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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]

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]

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]

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]