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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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A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs.

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A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs.

Protein Sci. 2020 Aug 17;:

Authors: Hanna R, Cardarelli L, Patel N, Blazer LL, Adams JJ, Sidhu SS

Abstract
Phage-displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full-length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate-limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage-displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage-displayed synthetic single-chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single-chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage-derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell-surface receptors, and scIgGs behave the same as conventional IgGs. This article is protected by copyright. All rights reserved.

PMID: 32803886 [PubMed - as supplied by publisher]



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Modeling the Control of TGF-β/Smad Nuclear Accumulation by the Hippo Pathway Effectors, Taz/Yap.

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Modeling the Control of TGF-β/Smad Nuclear Accumulation by the Hippo Pathway Effectors, Taz/Yap.

iScience. 2020 Jul 29;23(8):101416

Authors: Labibi B, Bashkurov M, Wrana JL, Attisano L

Abstract
Integration of transforming growth factor β (TGF-β) signals with those of other pathways allows for precise temporal and spatial control of gene expression patterns that drive development and homeostasis. The Hippo pathway nuclear effectors, Taz/Yap, interact with the TGF-β transcriptional mediators, Smads, to control Smad activity. Key to TGF-β signaling is the nuclear localization of Smads. Thus, to investigate the role of Taz/Yap in Smad nuclear accumulation, we developed mathematical models of Hippo and TGF-β cross talk. The models were based on experimental measurements of TGF-β-induced changes in Taz/Yap and Smad subcellular localization obtained using high-throughput immunofluorescence (IF) imaging in the mouse mammary epithelial cell line, EpH4. Bayesian MCMC DREAM parameter estimation was used to quantify the uncertainty in estimates of the kinetic parameters. Variation of the model parameters and statistical analysis show that our modeling predicts that Taz/Yap can alter TGF-β receptor activity and directly or indirectly act as nuclear retention factors.

PMID: 32798968 [PubMed - as supplied by publisher]



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Hydrogel-mediated co-transplantation of retinal pigmented epithelium and photoreceptors restores vision in an animal model of advanced retinal degeneration.

Hydrogel-mediated co-transplantation of retinal pigmented epithelium and photoreceptors restores vision in an animal model of advanced retinal degeneration.

Biomaterials. 2020 Jul 30;257:120233

Authors: Mitrousis N, Hacibekiroglu S, Ho MT, Sauvé Y, Nagy A, van der Kooy D, Shoichet MS

Abstract
We demonstrate a novel approach to reverse advanced stages of blindness using hydrogel-mediated delivery of retinal pigmented epithelium (RPE) and photoreceptors directly to the degenerated retina of blind mice. With sodium iodate (NaIO3) injections in mice, both RPE and photoreceptors degenerate, resulting in complete blindness and recapitulating the advanced retinal degeneration that is often observed in humans. We observed vision restoration only with co-transplantation of RPE and photoreceptors in a hyaluronic acid-based hydrogel, and not with transplantation of each cell type alone as determined with optokinetic head tracking and light avoidance assays. Both RPE and photoreceptors survived significantly better when co-transplanted than in their respective single cell type controls. While others have pursued transplantation of one of either RPE or photoreceptors, we demonstrate the importance of transplanting both cell types with a minimally-invasive hydrogel for vision repair in a degenerative disease model of the retina.

PMID: 32791386 [PubMed - as supplied by publisher]



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Leveraging an Open Science Drug Discovery Model to Develop CNS-Penetrant ALK2 Inhibitors for the Treatment of Diffuse Intrinsic Pontine Glioma.

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Leveraging an Open Science Drug Discovery Model to Develop CNS-Penetrant ALK2 Inhibitors for the Treatment of Diffuse Intrinsic Pontine Glioma.

