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Modulating cell state to enhance suspension expansion of human pluripotent stem cells.

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Modulating cell state to enhance suspension expansion of human pluripotent stem cells.

Proc Natl Acad Sci U S A. 2018 06 19;115(25):6369-6374

Authors: Lipsitz YY, Woodford C, Yin T, Hanna JH, Zandstra PW

Abstract
The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.

PMID: 29866848 [PubMed - indexed for MEDLINE]



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The role of biomaterials in overcoming barriers to regeneration in the central nervous system.

The role of biomaterials in overcoming barriers to regeneration in the central nervous system.

Biomed Mater. 2018 Jun 04;13(5):050201

Authors: Führmann T, Shoichet MS

PMID: 29864020 [PubMed - in process]



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Patient Similarity Networks for Precision Medicine.

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Patient Similarity Networks for Precision Medicine.

J Mol Biol. 2018 May 31;:

Authors: Pai S, Bader GD

Abstract
Clinical research and practice in the 21st century is poised to be transformed by analysis of computable electronic medical records and population-level genome-scale patient profiles. Genomic data captures genetic and environmental state, providing information about heterogeneity in disease and treatment outcome, but genomic-based clinical risk scores are limited. Achieving the goal of routine precision medicine that takes advantage of this rich genomics data will require computational methods that support heterogeneous data, have excellent predictive performance, and ideally, provide biologically-interpretable results. Traditional machine-learning approaches excel at performance, but often have limited interpretability. Patient similarity networks are an emerging paradigm for precision medicine, in which patients are clustered or classified based on their similarities in various features, including genomic profiles. This strategy is analogous to standard medical diagnosis, has excellent performance, is interpretable, and can preserve patient privacy. We review new methods based on patient similarity networks, including Similarity Network Fusion for patient clustering and netDx for patient classification. While these methods are already useful, much work is required to improve their scalability for contemporary genetic cohorts, optimize parameters, and incorporate a wide range of genomics and clinical data. The coming five years will provide an opportunity to assess the utility of network-based algorithms for precision medicine.

PMID: 29860027 [PubMed - as supplied by publisher]



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Antibody-Antisense Oligonucleotide Conjugate Downregulates a Key Gene in Glioblastoma Stem Cells.

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Antibody-Antisense Oligonucleotide Conjugate Downregulates a Key Gene in Glioblastoma Stem Cells.

Mol Ther Nucleic Acids. 2018 Jun 01;11:518-527

Authors: Arnold AE, Malek-Adamian E, Le PU, Meng A, Martínez-Montero S, Petrecca K, Damha MJ, Shoichet MS

Abstract
Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC invasion. We developed antibody-antisense oligonucleotide (AON) conjugates to target and reduce DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the GSCs, such as CD44 and EphA2, conjugated to chemically modified AONs against DRR/FAM107A, which were designed as chimeras of DNA and 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize, accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first example of an antibody-antisense strategy against cancer stem cells.

PMID: 29858087 [PubMed]



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Comparison of glycopyrronium versus tiotropium on the time to clinically important deteriorations in patients with COPD: a post-hoc analysis of randomized trials.

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Comparison of glycopyrronium versus tiotropium on the time to clinically important deteriorations in patients with COPD: a post-hoc analysis of randomized trials.

NPJ Prim Care Respir Med. 2018 May 24;28(1):18

Authors: D'Urzo A, Bader G, Shen S, Goyal P, Altman P

Abstract
Glycopyrronium is a once-daily, inhaled long-acting muscarinic antagonist (LAMA) demonstrating similar efficacy to inhaled tiotropium in patients with moderate-to-severe COPD; however, the benefit of LAMAs on COPD symptoms has been variable. COPD is a progressive disease in which many patients develop an acute or sustained deterioration. Data on the prevention of clinically important deteriorations (CID) using LAMAs are limited. A pooled analysis was performed on four Phase III trials (n = 2936) that compared the efficacy of glycopyrronium (n = 1859) with tiotropium (n = 1077). The primary endpoint was significant delay and/or reduction in the occurrence of CID. CID was defined as any of the following: ≥100 mL decrease from baseline in pre-dose forced expiratory volume in 1 second (FEV1), ≥4 point increase in St George's Respiratory Questionnaire score or a moderate-to-severe COPD exacerbation occurring after the first dose of study medication. A sustained CID was a CID occurring on ≥2 consecutive visits 4 weeks apart or for ≥50% of all available subsequent visits. Baseline characteristics for the overall population were similar. Patients had moderate (62%) or severe (38%) COPD. Mean post-bronchodilator FEV1 was approximately 55% predicted, and mean FEV1 reversibility was 16.7 and 18.6% in the glycopyrronium and tiotropium groups, respectively. Both glycopyrronium and tiotropium significantly reduced time to CID and sustained CID versus placebo (p < 0.001). No statistically significant differences were found between the glycopyrronium and tiotropium treatment groups in time to CID or sustained CID. Glycopyrronium is effective in delaying time to clinically important deteriorations, with similar efficacy to tiotropium.

