Dynamite extended: two new services to simplify protein dynamic analysis

We describe two additional services now available as part of the previously described Dynamite protein dynamics web service. Dynatraj provides principle component analysis and visualization of modes of motion for a user's own ensemble of protein structures, e.g. from Molecular Dynamics, NMR or experimental ensembles. Dynapocket predicts probable configurations of a protein pocket from a single known structure. Both have been provided in response to requests from users for additional functionality from the Dynamite server. Like Dynamite, both are available free of charge to all users.     

Cell spreading: the power to simplify

A eukaryotic cell spreads over a substrate in distinct stages, with the earliest events characterized by passive adhesion and cell deformation. Recent work suggests a common physical mechanism can explain the early stages of cell spreading for a wide range of cell types and substrates.     

A one-pot analysis approach to simplify measurements of protein stability and folding kinetics

To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as “one-pot analysis” which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.     

Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome

The discovery of classic parental imprinting came, at least in part, from the analysis of transgene expression in mice. It was noticed that some transgenes were only expressed following paternal transmission and that others sometimes showed differential patterns of methylation depending on the parent of origin. Here, we present evidence of a novel and more subtle form of parental imprinting by taking advantage of the highly sensitive detection of murine transgene expression afforded by flow cytometry. We have produced nine lines of transgenic mice carrying a GFP reporter linked to the human alpha-globin promoter and enhancer elements, which direct expression to erythroid cells. A high proportion of transgenic lines, four of the nine, display significantly lower levels of expression following maternal transmission. Both the percentage of expressing cells and the mean fluorescence in expressing cells are between 10% and 30% lower following maternal transmission. These effects are reversible upon passage through the opposite germline. This finding raises the possibility that differences in the epigenetic state of the maternal and paternal chromosomes in adult somatic cells are more widespread than was previously thought.     

GPAC: benchmarking the sensitivity of genome informatics analysis to genome annotation completeness

In view of the recent explosion in genome sequence data, and the 200 or more complete genome sequences currently available, the importance of genome-scale bioinformatics analysis is increasing rapidly. However, computational genome informatics analyses often lack a statistical assessment of their sensitivity to the completeness of the functional annotation. Therefore, a pre-analysis method to automatically validate the sensitivity of computational genome analyses with regard to genome annotation completeness is useful for this purpose. In this report we developed the Gene Prediction Accuracy Classification (GPAC) test, which provides statistical evidence of sensitivity by repeating the same analysis for five different gene groups (classified according to annotation accuracy level), and for randomly sampled gene groups, with the same number of genes as each of the five classified groups. Variability in these results is then assessed, and if the results vary significantly with different data subsets, the analysis is considered "sensitive" to annotation completeness, and careful selection of data is advised prior to the actual in silico analysis. The GPAC test has been applied to the analyses of Sakai et al., 2001, and Ohno et al., 2001, and it revealed that the analysis of Ohno et al. was more sensitive to annotation completeness. It showed that GPAC could be employed to ascertain the sensitivity of an analysis. The GPAC bendhmarking software is freely available in the latest G-language Genome Analysis Environment package, at http://www.g-language.org/.     

基于流式细胞技术的灵芝基因组大小估测

以药典规定的灵芝正品来源灵芝Ganoderma lucidum作为研究对象,利用已完成全基因组测序的黑曲霉Aspergillus niger作为内标,通过机械破碎菌丝体的方法获得合适浓度的细胞核悬液,碘化丙啶荧光染色后成功应用流式细胞术进行基因组大小估测。经过优化材料培养、样品制备、上机分析等实验条件,估测得出灵芝基因组大小(48.98±0.60)Mb,为灵芝基因组学研究提供重要数据。该方法简捷稳定,在蕈菌范围内,首次得到了全基因组测序数据与光学图谱结果验证,为蕈菌基因组学研究提供重要技术平台。     

Applications of the polymerase chain reaction to genome analysis

The objectives of the Human Genome Project are to create high-resolution genetic and physical maps, and ultimately to determine the complete nucleotide sequence of the human genome. The result of this initiative will be to localize the estimated 50,000-100,000 human genes, and acquire information that will enable development of a better understanding of the relationship between genome structure and function. To achieve these goals, new methodologies that provide more rapid, efficient, and cost effective means of genomic analysis will be required. From both conceptual and practical perspectives, the polymerase chain reaction (PCR) represents a fundamental technology for genome mapping and sequencing. The availability of PCR has allowed definition of a technically credible form that the final composite map of the human genome will take, as described in the sequence-tagged site proposal. Moreover, applications of PCR have provided efficient approaches for identifying, isolating, mapping, and sequencing DNA, many of which are amenable to automation. The versatility and power provided by PCR have encouraged its involvement in almost every aspect of human genome research, with new applications of PCR being developed on a continual basis.     

MitraClip step by step; how to simplify the procedure

The MitraClip system is a device for percutaneous edge-to-edge reconstruction of the mitral valve in patients with severe mitral regurgitation who are deemed at high risk for surgery. Studies have underlined the therapeutic benefit of the MitraClip system for patients at extreme and high risk for mitral valve surgery, suffering from either degenerative or functional mitral regurgitation. The MitraClip procedure shows low peri-procedural complication rates, and a significant reduction in mitral regurgitation, as well as an improvement in functional capacity and most importantly quality of life. It hereby widens the spectrum of mitral valve repair for the Heart Team. The current review underscores the efficacy of the procedure and describes the technique to simplify the procedure.     

