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Systematic analysis of bypass suppression of essential genes.

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Systematic analysis of bypass suppression of essential genes.

Mol Syst Biol. 2020 Sep;16(9):e9828

Authors: van Leeuwen J, Pons C, Tan G, Wang JZ, Hou J, Weile J, Gebbia M, Liang W, Shuteriqi E, Li Z, Lopes M, Ušaj M, Dos Santos Lopes A, van Lieshout N, Myers CL, Roth FP, Aloy P, Andrews BJ, Boone C

Abstract
Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project.

PMID: 32939983 [PubMed - in process]



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Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.

Sci Adv. 2020 Aug;6(34):

Authors: Hettiaratchi MH, O'Meara MJ, O'Meara TR, Pickering AJ, Letko-Khait N, Shoichet MS

Abstract
Maintaining biocatalyst stability and activity is a critical challenge. Chondroitinase ABC (ChABC) has shown promise in central nervous system (CNS) regeneration, yet its therapeutic utility is severely limited by instability. We computationally reengineered ChABC by introducing 37, 55, and 92 amino acid changes using consensus design and forcefield-based optimization. All mutants were more stable than wild-type ChABC with increased aggregation temperatures between 4° and 8°C. Only ChABC with 37 mutations (ChABC-37) was more active and had a 6.5 times greater half-life than wild-type ChABC, increasing to 106 hours (4.4 days) from only 16.8 hours. ChABC-37, expressed as a fusion protein with Src homology 3 (ChABC-37-SH3), was active for 7 days when released from a hydrogel modified with SH3-binding peptides. This study demonstrates the broad opportunity to improve biocatalysts through computational engineering and sets the stage for future testing of this substantially improved protein in the treatment of debilitating CNS injuries.

PMID: 32937356 [PubMed - as supplied by publisher]



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TheCellVision.org: A Database for Visualizing and Mining High-Content Cell Imaging Projects.

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TheCellVision.org: A Database for Visualizing and Mining High-Content Cell Imaging Projects.

G3 (Bethesda). 2020 Sep 15;:

Authors: Masinas MPD, Mattiazzi Usaj M, Usaj M, Boone C, Andrews BJ

Abstract
Advances in genome engineering and high throughput imaging technologies have enabled genome- scale screens of single cells for a variety of phenotypes, including subcellular morphology and protein localization. We constructed TheCellVision.org, a freely available and web-accessible image visualization and data browsing tool that serves as a central repository for fluorescence microscopy images and associated quantitative data produced by high-content screening experiments. Currently, TheCellVision.org hosts ~575,590 images and associated analysis results from two published high- content screening (HCS) projects focused on the budding yeast Saccharomyces cerevisiae TheCellVision.org allows users to access, visualize and explore fluorescence microscopy images, and to search, compare, and extract data related to subcellular compartment morphology, protein abundance, and localization. Each dataset can be queried independently or as part of a search across multiple datasets using the advanced search option. The website also hosts computational tools associated with the available datasets, which can be applied to other projects and cell systems, a feature we demonstrate using published images of mammalian cells. Providing access to HCS data through websites such as TheCelllVision.org enables new discovery and independent re-analyses of imaging data.

PMID: 32934016 [PubMed - as supplied by publisher]



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Attenuated diphtheria toxin mediates siRNA delivery.

Attenuated diphtheria toxin mediates siRNA delivery.

Sci Adv. 2020 May;6(18):

Authors: Arnold AE, Smith LJ, Beilhartz GL, Bahlmann LC, Jameson E, Melnyk RA, Shoichet MS

Abstract
Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 (ITGB1)-a gene that promotes invasion and metastasis-and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)-a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.

PMID: 32917630 [PubMed - as supplied by publisher]



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Tempora: Cell trajectory inference using time-series single-cell RNA sequencing data.

Tempora: Cell trajectory inference using time-series single-cell RNA sequencing data.

PLoS Comput Biol. 2020 Sep 09;16(9):e1008205

Authors: Tran TN, Bader GD

Abstract
Single-cell RNA sequencing (scRNA-seq) can map cell types, states and transitions during dynamic biological processes such as tissue development and regeneration. Many trajectory inference methods have been developed to order cells by their progression through a dynamic process. However, when time series data is available, most of these methods do not consider the available time information when ordering cells and are instead designed to work only on a single scRNA-seq data snapshot. We present Tempora, a novel cell trajectory inference method that orders cells using time information from time-series scRNA-seq data. In performance comparison tests, Tempora inferred known developmental lineages from three diverse tissue development time series data sets, beating state of the art methods in accuracy and speed. Tempora works at the level of cell clusters (types) and uses biological pathway information to help identify cell type relationships. This approach increases gene expression signal from single cells, processing speed, and interpretability of the inferred trajectory. Our results demonstrate the utility of a combination of time and pathway information to supervise trajectory inference for scRNA-seq based analysis.

