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作者： Edward Airey Stephanie Portelli Joicymara S. Xavier Yoo Chan Myung Michael Silk Malancha Karmakar João P. Velloso Carlos H. Rodrigues Hardik H. Parate Anjali Garg Raghad Al-Jarf Lucy Barr Juliana A. Geraldo Pâmela M. Rezende Douglas E. Pires David B. Ascher

Mutations in protein-coding regions can lead to large biological changes and are associated with genetic conditions, including cancers and Mendelian diseases, as well as drug resistance. Although whole genome and exome sequencing help to elucidate potential genotype–phenotype correlations, there is a large gap between the identification of new variants and deciphering their molecular consequences. A comprehensive understanding of these mechanistic consequences is crucial to better understand and treat diseases in a more personalized and effective way. This is particularly relevant considering estimates that over 80% of mutations associated with a disease are incorrectly assumed to be causative. A thorough analysis of potential effects of mutations is required to correctly identify the molecular mechanisms of disease and enable the distinction between disease-causing and non–disease-causing variation within a gene. Here we present an overview of our integrative mutation analysis platform, which focuses on refining the current genotype–phenotype correlation methods by using the wealth of protein structural information. less

关键词： Protein Structure Analysis Machine Learning Whole-exome sequencing Amino Acid Substitution Mutagenesis Homology Modeling Molecular Docking PyMol Ensembl programming language Python Homo sapiens Mutation Protein interactions Graph-based signatures mCSM Genotype–phenotype correlations

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Whole Transcriptome Sequencing Analysis of Cancer Stem/Progenitor Cells Obtained from Mouse Lung Adenocarcinomas

Whole Transcriptome Sequencing Analysis of Cancer Stem/Proge...

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作者： Ansam Sinjab Reem Daouk Wassim Abou-Kheir Humam Kadara

Interrogation and characterization of lung cancer stem cells (CSCs) that are implicated in lung oncogenesis is crucial for our understanding of inter- and intra-tumor heterogeneity and the aggressive nature of the disease, including tumor resistance and relapse in response to conventional therapy. Here, we describe an in vitro surrogate model, namely the “sphere-forming assay,” for the derivation, enrichment, and propagation of lung stem/progenitor cells with CSC properties, including self-renewal, tumor initiation capacity and propagation, and differentiation into cells of the tumor bulk, from a murine Kras-mutant lung adenocarcinoma cell line. Self-renewing cancer stem/progenitor cells, in the form of 3D in vitro spheres, can be phenotypically interrogated using downstream techniques such as gene expression analysis (e.g., whole transcriptome sequencing and quantitative real time PCR), which is the focus of this chapter. less

关键词： Real-time PCR RNA Extraction Gene Expression Analysis Trypsination In Vitro Assay Recombinant Protein Expression Immunofluorescence Whole-exome sequencing RNA-Seq Analysis Differential Expression Analysis Quantitative Reverse Transcription PCR Homogenization Cell And Tissue Culture RNA-seq Whole transcriptome sequencing Sphere-formation assay TruSeq Trimmomatic HISAT StringTie 3D Cell Culture programming language Mus musculus Bos taurus Self-renewal 3D spheres Cancer stem/progenitor cells

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Codon Resolution Analysis of Ribosome Profiling Data

Codon Resolution Analysis of Ribosome Profiling Data

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作者： Alexander Bartholomäus Zoya Ignatova

Translation is a central biological process in living cells. Ribosome profiling approach enables assessing translation on a global, cell-wide level. Extracting versatile information from the ribosome profiling data usually requires specialized expertise for handling the sequencing data that is not available to the broad community of experimentalists. Here, we provide an easy-to-use and modifiable workflow that uses a small set of commands and enables full data analysis in a standardized way, including precise positioning of the ribosome-protected fragments, for determining codon-specific translation features. The workflow is complemented with simple step-by-step explanations and is accessible to scientists with no computational background. less

关键词： Ribosomal Profiling Deep Sequencing Statistical Calculation Bowtie Illumina Sequencing Frameshift Detection Codon Usage Analysis Data analysis programming language bedtools Cutadapt Escherichia coli Escherichia coli str. K-12 substr. MG1655 Calibration Nucleotide resolution Codon Translation and regulation Analysis pipeline

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The Comprehensive and Reliable Detection of Secondary Metabolites in

The Comprehensive and Reliable Detection of Secondary Metabo...

