Computer-Aided,Rational Design of a Potent and Selective Small Peptide Inhibitor of Cyclooxygenase 2 (COX2)

Abstract

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors.

Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX' and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

Chronobiologic Evaluation of Drug Efficacy in Hypertension

Time-associated variations of blood pressure became accesible due to the modern development of pressure monitoring technology. The data collection and analysis should be standardized and formulated. Time-associated changes of pressure were evaluated not only by nonparametric and conventional but also by parametric and rhythmometric estimation. The reference data (chronodesms) are essential to define the deviant magnitude of pressure. This chronobiologic approach became feasible to quantitate the efficacy of antihypertensive agents in hypertensives.     

Accounting for data errors discovered from an audit in multiple linear regression

A data coordinating team performed onsite audits and discovered discrepancies between the data sent to the coordinating center and that recorded at sites. We present statistical methods for incorporating audit results into analyses. This can be thought of as a measurement error problem, where the distribution of errors is a mixture with a point mass at 0. If the error rate is nonzero, then even if the mean of the discrepancy between the reported and correct values of a predictor is 0, naive estimates of the association between two continuous variables will be biased. We consider scenarios where there are (1) errors in the predictor, (2) errors in the outcome, and (3) possibly correlated errors in the predictor and outcome. We show how to incorporate the error rate and magnitude, estimated from a random subset (the audited records), to compute unbiased estimates of association and proper confidence intervals. We then extend these results to multiple linear regression where multiple covariates may be incorrect in the database and the rate and magnitude of the errors may depend on study site. We study the finite sample properties of our estimators using simulations, discuss some practical considerations, and illustrate our methods with data from 2815 HIV-infected patients in Latin America, of whom 234 had their data audited using a sequential auditing plan.     

All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes

Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q_i site of the bacterial and bovine bc₁ complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q_i pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q_i pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc₁ complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc₁ complex and only rarely in the bovine bc₁ complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc₁ complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc₁ complex.     

浅谈网络分析法及其在古生物学中的应用

网络分析作为一种新的数据可视化途径和定量方法, 能简化复杂系统而发现元素间的关系模式。它在定量社会科学、计算机科学与机器学习等领域均有众多应用实例。近年来, 在古生物学, 特别是古生物地理学相关研究中逐渐受到关注。本文介绍了网络的基本构成、常见网络类型及其数据储存方式、以及网络分析中的重要参数及其定义, 同时给出了两种实现网络分析的方法及相关工具, 即Gephi软件与R语言平台。通过分析比较两种方法的步骤及结果, 发现Gephi虽成图简洁美观, 但算法功能有限, 且无法完成成图前的数据处理以及成图后的多元分析, 因而最终推荐使用R语言编程进行网络分析。本文以奥陶纪末大灭绝后复苏期全球腕足动物数据为例, 详细展现了利用R语言及其应用包“igraph”编程进行网络分析的过程, 并实现了古生物地理学数据资料的处理以及网络分析图件的绘制。希望对即将接触此类工具的古生物学科研人员在进行网络分析时提供借鉴与参考。     

额尔齐斯河流域垂枝桦林群落结构特征与物种多样性相关分析

为阐明植物群落结构特征和物种多样性之间的相互关系,选择额尔齐斯河流域白桦林国家森林公园的天然垂枝桦(Betula pendula)纯林、垂枝桦苦杨混交林、垂枝桦白柳混交林为研究对象,分林层调查群落的基本特征参数(高度、枝下高、冠幅、胸径、盖度等),计算物种重要值、丰富度指数、多样性指数、均匀度指数,并进行典范对应分析。结果表明:(1)垂枝桦白柳混交林的乔木层树高、枝下高和灌木层的地径、盖度均最高;3种群落的草本层特征参数(除基径)均具有显著差异;垂枝桦白柳林的盖度分别比垂枝桦苦杨林和垂枝桦纯林高19.1%和51.8%。(2)3种群落的乔木层重要值最高为垂枝桦,灌木层为疏花蔷薇(Rosa laxa)、阿尔泰山楂(Crataegus altaica),草本层为莎薹草(Carex bohemica)。(3)3种群落类型中乔木层的丰富度指数R、Simpson指数、Shannon-Wiener指数和灌木层的Pielou指数、Alatalo指数呈现出相同规律,即垂枝桦白柳混交林>垂枝桦苦杨混交林>垂枝桦纯林;草本层中除Alatalo指数之外,其他指数均呈现垂枝桦纯林>垂枝桦苦杨混交林>垂枝桦白柳混交林(P>0.05)。(4)CCA排序结果表明,不同垂枝桦林群落结构特征与物种多样性关系有差异。其中,垂枝桦纯林中,对物种多样性影响最大的是乔木枝下高、灌木株高、冠幅以及草本盖度;垂枝桦苦杨混交林中,对物种多样性影响最大的是乔木高度和冠幅、灌木冠幅和草本高度;垂枝桦白柳林中,对物种多样性影响最大的是乔木胸径、灌木冠幅、盖度以及草本高度。研究表明,乔木枝下高度、灌木冠幅、草本高度是影响3种群落类型物种多样性的主要因素。     

全球健康史项目亚洲模块——亚洲古代人群健康、疾病和生活方式的大数据

“全球健康史项目”探导新石器时期以来人类的疾病史,以前所未有的大数据研究方式回顾近代人类的历史,来衡量人类的生活质量和人类在充满挑战的生活条件下的适应能力。我们于2018年5月启动了全球健康史项目亚洲模块,将这个起源于美洲和欧洲的宏伟的项目扩展到亚洲。在全球健康史项目中纳入亚洲数据,不仅可以从进化的角度丰富近代人类演化历史上的第一手骨骼和口腔健康状况,还可以为健康政策制定的视野提供历史性的深度。     

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction¹, boosting the immune response², pheromone production³, as well as nutrition, including the synthesis of essential amino acids^4, among others.    Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing ¹³C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA⁵. The incorporation of ¹³C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (¹²C) one. In the end, the ¹³C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the ¹²C-unlabeled similar one⁶. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, ¹³C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.     

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.