Title: TPB-DMTP@S-CDs/MnO(2) Fluorescence Composite on a Dual-Emission-Capture Sensor Module for Fingerprint Recognition of Organophosphorus Pesticides Yuan L, Tian X, Fan Y, Sun Z, Zheng K, Zou X, Zhang W Ref: Analytical Chemistry, :, 2023 : PubMed
Residues of organophosphorus pesticides (OPs) raise considerable concern, while identifying OPs from unknown sources is still a challenge to onsite fluorescence techniques. Herein, a dual-emission-capture sensor module, based on a TPB-DMTP@S-CDs/MnO(2) fluorescence composite, is developed for OP fingerprint recognition. TPB-DMTP@S-CDs/MnO(2), synthesized by a hydrothermal method and self-assembly, is spectrographically validated as a dual-wavelength fluorescence source. OP-sensitive catalysis (acetylcholinesterase on acetylthiocholine chloride) is designed to regulate fluorescence by decomposing quenchable MnO(2). A flexibly fabricated sensor module supports the optimal dual-wavelength fluorescence excitations and captures and converts fluorescence emissions into equivalent photocurrents for feasible access. The most prominent finding is that dual-fluorescence emissions alternatively respond to levels, species, and multi-pH pretreatments of OPs due to varied MnO(2) sizes and distributions. Therefore, OP fingerprint recognition is conducted by refining the multidimensional information from fluorescence-triggered photocurrents and preset hydrolyzation using principal component analysis and the rule of maximum covariance. The recommended method provides a wide dynamic range (1 x 10(-6) - 12 microg mL(-1)), a good limit of detection (7.9 x 10(-7) microg mL(-1)), 15-day stability, and good selectivity to guarantee fingerprint recognition. For laboratory and natural samples, this method credibly identifies a single kind of OPs from multiple species at trace levels (10(-5) microg mL(-1)) and performs well in two-component and multicomponent analyses.
        
Title: Strategy to small intestine obstruction caused by Crohn's disease on the basis of transnasal ileus tube insertion Zuo L, Cao L, Ding C, Tu H, Wei C, Yuan L, Wang H, Zhang B Ref: BMC Surg, 22:183, 2022 : PubMed
BACKGROUND: Previous studies reported that transnasal ileus tube was a new and useful method for rapid relief of small intestinal obstruction. However, no study reported the impacts of the transnasal ileus tube for Crohn's disease combined with intestinal obstruction. We aimed to describe the strategy to the small intestine obstruction caused by Crohn's disease on the basis of transnasal ileus tube insertion. METHODS: From November 2019 to November 2021, the data of 6 hospitalized patients with CD, diagnosed and conservatively treated in The Second Hospital of Nanjing, were not relived and retrospectively collected. After the insertion of transnasal ileus tube, demographic information, clinical features and treatment data were extracted from medical records. RESULTS: Six Crohn's disease patients with intestinal obstruction were included. Half of them were male. The patients aged from 29 to 70 years. Five patients had chronic intestinal obstruction more than one year. Three patients had intestinal surgery history. One patient had colonic abdominal fistula and anastomotic fistula, when she took intermittent usage of sulfsalazine and steroid. On admission, all the patients had abdominal pain, distention and mass. Five patients had anemia, low albumin and cholinesterase. All CDAI scores were more than 400. Compared to 19 patients with incomplete intestinal obstruction improved by nasogastric decompression tube, 6 patients with intestinal obstruction catheter had significant difference in time for relieving abdominal pain and distension (p = 0.003), time for alleviating abnormal mass (p >= 0.01), drainage volume (p = 0.004), and preoperative CDAI score (p = 0.001). Compared with X-ray image before insertion, complete remission of obstruction of 5 patients were observed in intestinal cavity after insertion. After 1-2 months nutrition, all the patients had small intestine resection and ileostomy, half of them underwent colectomy and fistula repair, and 4 patients were performed enterolysis at the same time, the residual small intestine length ranging from 250 to 400 cm. 1 patient had permanent ileostomy;1 patient had abdominal infection after operation. The typical manifestations of acute and chronic inflammation, transmural inflammation, pseudopolyps and serous fiber hyperplasia could be seen in pathological findings of patients 1 to 5. All the patients continued enteral nutrition after surgery. Four patients were treated with infliximab or vedolizumab. CONCLUSION: The current intestinal obstruction catheter which is used to treat patients with Crohn's combined obstruction can afford quick clinical remission, longer nutrition time, and suitable preoperative CDAI score for operation, which is worthy of wildly being used.
        
