Title: Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method Li L, Ding L, Shao Y, Sun S, Wang M, Xiang J, Zhou J, Wu G, Song Z, Xin Z Ref: Foods, 12:1169, 2023 : PubMed
In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9-118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay revealed that the activity of DLFae4 was as high as 1550.796 U/mg and enhanced by 375.49% (5823.172 U/mg) toward 4-nitrophenyl acetate when residue Ala-341 was mutated to glycine (A341G), and the corresponding acyl transfer efficiency was increased by 7.7 times, representing the highest acyltransferase activity to date, and demonstrating that the WGG motif was pivotal for the acyltransferase activity in family VIII carboxylesterases. Further experiments indicated that DLFae4 and variant DLFae4 (A341G) could acylate cyanidin-3-O-glucoside effectively in aqueous solution. Taken together, our study suggested the effectiveness of error-prone PCR and site-directed saturation mutation to increase the specific activity of enzymes and may facilitate the practical application of this critical feruloyl esterase.
Title: Inhibition of Caspase-11-Mediated Pyroptosis Alleviates Acute Kidney Injury Associated with Severe Acute Pancreatitis in Rats Shao Y, Li C, Jiang Y, Li H, Tang X, Gao Z, Zhang D Ref: J Invest Surg, 36:1, 2023 : PubMed
Background: Acute kidney injury (AKI) is a common complication in patients with severe acute pancreatitis (SAP). Caspase-11-mediated pyroptosis is essential for the progression of multiple diseases, but its role in SAP-induced AKI remains unknown.Aims: This research investigated whether caspase-11-mediated pyroptosis is involved in SAP-induced AKI and whether inhibiting caspase-11-mediated pyroptosis improves SAP-induced AKI.Methods: A rat model of SAP with AKI was established by slowly injecting 5% sodium taurocholate into the biliopancreatic duct, then wedelolactone (25 or 50 mg/kg), an inhibitor of caspase-11, was injected through the intra-peritoneum 1 and 6 h after SAP induction. Serum biochemical indexes, including serum amylase, lipase, interleukin (IL)-6, blood urea nitrogen (BUN), tumor necrosis factor (TNF)-alpha, and creatinine (Cr) in rats, were evaluated using biochemical test kits. Caspase-11 and gasdermin D (GSDMD) expression in the kidney tissues was evaluated by western blotting and immunohistochemical staining. IL-1 and IL-18 levels in kidney tissues were detected by ELISA kits. Furthermore, histopathological alterations of pancreas and kidney were assessed by H&E staining.Results: The serum biochemical indexes and pyroptosis-related proteins in kidney tissues were significantly increased after SAP induction. Furthermore, wedelolactone decreased the expression of pyroptosis-linked proteins in kidney tissues, reduced serum lipase, amylase, IL-6, TNF-alpha, BUN, and Cr, and ameliorated the renal and pancreatic histological damage in SAP rats.Conclusion: Caspase-11-mediated pyroptosis contributes to SAP-induced AKI, and targeting caspase-11-mediated pyroptosis might be a novel treatment strategy for SAP-induced AKI.
Monoacylglycerol lipase (MAGL) constitutes a serine hydrolase that orchestrates endocannabinoid homeostasis and exerts its function by catalyzing the degradation of 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA). As such, selective inhibition of MAGL represents a potential therapeutic and diagnostic approach to various pathologies including neurodegenerative disorders, metabolic diseases and cancers. Based on a unique 4-piperidinyl azetidine diamide scaffold, we developed a reversible and peripheral-specific radiofluorinated MAGL PET ligand [(18)F]FEPAD. Pharmacokinetics and binding studies on [(18)F]FEPAD revealed its outstanding specificity and selectivity towards MAGL in brown adipose tissue (BAT) - a tissue that is known to be metabolically active. We employed [(18)F]FEPAD in PET studies to assess the abundancy of MAGL in BAT deposits of mice and found a remarkable degree of specific tracer binding in the BAT, which was confirmed by post-mortem tissue analysis. Given the negative regulation of endocannabinoids on the metabolic BAT activity, our study supports the concept that dysregulation of MAGL is likely linked to metabolic disorders. Further, we now provide a suitable imaging tool that allows non-invasive assessment of MAGL in BAT deposits, thereby paving the way for detailed mechanistic studies on the role of BAT in endocannabinoid system (ECS)-related pathologies.
