A novel tetrafunctional reagent for simultaneous intramolecular and intermolecular cross-linking of bovine hemoglobin

A novel tetrafunctional reagent, alpha,gamma,alpha',gamma'-tetra-succinimidyl-hexanediamide-di-glutamate ester (HDG(OSu)(4)), was successfully synthesized, and a well-defined cross-linked bovine hemoglobin (mainly 128 kDa) was prepared with this reagent. Due to the spatial structure of this cross-linking reagent, the intramolecular and intermolecular cross-linking of bovine hemoglobin was formed simultaneously in one reaction. Although the cross-linked bovine hemoglobin showed a slight decrease in half-saturated O(2) pressure value (P(50), from 28.1 mm Hg to 21.7 mm Hg) and Hill coefficient (from 2.5 to 2), due to the cross-linkage, it still performed well for O(2) delivery.     

ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints

ribosort is a computer package for convenient editing of automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) data. It is designed to eliminate the labourious task of manually classifying community fingerprints in microbial ecology studies. This program automatically assigns detected fragments and their respective relative abundances to appropriate ribotypes. It permits simultaneous sorting of multiple profiles and facilitates direct workflow from TRFLP and ARISA output through to community analyses. ribosort also provides several options to merge repeat profiles of a sample into a single composite profile. By creating a 'ribotypes by samples' matrix ready for statistical analyses, use of the package saves time and simplifies the preparation of DNA fingerprint data sets for statistical analysis. In addition, ribosort performs exploratory analysis on the data by creating multidimensional scaling plots that compare the similarity of sample profiles using the statistical software r.     

Microarray analysis of ageing-related signatures and their expression in tumors based on a computational biology approach

Ageing and cancer have been associated with genetic and genomic changes.The identification of common signatures between ageing and cancer can reveal shared molecular mechanisms underlying them.In this study,we collected ageing-related gene signatures from ten published studies involved in six different human tissues and an online resource.We found that most of these gene signatures were tissuespecific and a few were related to multiple tissues.We performed a genome-wide examination of the expression of these signatures in various human tumor types,and found that a large proportion of these signatures were universally differentially expressed among normal vs.tumor phenotypes.Functional analyses of the highly-overlapping genes between ageing and cancer using DAVID tools have identified important functional categories and pathways linking ageing with cancer.The convergent and divergent mechanisms between ageing and cancer are discussed.This study provides insights into the biology of ageing and cancer,suggesting the possibility of potential interventions aimed at postponing ageing and preventing cancer.     

Abacus: a computational tool for extracting and pre-processing spectral count data for label-free quantitative proteomic analysis

We describe Abacus, a computational tool for extracting spectral counts from MS/MS data sets. The program aggregates data from multiple experiments, adjusts spectral counts to accurately account for peptides shared across multiple proteins, and performs common normalization steps. It can also output the spectral count data at the gene level, thus simplifying the integration and comparison between gene and protein expression data. Abacus is compatible with the widely used Trans-Proteomic Pipeline suite of tools and comes with a graphical user interface making it easy to interact with the program. The main aim of Abacus is to streamline the analysis of spectral count data by providing an automated, easy to use solution for extracting this information from proteomic data sets for subsequent, more sophisticated statistical analysis.     

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm² from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.     

Capture–recapture analysis with a latent class model allowing for local dependence and observed heterogeneity

In epidemiology, capture–recapture models are commonly used to estimate the size of an unknown population based on several incomplete lists of individuals. The method operates under two main assumptions: independence between the lists (local independence) and homogeneity of capture probabilities of individuals. In practice, these assumptions are rarely satisfied. We introduce a multinomial latent class model that can account for both list dependence and heterogeneity. Parameter estimation is performed by maximizing the conditional likelihood function with the use of the EM algorithm. In addition, a new approach for evaluating the standard errors of the parameter estimates is discussed, which considerably reduces the computational burden associated with the evaluation of the variance of the population size estimate.     

A cautionary note on the robustness of latent class models for estimating diagnostic error without a gold standard

Modeling diagnostic error without a gold standard has been an active area of biostatistical research. In a majority of the approaches, model-based estimates of sensitivity, specificity, and prevalence are derived from a latent class model in which the latent variable represents an individual's true unobserved disease status. For simplicity, initial approaches assumed that the diagnostic test results on the same subject were independent given the true disease status (i.e., the conditional independence assumption). More recently, various authors have proposed approaches for modeling the dependence structure between test results given true disease status. This note discusses a potential problem with these approaches. Namely, we show that when the conditional dependence between tests is misspecified, estimators of sensitivity, specificity, and prevalence can be biased. Importantly, we demonstrate that with small numbers of tests, likelihood comparisons and other model diagnostics may not be able to distinguish between models with different dependence structures. We present asymptotic results that show the generality of the problem. Further, data analysis and simulations demonstrate the practical implications of model misspecification. Finally, we present some guidelines about the use of these models for practitioners.     