Title: Clinical Evaluation and Test of a Modified Lp-PLA2 Kit in Diagnosing Atherosclerosis Yuan L, Hou L, Zhang L, Qin Z, Yu C Ref: Clin Lab, 67:, 2021 : PubMed
BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been identified as an inflammatory marker tightly correlated with the onset of atherosclerosis. Although several methodologies have been developed to detect Lp-PLA2, including enzyme-linked immunosorbent assay, Lp-PLA2 detection is still time- and resource-consuming with poor antiinterference ability and low sensitivity. Thus, it is urgent to explore new methodology for Lp-PLA2 detection. METHODS: In the current study, we evaluated the clinical performance of a modified Lp-PLA2 quantitative assay kit based on magnetic particle chemiluminescence, and analyzed the levels of Lp-PLA2 in atherosclerosis patients using this kit. RESULTS: Our results showed that the magnetic particle chemiluminescence method could effectively dissociate Lp-PLA2 from lipoprotein and finish the test within 20 minutes with high accuracy and good repeatability, as demonstrated by the results of linear measurement range, precision, and recovery rate. Furthermore, our preliminary data revealed that serum Lp-PLA2 levels were correlated to the presence and degree of atherosclerotic plaques. CONCLUSIONS: Lp-PLA2 could be helpful in diagnosing atherosclerosis.
        
Title: Fluoride reactivation-enabled sensitive quantification of tabun adducts on human serum albumin by GC-MS/MS via isotope dilution Li XS, Wu JN, Yan L, Xing ZF, Liu CC, Chen B, Yuan L, Yang Y Ref: Bioanalysis, 11:2145, 2019 : PubMed
Organophosphorus nerve agents inhibit the cholinesterase activity by phosphylation of the active site serine. The resulting phosphylated cholinesterase and adducts on human serum albumin (HSA) are appropriate biomarkers for nerve agents exposure. Several methods have been developed for the detection of nerve agents, including fluoride reactivation or alkaline cleavage. It was previously thought that some nerve agents adducts to HSA could not be detected via fluoride regeneration. In our study, the results showed that tabun (GA) adducts of HSA could be detected by fluoride regeneration. The sample preparation included acetone precipitation, washing and SPE. Deuterated tabun (d5-GA) was applied as the internal standard. The product of regenerated fluorotabun is detected with a good linearity (R(2) > 0.997) in the concentration range from 0.02 to 100.0 ng/ml, small relative standard deviation (=6.89%) and favorable recoveries between 94.8 and 106.3%. The established preparation confirmed the fluorotabun was regenerated from the GA-HSA adducts.
        