As a serine hydrolase, monoacylglycerol lipase (MAGL) is principally responsible for the metabolism of 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS), leading to the formation of arachidonic acid (AA). Dysfunction of MAGL has been associated with multiple CNS disorders and symptoms, including neuroinflammation, cognitive impairment, epileptogenesis, nociception and neurodegenerative diseases. Inhibition of MAGL provides a promising therapeutic direction for the treatment of these conditions, and a MAGL positron emission tomography (PET) probe would greatly facilitate preclinical and clinical development of MAGL inhibitors. Herein, we design and synthesize a small library of fluoropyridyl-containing MAGL inhibitor candidates. Pharmacological evaluation of these candidates by activity-based protein profiling identified 14 as a lead compound, which was then radiolabeled with fluorine-18 via a facile S(N)Ar reaction to form 2-[(18)F]fluoropyridine scaffold. Good blood-brain barrier permeability and high in vivo specific binding was demonstrated for radioligand [(18)F]14 (also named as [(18)F]MAGL-1902). This work may serve as a roadmap for clinical translation and further design of potent (18)F-labeled MAGL PET tracers.
Monoacylglycerol lipase (MAGL) is a 33 kDa serine protease primarily responsible for hydrolyzing 2-arachidonoylglycerol into the proinflammatory eicosanoid precursor arachidonic acid in the central nervous system. Inhibition of MAGL constitutes an attractive therapeutic concept for treating psychiatric disorders and neurodegenerative diseases. Herein, we present the design and synthesis of multiple reversible MAGL inhibitor candidates based on a piperazinyl azetidine scaffold. Compounds 10 and 15 were identified as the best-performing reversible MAGL inhibitors by pharmacological evaluations, thus channeling their radiolabeling with fluorine-18 in high radiochemical yields and favorable molar activity. Furthermore, evaluation of [(18)F]10 and [(18)F]15 ([(18)F]MAGL-2102) by autoradiography and positron emission tomography (PET) imaging in rodents and nonhuman primates demonstrated favorable brain uptakes, heterogeneous radioactivity distribution, good specific binding, and adequate brain kinetics, and [(18)F]15 demonstrated a better performance. In conclusion, [(18)F]15 was found to be a suitable PET radioligand for the visualization of MAGL, harboring potential for the successful translation into humans.
Title: Evaluation of Sensitivity to Phoxim and Cypermethrin in an Endoparasitoid, Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae), and Its Parasitization Efficiency Under Insecticide Stress Sheng S, Wang J, Zhang XR, Liu ZX, Yan MW, Shao Y, Zhou JC, Wu FA Ref: J Insect Sci, 21:, 2021 : PubMed
Insecticides can have consequences for beneficial arthropods. Insect parasitoids can contact insecticides through direct exposure spray droplets or residues on crop foliage. Here, we focus on better understand the response of Meteorus pulchricornis (Wesmael), a parasitoid wasp of lepidopteran pests, and its detoxification mechanisms on stress caused by phoxim and cypermethrin. Hence, we determined the dose-mortality curves and estimating the sublethal concentrations (LC30 and LC50). Then, we applied the sublethal concentrations against adult parasitoids to assess its survival, parasitism efficacy, and also developmental and morphometric parameters of their offspring. Simultaneously, we check the activities of glutathione S-transferase (GST), acetylcholinesterase (AChE), and peroxidase (POD) after sublethal exposure of both insecticides, which has measured until 48 h after treatment. Overall, phoxim and cypermethrin exhibited acute lethal activity toward the parasitoid with LC50 values 4.608 and 8.570 mg/liter, respectively. Also, we detect that LC30 was able to trigger the enzymatic activity of GST, AChE, and POD, suggesting a potential detoxification mechanism. However, even when subjected to sublethal exposure, our results indicate strong negatives effects, in particular for phoxim, which has affected the parasitism efficacy and also the developmental and morphometric parameters of M. pulchricornis offspring. Therefore, it can be concluded that both phoxim and cypermethrin have negative impacts on M. pulchricornis and we suggest cautioning their use and the need for semifield and field assessments to confirm such an impact.
