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Quantifying the Biological Impact of Active Substances Using Causal Network Models

Quantifying the Biological Impact of Active Substances Using...

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作者： Alain Sewer Florian Martin Walter K. Schlage Julia Hoeng Manuel C. Peitsch

In this chapter a five-step strategy is described that provides comparative evaluations of the effects of biologically active substances. These evaluations constitute an integral part of the determination of the risks for the human population to exposure to these substances. The strategy is based on the concept of biological impact quantification for which novel computational methodologies have been developed in the past few years; these methodologies are reviewed in this chapter. The effects of the active substances are then described in terms of networks containing the biological mechanisms involved in the response to the exposure. As a consequence, the biological impact assessment represents a systems-wide metric of network-based perturbed biological mechanisms. The implementation of the strategy involves the generation of transcriptomics data following the exposure experiment and their evaluation in the context of causal network models. After the five-step strategy for biological impact quantification has been described in some detail, its application in a concrete case of a mouse smoking-cessation experiment is presented. The results show how mechanistic insights into the potential toxic effects of exposure to active substances can benefit the safety assessment.

关键词： Transcriptomics DNA Extraction Microarray Hybridization Proteomics Principal Component Analysis Statistical Calculation Data analysis programming language Biological impact of exposure Causal biological network models Network perturbations Mechanistic fingerprinting

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A Wearable Sensing System for Assessment of Exposures to Environmental Volatile Organic Compounds

A Wearable Sensing System for Assessment of Exposures to Env...

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作者： Cheng Chen Francis Tsow Xiaojun Xian Erica Forzani Nongjian Tao Raymond Tsui

A portable chemical sensing system that integrates sample preconcentration, separation and detection as well as wireless communication functionalities into a compact, wearable format can provide continuous and real-time monitoring of volatile organic compounds in the environment. The sensing modality relies on tuning forks coated with molecularly imprinted polymers that, in conjunction with sample preconcentration, offer selective detection down to parts-per-billion levels. The use of capillary columns allows individual components of complex mixtures to be detected at these highly sensitive levels even in the presence of interferents. The wireless capability facilitates the utilization of a paired smartphone as the user interface as well as a vehicle for additional processing and storage of the measured data. This integrated approach offers a cost-effective and reliable platform for personal exposure assessment. less

关键词： programming language Environmental monitoring Volatile organic compounds Chemical sensors Wearable detection system Wireless sensing Personal exposure assessment

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Large-Scale Parameter Studies of Cell-Based Models of Tissue Morphogenesis Using

Large-Scale Parameter Studies of Cell-Based Models of Tissue...

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作者： Margriet M. Palm Roeland M. Merks

Computational, cell-based models, such as the cellular Potts model (CPM), have become a widely used tool to study tissue formation. Most cell-based models mimic the physical properties of cells and their dynamic behavior, and generate images of the tissue that the cells form due to their collective behavior. Due to these intuitive parameters and output, cell-based models are often evaluated visually and the parameters are fine-tuned by hand. To get better insight into how in a cell-based model the microscopic scale (e.g., cell behavior, secreted molecular signals, and cell-ECM interactions) determines the macroscopic scale, we need to generate morphospaces and perform parameter sweeps, involving large numbers of individual simulations. This chapter describes a protocol and presents a set of scripts for automatically setting up, running, and evaluating large-scale parameter sweeps of cell-based models. We demonstrate the use of the protocol using a recent cellular Potts model of blood vessel formation model implemented in CompuCell3D. We show the versatility of the protocol by adapting it to an alternative cell-based modeling framework, VirtualLeaf. less

关键词： Statistical Calculation Python Data analysis programming language Quantification Angiogenesis Cellular Potts model CompuCell3D VirtualLeaf Cell-based model Parameter study

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Rapid reverse genetic screening using CRISPR in zebrafish

Rapid reverse genetic screening using CRISPR in zebrafish

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作者： Arish N Shah Crystal F Davey Alex C Whitebirch Adam C Miller Cecilia B Moens

Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. We used engineered CRISPR (clustered regularly interspaced short palindromic repeats) to efficiently mutate specific loci in zebrafish (Danio rerio) and screen for genes involved in vertebrate biological processes. We found that increasing CRISPR efficiency by injecting optimized amounts of Cas9-encoding mRNA and multiplexing single guide RNAs (sgRNAs) allowed for phenocopy of known mutants across many phenotypes in embryos. We performed a proof-of-concept screen in which we used intersecting, multiplexed pool injections to examine 48 loci and identified two new genes involved in electrical-synapse formation. By deep sequencing target loci, we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system. less