Extended CADLIVE: a novel graphical notation for design of biochemical network maps and computational pathway analysis

Biochemical network maps are helpful for understanding the mechanism of how a collection of biochemical reactions generate particular functions within a cell. We developed a new and computationally feasible notation that enables drawing a wide resolution map from the domain-level reactions to phenomenological events and implemented it as the extended GUI network constructor of CADLIVE (Computer-Aided Design of LIVing systEms). The new notation presents ‘Domain expansion’ for proteins and RNAs, ‘Virtual reaction and nodes’ that are responsible for illustrating domain-based interaction and ‘InnerLink’ that links real complex nodes to virtual nodes to illustrate the exact components of the real complex. A modular box is also presented that packs related reactions as a module or a subnetwork, which gives CADLIVE a capability to draw biochemical maps in a hierarchical modular architecture. Furthermore, we developed a pathway search module for virtual knockout mutants as a built-in application of CADLIVE. This module analyzes gene function in the same way as molecular genetics, which simulates a change in mutant phenotypes or confirms the validity of the network map. The extended CADLIVE with the newly proposed notation is demonstrated to be feasible for computational simulation and analysis.     

Pixel-based analysis of multiple images for the identification of changes: a novel approach applied to unravel proteome patterns [corrected] of 2-D electrophoresis gel images

A novel approach for revealing patterns of proteome variation among series of 2-DE gel images is presented. The approach utilises image alignment to ensure that each pixel represents the same information across all gels. Gel images are normalised, and background corrected, followed by unfolding of the images to 1-D pixel vectors and analysing pixel vectors by multivariate data modelling. Information resulting from the data analysis is refolded back to the image domain for visualisation and interpretation. The method is rapid and suitable for automatic routines applied after the gel alignment. The approach is compared with spot volume analysis to illustrate how this approach can solve persistent problems like mismatch of protein spots, erroneous missing values and failure to detect variation in overlapping proteins. The method may also detect variation in the border area of saturated proteins. The approach is given the name pixel-based analysis of multiple images for the identification of changes (PMC). The method can be used for multiple images in general. Effects of pretreatment of the images are discussed.     

The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function

Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.     

Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase

Hormone-sensitive lipase (HSL) plays an important role in the regulation of rodent fat cell lipolysis. It is regarded as an adipose tissue-specific enzyme whose sole metabolic role is the catalysis of hormone-stimulated lipolysis in mammalian cells. In this report we describe the functional and structural analysis of an EstE5 protein from a soil metagenome library. Function analysis results indicated that EstE5 preferentially hydrolyzes short-chain ester compounds, and our kinetic studies revealed the optimal pH and temperature. Based on the structural analysis, we defined the active site and the binding pocket. Structurally, EstE5 belongs to the HSL family and these structural studies may have applications in the production of value-added products, including pharmaceuticals.     

基于SSR分子标记的地被菊遗传多样性分析和指纹图谱构建

为了从分子水平上研究地被菊(Chrysanthemum×morifolium)种质资源的遗传多样性并建立地被菊品种的指纹图谱库,筛选出多态性高的引物用于地被菊品种间鉴定、亲缘关系分析和分子标记辅助选种体系的建立,本研究利用多态性好、条带清晰、重复性好的12对引物对91份地被菊材料和14份菊属近缘种材料进行简单重复序列(simple sequence repeat,SSR)分子标记和遗传多样性分析,从12对引物中筛选出9对核心引物对受试材料进行指纹图谱构建。结果显示,在105个样品中,12对引物检测出104个等位位点,范围为2−26,平均每个点位检测出9.25个等位基因数,平均每个点位检测得到的有效等位基因数(number of effective alleles,Ne)为2.7456,范围为1.2760−4.7425;Shannon信息指数(Shannon genetic diversity index,I)变化范围是0.5133−2.2399,均值是1.2090;Nei’s基因多样性指数(Nei’s gene diversity index,H)范围是0.2163−0.7891,均值是0.5780;观测杂合度(observed heterozygosity,Ho)的范围是0.2233−0.8952,均值是0.5575;期望杂合度(expected heterozygosity,He)的范围是0.2174−0.7933,均值是0.5808;多态信息含量(polymorphism information content,PIC)值变化范围是0.2115−0.7740,均值是0.5329;遗传相似性系数(genetic similarity,GS)范围为0.2285−1.0000,均值是0.6083。聚类分析表明,在遗传距离(genetic distance,GD)=0.30时,受试材料可以分为两个类群。Structure群体结构分析将受试材料分为3个种群和1个混合种群。从12对引物中筛选出可完全区分105份受试材料的9对核心引物,构建了91份地被菊材料和14份菊属近缘种材料的指纹图谱。地被菊材料之间具有显著的遗传差异和丰富的遗传多样性,对于地被菊的园林应用和品种选育具有重要意义。地被菊品种和菊属近缘种的指纹图谱库的构建,一定程度上揭示了105份实验材料的亲缘关系,为今后地被菊品种鉴定与筛选体系的研究提供了技术支撑。     