Title: Characterization of a novel carboxylesterase with catalytic activity toward di(2-ethylhexyl) phthalate from a soil metagenomic library Yan Z, Ding L, Zou D, Qiu J, Shao Y, Sun S, Li L, Xin Z Ref: Sci Total Environ, 785:147260, 2021 : PubMed
A novel carboxylesterase gene estyz5 was isolated from a soil metagenomic library. The recombinant enzyme EstYZ5 is 298 amino acids in length with a predicted molecular weight of 32 kDa. Sequence alignment and phylogenetic analysis revealed that EstYZ5 belongs to the hormone-sensitive lipase (HSL) family with a deduced catalytic triad of Ser144-Glu238-His268. EstYZ5 contains two conserved motifs, a pentapeptide motif GDSAG and a HGGG motif, which are typically found in members of the HSL family. Esterolytic activity of the recombinant enzyme was optimal at 30 degreesC and pH 8.0, and the kcat/Km value of the enzyme for the optimum substrate p-nitrophenyl butyrate was as high as 1272 mM(-)(1).s(-1). Importantly, EstYZ5 showed activity toward di(2-ethylhexyl) phthalate with complex side chains, which is rare for HSLs. Molecular docking simulations revealed that the catalytic triad and an oxyanion hole likely play vital roles in enzymatic activity and specificity. The phthalate-degrading activity of EstYZ5, combined with its high levels of esterolytic activity, render this new enzyme a candidate for biotechnological applications.
Title: Identification and characterization of a novel phthalate-degrading hydrolase from a soil metagenomic library Qiu J, Zhang Y, Shi Y, Jiang J, Wu S, Li L, Shao Y, Xin Z Ref: Ecotoxicology & Environmental Safety, 190:110148, 2020 : PubMed
Phthalate esters have raised public concerns owing to their effects on the environment and human health. We identified a novel phthalate-degrading hydrolase, EstJ6, from a metagenomic library using function-driven screening. Phylogenetic analysis indicated that EstJ6 is a member of family IV esterases. EstJ6 hydrolyzed various dialkyl and monoalkyl phthalate esters, and exhibited high hydrolytic activity (128 U/mg) toward dibutyl phthalate at 40 degrees C and pH 7.5. EstJ6 hydrolyzed not only common phthalate esters with simple side chains but also diethylhexyl phthalate and monoethylhexyl phthalate, which have complex and long side chains. Site-directed mutagenesis indicated that the catalytic triad residues of EstJ6 consists of Ser146, Glu240, and His270. EstJ6 is therefore a promising biodegradation enzyme, and our study illustrates the advantages of a metagenomic approach in identifying enzyme-coding genes for agricultural, food, and biotechnological applications.
A fosmid metagenomic library containing 9.7 x 10(4) clones was constructed. A novel esterase, XtjR8, was isolated through functional screening. XtjR8 shared the maximum amino acid identity (44%) with acetyl-hydrolase from Streptomyces hygroscopicus, and was classified into family IV esterase. XtjR8 exhibited the highest hydrolytic activity for p-nitrophenyl acetate at 40 degreesC and pH 8.0, and presented more than 40% activity from 20 degreesC to 80 degreesC. More importantly, XtjR8 displayed the ability to hydrolyze both phthalate monoesters and diesters, this feature is extremely rare among previously reported esterases. Site-directed mutagenesis experiments revealed that the catalytic triad residues were Ser152, Glu246, and His276. Among them, Ser152 formed a hydrogen bond with dibutyl phthalate (DBP) by molecular docking, Gly84, Gly85, and Leu248 of conserved motifs formed hydrophobic interactions with DBP, respectively, which were important for the catalytic activity. Considering its wide range of temperature and hydrolytic potential toward phthalate esters, XtjR8 will be served as an interesting candidate for biodegradation and industrial applications.
Monoacylglycerol lipase (MAGL) is a serine hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with (11)C or (18)F. [(11)C]8 ([(11)C]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [(11)C]17 ([(11)C]PAD) and [(18)F]37 ([(18)F]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.