关键词： DNA Extraction Immunohistochemistry CRISPR In Vitro Transcription Real-time PCR Deep Sequencing HPLC Mutagenesis Illumina Sequencing Fiji ImageJ Data analysis programming language Danio rerio Mutation

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X-Ray Diffraction of Lipid Model Membranes

X-Ray Diffraction of Lipid Model Membranes

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作者： Arwen I. Tyler Robert V. Law John M. Seddon

In this chapter the use of X-ray diffraction to study the structure of lyotropic phases and lipid model membranes is described. Determination of the phase symmetry and lattice parameters from small-angle X-ray scattering (SAXS), and of the nature of the hydrocarbon chain packing from wide-angle X-ray scattering (WAXS), are discussed. Methods by which the sign of the interfacial curvature of non-lamellar phases may be determined are then presented. Finally, the calculation of electron density profiles from the intensities of the observed Bragg peaks is described, for the lamellar phase and for the inverse hexagonal phase. less

关键词： X-ray Diffraction Membrane Modeling Wide-angle X-ray Scattering Small-angle X-ray Scattering (SAXS) Freeze-thaw Method HPLC Small-angle X-ray Diffraction MATLAB Data analysis programming language SAXS X-ray Phase WAXS

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e-DNA Meta-Barcoding: From NGS Raw Data to Taxonomic Profiling

e-DNA Meta-Barcoding: From NGS Raw Data to Taxonomic Profili...

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作者： Fosso Bruno Marzano Marinella Monica Santamaria

In recent years, thanks to the essential support provided by the Next-Generation Sequencing (NGS) technologies, Metagenomics is enabling the direct access to the taxonomic and functional composition of mixed microbial communities living in any environmental niche, without the prerequisite to isolate or culture the single organisms. This approach has already been successfully applied for the analysis of many habitats, such as water or soil natural environments, also characterized by extreme physical and chemical conditions, food supply chains, and animal organisms, including humans. A shotgun sequencing approach can lead to investigate both organisms and genes diversity. Anyway, if the purpose is limited to explore the taxonomic complexity, an amplicon-based approach, based on PCR-targeted sequencing of selected genetic species markers, commonly named “meta-barcodes”, is desirable. Among the genomic regions most widely used for the discrimination of bacterial organisms, in some cases up to the species level, some hypervariable domains of the gene coding for the 16S rRNA occupy a prominent place. The amplification of a certain meta-barcode from a microbial community through the use of PCR primers able to work in the entire considered taxonomic group is the first task after the extraction of the total DNA. Generally, this step is followed by the high-throughput sequencing of the resulting amplicons libraries by means of a selected NGS platform. Finally, the interpretation of the huge amount of produced data requires appropriate bioinformatics tools and know-how in addition to efficient computational resources. Here a computational methodology suitable for the taxonomic characterization of 454 meta-barcode sequences is described in detail. In particular, a dataset covering the V1–V3 region belonging to the bacterial 16S rRNA coding gene and produced in the Human Microbiome Project (HMP) from a palatine tonsils sample is analyzed. The proposed exercise includes the basic

关键词： Metagenomics Pyrosequencing Shotgun Sequencing PCR High-Throughput Sequencing Statistical Calculation Python Mothur Data analysis programming language Homo sapiens Microbiome 16S rRNA Taxonomy e-DNA Meta-barcoding

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Gene Expression-Based Approaches to Understanding Huntington’s Disease and New Tools for the Interpretation of Expression Datasets

Gene Expression-Based Approaches to Understanding Huntington...

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作者： Alexandre Kuhn Alberto Capurro Ruth Luthi-Carter

Expression profiling has become a well-established and widely utilized approach to generate and support hypotheses regarding biological processes. Etiopathologic mechanisms of Huntingtin toxicity include transcriptional dysregulation [reviewed in Luthi-Carter (Drug Dis Today Dis Mech 4:111–119, 2007)], and a strikingly characteristic set of HD-related changes in gene expression have been informative in assessing model systems and underlying pathological (and compensatory) mechanisms [reviewed in Seredenina and Luthi-Carter (Neurobiol Dis 45:83–98, 2012)]. Here we provide details of two strategies that we have developed to improve gene expression analyses in Huntington’s disease (HD). The first subsection of this chapter describes an approach we have termed “Population-Specific Expression Analysis” (PSEA). This computational method was developed to be able to assign expression measures to specific cell types within complex tissues. The second subsection demonstrates the implementation of a simple measure that we have termed the “concordance coefficient” to compare the similarities of disease-related effects across different systems. These methods are both applicable to a variety of experimental contexts and molecular data types. less

