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Recent Publications

Endothelialized collagen based pseudo-islets enables tuneable subcutaneous diabetes therapy.

Endothelialized collagen based pseudo-islets enables tuneable subcutaneous diabetes therapy.

Biomaterials. 2019 Dec 26;232:119710

Authors: Vlahos AE, Kinney SM, Kingston BR, Keshavjee S, Won SY, Martyts A, Chan WWC, Sefton MV

Abstract
Pancreatic islets are fragile cell clusters and many isolated islets are not suitable for transplantation. Furthermore, following transplantation, islets will experience a state of hypoxia and poor nutrient diffusion before revascularization, which is detrimental to islet survival; this is affected by islet size and health. Here we engineered tuneable size-controlled pseudo-islets created by dispersing de-aggregated islets in an endothelialized collagen scaffold. This supported subcutaneous engraftment, which returned streptozotocin-induced diabetic mice to normoglycemia. Whole-implant imaging after tissue clearing demonstrated pseudo-islets regenerated their vascular architecture and insulin-secreting β-cells were within 5 μm of a perfusable vessel - a feature unique to this approach. By using an endothelialized collagen scaffold, this work highlights a novel "bottom-up" approach to islet engineering that provides control over the size and composition of the constructs, while enabling the critical ability to revascularize and engraft when transplanted into the clinically useful subcutaneous space.

PMID: 31901691 [PubMed - as supplied by publisher]



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When robotics met fluidics.

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When robotics met fluidics.

Lab Chip. 2020 Jan 02;:

Authors: Zhong J, Riordon J, Wu TC, Edwards H, Wheeler AR, Pardee K, Aspuru-Guzik A, Sinton D

Abstract
High-throughput fluidic technologies have increased the speed and accuracy of fluid processing to the extent that unlocking further gains will require replacing the human operator with a robotic counterpart. Recent advances in chemistry and biology, such as gene editing, have further exacerbated the need for smart, high-throughput experimentation. A growing number of innovations at the intersection of robotics and fluidics illustrate the tremendous opportunity in achieving fully self-driving fluid systems. We envision that the fields of synthetic chemistry and synthetic biology will be the first beneficiaries of AI-directed robotic and fluidic systems, and largely fall within two modalities: complex integrated centralized facilities that produce data, and distributed systems that synthesize products and conduct disease surveillance.

PMID: 31895394 [PubMed - as supplied by publisher]



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Induction of Rod and Cone Photoreceptor-Specific Progenitors from Stem Cells.

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Induction of Rod and Cone Photoreceptor-Specific Progenitors from Stem Cells.

Adv Exp Med Biol. 2019;1185:551-555

Authors: Ballios BG, Khalili S, Shoichet MS, van der Kooy D

Abstract
Retinal degeneration includes a variety of diseases for which there is no regenerative therapy. Cellular transplantation is one potential approach for future therapy for retinal degeneration, and stem cells have emerged as a promising source for future cell therapeutics. One major barrier to therapy is the ability to specify individual photoreceptor lineages from a variety of stem cell sources. In this review, we focus on photoreceptor genesis from progenitor populations in the developing embryo and how this understanding has given us the tools to manipulate cultures to specific unique rod and cone lineages from adult stem cell populations. We discuss experiments and evidence uncovering the lineage mechanisms at play in the establishment of fate-specific rod and cone photoreceptor progenitors. This may lead to an improved understanding of retinal development in vivo, as well as new cell sources for transplantation.

PMID: 31884669 [PubMed - indexed for MEDLINE]



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IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers.

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IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers.

J Cell Biol. 2020 Feb 03;219(2):

Authors: Mis M, O'Brien S, Steinhart Z, Lin S, Hart T, Moffat J, Angers S

Abstract
Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

PMID: 31881079 [PubMed - in process]



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Cored in the act: the use of models to understand core myopathies.

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Cored in the act: the use of models to understand core myopathies.

Dis Model Mech. 2019 12 19;12(12):

Authors: Fusto A, Moyle LA, Gilbert PM, Pegoraro E

Abstract
The core myopathies are a group of congenital myopathies with variable clinical expression - ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity - that are identified by characteristic 'core-like' lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.

PMID: 31874912 [PubMed - indexed for MEDLINE]



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Protein Structure from Experimental Evolution.

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Protein Structure from Experimental Evolution.