Dysfunction of monoacylglycerol lipase (MAGL) is associated with several psychopathological disorders, including drug addiction and neurodegenerative diseases. Herein we design, synthesize, and evaluate several irreversible fluorine-containing MAGL inhibitors for positron emission tomography (PET) ligand development. Compound 6 (identified from a therapeutic agent) was advanced for (18)F-labeling via a novel spirocyclic iodonium ylide (SCIDY) strategy, which demonstrated high brain permeability and excellent specific binding. This work supports further development of novel (18)F-labeled MAGL PET probes.
Title: Catalpol prevents denervated muscular atrophy related to the inhibition of autophagy and reduces BAX/BCL2 ratio via mTOR pathway Wang Y, Shao Y, Gao Y, Wan G, Wan D, Zhu H, Qiu Y, Ye X Ref: Drug Des Devel Ther, 13:243, 2019 : PubMed
Aim: To investigate the effects of catalpol on muscular atrophy induced by sciatic nerve crush injury (SNCI). Methods: Seventy male Kunming mice were randomized into five groups (n=10): model, sham, catalpol (Cat), rapamycin (Rapa), and catalpol+rapamycin (Rapa+Cat). The ratio of gastrocnemius muscle wet weight (right/left, R/L) between the operated leg (right) and the normal leg (left) was calculated, and acetylcholinesterase (AChE) immunohistochemistry assays were performed to observe the change of motor end plate (MEP), along with the sizes of denervated and innervated muscle fibers. The expression levels of LC3II, TUNEL, BAX/BCL-2, LC3II/LC3I and P62, Beclin1, mTOR, and p-mTOR (ser2448) proteins in muscle were examined by fluorescence immunohistochemistry or Western blotting. Results: Results show that catalpol improved the results of the grid walking tests by reducing the percentage of foot slips, which increased the gastrocnemius muscle wet weight (R/L), enhanced AChE expression at the MEP, and enlarged the section area of the muscle. The expression of LC3II and TUNEL was significantly inhibited by catalpol. The BAX/BCL-2 ratio was significantly increased in muscles of denervated and control groups. Lower LC3II/LC3I and BAX/BCL-2 ratios in denervated muscles were also detected after catalpol treatment. Conclusion: These results indicated that apoptosis and autophagy play a role in the regulation of denervation-induced muscle atrophy after SNCI, and catalpol alleviates muscle atrophy through the regulation of muscle apoptosis and autophagy via the mTOR signaling pathway.
Title: Self-reduction bimetallic nanoparticles on ultrathin MXene nanosheets as functional platform for pesticide sensing Zhao F, Yao Y, Jiang C, Shao Y, Barcelo D, Ying Y, Ping J Ref: J Hazard Mater, 384:121358, 2019 : PubMed
Two-dimensional (2D) transition metal carbides and nitrides, named MXene, appear promising application prospects in sensor filed. Metal nanoparticles, especially bimetallic nanoparticles, are the superior nanocatalyst, which process excellent features due to the high specific surface area and synergistic catalytic capacity. Using ultrathin MXene nanosheets as the natural reducing agent and support, we prepare the shape-controlled Au-Pd bimetallic nanoparticles via a self-reduction process at room temperature in a short time, which can well enhance the catalytic performance and are benefit for the acetylcholinesterase immobilization. Based on their desired properties, we propose a disposable electrochemical biosensor for the detection of organophosphorus pesticide using the multi-dimensional nanocomposites (MXene/Au-Pd) as the functional platform. Under the optimized conditions, our fabricated biosensor exhibits a favorable linear relationship with the concentration of paraoxon from 0.1 to 1000mugL(-1), with a low detection limit of 1.75ng L(-1). Furthermore, the biosensor can be applied for paraoxon detection in pear and cucumber samples, providing an effective and useful avenue for the applicability of novel 2D nanomaterials in biosensing field.