A soil microscale study to reveal the heterogeneity of Hg(II) impact on indigenous bacteria by quantification of adapted phenotypes and analysis of community DNA fingerprints

The short term impact of 50 μM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the outer fraction, containing loosely associated bacteria, and the inner fraction, containing bacteria retained into aggregates, followed by a physical fractionation of the inner fraction to separate aggregates according to their size (size fractions). Indirect enumerations of viable heterotrophic (VH) and resistant (Hg(R)) bacteria were performed before and 30 days after mercury spiking. A ribosomal intergenic spacer analysis (RISA), combined with multivariate analysis, was used to compare modifications at the community level in the unfractionated soil and in the microenvironments. The spatial heterogeneity of the mercury impact was revealed by a higher increase of Hg(R) numbers in the outer fraction and in the coarse size fractions. Furthermore, shifts in RISA patterns of total community DNA indicated changes in the composition of the dominant bacterial populations in response to Hg(II) stress in the outer and in the clay size fractions. The heterogeneity of metal impact on indigenous bacteria, observed at a microscale level, is related to both the physical and chemical characteristics of the soil microenvironments governing mercury bioavailability and to the bacterial composition present before spiking.     

植物一个新box H/ACA snoRNA的鉴定及功能分析

通过生物信息学方法对拟南芥基因组序列进行搜索,发现两个新的非编码小分子RNA基因,分别命名为AthsnoR206 a和AthsnoR206b。它们相距约170nt,位于蛋白质基因间隔区。MFOLD二级结构预测这两个RNA均具有典型的box H/ACAsnoRNA"发夹-铰链-发夹-尾巴"结构,符合box H/ACA snoRNA的判定标准;两个RNA分子的反义序列一致,可以判定它们为同一基因的两个拷贝。分析预测snoR206的两段反义序列分别指导拟南芥rRNA小亚基U1717位点和大亚基U2181位点的假尿嘧啶化修饰。在其它13种包括单子叶植物和双子叶植物在内的植物搜索到14个snoR206同源分子,其中12个发现于表达序列标签中,表明该snoRNA在植物中表达且广泛存在。具有双功能的snoR206在人和酵母中的部分功能同源分子分别为U70和snR32,表明其祖先分子在进化过程中存在分子重组。     

StatFingerprints: a friendly graphical interface program for processing and analysis of microbial fingerprint profiles

Molecular fingerprint methods are widely used to compare microbial communities in various habitats. The free program StatFingerprints can import, process, and display fingerprint profiles and perform numerous statistical analyses on them, and also estimate diversity indexes. StatFingerprints works with the free program R, providing an environment for statistical computing and graphics. No programming knowledge is required to use StatFingerprints, thanks to its friendly graphical user interface. StatFingerprints is useful for analysing the effect of a controlled factor on the microbial community and for establishing the relationships between the microbial community and the parameters of its environment. Multivariate analyses include ordination, clustering methods and hypothesis-driven tests like 50-50 multivariate analysis of variance, analysis of similarity or similarity percentage procedure and the program offers the possibility of plotting ordinations as a three-dimensional display.     

A fluorimetric method for the determination of trace pentachlorophenol, based on its inhibitory effect on the redox reaction between the improved Fenton reagent and rhodamine B.

A sensitive fluorimetric method is presented and discussed for the determination of pentachlorophenol in aqueous solutions. This method is based on the inhibitory effect of pentachlorophenol on the reaction of conventional Fenton [Fe(III) + H(2)O(2)] reagent with rhodamine B in the medium of perchloric acid, which results in the fluorescence quenching of rhodamine B. It was further found that the sensitivity for the determination was improved significantly when the molecular ligand EDTA was added. This improved system was therefore presented for the determination of pentachlorophenol. The characteristics of the excitation and emission spectra, optimization of the experimental conditions, the stability of the system and the influence of foreign matter have all been investigated. Under optimal conditions, the linear range for the determination of pentachlorophenol is 12-480 ng/mL with a 3sigma limit of detection of 0.96 ng/mL. Compared with the conventional Fenton system, the improved system shows obvious advantages in both sensitivity and selectivity. By combination with the pretreatment of samples using ion exchange resins and XDA-1 absorption resin, the improved Fenton method was used for the first time for the determination of pentachlorophenol in synthetic samples and natural water samples, and satisfactory results, in agreement with those of the HPLC method, were achieved. The possible mechanism of the reactions has also been discussed. Copyright (c) 2007 John Wiley & Sons, Ltd.