Cell Syst. 2019 Dec 10;:

Authors: Stiffler MA, Poelwijk FJ, Brock KP, Stein RR, Riesselman A, Teyra J, Sidhu SS, Marks DS, Gauthier NP, Sander C

Abstract
Natural evolution encodes rich information about the structure and function of biomolecules in the genetic record. Previously, statistical analysis of co-variation patterns in natural protein families has enabled the accurate computation of 3D structures. Here, we explored generating similar information by experimental evolution, starting from a single gene and performing multiple cycles of in vitro mutagenesis and functional selection in Escherichia coli. We evolved two antibiotic resistance proteins, β-lactamase PSE1 and acetyltransferase AAC6, and obtained hundreds of thousands of diverse functional sequences. Using evolutionary coupling analysis, we inferred residue interaction constraints that were in agreement with contacts in known 3D structures, confirming genetic encoding of structural constraints in the selected sequences. Computational protein folding with interaction constraints then yielded 3D structures with the same fold as natural relatives. This work lays the foundation for a new experimental method (3Dseq) for protein structure determination, combining evolution experiments with inference of residue interactions from sequence information. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

PMID: 31838147 [PubMed - as supplied by publisher]



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Genome Gerrymandering: optimal division of the genome into regions with cancer type specific differences in mutation rates.

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Genome Gerrymandering: optimal division of the genome into regions with cancer type specific differences in mutation rates.

Pac Symp Biocomput. 2020;25:274-285

Authors: Young A, Chmura J, Park Y, Morris Q, Atwal G

Abstract
The activity of mutational processes differs across the genome, and is influenced by chromatin state and spatial genome organization. At the scale of one megabase-pair (Mb), regional mutation density correlate strongly with chromatin features and mutation density at this scale can be used to accurately identify cancer type. Here, we explore the relationship between genomic region and mutation rate by developing an information theory driven, dynamic programming algorithm for dividing the genome into regions with differing relative mutation rates between cancer types. Our algorithm improves mutual information when compared to the naive approach, effectively reducing the average number of mutations required to identify cancer type. Our approach provides an efficient method for associating regional mutation density with mutation labels, and has future applications in exploring the role of somatic mutations in a number of diseases.

PMID: 31797603 [PubMed - in process]



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TrackSigFreq: subclonal reconstructions based on mutation signatures and allele frequencies.

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TrackSigFreq: subclonal reconstructions based on mutation signatures and allele frequencies.

Pac Symp Biocomput. 2020;25:238-249

Authors: Harrigan CF, Rubanova Y, Morris Q, Selega A

Abstract
Mutational signatures are patterns of mutation types, many of which are linked to known mutagenic processes. Signature activity represents the proportion of mutations a signature generates. In cancer, cells may gain advantageous phenotypes through mutation accumulation, causing rapid growth of that subpopulation within the tumour. The presence of many subclones can make cancers harder to treat and have other clinical implications. Reconstructing changes in signature activities can give insight into the evolution of cells within a tumour. Recently, we introduced a new method, TrackSig, to detect changes in signature activities across time from single bulk tumour sample. By design, TrackSig is unable to identify mutation populations with different frequencies but little to no difference in signature activity. Here we present an extension of this method, TrackSigFreq, which enables trajectory reconstruction based on both observed density of mutation frequencies and changes in mutational signature activities. TrackSigFreq preserves the advantages of TrackSig, namely optimal and rapid mutation clustering through segmentation, while extending it so that it can identify distinct mutation populations that share similar signature activities.

PMID: 31797600 [PubMed - in process]



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Identifying Transitional High Cost Users from Unstructured Patient Profiles Written by Primary Care Physicians.

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Identifying Transitional High Cost Users from Unstructured Patient Profiles Written by Primary Care Physicians.

Pac Symp Biocomput. 2020;25:127-138

Authors: Zhang H, Candido E, Wilton AS, Duchen R, Jaakkimainen L, Wodchis W, Morris Q

Abstract
Identification and subsequent intervention of patients at risk of becoming High Cost Users (HCUs) presents the opportunity to improve outcomes while also providing significant savings for the healthcare system. In this paper, the 2016 HCU status of patients was predicted using free-form text data from the 2015 cumulative patient profiles within the electronic medical records of family care practices in Ontario. These unstructured notes make substantial use of domain-specific spellings and abbreviations; we show that word embeddings derived from the same context provide more informative features than pre-trained ones based on Wikipedia, MIMIC, and Pubmed. We further demonstrate that a model using features derived from aggregated word embeddings (EmbEncode) provides a significant performance improvement over the bag-of-words representation (82.48±0.35% versus 81.85±0.36% held-out AUROC, p = 3.2 × 10-4), using far fewer input features (5,492 versus 214,750) and fewer non-zero coefficients (1,177 versus 4,284). The future HCUs of greatest interest are the transitional ones who are not already HCUs, because they provide the greatest scope for interventions. Predicting these new HCU is challenging because most HCUs recur. We show that removing recurrent HCUs from the training set improves the ability of EmbEncode to predict new HCUs, while only slightly decreasing its ability to predict recurrent ones.

PMID: 31797592 [PubMed - in process]



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Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

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Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device.

Acta Crystallogr D Struct Biol. 2019 Dec 01;75(Pt 12):1063-1070

Authors: Rubinstein JL, Guo H, Ripstein ZA, Haydaroglu A, Au A, Yip CM, Di Trani JM, Benlekbir S, Kwok T

Abstract
Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

PMID: 31793900 [PubMed - indexed for MEDLINE]



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