Title: Neuroligin 3 R451C mutation alters electroencephalography spectral activity in an animal model of autism spectrum disorders Liu JJ, Grace KP, Horner RL, Cortez MA, Shao Y, Jia Z Ref: Mol Brain, 10:10, 2017 : PubMed
Human studies demonstrate that sleep impairment is a concurrent comorbidity of autism spectrum disorders (ASD), but its etiology remains largely uncertain. One of the prominent theories of ASD suggests that an imbalance in synaptic excitation/inhibition may contribute to various aspects of ASD, including sleep impairments. Following the identification of Nlgn3R451C mutation in patients with ASD, its effects on synaptic transmission and social behaviours have been examined extensively in the mouse model. However, the contributory role of this mutation to sleep impairments in ASD remains unknown. In this study, we showed that Nlgn3R451C knock-in mice, an established genetic model for ASD, exhibited normal duration and distribution of sleep/wake states but significantly altered electroencephalography (EEG) power spectral profiles for wake and sleep.
The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from -3.6 to 9.4 (at pH7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1ngL-1 to 2400ngL-1). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes.
Surface waters can contain a range of micropollutants from point sources, such as wastewater effluent, and diffuse sources, such as agriculture. Characterizing the source of micropollutants is important for reducing their burden and thus mitigating adverse effects on aquatic ecosystems. In this study, chemical analysis and bioanalysis were applied to assess the micropollutant burden during low flow conditions upstream and downstream of three wastewater treatment plants (WWTPs) discharging into small streams in the Swiss Plateau. The upstream sites had no input of wastewater effluent, allowing a direct comparison of the observed effects with and without the contribution of wastewater. Four hundred and five chemicals were analyzed, while the applied bioassays included activation of the aryl hydrocarbon receptor, activation of the androgen receptor, activation of the estrogen receptor, photosystem II inhibition, acetylcholinesterase inhibition and adaptive stress responses for oxidative stress, genotoxicity and inflammation, as well as assays indicative of estrogenic activity and developmental toxicity in zebrafish embryos. Chemical analysis and bioanalysis showed higher chemical concentrations and effects for the effluent samples, with the lowest chemical concentrations and effects in most assays for the upstream sites. Mixture toxicity modeling was applied to assess the contribution of detected chemicals to the observed effect. For most bioassays, very little of the observed effects could be explained by the detected chemicals, with the exception of photosystem II inhibition, where herbicides explained the majority of the effect. This emphasizes the importance of combining bioanalysis with chemical analysis to provide a more complete picture of the micropollutant burden. While the wastewater effluents had a significant contribution to micropollutant burden downstream, both chemical analysis and bioanalysis showed a relevant contribution of diffuse sources from upstream during low flow conditions, suggesting that upgrading WWTPs will not completely reduce the micropollutant burden, but further source control measures will be required.
Title: Effects of Light Intensity and Color on the Biomass, Extracellular Red Pigment, and Citrinin Production of Monascus ruber Wang L, Dai Y, Chen W, Shao Y, Chen F Ref: Journal of Agricultural and Food Chemistry, 64:9506, 2016 : PubMed
Light is a crucial environmental signal for fungi. In this work, the effects of different light intensities and colors on biomass, Monascus pigments (MPs) and citrinin production of Monascus ruber M7 were investigated. We have demonstrated that low intensity of blue light (500 lx) decreased Monascus biomass, increased MPs accumulation via upregulation of MpigA, MpigB, and MpigJ genes expression, but had no significant influence on citrinin production. High intensity of blue light (1500 lx) decreased citrinin accumulation but had no significant influence on biomass and MPs production after 14 days cultivation. Low intensity of green light (500 lx) stimulated citrinin production via upregulation of pksCT, mrl1, mrl2, and ctnA genes expression. One putative red light photoreceptor and two putative green light photoreceptors were identified in M. ruber M7. These observations will not only guide the practical production of Monascus but also contribute to our understanding light effects on Monascus.
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.
