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...
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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
Several animal models have been developed to study atherosclerosis. Here we present a rabbit atherosclerosis model generated by surgical denudation of the aortic endothelium in combination with a high-fat and choleste...
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Several animal models have been developed to study atherosclerosis. Here we present a rabbit atherosclerosis model generated by surgical denudation of the aortic endothelium in combination with a high-fat and cholesterol-enriched diet. This model is characterized by the formation of vascular lesions that exhibit several hallmarks of human atherosclerosis. Due to the rabbit’s relative large size, as compared to rodents, this model is suited for the imaging-guided evaluation of novel therapeutic strategies using clinical scanners. In this chapter, we present an extensive outline of the procedures to induce aortic atherosclerotic lesions in rabbits as well as methods to evaluate the disease, including noninvasive in vivo multiparametric imaging and histopathology. less
Sequence comparison tools for metagenome-assembled genomes (MAGs) struggle with high-volume or low-quality data. We present skani (https://***/bluenote-1577/skani ), a method for determining average nucleot...
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Sequence comparison tools for metagenome-assembled genomes (MAGs) struggle with high-volume or low-quality data. We present skani (https://***/bluenote-1577/skani ), a method for determining average nucleotide identity (ANI) via sparse approximate alignments. skani outperforms FastANI in accuracy and speed (>20× faster) for fragmented, incomplete MAGs. skani can query genomes against >65,000 prokaryotic genomes in seconds and 6 GB memory. skani unlocks higher-resolution insights for extensive, noisy metagenomic *** comparison tools for metagenome-assembled genomes (MAGs) struggle with high-volume or low-quality data. We present skani (https://***/bluenote-1577/skani ), a method for determining average nucleotide identity (ANI) via sparse approximate alignments. skani outperforms FastANI in accuracy and speed (>20× faster) for fragmented, incomplete MAGs. skani can query genomes against >65,000 prokaryotic genomes in seconds and 6 GB memory. skani unlocks higher-resolution insights for extensive, noisy metagenomic datasets.
Teravoxel volume electron microscopy data sets from neural tissue can now be acquired in weeks, but data analysis requires years of manual labor. We developed the SyConn framework, which uses deep convolutional neural...
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Teravoxel volume electron microscopy data sets from neural tissue can now be acquired in weeks, but data analysis requires years of manual labor. We developed the SyConn framework, which uses deep convolutional neural networks and random forest classifiers to infer a richly annotated synaptic connectivity matrix from manual neurite skeleton reconstructions by automatically identifying mitochondria, synapses and their types, axons, dendrites, spines, myelin, somata and cell types. We tested our approach on serial block-face electron microscopy data sets from zebrafish, mouse and zebra finch, and computed the synaptic wiring of songbird basal ganglia. We found that, for example, basal-ganglia cell types with high firing rates in vivo had higher densities of mitochondria and vesicles and that synapse sizes and quantities scaled systematically, depending on the innervated postsynaptic cell types. less
The accurate and precise analysis of isotopologue and tandem mass isotopologue ratios in heavy stable isotope labeling experiments is a critical part of assessing absolute intracellular metabolic fluxes. Resulting fro...
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The accurate and precise analysis of isotopologue and tandem mass isotopologue ratios in heavy stable isotope labeling experiments is a critical part of assessing absolute intracellular metabolic fluxes. Resulting from feeding the organism of interest with a specifically isotope-labeled substrate, the principal characteristics of these labeling experiments are the metabolites’ non-naturally distributed isotopologue patterns. For the purpose of inferring metabolic rates by maximizing the fit between a priori simulated and experimentally obtained labeling patterns, 13C is the preferred stable isotope of use. The analysis of the obtained labeling patterns can be approached by different mass spectrometric approaches. Gas chromatography (GC) features broad metabolite coverage and excellent separation efficiency of biologically relevant isomers. These advantages compensate for laborious derivatization steps and the resulting need for interference correction for natural abundant isotopes. Here, we describe a workflow based on GC-high resolution mass spectrometry with chemical ionization for the analysis of carbon-isotopologue distributions and some positional labeling information of primary metabolites. To study the associated measurement uncertainty of the resulting 13C labeling patterns, guidance to uncertainty estimation according to the EURACHEM guidelines with Monte-Carlo simulation is provided. less
Metabolomic analyses can provide valuable information about the internal metabolism of an organism; however, these studies can become quickly complicated by the large number of metabolites that are often detected. Ove...