Title: High NDRG3 expression facilitates HCC metastasis by promoting nuclear translocation of beta-catenin Shi J, Zheng H, Yuan L Ref: BMB Rep, 52:451, 2019 : PubMed
NDRG1 has been reported to exert pivotal roles in tumor progression and metastasis via Wnt/beta-catenin signaling pathway. However, little is known about the role of NDRG3 in hepatocarcinogenesis despite its classification in the same subfamily of NDRG1. The present study was aimed to characterize the expression pattern and understand the biological roles of NDRG3 in hepatocarcinogenesis, as a means to exploit its therapeutic potential. It was observed that NDRG3 was up-regulated in HCC tissues and higher NDRG3 expression was associated with significantly shorter overall survival. Furthermore, a lower level of NDRG3 exhibited marked positive correlation with metastasis-free survival. In vitro and in vivo experiments revealed that knock-down of NDRG3 inhibits HCC metastasis and angiogenesis. We further demonstrated that activation of WNT/beta-catenin signaling and enhanced CSC-like properties were responsible for NDRG3- mediated promoting effect on HCC. In conclusion, the principal findings demonstrated that high NDRG3 expression facilitates HCC metastasis via regulating the turnover of beta-catenin, as well as provides a potential therapeutic target for future therapeutic interventions. [BMB Reports 2019; 52(7): 451-456].
In this study, the influence of bisphenol F (BPF) toward central nervous system (CNS) was assessed using zebrafish embryos. We found that BPF could induce significant neurotoxicity toward zebrafish embryos, including inhibited locomotion, reduced moving distance, and CNS cell apoptosis at an effective concentration of 0.0005 mg/L. Immunofluorescence assay showed that both microglia and astrocyte in zebrafish brain were significantly activated by BPF, indicating the existence of neuroinflammatory response. Peripheral motor neuron development was significantly inhibited by BPF at 72 hpf. RNA-seq data indicated that neuronal developmental processes and cell apoptosis pathways were significantly affected by BPF exposure, which was consistent with the phenotypic results. Chip-seq assay implied that the transcriptional changes were not mediated by ERalpha. Additionally, no significant change was found in neurotransmitter levels (5-hydroxytryptamine, dopamine, and acetylcholine) or acetylcholinesterase (Ache) enzyme activity after BPF exposure, indicating that BPF may not affect neurotransmission. In conclusion, BPF could lead to abnormal neural outcomes during zebrafish early life stage through inducing neuroinflammation and CNS cell apoptosis even at environmentally relevant concentration.
        
Title: Genetic analysis of the RIC3 gene in Han Chinese patients with Parkinson's disease He D, Hu P, Deng X, Song Z, Yuan L, Yuan X, Deng H Ref: Neuroscience Letters, 653:351, 2017 : PubMed
Parkinson's disease (PD) is the second-most common etiologically complex neurodegenerative disease. Genetic abnormalities are thought to play an important role in the development of PD. Recently, mutations in the resistance to inhibitors of cholinesterase 3 gene (RIC3) have been reported to cause autosomal-dominant PD in Indian population. To determine whether RIC3 gene coding variant(s) are associated with PD in Han Chinese population, the RIC3 gene coding region in 218 mainland Han Chinese patients with PD and the identified variants in 242 normal controls were examined using direct sequencing analysis. Four known single nucleotide variants (c.354C>A, p.L118L, rs10839976; c.389G>A, p.C130Y, rs55990541; c.403C>T, p.P135S, rs73411617; and c.1054G>A, p.D352N, rs11826236) were identified in the RIC3 gene coding region. No significant differences were observed in either genotypic or allelic distributions between the PD patients and the normal controls (all P>0.05) for these four variants. Haplotype analysis showed that the presence of haplotype A-G-C-G (rs10839976-rs55990541-rs73411617-rs11826236) was associated with a 0.764-fold decreased risk (P=0.049, OR=0.764, 95% CI=0.585-0.999) for PD, whereas the presence of haplotype C-A-C-A was associated with a 2.143-fold increased risk (P=0.039, OR=2.143, 95% CI=1.023-4.488) for PD. The findings indicate that four variants: rs10839976, rs55990541, rs73411617 and rs11826236 in the RIC3 gene coding region may play little or no role in the development of PD. Two RIC3 gene haplotypes of four variants: A-G-C-G, and C-A-C-A might relate to either protection against or increased susceptibility to PD in the Han Chinese population, respectively.
        
Title: Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS Liu CC, Huang GL, Xi HL, Liu SL, Liu JQ, Yu HL, Zhou SK, Liang LH, Yuan L Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 1036-1037:57, 2016 : PubMed
This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2>/=0.995), and 3.00-200ngmL-1 for BChE NP (R2>/=0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.
        
