Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and mot...
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Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum. less
Retinal development is a dynamic process both anatomically and functionally. High-resolution imaging and dynamic monitoring of photoreceptors and inner neurons can provide important information regarding the structure...
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Retinal development is a dynamic process both anatomically and functionally. High-resolution imaging and dynamic monitoring of photoreceptors and inner neurons can provide important information regarding the structure and function of the developing retina. In this chapter, we describe intrinsic optical signal (IOS) imaging as a high spatiotemporal resolution method for functional study of living retinal tissues. IOS imaging is based on near infrared (NIR) light detection of stimulus-evoked transient change of inherent optical characteristics of the cells. With no requirement for exogenous biomarkers, IOS imaging is totally noninvasive for functional mapping of stimulus-evoked spatiotemporal dynamics of the photoreceptors and inner retinal neurons. less
Bacterial metabolism is an important source of novel products/processes for everyday life and strong efforts are being undertaken to discover and exploit new usable substances of microbial origin. Computational modeli...
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Bacterial metabolism is an important source of novel products/processes for everyday life and strong efforts are being undertaken to discover and exploit new usable substances of microbial origin. Computational modeling and in silico simulations are powerful tools in this context since they allow the exploration and a deeper understanding of bacterial metabolic circuits. Many approaches exist to quantitatively simulate chemical reaction fluxes within the whole microbial metabolism and, regardless of the technique of choice, metabolic model reconstruction is the first step in every modeling pipeline. Reconstructing a metabolic network consists in drafting the list of the biochemical reactions that an organism can carry out together with information on cellular boundaries, a biomass assembly reaction, and exchange fluxes with the external environment. Building up models able to represent the different functional cellular states is universally recognized as a tricky task that requires intensive manual effort and much additional information besides genome sequence. In this chapter we present a general protocol for metabolic reconstruction in bacteria and the main challenges encountered during this process. less
Physiologically based pharmacokinetic (PBPK) models differ from conventional compartmental pharmacokinetic models in that they are based to a large extent on the actual physiology of the organism. The application of p...
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Physiologically based pharmacokinetic (PBPK) models differ from conventional compartmental pharmacokinetic models in that they are based to a large extent on the actual physiology of the organism. The application of pharmacokinetics to toxicology or risk assessment requires that the toxic effects in a particular tissue are related in some way to the concentration time course of an active form of the substance in that tissue. The motivation for applying pharmacokinetics is the expectation that the observed effects of a chemical will be more simply and directly related to a measure of target tissue exposure than to a measure of administered dose. The goal of this work is to provide the reader with an understanding of PBPK modeling and its utility as well as the procedures used in the development and implementation of a model to chemical safety assessment using the styrene PBPK model as an example. less
A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of ...
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A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5ʹ end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2′-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks. Using EdUseq-HU, the spatiotemporal program of DNA replication in human cell lines can be interrogated in <2 weeks. The protocol requires basic cell culture and molecular biology skills, as well as familiarity with the Perl programming language and the Linux operating system. less
Reliable identification and assignment of cis-regulatory elements in promoter regions is a challenging problem in biology. The sophistication of transcriptional regulation in higher eukaryotes, particularly in metazoa...
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Reliable identification and assignment of cis-regulatory elements in promoter regions is a challenging problem in biology. The sophistication of transcriptional regulation in higher eukaryotes, particularly in metazoans, could be an important factor contributing to their organismal complexity. Here we present an integrated approach where networks of co-expressed genes are combined with gene ontology–derived functional networks to discover clusters of genes that share both similar expression patterns and functions. Regulatory elements are identified in the promoter regions of these gene clusters using a Gibbs sampling algorithm implemented in the A-GLAM software package. Using this approach, we analyze the cell-cycle co-expression network of the yeast Saccharomyces cerevisiae, showing that this approach correctly identifies cis-regulatory elements present in clusters of co-expressed genes. less
In this chapter, we present a computational method, TarPmiR, for miRNA target prediction. TarPmiR is based on emerging features of miRNA–target interactions learned from CLASH (crosslinking, ligation and sequencing o...
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In this chapter, we present a computational method, TarPmiR, for miRNA target prediction. TarPmiR is based on emerging features of miRNA–target interactions learned from CLASH (crosslinking, ligation and sequencing of hybrids) data. First, we introduce miRNA target prediction, delineate existing methods for miRNA target prediction, and discuss their usage and limitations. Next, we describe available CLASH data, the learning of new miRNA binding features from CLASH data, and the usage of CLASH features in miRNA target prediction. Finally, we detail the computational pipeline of TarPmiR, discuss its performance compared with existing computational methods for miRNA target prediction, and present its installation and usage for miRNA target prediction. This chapter will facilitate the common understanding of CLASH data, new characteristics of miRNA–target interactions, and the use of the CLASH based miRNA target prediction tool TarPmiR. less
Scaffolds are often used in bioengineering to replace damaged tissues. They promote cell ingrowth and provide mechanical support until cells regenerate. Such scaffolds are often made using the additive manufacturing p...
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Scaffolds are often used in bioengineering to replace damaged tissues. They promote cell ingrowth and provide mechanical support until cells regenerate. Such scaffolds are often made using the additive manufacturing process, given its ability to create complex shapes, affordability, and the potential for patient-specific solutions. The success of the implant is closely related to the match of the scaffold mechanical properties to those of the host tissue. Many biological tissues show properties that vary in space. Therefore, the aim is to manufacture materials with variable properties, commonly referred to as functionally graded materials. Here we present a novel technique used to manufacture porous films with functionally graded properties using 3D printers. Such an approach exploits the control of a process parameter, without any hardware modification. The mechanical properties of the manufactured films have been experimentally tested and analytically characterized. less
Current methods to identify genomic alterations using whole-genome sequencing (WGS) data are often limited to single nucleotide polymorphismsSingle nucleotide polymorphism (SNP) and insertions and deletions that are l...
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Current methods to identify genomic alterations using whole-genome sequencing (WGS) data are often limited to single nucleotide polymorphismsSingle nucleotide polymorphism (SNP) and insertions and deletions that are less than 10 bp in length. These limitations are largely due to challenges in accurately mapping short sequencing reads that significantly diverge from the reference genome. Newer sequencing-based methods have been developed to define and characterize larger DNA structural elements. This is achieved by enriching for and sequencing regions of the genome that contain a specific element, followed by identifying genomic regions with high densities of mapped short reads that designate the location of these elements. This process essentially aggregates short read data into larger structural units for further characterization. Here, we describe protocols for identifying various types of genomic alterations using differential analysis of these structural units. We focus on changes in DNA accessibility, protein–DNA interactions, and chromosomal contacts as measured by ATAC-Seq, ChIP-Seq, and Hi-C respectively. As many protocols have been published describing the generation and processing of these data, we focus on simple methods that can be used to identify mutations in these data, and can be executed by someone with limited computational expertise. less
High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that...
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High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that are aligned to a reference genome allowing the comprehensive characterization of the transcripts in a cell. RNA-Seq has a wide variety of applications which lead to a pervasive adoption of this method well beyond the genomics community and a deployment of this technique as a standard part of the toolkit applied in life sciences. This chapter describes a protocol to perform mRNA sequencing using the Illumina NextSeq or MiSeq platforms, presents sequencing data quality metrics, and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, identification of gene fusions, detection of transcript isoforms, description and annotation of genetic variants, and de novo immunoglobulin gene assembly. less
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