-PLA(2) activity were 1.27 (95% CI, 1.07-1.50) and 1.27 (95% CI, 1.05-1.54), respectively. Both single SNP analysis and haplotype analysis showed that the V279F and I198T polymorphisms were significantly associated with the reduced Lp-PLA(2) activity, but neither was associated with increased CHD risk. Both univariate and multivariate analyses, adjusting effects of conventional factors, indicated that the rs13210554 T allele increased the risk of MI in this Chinese Han population. In summary, an independent association of increased plasma Lp-PLA(2) activity with CHD and MI existed in this Chinese Han Population. Although V279F and I198T mutations significantly decreased the activity of Lp-PLA(2), only the promoter rs13210554 polymorphism was associated with MI. Lp-PLA(2) activity appears to influence the CHD and MI risk in Chinese Han population.
The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.
Title: Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp Lu X, Zhang W, Jian J, Wu Q, Chen GQ Ref: FEMS Microbiology Letters, 243:149, 2005 : PubMed
Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.
Title: The strength of dehalogenase-substrate hydrogen bonding correlates with the rate of Meisenheimer intermediate formation Dong J, Lu X, Wei Y, Luo L, Dunaway-Mariano D, Carey PR Ref: Biochemistry, 42:9482, 2003 : PubMed
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site aspartate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion which forms the arylated intermediate (EAr). This is then hydrolyzed to the product. In this paper, we explore the relationship between active site polarizing forces acting on the benzoyl carbonyl and the rate of formation of the Meisenheimer complex. The polarizing forces at the C[double bond]O group were modulated by introducing site-selected mutations (A112V, Y65D, G113A, G113S, G113N, and F64P), near the C[double bond]O binding site. Using either the substrate, 4-CBA-CoA, or the substrate analogue, 4-methylbenzoyl-CoA (4-MBA-CoA), Raman difference spectroscopy provided the position of the C[double bond]O stretching frequency (nu(C)[double bond](O)) for a total of 10 enzyme-ligand complexes. In turn, the values of the C[double bond]O frequencies could be converted to differences in effective hydrogen bonding strengths between members of the series, based on earlier model studies [Clarkson, J., Tonge, P. J., Taylor, K. L., Dunaway-Mariano, D., and Carey, P. (1997) Biochemistry 36, 10192-10199]. Catalysis in the F64P, G113A, G113S, and G113N dehalogenase mutants was very slow with k(cat) values ranging from 8 x 10(-3) to 7.6 x 10(-6) s(-1). The EAr intermediate did not accumulate to a detectable level on these enzymes during a single turnover. Catalysis in the Y65D and A112V dehalogenase mutants were almost as efficient as catalysis in wild-type dehalogenase with k(cat) values of 0.1-0.6 s(-1). In wild-type dehalogenase, 22% of the bound substrate accumulated as the EAr intermediate during a single turnover (k(obs) for EAr formation = 24 s(-(1)); in the Y65D mutant, the level of accumulation is 17% (k(obs) for EAr formation = 3 s(-1)), and in the A112V mutant, the level is 23% (k(obs) for EAr formation = 17 s(-1)). The k(obs) for EAr formation in wild-type dehalogenase and the more active dehalogenase mutants (Y65D and A112V) was taken to be an estimate of the k for EMc formation, and the k(obs) for EP formation in a single turnover was taken to be an estimate of the k for EMc formation in the severely impaired mutants (F64P, G113A, G113S, and G113N). A plot of the log k(obs) for EMc formation versus the C[double bond]O stretching frequency of bound 4-CBA-CoA (or 4-MBA-CoA) is a straight line (R(2) = 0.9584). Throughout the series, nu(C)[double bond](O) varied by 61 cm(-1), corresponding to the change in hydrogen bonding enthalpy of 67 kJ/mol. The results show that changes in polarizing forces at the benzoyl carbonyl are transmitted to the benzoyl (4) position and correlate with the rate of aromatic nucleophilic addition five chemical bonds away. Interestingly, the relationship between effective polarizing forces and reactivity seen here for dehalogenase is similar to that reported for the addition-elimination reaction involving the hydrolysis of a series of acyl serine proteases.
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).