Title: Targeting neurotrophic factors and their receptors, but not cholinesterase or neurotransmitter, in the neurotoxicity of TDCPP in Chinese rare minnow adults (Gobiocypris rarus) Yuan L, Li J, Zha J, Wang Z Ref: Environ Pollut, 208:670, 2016 : PubMed
Organophosphate flame retardants (OPFRs) have been detected at high concentrations in various environmental and biotic samples, but little is known about their toxicity. In this study, the potential neurotoxicity of three OPFRs (TCEP, TDCPP, and TPP) and Chlorpyrifos (CPF, an organophosphate pesticide) were compared in Chinese rare minnow using an acute toxicity test and a 21-day fish assay. The acute test demonstrated significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) by CPF. Although significant AChE inhibition at high concentration of TPP was also observed, none of the OPFRs had effects similar to CPF on these enzymes, indicating that their acute toxicities to Chinese rare minnow may be unrelated to cholinesterase inhibition. In addition, the 21-day fish assay with TDCPP demonstrated no significant effects on cholinesterase activities or neurotransmitter levels. Nonetheless, this OPFR exhibited widespread effects on the neurotrophic factors and their receptors (e.g., ntf3, ntrk1, ntrk2, ngfr, and fgf2, fgf11, fgf22, fgfr4), indicating that TDCPP or other OPFRs may elicit neurological effects by targeting neurotrophic factors and their receptors in Chinese rare minnow.
        
Title: Inhibition of Acetylcholinesterase (AChE): A Potential Therapeutic Target to Treat Alzheimer's Disease Li Y, Zhang XX, Jiang LJ, Yuan L, Cao TT, Li X, Dong L, Yin SF Ref: Chemical Biology Drug Des, 86:776, 2015 : PubMed
A new series of icariin derivatives were synthesized and evaluated for their in vitro acetylcholinesterase (AChE) inhibitory activity. Most of the tested compounds exhibited high AChE inhibition and low toxicity, and among which compounds 1, 2, and 10 were the most potent (IC50 = 71.52 +/- 22.43, 8.28 +/- 1.45, 5.830 +/- 1.78 nm, respectively).
        
Title: Fast identification of lipase inhibitors in oolong tea by using lipase functionalised Fe3O4 magnetic nanoparticles coupled with UPLC-MS/MS Zhu YT, Ren XY, Yuan L, Liu YM, Liang J, Liao X Ref: Food Chem, 173:521, 2015 : PubMed
Oolong tea is an important member in tea family, which claims for various health benefits such as preventing obesity and improving lipid metabolism. In this work, using pancreatic lipase (PL) functionalised magnetic nanoparticles (PL-MNPs) as solid phase extraction absorbent in combination with ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), we developed a method for rapid screening and identification of lipase inhibitors from oolong tea. Three PL ligands were selectively extracted and identified as (-)-epigallocatechin-3-O-gallate (EGCG), (-)-gallocatechin-3-O-gallate (GCG) and (-)-epicatechin-3-O-gallate (ECG). Their lipase inhibitory activities were significantly higher than those non-ligands. Structure-activity analysis revealed that the presence of a galloyl moiety in the structure was required for binding to PL-MNPs, and therefore, exhibiting a strong inhibition on the enzyme. Taking advantages of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for fast screening of lipase inhibitors from natural resources.
        
