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The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD.

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The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD.

J Virol. 2019 Aug 28;:

Authors: Salamun SG, Sitz J, De La Cruz-Herrera CF, Yockteng-Melgar J, Marcon E, Greenblatt J, Fradet-Turcotte A, Frappier L

Abstract
The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex, and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.

PMID: 31462557 [PubMed - as supplied by publisher]



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Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

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Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Elife. 2019 08 27;8:

Authors: Tao Y, Mis M, Blazer L, Ustav M, Steinhart Z, Chidiac R, Kubarakos E, O'Brien S, Wang X, Jarvik N, Patel N, Adams J, Moffat J, Angers S, Sidhu SS

Abstract
Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.

PMID: 31452509 [PubMed - indexed for MEDLINE]



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Extracellular phosphorylation drives the formation of neuronal circuitry.

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Extracellular phosphorylation drives the formation of neuronal circuitry.

Nat Chem Biol. 2019 11;15(11):1035-1042

Authors: Harada H, Farhani N, Wang XF, Sugita S, Charish J, Attisano L, Moran M, Cloutier JF, Reber M, Bremner R, Monnier PP

Abstract
Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

PMID: 31451763 [PubMed - indexed for MEDLINE]



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Decyl gallate as a possible inhibitor of N-glycosylation process in Paracoccidioides lutzii.

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Decyl gallate as a possible inhibitor of N-glycosylation process in Paracoccidioides lutzii.

Antimicrob Agents Chemother. 2019 Aug 26;:

Authors: de Paula E Silva ACA, de Oliveira HC, Scorzoni L, Marcos CM, Dos Santos CT, Fusco-Almeida AM, Salina ACG, Medeiros AI, Almeida F, Li SC, Boone C, Mendes-Giannini MJS

Abstract
The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and wide spectra antifungal activity. Previously, our group demonstrated the promising anti-Paracoccidioides activity of G14. In this work, aiming to evaluate the antifungal characteristics of G14 for P. lutzii, chemical-genetic interaction analysis was conducted on Saccharomyces cerevisiae model. N-glycosylation and/or the unfolded protein response pathways were identified as high confidence target process prediction. The over activation of unfolded protein response (UPR) signaling was confirmed using this model with green fluorescent protein (GFP) tagged IRE1/ATF6/PERK genes. In P. lutzii, this prediction was confirmed by the low activity of glycosylated enzymes (α-(1,3)-glucanase, N-Acetyl-β-D-glucosaminidase, α-(1,4)-Amylase), by hyperexpresion of genes involved with the UPR and glycosylated enzymes, and by the reduction of glycosylated proteins and chitin amount. All these components are involved in fungal cell wall integrity and are depended of N-glycosylation process. This loss was confirmed by the reduction of mitochondrial activity, the impaired budding, the enhance of wall permeability and the decrease of viability. These events lead to a reduction of the ability of fungi to adhere on lung epithelial human cell (A549) in vitro Therefore, G14 may to have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering on microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti-Paracoccidioides compound.

PMID: 31451502 [PubMed - as supplied by publisher]



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Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

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Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

Angew Chem Int Ed Engl. 2019 Aug 26;:

Authors: Wu B, von der Ecken S, Swyer I, Li C, Jenne A, Vincent F, Schmidig D, Kuehn T, Busse F, Stronks H, Soong R, Wheeler AR, Simpson AJ

Abstract
Microcoil Nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR to initiate reactions while using sub-microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF-NMR. The study represents a key first step towards larger DMF-NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.

PMID: 31449724 [PubMed - as supplied by publisher]



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Differential contribution of steady-state RNA and active transcription in chromatin organization.

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Differential contribution of steady-state RNA and active transcription in chromatin organization.

EMBO Rep. 2019 Aug 26;:e48068

Authors: Barutcu AR, Blencowe BJ, Rinn JL

Abstract
Nuclear RNA and the act of transcription have been implicated in nuclear organization. However, their global contribution to shaping fundamental features of higher-order chromatin organization such as topologically associated domains (TADs) and genomic compartments remains unclear. To investigate these questions, we perform genome-wide chromatin conformation capture (Hi-C) analysis in the presence and absence of RNase before and after crosslinking, or a transcriptional inhibitor. TAD boundaries are largely unaffected by RNase treatment, although a subtle disruption of compartmental interactions is observed. In contrast, transcriptional inhibition leads to weaker TAD boundary scores. Collectively, our findings demonstrate differences in the relative contribution of RNA and transcription to the formation of TAD boundaries detected by the widely used Hi-C methodology.