PMID: 32903255 [PubMed - as supplied by publisher]



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A framework for designing delivery systems.

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A framework for designing delivery systems.

Nat Nanotechnol. 2020 Sep 07;:

Authors: Poon W, Kingston BR, Ouyang B, Ngo W, Chan WCW

Abstract
The delivery of medical agents to a specific diseased tissue or cell is critical for diagnosing and treating patients. Nanomaterials are promising vehicles to transport agents that include drugs, contrast agents, immunotherapies and gene editors. They can be engineered to have different physical and chemical properties that influence their interactions with their biological environments and delivery destinations. In this Review Article, we discuss nanoparticle delivery systems and how the biology of disease should inform their design. We propose developing a framework for building optimal delivery systems that uses nanoparticle-biological interaction data and computational analyses to guide future nanomaterial designs and delivery strategies.

PMID: 32895522 [PubMed - as supplied by publisher]



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Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.

Read Full Article on External Site Related Articles

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability.

Sci Adv. 2020 Aug;6(34):eabc6378

Authors: Hettiaratchi MH, O'Meara MJ, O'Meara TR, Pickering AJ, Letko-Khait N, Shoichet MS

Abstract
Maintaining biocatalyst stability and activity is a critical challenge. Chondroitinase ABC (ChABC) has shown promise in central nervous system (CNS) regeneration, yet its therapeutic utility is severely limited by instability. We computationally reengineered ChABC by introducing 37, 55, and 92 amino acid changes using consensus design and forcefield-based optimization. All mutants were more stable than wild-type ChABC with increased aggregation temperatures between 4° and 8°C. Only ChABC with 37 mutations (ChABC-37) was more active and had a 6.5 times greater half-life than wild-type ChABC, increasing to 106 hours (4.4 days) from only 16.8 hours. ChABC-37, expressed as a fusion protein with Src homology 3 (ChABC-37-SH3), was active for 7 days when released from a hydrogel modified with SH3-binding peptides. This study demonstrates the broad opportunity to improve biocatalysts through computational engineering and sets the stage for future testing of this substantially improved protein in the treatment of debilitating CNS injuries.

PMID: 32875119 [PubMed - in process]



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Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact.

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Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact.

Elife. 2020 Sep 01;9:

Authors: Chiasson MA, Rollins NJ, Stephany JJ, Sitko KA, Matreyek KA, Verby M, Sun S, Roth FP, DeSloover D, Marks DS, Rettie AE, Fowler DM

Abstract
Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

PMID: 32870157 [PubMed - as supplied by publisher]



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Dynamics of the cell-free DNA methylome of metastatic prostate cancer during androgen-targeting treatment.

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Dynamics of the cell-free DNA methylome of metastatic prostate cancer during androgen-targeting treatment.

Epigenomics. 2020 Sep 01;:

Authors: Peter MR, Bilenky M, Isserlin R, Bader GD, Shen SY, De Carvalho DD, Hansen AR, Hu P, Fleshner NE, Joshua AM, Hirst M, Bapat B

Abstract
Aim: We examined methylation changes in cell-free DNA (cfDNA) in metastatic castration-resistant prostate cancer (mCRPC) during treatment. Patients & methods: Genome-wide methylation analysis of sequentially collected cfDNA samples derived from mCRPC patients undergoing androgen-targeting therapy was performed. Results: Alterations in methylation states of genes previously implicated in prostate cancer progression were observed and patients that maintained methylation changes throughout therapy tended to have a longer time to clinical progression. Importantly, we also report that markers associated with a highly aggressive form of the disease, neuroendocrine-CRPC, were associated with a faster time to clinical progression. Conclusion: Our findings highlight the potential of monitoring the cfDNA methylome during therapy in mCRPC, which may serve as predictive markers of response to androgen-targeting agents.

PMID: 32867540 [PubMed - as supplied by publisher]



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Stem cell heterogeneity and regenerative competence: the enormous potential of rare cells.

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Stem cell heterogeneity and regenerative competence: the enormous potential of rare cells.

Neural Regen Res. 2021 Feb;16(2):285-286

Authors: Gilbert EAB, Morshead CM

PMID: 32859777 [PubMed - as supplied by publisher]



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