Title: Use of a carboxylesterase inhibitor of phenylmethanesulfonyl fluoride to stabilize epothilone D in rat plasma for a validated UHPLC-MS/MS assay Yuan L, Fu Y, Zhang D, Xia YQ, Peng Q, Aubry AF, Arnold ME Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 969C:60, 2014 : PubMed
A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x2 weighted linear regression model. The intra-assay precision was within +/-3.6% CV and inter-assay precision was within +/-4.2% CV. The assay accuracy was within +/-8.3% of the nominal values. Assay recovery of EpoD was high ( approximately 90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20 degrees C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.
Recently, 5 single nucleotide polymorphisms (SNPs), rs2306677 in the inositol 1,4,5-triphosphate receptor 2 gene (ITPR2), rs1541160 in the kinesin-association protein 3 gene (KIFAP3), rs6690993 and rs6700125 in the FLJ10986 gene, and rs10260404 in the dipeptidyl-peptidase 6 gene (DPP6) have been reported to be associated with the risk of developing sporadic amyotrophic lateral sclerosis (SALS) in Caucasian populations. However, this association is not consistent among different studies and yet to be tested in Chinese SALS patients. We examined the above SNPs in a large cohort consisting of 395 SALS patients and 288 controls from Southwest China. Our results suggest that these SNPs are unlikely to be a common cause of SALS in Chinese populations.
BACKGROUND: Hazelnut (Corylus avellana) decline disease in Greece and Italy is caused by the convergent evolution of two distantly related lineages of Pseudomonas syringae pv. avellanae (Pav). We sequenced the genomes of three Pav isolates to determine if their convergent virulence phenotype had a common genetic basis due to either genetic exchange between lineages or parallel evolution. RESULTS: We found little evidence for horizontal transfer (recombination) of genes between Pav lineages, but two large genomic islands (GIs) have been recently acquired by one of the lineages. Evolutionary analyses of the genes encoding type III secreted effectors (T3SEs) that are translocated into host cells and are important for both suppressing and eliciting defense responses show that the two Pav lineages have dramatically different T3SE profiles, with only two shared putatively functional T3SEs. One Pav lineage has undergone unprecedented secretome remodeling, including the acquisition of eleven new T3SEs and the loss or pseudogenization of 15, including five of the six core T3SE families that are present in the other Pav lineage. Molecular dating indicates that divergence within both of the Pav lineages predates their observation in the field. This suggest that both Pav lineages have been cryptically infecting hazelnut trees or wild relatives for many years, and that the emergence of hazelnut decline in the 1970s may have been due to changes in agricultural practice. CONCLUSIONS: These data show that divergent lineages of P. syringae can converge on identical disease etiology on the same host plant using different virulence mechanisms and that dramatic shifts in the arsenal of T3SEs can accompany disease emergence.
Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.
To examine the protective efficacy of paraoxonase-1 (PON1) against tissue damage caused by dichlorvos, purified rabbit PON1 was injected intravenously into rats 30min before they were given dichlorvos, while dichlorvos administration group and corn coil administration group were conducted to compare. Blood was collected at different time points after dichlorvos administration to examine the acetyl cholinesterase (AChE) inhibition level and clinical signs were observed after poisoning. 72h later, animals were anesthetized and the hippocampus, liver, lung and kidney were removed for observation of ultrastructure. AChE activities in PON1 pretreament group were statistically significant from dichlorvos administration group (P<0.01). The clinical signs were alleviated by PON1 significantly (P<0.05). The most common change of organophosphorus poisoning damage to liver was small lipid-like structures could be seen throughout the liver structure. In kidney, dense bodies were seen. The most significant changes in lung were lost of lamellar structure of lamellar bodies in type II alveolar epithelial cell. As for changes of hippocampus, demyaliation takes place after acute organophosphorus, but neural edema was not improved significantly in our study. In conclusion, PON1 can decrease the AChE inhibition, and alleviated clinical signs and tissue damage caused by dichlorvos.
We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.
        
Title: The catalytic cysteine and histidine in the plant acyl-acyl carrier protein thioesterases Yuan L, Nelson BA, Caryl G Ref: Journal of Biological Chemistry, 271:3417, 1996 : PubMed
The plant acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of their utilities in the genetic engineering of plant seed oils. Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis, suggesting that these enzymes activate the hydrolysis of the thioester bond using the same basic catalytic machinery as those of proteases and lipases. To identify the cysteine and histidine residues that are critical in catalysis we substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine (Cys-320) to an Ala or a Ser, and three conserved histidines (His-140, His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a substantial loss of enzyme activity. However, only C320A and H285A completely inactivated the enzyme, indicating that these two residues are essential for catalysis. Considerable activity (>60%) still remained when Cys-320 was converted to a Ser, but this mutant (C320S) displayed a reversed sensitivity toward thiol or serine hydroxyl inhibitors compared with the wild-type enzyme. A pH optimal study demonstrates that while the wild-type enzyme has the highest activity between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to higher pH and a significant increase of activity around pH 12. This result suggests that Arg-285 (pKa 12) is deprotonated at high pH, thus partially mimicking the role of His-285 for proton abstraction in the wild-type enzyme. We conclude that the Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can attack the thioester bond of the substrate 12:0 ACP, assisted by His-285. Because plant TEs are highly conserved in length and sequence and the residues investigated here are completely conserved in all available TEs, it is reasonable to believe that homologues of Cys-320 and His-285 are present in the active sites of all plant acyl-ACP TEs.