Current approaches to whole genome phylogenetic analysis

It has long been known that evolutionary trees (phylogenies) can be estimated by comparing the DNA or protein sequences of homologous genes across different organisms. More recently, attempts have been made to estimate phylogenies by comparing entire genomes. These attempts have focused largely on comparisons of gene content and gene order. Many different methods have been proposed for making these comparisons. These include primarily maximum parsimony and distance methods, although more recently maximum likelihood and Bayesian methods are being developed. This paper discusses each of these approaches in turn, including their merits and limitations, and any software which is available to make use of them.     

A device to simplify the assay of ligand binding to cell surfaces

A sensitive and reproducible technique for characterization of the binding of ¹²⁵I-labeled protein ligands to cell surfaces is described. The physical separation of cell-bound and free ligand was accomplished by centrifugation-filtration using an assembly of plastic micro test tubes. This assembly allowed rapid and efficient separation of free and cell-bound ligands with minimal manipulation of the cells.     

Computational aspects underlying genome to phenome analysis in plants

Recent advances in genomics technologies have greatly accelerated the progress in both fundamental plant science and applied breeding research. Concurrently, high‐throughput plant phenotyping is becoming widely adopted in the plant community, promising to alleviate the phenotypic bottleneck. While these technological breakthroughs are significantly accelerating quantitative trait locus (QTL) and causal gene identification, challenges to enable even more sophisticated analyses remain. In particular, care needs to be taken to standardize, describe and conduct experiments robustly while relying on plant physiology expertise. In this article, we review the state of the art regarding genome assembly and the future potential of pangenomics in plant research. We also describe the necessity of standardizing and describing phenotypic studies using the Minimum Information About a Plant Phenotyping Experiment (MIAPPE) standard to enable the reuse and integration of phenotypic data. In addition, we show how deep phenotypic data might yield novel trait?trait correlations and review how to link phenotypic data to genomic data. Finally, we provide perspectives on the golden future of machine learning and their potential in linking phenotypes to genomic features.     

landgenreport: a new r function to simplify landscape genetic analysis using resistance surface layers

We describe functions recently added to the r package popgenreport that can be used to perform a landscape genetic analysis (LGA) based on landscape resistance surfaces, which aims to detect the effect of landscape features on gene flow. These functions for the first time implement a LGA in a single framework. Although the approach has been shown to be a valuable tool to study gene flow in landscapes, it has not been widely used to date, despite the type of data being widely available. In part, this is likely due to the necessity to use several software packages to perform landscape genetic analyses. To apply LGA functions, two types of data sets are required: a data set with spatially referenced and genotyped individuals, and a resistance layer representing the effect of the landscape. The function outputs three pairwise distance matrices from these data: a genetic distance matrix, a cost distance matrix and a Euclidean distance matrix. Statistical tests are performed to test whether the cost matrix contributes to the understanding of the observed population structure. A full report on the analysis and outputs in the form of plots and tables of all intermediate steps of the LGA is produced. It is possible to customize the LGA to allow for different cost path approaches and measures of genetic distances. The package is written in the r language and is available through the Comprehensive r Archive. Comprehensive tutorials and information on how to install and use the package are provided at the authors’ website ( www.popgenreport.org ).     

Genetic analysis of theT-H-2 region in non-t Chromosomes

A general breeding protocol useful in the construction of congenic lines of mice disparate in the 15 cMT-H-2 region of chromosome 17 in non-t chromosomes is described. Two such congenic lines, B6.TC2/Rn and B6.TC3/Rn, were derived from the C57BL/6J and B6.C-H-2 ^d/By strains using this protocol. Both B6.TC2 and B6.TC3 dissociate the quantitative activity locus for glyoxalase I (Qglo-1) from theH-2 complex, and hence possess BALB/cBy DNA centromeric toH-2. However, neither new strain is able to map anyH-2-associated restriction fragment length polymorphism with anH-2 cDNA probe even though both strains are recombinant in the 2 cMQglo-1-H-2K interval.     

Statistical analysis of nuclear genome size of plants with flow cytometer data.

BACKGROUND: There is a mismatch between the sophistication of the cytometer and the resulting data, made possible by the computing power of today, and the traditional statistical methods based on the computing power of the early 1930s. The purpose here is to apply modern statistical techniques that similarly take advantage of this computer power. METHODS: Likelihood functions and their graphs are introduced as direct measures of plausibility of the parameters of interest. These methods are valid for samples of any size. They are exemplified on an experimental plant flow cytometer data set with n = 2 replications. RESULTS: The likelihood functions revealed important features of the data that would have been missed by the traditional methods, and in fact would invalidate them. CONCLUSIONS: The likelihood function produced highly informative graphs that allow quantitative comparisons of different aspects of 2C DNA nuclear contents among different groups or varieties of plants.     

Long‐term effects of flow regulation by dams simplify fish functional diversity

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