Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Is optimal solution of every NP-complete or NP-hard problem determined from its characteristic for DNA-based computing

Cook's Theorem [Cormen, T.H., Leiserson, C.E., Rivest, R.L., 2001. Introduction to Algorithms, second ed., The MIT Press; Garey, M.R., Johnson, D.S., 1979. Computer and Intractability, Freeman, San Fransico, CA] is that if one algorithm for an NP-complete or an NP-hard problem will be developed, then other problems will be solved by means of reduction to that problem. Cook's Theorem has been demonstrated to be correct in a general digital electronic computer. In this paper, we first propose a DNA algorithm for solving the vertex-cover problem. Then, we demonstrate that if the size of a reduced NP-complete or NP-hard problem is equal to or less than that of the vertex-cover problem, then the proposed algorithm can be directly used for solving the reduced NP-complete or NP-hard problem and Cook's Theorem is correct on DNA-based computing. Otherwise, a new DNA algorithm for optimal solution of a reduced NP-complete problem or a reduced NP-hard problem should be developed from the characteristic of NP-complete problems or NP-hard problems.     

城市景观功能的区域协调规划——以深圳市为例

城市景观功能的完善是城市可持续发展的重要保障之一,其与区域景观功能的相互协调是城市规划的重要组成部分,有利于城市和区域的整体持续发展。在分析城市景观基本特征的基础上,探讨了城市景观功能区域协调规划的基本思路,以城市景观为规划对象,以人类社会的功能需求为立足点,依据景观生态学理论,将城市景观的功能划分为生物生产功能、环境服务功能、文化支持功能和信息输运功能,对体现这些基本功能的景观类型进行区域协调规划。以深圳市为例,实证分析了其景观功能在珠江三角洲地区的协调规划。     

人谷胱甘肽S-转移酶pi(GSTp)中半胱氨酸对其在细胞氧化应激下功能的影响

利用PCR定点突变技术构建人GSTp三种半胱氨酸突变体C~(47/101)、C~(14/47/101)和C~(14/47/101/169)。将CSTp 野生型和突变体表达质粒转染293细胞,以CDNB 为底物测定胞内GST 的转移酶活性,结果显示各类突变体均明显抑制了细胞内源性CST 的催化活性,具有显著的负显性(dominant nega-tive)突变体的功能;将CSTp 野生型和突变体与c-Jun、NF-kB 和p53的报告基因载体共转染,通过萤光素酶活性测定发现突变体C~(14/47/101)和C~(14/47/101/169)能明显激活c-Jun 和p21的转录活性;Western印迹分析显示突变体均能上调细胞内p21蛋白的水平,细胞存活率的测定表明GSTp 突变体能增强293细胞对H_2O_2刺激的敏感性;实验结果表明半胱氨酸残基对于维持GSTp 在对抗细胞氧化应激过程中的保护作用至关重要。     

植酸酶酵母工程菌的构建*

从无花果曲霉(Aspergillus ficuum)3．4322中用RT-PCR方法扩增出一条约1．4kb的特异性条带,DNA序列测定表明,目的片段为不含信号肽的植酸酶编码序列,全长1347bp。无花果曲霉(Aspergillus ficuum)3．4322phyA基因序列已在GenBank注册(注册号为：AF537344)。将该基因克隆到酵母表达载体pYES2中,构建成不带信号肽phyA基因的重组表达载体pYPA2。用醋酸锂法将pYPA2转进urd缺陷型的酿酒酵母(s．oeraisiae INVSc1),筛选获得含植酸酶基因的酵母转化子。经半乳糖诱导表达后,用磷钼蓝显色(AMES)法对酵母菌体进行酶活测定,测出了明显的植酸酶活性,pYPA2胞内植酸酶活性约11．55IU／mL,表明无花果曲霉(Aspergillus ficuum)3．4322phyA基因能在酿酒酵母中表达。     

不同时辰电针对大鼠杏仁核一氧化氮合酶表达的影响

目的观察不同时辰电针对大鼠杏仁核一氧化氮合酶(NOS)表达的影响.方法采用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)法,观察不同时辰电针大鼠一侧"足三里"穴对杏仁核NOS表达的影响.结果电针对大鼠杏仁皮质内侧核群、基底外侧核群NOS的表达有上调作用,并存在时辰差异(P<0.05);电针对大鼠杏仁中央核NOS表达无明显作用(P>0.05).结论电针对大鼠杏仁核NOS表达的影响有时辰差异.     

人乳头状瘤病毒协同60钴照射促进食管上皮细胞恶性转化

为探明人乳头状瘤病毒(HPV)和促癌剂对食管上皮致癌作用,人胚食管上皮细胞转染HPV协同60钴(60Co)放射观察其恶性转化.用HPV18E6E7AAV转染的人胚食管上皮(SHEE),培养至13代,分为4组,实验组分别用60Co2、4、8Gy照射,每周1次共4周;SHEE未经照射为对照组.细胞形态用相差显微镜观察;细胞DNA合成和定量用3H-TdR掺入和用流式细胞仪分析;染色体众数用常规方法分析;致瘤性用软琼脂培养和裸小鼠接种;HPVDNA用PCR检测.经60Co照射后细胞呈凋亡和坏死(危象期).8周后SHEE 4Gy组细胞增殖,增殖指数(34%)和3H-TdR摄入增高,软琼脂培养和裸鼠接种出现致瘤性.对照组SHEE组细胞增殖指数24%,伴有少数3H-TdR掺入,裸鼠未成瘤.染色体众数:对照组,58～62;4Gy组,63～65;两组HPV18E6E7 PCR呈阳性条带.此结果表明,用HPV18E6E7协同60Coγ射线可以使人胚食管上皮恶性转化,60Co γ射线有加速食管上皮细胞恶性转化作用.