Title: Inhibition of miR-134 Protects Against Hydrogen Peroxide-Induced Apoptosis in Retinal Ganglion Cells Shao Y, Yu Y, Zhou Q, Li C, Yang L, Pei CG Ref: Journal of Molecular Neuroscience, 56:461, 2015 : PubMed
MicroRNAs (miRNAs) have been suggested to play an important role in neurological diseases. Particularly, miR-134 is reportedly involved in regulating neuron survival. However, the association between miR-134 and retinal ganglion cell (RGC) survival under adverse stimulus has not been extensively investigated. In this study, we aimed to explore the role and underlying mechanism of miR-134 in regulating RGC apoptosis in response to hydrogen peroxide (H2O2) treatment. Results showed that the expression of miR-134 dose- and time-dependently increased in RGC after H2O2 treatment. H2O2-induced RGC apoptosis was significantly attenuated by the inhibition of miR-134 expression by antagomiR-134 and was enhanced by miR-134 overexpression. Luciferase reporter assay revealed a direct interaction between miR-134 and the 3'-untranslated region of cyclic AMP-response element-binding protein (CREB), a critical transcription factor for neuronal protection. In H2O2-treated RGCs, the inhibition of miR-134 significantly elevated the expression of CREB and its downstream genes, including brain-derived neurotrophic factor (BDNF) and Bcl-2. Furthermore, the inhibition of miR-134 also increased the expression of miR-132, a rapid response gene downstream of CREB. In addition, the target gene of miR-132, acetylcholinesterase was expectedly decreased by miR-134 inhibition. However, the overexpression of miR-134 exerted an opposite effect. The knockdown of CREB apparently abolished the protective effect of miR-134 inhibition against H2O2-induced RGC apoptosis. The increased expression of BDNF and Bcl-2 induced by miR-134 inhibition was also abrogated by CREB knockdown. Overall, our results suggested that the downregulation of miR-134 can effectively protect against H2O2-induced RGC apoptosis by negatively modulating CREB expression.
Title: The protective role of tacrine and donepezil in the retina of acetylcholinesterase knockout mice Yi YM, Cai L, Shao Y, Xu M, Yi JL Ref: Int J Ophthalmol, 8:884, 2015 : PubMed
AIM: To determine the effect of different concentrations of the acetylcholinesterase (AChE) inhibitors tacrine and donepezil on retinal protection in AChE(+/-) mice (AChE knockout mice) of various ages. METHODS: Cultured ARPE-19 cells were treated with hydrogen peroxide (H2O2) at concentrations of 0, 250, 500, 1000 and 2000 micromol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil (0.1 mg/mL, 0.2 mg/mL and 0.4 mg/mL) were respectively given to AChE(+/-) mice aged 2mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline (PBS) was administered to the control group. The mice were sacrificed after 30d by in vitro cardiac perfusion and retinal samples were taken. AChE-deficient mice were identified by polymerase chain reaction (PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website. H&E staining, immunofluorescence and Western blot were performed to observe AChE protein expression changes in the retinal pigment epithelial (RPE) cell layer. RESULTS: Different concentrations of H2O2 induced AChE expression during RPE cell apoptosis. AChE(+/-) mice retina were thinner than those in wild-type mice (P<0.05); the retinal structure was still intact at 2mo but became thinner with increasing age (P<0.05); furthermore, AChE(+/-) mice developed more slowly than wild-type mice (P<0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology (P>0.05). CONCLUSION: In vivo, tacrine and donepezil can inhibit the expression of AChE; the decrease of AChE expression in the retina is beneficial for the development of the retina.
Title: Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides Zhang Y, Shao Y, Jiang F, Li J, Liu Z Ref: Insect Biochemistry & Molecular Biology, 46C:25, 2014 : PubMed
Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, Vmax and Km, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in Ki ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.
Title: Acetylcholinesterase function in apoptotic retina pigment epithelial cells induced by H2O2 Cai L, Liao HF, Zhang XJ, Shao Y, Xu M, Yi JL Ref: Int J Ophthalmol, 6:772, 2013 : PubMed
AIM: To investigate the acetylcholinesterase (AChE) expression involved in retina pigment epithelial (RPE) apoptosis induced by higher concentrations H2O2. METHODS: The human retinal pigment epithelium cell line ARPE-19 was from ATCC (Rockville, MD). Cultured ARPE-19 cells were treated with H2O2 at 0, 250, 500, 1 000, 2 000micromol/L and cell viability was measured with MTT assay. AChE expression and DNA fragments were analyzed by immunocytochemistry, TUNEL and PARP-1 Western blotting. RESULTS: Immunofluorescence detected AChE exist in the normal human retinal tissue. When H2O2 >500micromol/L, AChE expression showed an increase after 2h, and this concentration was selected for the present study. RPE cell was induced with 1 000micromol/L H2O2 for 2h, compared to the control group, cell activity decline detected by MTT, AChE and PARP-1 protein expression was significantly increased detected by Western blotting. AChE immunofluorescence staining was positive in RPE cell after H2O2 incubate 2h. In addition, pretreatment with 100micromol/L epigallocatechin gallate (EGCG), cell viability increased from 31.20%+/-3.90% to 70.23%+/-12.96%. CONCLUSION: AChE is weakly expressed in normal human RPE cells. Stimulation with H2O2 caused the stable increase of AChE expression in RPE cells, which may indicate that AChE may be an important role in AMD.