J Med Chem. 2020 09 10;63(17):10061-10085

Authors: Smil D, Wong JF, Williams EP, Adamson RJ, Howarth A, McLeod DA, Mamai A, Kim S, Wilson BJ, Kiyota T, Aman A, Owen J, Poda G, Horiuchi KY, Kuznetsova E, Ma H, Hamblin JN, Cramp S, Roberts OG, Edwards AM, Uehling D, Al-Awar R, Bullock AN, O'Meara JA, Isaac MB

Abstract
There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.

PMID: 32787083 [PubMed - indexed for MEDLINE]



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Single-Cell Profiling Shows Murine Forebrain Neural Stem Cells Reacquire a Developmental State when Activated for Adult Neurogenesis.

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Single-Cell Profiling Shows Murine Forebrain Neural Stem Cells Reacquire a Developmental State when Activated for Adult Neurogenesis.

Cell Rep. 2020 Aug 11;32(6):108022

Authors: Borrett MJ, Innes BT, Jeong D, Tahmasian N, Storer MA, Bader GD, Kaplan DR, Miller FD

Abstract
The transitions from developing to adult quiescent and activated neural stem cells (NSCs) are not well understood. Here, we use single-cell transcriptional profiling and lineage tracing to characterize these transitions in the murine forebrain. We show that the two forebrain NSC parental populations, embryonic cortex and ganglionic eminence radial precursors (RPs), are highly similar even though they make glutamatergic versus gabaergic neurons. Both RP populations progress linearly to transition from a highly active embryonic to a dormant adult stem cell state that still shares many similarities with embryonic RPs. When adult NSCs of either embryonic origin become reactivated to make gabaergic neurons, they acquire a developing ganglionic eminence RP-like identity. Thus, transitions from embryonic RPs to adult NSCs and back to neuronal progenitors do not involve fundamental changes in cell identity, but rather reflect conversions between activated and dormant NSC states that may be determined by the niche environment.

PMID: 32783944 [PubMed - in process]



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Functional characterization of a PROTAC directed against BRAF mutant V600E.

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Functional characterization of a PROTAC directed against BRAF mutant V600E.

Nat Chem Biol. 2020 11;16(11):1170-1178

Authors: Posternak G, Tang X, Maisonneuve P, Jin T, Lavoie H, Daou S, Orlicky S, Goullet de Rugy T, Caldwell L, Chan K, Aman A, Prakesch M, Poda G, Mader P, Wong C, Maier S, Kitaygorodsky J, Larsen B, Colwill K, Yin Z, Ceccarelli DF, Batey RA, Taipale M, Kurinov I, Uehling D, Wrana J, Durocher D, Gingras AC, Al-Awar R, Therrien M, Sicheri F

Abstract
The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.

PMID: 32778845 [PubMed - indexed for MEDLINE]



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The dose threshold for nanoparticle tumour delivery.

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The dose threshold for nanoparticle tumour delivery.

Nat Mater. 2020 Aug 10;:

Authors: Ouyang B, Poon W, Zhang YN, Lin ZP, Kingston BR, Tavares AJ, Zhang Y, Chen J, Valic MS, Syed AM, MacMillan P, Couture-Senécal J, Zheng G, Chan WCW

Abstract
Nanoparticle delivery to solid tumours over the past ten years has stagnated at a median of 0.7% of the injected dose. Varying nanoparticle designs and strategies have yielded only minor improvements. Here we discovered a dose threshold for improving nanoparticle tumour delivery: 1 trillion nanoparticles in mice. Doses above this threshold overwhelmed Kupffer cell uptake rates, nonlinearly decreased liver clearance, prolonged circulation and increased nanoparticle tumour delivery. This enabled up to 12% tumour delivery efficiency and delivery to 93% of cells in tumours, and also improved the therapeutic efficacy of Caelyx/Doxil. This threshold was robust across different nanoparticle types, tumour models and studies across ten years of the literature. Our results have implications for human translation and highlight a simple, but powerful, principle for designing nanoparticle cancer treatments.

PMID: 32778816 [PubMed - as supplied by publisher]



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