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Metabolomic analyses can provide valuable information about the internal metabolism of an organism; however, these studies can become quickly complicated by the large number of metabolites that are often detected. Overcoming this limitation requires high-resolution analytical separation techniques, coupled with high-power deconvolution software. Additionally, much care must be taken in metabolomic sample preparation to quench active enzymes and avoid artifactual changes in the metabolome. Here we present a relatively simple and straightforward technique, exometabolome mapping, which bypasses each of these concerns, is noninvasive, and provides a concise summary of the key metabolic processes operative in an organism. We illustrate our method using the nematode C. elegans, an organism which has been widely exploited in aging studies; however, with only minimal modification, our technique is extendible to other sample types, and indeed we have successfully used it both to perform yeast footprinting and to study the excreted metabolic end products of human kidney cancer cell lines. less
The simulation of protein aggregation poses several computational challenges due to the disparate time and lengths scales that are involved. This chapter focuses on the use of atomistically detailed simulations to pro...
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The simulation of protein aggregation poses several computational challenges due to the disparate time and lengths scales that are involved. This chapter focuses on the use of atomistically detailed simulations to probe the initial steps of aggregation, with an emphasis on the Tau peptide as a model system, run under a replica exchange molecular dynamics protocol. less
We describe a computational protocol to identify functional modules and pathway relationship of chaperones based on physical interaction data derived from high-throughput proteomic experiments. The protocol first iden...
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We describe a computational protocol to identify functional modules and pathway relationship of chaperones based on physical interaction data derived from high-throughput proteomic experiments. The protocol first identifies interacting proteins shared by the different chaperone systems to organize the chaperones into functional modules. The chaperone functional modules represent groups of chaperones that are involved in mediating the folding of the shared interacting proteins. Either the chaperones in a module can function along a single folding pathway of a given substrate protein or the substrate protein might have two or more different folding pathways that the chaperones act on independently. As described in our computational protocol, probabilities of these pathway relationships between two chaperones in a two-component chaperone module can be determined using whole-genome expression and cellular pathways as reference. This protocol is potentially useful for identifying functional modules and pathway relationships in other biological systems that involve multiple proteins with many identified *** describe a computational protocol to identify functional modules and pathway relationship of chaperones based on physical interaction data derived from high-throughput proteomic experiments. The protocol first identifies interacting proteins shared by the different chaperone systems to organize the chaperones into functional modules. The chaperone functional modules represent groups of chaperones that are involved in mediating the folding of the shared interacting proteins. Either the chaperones in a module can function along a single folding pathway of a given substrate protein or the substrate protein might have two or more different folding pathways that the chaperones act on independently. As described in our computational protocol, probabilities of these pathway relationships between two chaperones in a two-component chaperone module can be determined
microRNA molecules have been shown to play various significant roles in many physiological and pathophysiological processes in living organisms. The tremendous interest in these molecules has led to the significant de...
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microRNA molecules have been shown to play various significant roles in many physiological and pathophysiological processes in living organisms. The tremendous interest in these molecules has led to the significant development and constant release of a number of computational tools useful for basic as well as advanced miRNA-related analyses. These approaches have various constantly evolving utilities, such as detection, target prediction, functional annotation, and many others. In this chapter, we provide an overview of several computational tools useful for broadly defined plant miRNA analysis. less
Software agents are particularly suitable for engineering models and simulations of cellular systems. In a very natural and intuitive manner, individual software components are therein delegated to reproduce “in sil...
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Software agents are particularly suitable for engineering models and simulations of cellular systems. In a very natural and intuitive manner, individual software components are therein delegated to reproduce “in silico” the behavior of individual components of alive systems at a given level of resolution. Individuals’ actions and interactions among individuals allow complex collective behavior to emerge. In this chapter we first introduce the readers to software agents and multi-agent systems, reviewing the evolution of agent-based modeling of biomolecular systems in the last decade. We then describe the main tools, platforms, and methodologies available for programming societies of agents, possibly profiting also of toolkits that do not require advanced programming skills. less
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