Streptococcus thermophilus strain ND03 is a Chinese commercial dairy starter used for the manufacture of yogurt. It was isolated from naturally fermented yak milk in Qinghai, China. We present here the complete genome sequence of ND03 and compare it to three other published genomes of Streptococcus thermophilus strains.
Title: Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid Li J, Shao Y, Ding Z, Bao H, Liu Z, Han Z, Millar NS Ref: Insect Biochemistry & Molecular Biology, 40:17, 2010 : PubMed
Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [(3)H]imidacloprid binding sites with different affinities (K(d) = 3.5 +/- 0.6 pM and 1.5 +/- 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlalpha1, Nlalpha2 and Nlbeta1 subunits into one receptor complex and of Nlalpha3, Nlalpha8 and Nlbeta1 into another. Immunodepletion of Nlalpha1 or Nlalpha2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlalpha3 or Nlalpha8 caused the selective loss of the high-affinity site. Immunodepletion of Nlbeta1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlalpha4, Nlalpha6, Nlalpha7 and Nlbeta2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlalpha1, Nlalpha2 and Nlbeta1 constitute the lower affinity binding site, whereas nAChRs containing Nlalpha3, Nlalpha8 and Nlbeta1 constitute the higher affinity binding site for imidacloprid in N. lugens.
Title: Functional co-expression of two insect nicotinic receptor subunits (Nlalpha3 and Nlalpha8) reveals the effects of a resistance-associated mutation (Nlalpha3) on neonicotinoid insecticides Yixi Z, Liu Z, Han Z, Song F, Yao X, Shao Y, Li J, Millar NS Ref: Journal of Neurochemistry, 110:1855, 2009 : PubMed
Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens. Although this mutation has been identified in two different N. lugens nAChR subunits (Nlalpha1 and Nlalpha3) because of difficulties in heterologous expression of Nlalpha3; its influence on agonist potency has been examined only in Nlalpha1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlalpha8), together with evidence for its co-assembly with Nlalpha3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlalpha3(Y151S) mutation to be examined. The Nlalpha3(Y151S) mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I(max) values and increasing EC(50) values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlalpha3-containing, rather than Nlalpha1-containing nAChR. We conclude that Nlalpha3- and Nlalpha1-containing nAChRs may be representative of two distinct insect nAChR populations.
The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.
The complete DNA sequence of pO157, the large virulence plasmid of EHEC strain O157:H7 EDL 933, is presented. The 92 kb F-like plasmid is composed of segments of putative virulence genes in a framework of replication and maintenance regions, with seven insertion sequence elements, located mostly at the boundaries of the virulence segments. One hundred open reading frames (ORFs) were identified, of which 19 were previously sequenced potential virulence genes. Forty-two ORFs were sufficiently similar to known proteins for suggested functions to be assigned, and 22 had no convincing similarity with any known proteins. Of the newly identified genes, an unusually large ORF of 3169 amino acids has a putative cytotoxin active site shared with the large clostridial toxin (LCT) family and proteins such as ToxA and B of Clostridium difficile . A conserved motif was detected that links the large ORF and the LCT proteins with the OCH1 family of glycosyltransferases. In the complete sequence, the mosaic form can be observed at the levels of base composition, codon usage and gene organization. Insights were obtained from patterns of DNA composition as well as the pathogenic and 'housekeeping' gene segments. Evolutionary trees built from shared plasmid maintenance genes show that even these genes have heterogeneous origins.
The 4,639,221-base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families; many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication; guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer.
