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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

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On-demand serum-free media formulations for human hematopoietic cell expansion using a high dimensional search algorithm.

Commun Biol. 2019 Feb 01;2(1):48

Authors: Kim MM, Audet J

Abstract
Substitution of serum and other clinically incompatible reagents is requisite for controlling product quality in a therapeutic cell manufacturing process. However, substitution with chemically defined compounds creates a complex, large-scale optimization problem due to the large number of possible factors and dose levels, making conventional process optimization methods ineffective. We present a framework for high-dimensional optimization of serum-free formulations for the expansion of human hematopoietic cells. Our model-free approach utilizes evolutionary computing principles to drive an experiment-based feedback control platform. We validate this method by optimizing serum-free formulations for first, TF-1 cells and second, primary T-cells. For each cell type, we successfully identify a set of serum-free formulations that support cell expansions similar to the serum-containing conditions commonly used to culture these cells, by experimentally testing less than 1 × 10-5 % of the total search space. We also demonstrate how this iterative search process can provide insights into factor interactions that contribute to supporting cell expansion.

PMID: 31924921 [PubMed - in process]



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A community effort to create standards for evaluating tumor subclonal reconstruction.

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A community effort to create standards for evaluating tumor subclonal reconstruction.

Nat Biotechnol. 2020 Jan;38(1):97-107

Authors: Salcedo A, Tarabichi M, Espiritu SMG, Deshwar AG, David M, Wilson NM, Dentro S, Wintersinger JA, Liu LY, Ko M, Sivanandan S, Zhang H, Zhu K, Ou Yang TH, Chilton JM, Buchanan A, Lalansingh CM, P'ng C, Anghel CV, Umar I, Lo B, Zou W, DREAM SMC-Het Participants, Simpson JT, Stuart JM, Anastassiou D, Guan Y, Ewing AD, Ellrott K, Wedge DC, Morris Q, Van Loo P, Boutros PC

Abstract
Tumor DNA sequencing data can be interpreted by computational methods that analyze genomic heterogeneity to infer evolutionary dynamics. A growing number of studies have used these approaches to link cancer evolution with clinical progression and response to therapy. Although the inference of tumor phylogenies is rapidly becoming standard practice in cancer genome analyses, standards for evaluating them are lacking. To address this need, we systematically assess methods for reconstructing tumor subclonality. First, we elucidate the main algorithmic problems in subclonal reconstruction and develop quantitative metrics for evaluating them. Then we simulate realistic tumor genomes that harbor all known clonal and subclonal mutation types and processes. Finally, we benchmark 580 tumor reconstructions, varying tumor read depth, tumor type and somatic variant detection. Our analysis provides a baseline for the establishment of gold-standard methods to analyze tumor heterogeneity.

PMID: 31919445 [PubMed - in process]



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Modular Mimicry and Engagement of the Hippo Pathway by Marburg Virus VP40: Implications for Filovirus Biology and Budding.

Modular Mimicry and Engagement of the Hippo Pathway by Marburg Virus VP40: Implications for Filovirus Biology and Budding.

PLoS Pathog. 2020 Jan 06;16(1):e1008231

Authors: Han Z, Dash S, Sagum CA, Ruthel G, Jaladanki CK, Berry CT, Schwoerer MP, Harty NM, Freedman BD, Bedford MT, Fan H, Sidhu SS, Sudol M, Shtanko O, Harty RN

Abstract
Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

PMID: 31905227 [PubMed - as supplied by publisher]



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Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Dev Cell. 2019 Dec 26;:

Authors: Storer MA, Mahmud N, Karamboulas K, Borrett MJ, Yuzwa SA, Gont A, Androschuk A, Sefton MV, Kaplan DR, Miller FD

Abstract
Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

PMID: 31902657 [PubMed - as supplied by publisher]



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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.

Read Full Article on External Site Related Articles

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 Dec 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 - in process]



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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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