Accurate SAXS Profile Computation and its Assessment by Contrast Variation Experiments

A major challenge in structural biology is to characterize structures of proteins and their assemblies in solution. At low resolution, such a characterization may be achieved by small angle x-ray scattering (SAXS). Because SAXS analyses often require comparing profiles calculated from many atomic models against those determined by experiment, rapid and accurate profile computation from molecular structures is needed. We developed fast open-source x-ray scattering (FoXS) for profile computation. To match the experimental profile within the experimental noise, FoXS explicitly computes all interatomic distances and implicitly models the first hydration layer of the molecule. For assessing the accuracy of the modeled hydration layer, we performed contrast variation experiments for glucose isomerase and lysozyme, and found that FoXS can accurately represent density changes of this layer. The hydration layer model was also compared with a SAXS profile calculated for the explicit water molecules in the high-resolution structures of glucose isomerase and lysozyme. We tested FoXS on eleven protein, one DNA, and two RNA structures, revealing superior accuracy and speed versus CRYSOL, AquaSAXS, the Zernike polynomials-based method, and Fast-SAXS-pro. In addition, we demonstrated a significant correlation of the SAXS score with the accuracy of a structural model. Moreover, FoXS utility for analyzing heterogeneous samples was demonstrated for intrinsically flexible XLF-XRCC4 filaments and Ligase III-DNA complex. FoXS is extensively used as a standalone web server as a component of integrative structure determination by programs IMP, Chimera, and BILBOMD, as well as in other applications that require rapidly and accurately calculated SAXS profiles.     

Replication, Variation and Normalisation in Microarray Experiments

INTRODUCTION: Microarray experiments often have complex designs that include sample pooling, biological and technical replication, sample pairing and dye-swapping. This article demonstrates how statistical modelling can illuminate issues in the design and analysis of microarray experiments, and this information can then be used to plan effective studies. METHODS: A very detailed statistical model for microarray data is introduced, to show the possible sources of variation that are present in even the simplest microarray experiments. Based on this model, the efficacy of common experimental designs, normalisation methodologies and analyses is determined. RESULTS: When the cost of the arrays is high compared with the cost of samples, sample pooling and spot replication are shown to be efficient variance reduction methods, whereas technical replication of whole arrays is demonstrated to be very inefficient. Dye-swap designs can use biological replicates rather than technical replicates to improve efficiency and simplify analysis. When the cost of samples is high and technical variation is a major portion of the error, technical replication can be cost effective. Normalisation by centreing on a small number of spots may reduce array effects, but can introduce considerable variation in the results. Centreing using the bulk of spots on the array is less variable. Similarly, normalisation methods based on regression methods can introduce variability. Except for normalisation methods based on spiking controls, all normalisation requires that most genes do not differentially express. Methods based on spatial location and/or intensity also require that the nondifferentially expressing genes are at random with respect to location and intensity. Spotting designs should be carefully done so that spot replicates are widely spaced on the array, and genes with similar expression patterns are not clustered. DISCUSSION: The tools for statistical design of experiments can be applied to microarray experiments to improve both efficiency and validity of the studies. Given the high cost of microarray experiments, the benefits of statistical input prior to running the experiment cannot be over-emphasised.     

采用RAPD和ISSR标记探讨中国疣粒野生稻的遗传多样性

用随机扩增多态ＤＮＡ（ＲＡＰＤ）和ｉｎｔｅｒ－简单重复序列（ＩＳＳＲ０标记对分别来自中国海南和云南２０个居群的疣料野生稻（Ｏｒｙａｚ ｇｒａｎｕｌａｔａ （Ｎｅｅｓ ｅｔ Ａｒｎ． ｅｘ Ｗａｔｔ．））混合样品,以及海南（Ｍ５）和云南（Ｍ２７）２个居群各２０个植株的遗传多生进行了检测。在混合取样的居群中,２０个ＲＡＰＤ引物和１２个ＩＳＳＲ引物分别扩增出２０９个１２２条带,多态条带比率９ＰＰＢ）分别     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

