PubMed

Recent Publications

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.

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

PLoS Comput Biol. 2020 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 - indexed for MEDLINE]



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A framework for designing delivery systems.

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A framework for designing delivery systems.

Nat Nanotechnol. 2020 10;15(10):819-829

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 - indexed for MEDLINE]



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

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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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Methacrylic acid copolymer coating of polypropylene mesh chamber improves subcutaneous islet engraftment.

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Methacrylic acid copolymer coating of polypropylene mesh chamber improves subcutaneous islet engraftment.

Biomaterials. 2020 Aug 15;259:120324

Authors: Coindre VF, Kinney SM, Sefton MV

Abstract
Subcutaneous devices can be used to house therapeutic cells such as pancreatic islets so that the cells can be retrieved. However, a high number of cells may be required to reverse diabetes, since a portion of the graft can be lost after transplantation due to ischemia and therefore the right device design is important. Increasing the vascularity of the subcutaneous space prior to cell transplantation is a strategic goal for cell transplantation, as it promotes islet survival, glucose-sensing and insulin secretion. In this study, a porous cell transplantation device was coated with 40% methacrylic acid-co-isodecyl acrylate (MAA-co-IDA), a biomaterial which promotes a vascular response without additional biologics. Three weeks after device implantation, the vessel density surrounding the device was double that of an uncoated device. The vasculature was mature and connected to the host bloodstream, as demonstrated by perfusion studies and histology. The tissue response to coated devices demonstrated lower levels of inflammation, measured by reduced gene expression of i-NOS and IL1β, and increased expression of IL4. Syngeneic islets (300 islet equivalents) transplanted into the prevascularized coated device were able to return diabetic animals to normoglycemia for up to 11 weeks and resolve a glucose bolus similarly to non-diabetic mice by 3 weeks post-transplantation. We expect that the vessels and microenvironment resulting from the device coating are permissive to islet survival and thus enabled islets to reverse diabetes.

PMID: 32858417 [PubMed - as supplied by publisher]



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Retrons: Complementing CRISPR in Phage Defense.

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Retrons: Complementing CRISPR in Phage Defense.

CRISPR J. 2020 08;3(4):226-227

Authors: Maxwell KL

PMID: 32833529 [PubMed - indexed for MEDLINE]



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Cell invasion in digital microfluidic microgel systems.

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Cell invasion in digital microfluidic microgel systems.

Sci Adv. 2020 Jul;6(29):eaba9589

Authors: Li BB, Scott EY, Chamberlain MD, Duong BTV, Zhang S, Done SJ, Wheeler AR

Abstract
Microfluidic methods for studying cell invasion can be subdivided into those in which cells invade into free space and those in which cells invade into hydrogels. The former techniques allow straightforward extraction of subpopulations of cells for RNA sequencing, while the latter preserve key aspects of cell interactions with the extracellular matrix (ECM). Here, we introduce "cell invasion in digital microfluidic microgel systems" (CIMMS), which bridges the gap between them, allowing the stratification of cells on the basis of their invasiveness into hydrogels for RNA sequencing. In initial studies with a breast cancer model, 244 genes were found to be differentially expressed between invading and noninvading cells, including genes correlating with ECM-remodeling, chemokine/cytokine receptors, and G protein transducers. These results suggest that CIMMS will be a valuable tool for probing metastasis as well as the many physiological processes that rely on invasion, such as tissue development, repair, and protection.

PMID: 32832633 [PubMed - as supplied by publisher]



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