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Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry
In pharmaceutical research, high‐content screening is an integral part of lead candidate development. Measuring drug response in vitro by examining over 40 parameters, including biomarkers, signaling molecules, cell morphological changes, proliferation indices, and toxicity in a single sample, could significantly enhance discovery of new therapeutics. As a proof of concept, we present here a workflow for multidimensional Imaging Mass Cytometry™ (IMC™) and data processing with open source computational tools. CellProfiler was used to identify single cells through establishing cellular boundaries, followed by histoCAT™ (histology topography cytometry analysis toolbox) for extracting single‐cell quantitative information visualized as t‐SNE plots and heatmaps. Human breast cancer‐derived cell lines SKBR3, HCC1143, and MCF‐7 were screened for expression of cellular markers to generate digital images with a resolution comparable to conventional fluorescence microscopy. Predicted pharmacodynamic effects were measured in MCF‐7 cells dosed with three target‐specific compounds: growth stimulatory EGF, microtubule depolymerization agent nocodazole, and genotoxic chemotherapeutic drug etoposide. We show strong pairwise correlation between nuclear markers pHistone3S28, Ki‐67, and p4E‐BP1T37/T46 in classified mitotic cells and anticorrelation with cell surface markers. Our study demonstrates that IMC data expand the number of measured parameters in single cells and brings higher‐dimension analysis to the field of cell‐based screening in early lead compound discovery. Imaging Mass Cytometry™ is an emerging technology which combines metal labeled reagents, laser ablation, and mass cytometry for the spatial analysis of 40 parameters within single cells or tissue sections on slide. Our method describes a comprehensive workflow for sample preparation, acquisition, and analysis of breast cancer cell line models in the context of in vitro drug screening assays.
Journal Article
Networks of High Mutual Information Define the Structural Proximity of Catalytic Sites: Implications for Catalytic Residue Identification
Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.
Journal Article
Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3
Culture-independent analyses of microbial communities have progressed dramatically in the last decade, particularly due to advances in methods for biological profiling via shotgun metagenomics. Opportunities for improvement continue to accelerate, with greater access to multi-omics, microbial reference genomes, and strain-level diversity. To leverage these, we present bioBakery 3, a set of integrated, improved methods for taxonomic, strain-level, functional, and phylogenetic profiling of metagenomes newly developed to build on the largest set of reference sequences now available. Compared to current alternatives, MetaPhlAn 3 increases the accuracy of taxonomic profiling, and HUMAnN 3 improves that of functional potential and activity. These methods detected novel disease-microbiome links in applications to CRC (1262 metagenomes) and IBD (1635 metagenomes and 817 metatranscriptomes). Strain-level profiling of an additional 4077 metagenomes with StrainPhlAn 3 and PanPhlAn 3 unraveled the phylogenetic and functional structure of the common gut microbe Ruminococcus bromii , previously described by only 15 isolate genomes. With open-source implementations and cloud-deployable reproducible workflows, the bioBakery 3 platform can help researchers deepen the resolution, scale, and accuracy of multi-omic profiling for microbial community studies.
Journal Article
A Computational Study of High-Speed Microdroplet Impact onto a Smooth Solid Surface
Numerical solutions of high-speed microdroplet impact onto a smooth solid surface are computed, using the interFoam VoF solver of the OpenFOAM(R) CFD package. Toward the solid surface, the liquid microdroplet is moving with an impinging gas flow, simulating the situation of ink droplets being deposited onto substrate with a collimated mist jet in the Optomec Aerosol Jet(R) printing process. For simplicity and computational efficiency, axisymmetric incompressible flow is assumed here for the free-surface fluid dynamic problem. The computed values of maximum spread factor [xi], for the range of parameters relevant to Aerosol Jet(R) printing, are found in good agreement with some of the correlation formulas proposed by previous authors in the literature. A formula of improved accuracy is then obtained for evaluating [xi] of Aerosol Jet(R) deposited droplets, by combining selected formulas from different authors with appropriate modifications. The computational results also illustrate droplet impact dynamics with lamella shape evolution throughout the spreading, receding-relaxation, and wetting equilibrium phases, consistent with that observed and described by many authors. This suggests a scale-invariant nature of the basic droplet impact behavior such that experiments with larger droplets at the same nondimensional parameter values may be applicable for studying microdroplet impact dynamics. Significant free surface oscillations can be observed with low viscosity droplets. The border line between free surface oscillations and aperiodic creeping to the capillary equilibrium shape appears at Oh ~ 0.25. Droplet bouncing after receding is prompted with large contact angles at solid surface (as consistent with findings reported in the literature), but can be suppressed by increasing the droplet viscosity.
Journal Article
Synthesis and Magnetic Properties of Stable Radical Derivatives Carrying a Phenylacetylene Unit
A nitronyl nitroxide derivative, 2-phenylethynyl-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxyl-3-oxide (1), and two verdazyl derivatives carrying a phenylacetylene unit, 1,5-diphenyl-3-phenylethynyl-6-oxo-1,2,4,5-tetrazin-2-yl (2) and 1,5-diisopropyl-3-phenylethynyl-6-oxo-1,2,4,5-tetrazin-2-yl (3), were synthesized and their packing structures were studied by X-ray crystallographic analysis and magnetically characterized in the solid state. While 1 and 3 had an isolated doublet spin state, 2 formed an antiferromagnetically coupled pair (2J/kB = −118 K). Density functional theory (DFT) calculations reveal that the spin density polarized in the phenyl group decreases as the dihedral angle between the phenyl ring and radical plane increases.
Journal Article