采用RAPD标记探讨瓣蕊唐松草(毛茛科唐松草属)的遗传多样性及其地理分布格局(英文)

Random amplified polymerphic DNA(RAPD)method was applied to assessg enetic variation and population structure of Thahctrum petalotdeum L(Ranunoulaceae),Two hundred and forty-six individuals from 11 populations of the species were investigated by RAPD profiles Twenty selected RAPD primers generated 125 bands．in which 120 were polymorphic Ther esults revealed a high level of genetic variation(ercentage of polymorphIc bands(PPB was 96％．Nei’s gene diversity(りwas 03502 and shannon’s information index(I) was 0．5199 at the species level) The differentiation among the populations was high(Gst=0．3511)in this species．Result of analyzing of molecularvariance(AMOVA)showedthat38．88％of genetic variance was found among the populations Positive correlation withr r=01945(P=00002)was found between genetic distance and geographic distance amongpo pulations Two populations distributed in the drainage basin of YanELz River affined genedcally and formed one clada and the rest nine populations formed the other clade in both unweighted pair-group method using arithmetic average(UPGMA)trees made by two different method different methods. It was yen/clear that these two populations were very special, andmust be closely related in history, despite the fact that they now share quite weak link to the restpopulations through gene communication.     

Immune-Related Gene Expression Profile in Laboratory Common Marmosets Assessed by an Accurate Quantitative Real-Time PCR Using Selected Reference Genes

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.     

Improving Assessment of Lipoprotein Profile in Type 1 Diabetes by 1H NMR Spectroscopy

Patients with type 1 diabetes (T1D) present increased risk of cardiovascular disease (CVD). The aim of this study is to improve the assessment of lipoprotein profile in patients with T1D by using a robust developed method 1H nuclear magnetic resonance spectroscopy (1H NMR), for further correlation with clinical factors associated to CVD. Thirty patients with T1D and 30 non-diabetes control (CT) subjects, matched for gender, age, body composition (DXA, BMI, waist/hip ratio), regular physical activity levels and cardiorespiratory capacity (VO_2peak), were analyzed. Dietary records and routine lipids were assessed. Serum lipoprotein particle subfractions, particle sizes, and cholesterol and triglycerides subfractions were analyzed by 1H NMR. It was evidenced that subjects with T1D presented lower concentrations of small LDL cholesterol, medium VLDL particles, large VLDL triglycerides, and total triglycerides as compared to CT subjects. Women with T1D presented a positive association with HDL size (p<0.005; R = 0.601) and large HDL triglycerides (p<0.005; R = 0.534) and negative (p<0.005; R = -0.586) to small HDL triglycerides. Body fat composition represented an important factor independently of normal BMI, with large LDL particles presenting a positive correlation to total body fat (p<0.005; R = 0.505), and total LDL cholesterol and small LDL cholesterol a positive correlation (p<0.005; R = 0.502 and R = 0.552, respectively) to abdominal fat in T1D subjects; meanwhile, in CT subjects, body fat composition was mainly associated to HDL subclasses. VO_2peak was negatively associated (p<0.005; R = -0.520) to large LDL-particles only in the group of patients with T1D. In conclusion, patients with T1D with adequate glycemic control and BMI and without chronic complications presented a more favourable lipoprotein profile as compared to control counterparts. In addition, slight alterations in BMI and/or body fat composition showed to be relevant to provoking alterations in lipoproteins profiles. Finally, body fat composition appears to be a determinant for cardioprotector lipoprotein profile.     

Assessment of GABARAP self-association by its diffusion properties

Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) belongs to a family of small ubiquitin-like adaptor proteins implicated in intracellular vesicle trafficking and autophagy. We have used diffusion-ordered nuclear magnetic resonance spectroscopy to study the temperature and concentration dependence of the diffusion properties of GABARAP. Our data suggest the presence of distinct conformational states and provide support for self-association of GABARAP molecules. Assuming a monomer–dimer equilibrium, a temperature-dependent dissociation constant could be derived. Based on a temperature series of ¹H¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance spectra, we propose residues potentially involved in GABARAP self-interaction. The possible biological significance of these observations is discussed with respect to alternative scenarios of oligomerization.     