关键词： Proteomics Transcriptomics Microarray RNA-seq Gene Expression Profiling Affymetrix SNP Array Statistical Modeling Statistical Calculation programming language Data analysis Gene expression Polyglutamine disease

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Designing “High-Affinity, High-Specificity” Glycosaminoglycan Sequences Through Computerized Modeling

Designing “High-Affinity, High-Specificity” Glycosaminogly...

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作者： Nehru Viji Sankaranarayanan Aurijit Sarkar Umesh R. Desai Philip D. Mosier

The prediction of high-affinity and/or high-specificity protein–glycosaminoglycan (GAG) interactions is an inherently difficult task, due to several factors including the shallow nature of the typical GAG-binding site and the inherent size, flexibility, diversity, and polydisperse nature of the GAG molecules. Here, we present a generally applicable methodology termed Combinatorial Library Virtual Screening (CVLS) that can identify potential high-affinity, high-specificity protein–GAG interactions from very large GAG combinatorial libraries and a suitable GAG-binding protein. We describe the CVLS approach along with the rationale behind it and provide validation for the method using the well-known antithrombin–thrombin–heparin system. less

关键词： Virtual Screening Homology Modeling Molecular Modeling Molecular Docking Protein Structure Analysis Site-directed Mutagenesis programming language Specificity Docking Molecular interaction Glycosaminoglycan (GAG) Virtual library Genetic algorithm (GA) Antithrombin

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Analysis and Application of Viroid-Specific Small RNAs Generated by Viroid-Inducing RNA Silencing

Analysis and Application of Viroid-Specific Small RNAs Gener...

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作者： Charith Raj Adkar-Purushothama Zhixiang Zhang Shifang Li Teruo Sano

Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing—a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21–24-nucleotides by cleaving (or “dicing”) entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant. The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid–host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species. less

关键词： Deep Sequencing Reverse Transcription PCR PCR Heat Denaturation Restriction Enzyme Digestion Agarose Gel Electrophoresis DNA Extraction Ultraviolet Spectrophotometry Fractionation Cross-linking ELISA Sequence Analysis PAGE DNA Sequencing Statistical Calculation Transcription Profiling Genomics Tag Mapping CDNA Amplification DNA Ligation programming language Trimmomatic Cutadapt GenBank Viroids Saccharomyces cerevisiae Escherichia coli Bioinformatics Next-generation sequencing RNA silencing Mechanical inoculation Host interaction Infectious cDNA clones Viroid-specific small RNAs RNA gel-blot hybridization Anti-DIG

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Melanoma and Other Skin Lesion Detection Using Smart Handheld Devices

Melanoma and Other Skin Lesion Detection Using Smart Handhel...

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作者： George Zouridakis Tarun Wadhawan Ning Situ Rui Hu Xiaojing Yuan Keith Lancaster Courtney M. Queen

Smartphones of the latest generation featuring advanced multicore processors, dedicated microchips for graphics, high-resolution cameras, and innovative operating systems provide a portable platform for running sophisticated medical screening software and delivering point-of-care patient diagnostic services at a very low cost. In this chapter, we present a smartphone digital dermoscopy application that can analyze high-resolution images of skin lesions and provide the user with feedback about the likelihood of malignancy. The same basic procedure has been adapted to evaluate other skin lesions, such as the flesh-eating bacterial disease known as Buruli ulcer. When implemented on the iPhone, the accuracy and speed achieved by this application are comparable to that of a desktop computer, demonstrating that smartphone applications can combine portability and low cost with high performance. Thus, smartphone-based systems can be used as assistive devices by primary care physicians during routine office visits, and they can have a significant impact in underserved areas and in developing countries, where health-care infrastructure is limited. less

关键词： Dermoscopy Image Analysis Multispectral Imaging Transillumination Machine Learning Data Mining Image Processing Data analysis programming language Homo sapiens Malus domestica Skin cancer detection Melanoma screening Buruli ulcer Smartphones in health care Handheld devices

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