PMID: 29795478 [PubMed - in process]



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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity.

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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity.

J Vis Exp. 2018 05 01;(135):

Authors: Huang G, Zhong Z, Miersch S, Sidhu SS, Hou SC, Wu D

Abstract
Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

PMID: 29782009 [PubMed - indexed for MEDLINE]



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Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

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Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

J Mol Biol. 2018 May 17;:

Authors: Nelson B, Adams J, Kuglstatter A, Li Z, Harris SF, Liu Y, Bohini S, Ma H, Klumpp K, Gao J, Sidhu SS

Abstract
Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block Hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop (LEL) in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 LEL that equated to increased efficacy while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.

PMID: 29778602 [PubMed - as supplied by publisher]



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Author Correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

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Author Correction: A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies.

Nature. 2018 May 16;:

Authors: Bahr C, von Paleske L, Uslu VV, Remeseiro S, Takayama N, Ng SW, Murison A, Langenfeld K, Petretich M, Scognamiglio R, Zeisberger P, Benk AS, Amit I, Zandstra PW, Lupien M, Dick JE, Trumpp A, Spitz F

Abstract
In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

PMID: 29769714 [PubMed - as supplied by publisher]



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A Screen for Epstein-Barr Virus Proteins that Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein.

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A Screen for Epstein-Barr Virus Proteins that Inhibit the DNA Damage Response Reveals a Novel Histone Binding Protein.

J Virol. 2018 May 09;:

Authors: Ho TH, Sitz J, Shen Q, Leblanc-Lacroix A, Campos EI, Borozan I, Marcon E, Greenblatt J, Fradet-Turcotte A, Jin DY, Frappier L

Abstract
To replicate and persist in human cells, linear double-stranded (ds) DNA viruses, such as Epstein-Barr virus (EBV), must overcome the host DNA damage response (DDR) that is triggered by the viral genomes. Since this response is necessary to maintain cellular genome integrity, its inhibition by EBV is likely an important factor in the development of cancers associated with EBV infection, including gastric carcinoma. Here we present the first extensive screen of EBV proteins that inhibit dsDNA break signaling. We identify the BKRF4 tegument protein as a DDR inhibitor that interferes with histone ubiquitylation at dsDNA breaks and recruitment of the RNF168 histone ubiquitin ligase. We further show that BKRF4 binds directly to histones through an acidic domain that targets BKRF4 to cellular chromatin and is sufficient to inhibit dsDNA break signaling. BKRF4 transcripts were detected in EBV-positive gastric carcinoma cells (AGS-EBV) and these increased in lytic infection. Silencing of BKRF4 in both latent and lytic AGS-EBV cells (but not in EBV-negative AGS cells) resulted in increased dsDNA break signaling, confirming a role for BKRF4 in DDR inhibition in the context of EBV infection and suggesting that BKRF4 is expressed in latent cells. BKRF4 was also found to be consistently expressed in EBV-positive gastric tumours in the absence of a full lytic infection. The results suggest that BKRF4 plays a role in inhibiting the cellular DDR in latent and lytic EBV infection, and that the resulting accumulation of DNA damage might contribute to development of gastric carcinoma.IMPORTANCE Epstein-Barr virus (EBV) infects most people worldwide and is causatively associated with several types of cancer, including ∼10% of gastric carcinoma. EBV encodes ∼80 proteins many of which are believed to manipulate cellular regulatory pathways but are poorly characterized. The DNA damage response (DDR) is one such pathway that is critical for maintaining genome integrity and preventing cancer-associated mutations. Here a screen for EBV proteins that inhibit the DDR identified BKRF4 as a DDR inhibitor that binds histones and block their ubiquitylation at the DNA damage sites. We also present evidence that BKRF4 is expressed in both latent and lytic forms of EBV infection, where it down-regulates the DDR, as well as in EBV-positive gastric tumours. The results suggest that BKRF4 could contribute to the development of gastric carcinoma through its ability to inhibit the DDR.

PMID: 29743367 [PubMed - as supplied by publisher]



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Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner.

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Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner.

Nucleic Acids Res. 2018 May 07;:

Authors: Becker JR, Gallo D, Leung W, Croissant T, Thu YM, Nguyen HD, Starr TK, Brown GW, Bielinsky AK

Abstract
Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.

PMID: 29741650 [PubMed - as supplied by publisher]



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