东俄洛橐吾遗传变异与分化的ISSR分析

应用ISSR标记对东俄洛橐吾（Ligularia tongolensis）的遗传多样性进行了研究。从100个引物中筛选出8个用于正式扩增。在所研究的8个居群共150个个体中检测到148个多态位点。在居群水平上,多态位点百分率（PPB）为50.45%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.1595和0.2440。在物种水平上,多态位点百分率（PPB）为88.10%,Nei′s基因多样性指数（H）和Shannon信息指数（I）分别为0.2811和0.4279。居群间的遗传分化系数（Gst）达0.4355。研究结果表明东俄洛橐吾的遗传多样性水平很高,居群间遗传分化较大。这与其多样化的生态环境是有必然联系的。因适应其多样化的生态环境而形成了遗传多样性;且因其生态环境的不连续性阻碍了居群间的基因交流而产生了遗传分化,即东俄洛橐吾高水平的遗传多样性和遗传分化是适应其分布区多样化生态环境的结果。     

基于PCR的长片段DNA精确合成及应用

长片段DNA的组装及基于PCR的长片段DNA的精确合成(PAS)是基因异源表达时简单快速合成长片段DNA的方法,适于合成含有较高GC含量、重复序列和复杂二级结构的长片段DNA分子(5～6 kb)。简要综述了PAS技术用于长片段DNA序列精确合成的基本原理、操作步骤、常见问题和解决方法,以及该技术的应用。     

Observer Variation in the Clinical and Radiological Assessment of Hepatosplenomegaly

The size of the liver and spleen of 32 patients was assessed by four clinicians from a clinical examination and by four radiologists from a plain radiograph of the abdomen. The latter assessment was found to be subject to less variation than the former, particularly in regard to the liver. Large livers and spleens were more easily seen radiologically than small ones. Most of the variation among the radiologists arose because of the 6% and 24% of cases in which the liver and spleen, respectively, were poorly seen on the radiograph. It is concluded that a plain radiograph of the abdomen, including the diaphragm and with the costal margin indicated, is a useful adjunct to clinical examination.     

The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure

Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.     

Uncertainty in Diatom Assessment: Sampling, Identification and Counting Variation

Despite the widespread application of periphytic diatoms to water quality assessment at a regional level, there is no standard European sampling protocol or associated assessment metrics. Furthermore, relatively little is known about the uncertainty in the results of such assessments. One of the objectives of the European project for the Standardisation of River Classifications (STAR) is to improve and standardise diatom assessment methods. An extensive diatom ring test, together with an audit of the project results, provided a better understanding and quantification of the uncertainty in quality assessment of running waters using diatoms. The variation in multimetric analysis shows that the choice of site and substrate for sampling, the inter-operator differences in diatom taxonomy and the counting techniques are the primary sources of uncertainty. To some extent, this variation also reveals the robustness of specific metrics in relation to the sources of uncertainty. Of the three most common substrate types tested (stone, macrophyte and sediment), macrophytes emerge as the most preferred substrate for diatom sampling when performing multimetric water quality assessment.     

Conformational study of proteins by SAXS and EDXD: the case of trypsin and trypsinogen

The radius of gyration (Rg) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique (EDXD) and by small-angle X-ray scattering (SAXS), under different solvent conditions. Both techniques gave superimposable results. The experimental evidence demonstrated that: (1) no structural modifications and/or damage occurred during the data acquisition by EDXD; (2) at pH 4 the active enzyme has one class of chloride binding sites in common with the zymogen, whereas the latter protease shows an additional class able to reverse the effects on Rg induced by chloride at low concentration; and (3) the pH profile of the Rg of both proteases does not resemble at all the pH effect on beta-trypsin activity, a result in line with the finding that the electrical potentials induced by surface charge are small in absolute magnitude and produce no gradient across the active site.     