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作者： Bernhard Seidl Christoph Bueschl Rainer Schuhmacher

A method based on reversed phase high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (RP-HPLC-ESI-HRMS) for the comprehensive and reliable detection of secondary metabolites of Trichoderma reesei cultured in synthetic minimal liquid medium is presented. A stable isotope-assisted (SIA) workflow is used, which allows the automated, comprehensive extraction of truly fungal metabolite-derived LC-MS signals from the acquired chromatographic data. The subsequent statistical data analysis and a typical outcome of such a metabolomics data evaluation are shown by way of example in a previously published study on the influence of the pleiotropic regulator transcription factor Xylanase promoter binding protein 1 (Xpp1) in T. reesei on secondary metabolism. less

关键词： Metabolomics Statistical Calculation Isotope Labeling Mass Spectrometry Reversed-phase HPLC Electrospray Ionization (ESI) High Resolution Mass Spectrometry Liquid Chromatography-mass Spectrometry Freeze-thaw Method Principal Component Analysis Database Comparison Database Search HPLC Data analysis programming language Trichoderma Stable isotope-assisted workflow

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Ontology-based code snippets management in a cloud environment

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JOURNAL OF AMBIENT INTELLIGENCE AND HUMANIZED COMPUTING 2019年第8期10卷 2971-2985页

作者： Shi, Jianjun Ji, Weixing Gao, Zhiwei Gao, Yujin Wang, Yizhuo Liao, Xinyi Shi, Feng Beijing Inst Technol Sch Comp Sci & Technol 5 South Zhongguancun St Beijing 100081 Peoples R China

A code snippet is a small region of reusable source code that is common to many functions. Skilled management and reuse of snippets can improve programming efficiency in practice. However, we find that most snippets are posted to online blogs or snippet management systems with little description and tagging, which leads to an embarrassing situation in which existing snippets are difficult to reuse. This occurs because snippets are usually hastilypushed to online services or saved in text files by programmers and also because existing management systems do not provide efficient labeling and reusing frameworks. In this paper, we propose to annotate snippets with a well-formed domain-specific ontology-programming ontology. With a thorough investigation of real world snippets, we designed a programming ontology to annotate and recommend snippets. We show how to annotate a snippet with ontology terms based on text classification models. In addition, we built a snippet management system that stores user snippets in the cloud and automatically recommends snippets, so that the retrieval of snippets becomes trivial in popular integrated developing environments, such as Eclipse and Visual Studio. Our evaluation results demonstrate that theontology annotation algorithm is able to automatically annotate a snippet with a high degree of accuracy. The shared domain knowledge also makes it possible to share snippets among programmers and systems. As the number of labeled snippets increases, deep learning models can be trained and used to annotate code snippets with high accuracy.

关键词： Ontology Code snippet programming language Cloud computing Deep learning

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Using GRO-Seq to Measure Circadian Transcription and Discover Circadian Enhancers

Using GRO-Seq to Measure Circadian Transcription and Discove...

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作者： Bin Fang Dongyin Guan Mitchell A. Lazar

Circadian gene transcription transmits timing information and drives cyclic physiological processes across various tissues. Recent studies indicate that oscillating enhancer activity is a major driving force of rhythmic gene transcription. Functional circadian enhancers can be identified in an unbiased manner by correlation with the rhythms of nearby gene transcription. Global run-on sequencing (GRO-seq) measures nascent transcription of both pre-mRNAs and enhancer RNAs (eRNAs) at a genome-wide level, making it a unique tool for unraveling complex gene regulation mechanisms in vivo. Here, we describe a comprehensive protocol, ranging from wet lab to in silico analysis, for detecting and quantifying circadian transcription of genes and eRNAs. Moreover, using gene-eRNA correlation, we detail the steps necessary to identify functional enhancers and transcription factors (TFs) that control circadian gene expression in vivo. While we use mouse liver as an example, this protocol is applicable for multiple tissues. less

关键词： Run-on Sequencing GRO-seq Immunoprecipitation Library Preparation PCR Peak Calling ChIP-seq ChIP Assay DNA Sequencing Bowtie bedtools SAMtools programming language Escherichia coli Mus musculus Circadian Transcription factor Enhancer RNA Nascent RNA

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Epitope Mapping of Food Allergens Using Noncontact Piezoelectric Microarray Printer

Epitope Mapping of Food Allergens Using Noncontact Piezoelec...