A Modified Cooling Method and its Application in Drosophila Experiments

Chilling is a cost-effective and safe method of immobilising flies in Drosophila experiments. However, should condensation form on the plate, it would be fatal to the flies. Here we describe a modified cooling method using reusable commercial ice pack(s) (ca. 400 ml, 2–3 cm tall) rather than crushed ice. The ice pack is covered with a piece of filter paper which absorbs the condensed water formed on the cold surface, and the moistened filter paper does not wet the wings of flies inverted onto it. An ice pack (ca. 400 ml) takes effect in at least 1 h, and its height is shorter than the maximum specimen height of a dissecting microscope, so that flies can be checked while they remain on the cold surface. This method overcomes the disadvantages of the indigenous cooling anaesthetising approach and makes it practically feasible. We also describe an easy-to-make fly-transferring toolkit which will greatly facilitate fly transferral when it is combined with our chilling method. In our design, the flies were directed out of a vial by a blue pipette tip, which is inserted in a foam stopper and channelled down into another vial by a glass funnel inserted in another foam stopper which is escape-proof.     

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 °C. SAXS data show the presence of a broad peak around q ∼ 0.12 Å^− 1 at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d = 90-100 Å is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after ∼ 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 degrees C. SAXS data show the presence of a broad peak around q approximately 0.12 A(-1) at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d=90-100 A is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after approximately 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Variation of oleate/cis-vaccenate ratios and its regulation by substrates in hepatic tissues

The oleate (delta 9-18:1)/cis-vaccenate (delta 11-18:1) ratios in phospholipids increased in the order of normal liver, host liver and hepatoma in rats. The amount of oleate increased in phospholipids and decreased in triacylglycerol in the same order, whereas the distributions of cis-vaccenate among the major lipid classes were relatively unchanged among the three kinds of cells. Biochemical bases for these differences were sought by characterizing the microsomal elongation system and by analyzing the elongation and desaturation products in vitro. Kinetic parameters for the elongations of palmitoyl-CoA and palmitoleoyl-CoA did not account for the observed differences in the oleate/cis-vaccenate ratios in these cells. However, the oleate/cis-vaccenate ratios varied depending on the availability of substrates, NADH, NADPH, and malonyl-CoA, in the elongation and desaturation of palmitoyl-CoA. Based on the results, it is proposed that differences in the concentrations of substrates for the elongation and desaturation systems might account at least in part for the differences in the oleate/cis-vaccenate ratios among the three kinds of cells.     

Phylodynamic Inference and Model Assessment with Approximate Bayesian Computation: Influenza as a Case Study

A key priority in infectious disease research is to understand the ecological and evolutionary drivers of viral diseases from data on disease incidence as well as viral genetic and antigenic variation. We propose using a simulation-based, Bayesian method known as Approximate Bayesian Computation (ABC) to fit and assess phylodynamic models that simulate pathogen evolution and ecology against summaries of these data. We illustrate the versatility of the method by analyzing two spatial models describing the phylodynamics of interpandemic human influenza virus subtype A(H3N2). The first model captures antigenic drift phenomenologically with continuously waning immunity, and the second epochal evolution model describes the replacement of major, relatively long-lived antigenic clusters. Combining features of long-term surveillance data from the Netherlands with features of influenza A (H3N2) hemagglutinin gene sequences sampled in northern Europe, key phylodynamic parameters can be estimated with ABC. Goodness-of-fit analyses reveal that the irregularity in interannual incidence and H3N2''s ladder-like hemagglutinin phylogeny are quantitatively only reproduced under the epochal evolution model within a spatial context. However, the concomitant incidence dynamics result in a very large reproductive number and are not consistent with empirical estimates of H3N2''s population level attack rate. These results demonstrate that the interactions between the evolutionary and ecological processes impose multiple quantitative constraints on the phylodynamic trajectories of influenza A(H3N2), so that sequence and surveillance data can be used synergistically. ABC, one of several data synthesis approaches, can easily interface a broad class of phylodynamic models with various types of data but requires careful calibration of the summaries and tolerance parameters.