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作者： Javier Martínez-Botas Carlos Fernández-Lozano Alberto Rodríguez-Alonso Laura Sánchez-Ruano Belén de la Hoz

Peptide microarrays have been used to study protein-protein interaction, enzyme-substrate profiling, epitope mapping, vaccine development, and immuno-profiling. Unlike proteins, peptides are cheap to produce, and can be produced in a high-throughput manner, in a reliable and consistent procedure that reduces batch-to-batch variability. All this provides the peptide microarrays a great potential in the development of new diagnostic tools. Noncontact printing, such as piezoelectric systems, results in a considerable advance in protein and peptide microarray production. In particular, they improve drop deposition, sample distribution, quality control, and flexibility in substrate deposition and eliminate cross-contamination and carryover. These features contribute to creating reproducible assays and generating more reliable data. Here we describe the methods and materials for epitope mapping of food allergens using peptide microarrays produced with a noncontact piezoelectric microarray printer. less

关键词： Epitope Mapping Microarray Hybridization Microarray Peptide Microarray Data Mining Machine Learning HPLC Mass Spectrometry Data analysis programming language Homo sapiens Bos taurus Food allergies Anti-rat Anti-human IgE Anti-rat IgG (H + L) Anti-human

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An unsupervised method for physical cell interaction profiling of complex tissues

An unsupervised method for physical cell interaction profili...

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作者： Nathanael Andrews Jason T. Serviss Natalie Geyer Agneta B. Andersson Ewa Dzwonkowska Iva Šutevski Rosan Heijboer Ninib Baryawno Marco Gerling Martin Enge

Cellular identity in complex multicellular organisms is determined in part by the physical organization of cells. However, large-scale investigation of the cellular interactome remains technically challenging. Here we develop cell interaction by multiplet sequencing (CIM-seq), an unsupervised and high-throughput method to analyze direct physical cell–cell interactions between cell types present in a tissue. CIM-seq is based on RNA sequencing of incompletely dissociated cells, followed by computational deconvolution into constituent cell types. CIM-seq estimates parameters such as number of cells and cell types in each multiplet directly from sequencing data, making it compatible with high-throughput droplet-based methods. When applied to gut epithelium or whole dissociated lung and spleen, CIM-seq correctly identifies known interactions, including those between different cell lineages and immune cells. In the colon, CIM-seq identifies a previously unrecognized goblet cell subtype expressing the wound-healing marker Plet1, which is directly adjacent to colonic stem cells. Our results demonstrate that CIM-seq is broadly applicable to unsupervised profiling of cell-type interactions in different tissue types. less

关键词： Gene Expression Analysis RNA-seq Cell Separation And Isolation Single-cell RNA-seq Immunofluorescence Statistical Calculation Light Microscopy FACS Phase Contrast Microscopy Library Preparation Principal Component Analysis Genomics Paraffin Embedding Snap Freeze ImageJ programming language Seurat RNAscope Stem-cell niche Anti-CD326 Anti-CD24 Anti-CD45

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Computational Resources and Strategies to Construct Single-Molecule Models of FISH

Computational Resources and Strategies to Construct Single-M...

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作者： Beatriz T. Magalhães Rita S. Santos Nuno F. Azevedo Anália Lourenço

Currently, the interactions occurring between oligonucleotides and the cellular envelope of bacteria are not fully resolved at the molecular level. Understanding these interactions is essential to gain insights on how to improve the internalization of the tagged oligonucleotides during fluorescence in situ hybridization (FISH). Agent-based modeling (ABM) is a promising in silico tool to dynamically simulate FISH and bring forward new knowledge on this process. Notably, it is important to simulate the whole bacterial cell, including the different layers of the cell envelope, given that the oligonucleotide must cross the envelope to reach its target in the cytosol. In addition, it is also important to characterize other molecules in the cell to best emulate the cell and represent molecular crowding. Here, we review the main information that should be compiled to construct an ABM on FISH and provide a practical example of an oligonucleotide targeting the 23S rRNA of Escherichia coliEscherichia coli. less

关键词： FISH FRAP programming language Computational modeling Agent-based modeling Biological simulation Molecular diffusion

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Metabolomic profiling of single enlarged lysosomes

Metabolomic profiling of single enlarged lysosomes

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作者： Hongying Zhu Qianqian Li Tiepeng Liao Xiang Yin Qi Chen Ziyi Wang Meifang Dai Lin Yi Siyuan Ge Chenjian Miao Wenping Zeng Lili Qu Zhenyu Ju Guangming Huang Chunlei Cang Wei Xiong

Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes. less

关键词： Mass Spectrometry Metabolomics Patch Clamp Technique Cell And Tissue Culture Western Blot Statistical Calculation CRISPR Transfection Machine Learning Tandem Mass Spectrometry Electrospray Ionization (ESI) PCR ImageJ Scanning Electron Microscopy (SEM) Seurat Python GraphPad Prism programming language Analytical biochemistry Anti-LC3B Anti-GAPDH

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