PMID: 31448565 [PubMed - as supplied by publisher]



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Identifying chemogenetic interactions from CRISPR screens with drugZ.

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Identifying chemogenetic interactions from CRISPR screens with drugZ.

Genome Med. 2019 08 22;11(1):52

Authors: Colic M, Wang G, Zimmermann M, Mascall K, McLaughlin M, Bertolet L, Lenoir WF, Moffat J, Angers S, Durocher D, Hart T

Abstract
BACKGROUND: Chemogenetic profiling enables the identification of gene mutations that enhance or suppress the activity of chemical compounds. This knowledge provides insights into drug mechanism of action, genetic vulnerabilities, and resistance mechanisms, all of which may help stratify patient populations and improve drug efficacy. CRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells, but until recently has primarily been used to screen only for resistance mechanisms.
RESULTS: We present drugZ, an algorithm for identifying both synergistic and suppressor chemogenetic interactions from CRISPR screens. DrugZ identifies synthetic lethal interactions between PARP inhibitors and both known and novel members of the DNA damage repair pathway, confirms KEAP1 loss as a resistance factor for ERK inhibitors in oncogenic KRAS backgrounds, and defines the genetic context for temozolomide activity.
CONCLUSIONS: DrugZ is an open-source Python software for the analysis of genome-scale drug modifier screens. The software accurately identifies genetic perturbations that enhance or suppress drug activity. Interestingly, analysis of new and previously published data reveals tumor suppressor genes are drug-agnostic resistance genes in drug modifier screens. The software is available at github.com/hart-lab/drugz .

PMID: 31439014 [PubMed - indexed for MEDLINE]



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Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

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Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

Acc Chem Res. 2019 Aug 20;:

Authors: Malekjahani A, Sindhwani S, Syed AM, Chan WCW

Abstract
Mobile phone technology is a perfect companion for point-of-care diagnostics as they come equipped with advanced processors, high resolution cameras, and network connectivity. Despite several academic pursuits, only a few mobile phone diagnostics have been tested in the field, commercialized or achieved regulatory approval. This review will address the challenges associated with developing mobile diagnostics and suggest strategies to overcome them. We aim to provide a resource for researchers to accelerate the development of new diagnostics. Our Account includes an overview of published mobile phone diagnostics and highlights lessons learned from their approach to diagnostic development. Also, we have included recommendations from regulatory and public health agencies, such as the U.S. Food and Drug Administration and World Health Organization, to further guide researchers. We believe that the development of mobile phone point-of-care diagnostics takes place in four distinct steps: (1) Needs and Value Assessment, (2) Technology Development, (3) Preclinical Verification, and (4) Clinical Validation and Field Trials. During each step, we outline developmental strategies to help researchers avoid potential challenges. (1) Researchers commonly develop devices to maximize technical parameters such as sensitivity and time which do not necessarily translate to increased clinical impact. Researchers must focus on assessing specific diagnostic needs and the value which a potential device would offer. (2) Often, researchers claim they have developed devices for feasible implementation at the point-of-care, yet they rely on laboratory resources. Researchers must develop equipment-free devices which are agnostic to any mobile phone. (3) Another challenge researchers face is decreased performance during field evaluations relative to initial laboratory verification. Researchers must ensure that they simulate the field conditions during laboratory verification to achieve successful translation. (4) Finally, proper field testing of devices must be performed in conditions which match that of the final intended use. The future of mobile phone point-of-care diagnostic devices is bright and has the potential to radically change how patients are diagnosed. Before we reach this point, researchers must take a step backward and focus on the first-principles of basic research. The widespread adoption and rapid scaling of these devices can only be achieved once the fundamentals have been considered. The insights and strategies provided here will help researchers avoid pitfalls, streamline development and make better decisions during the development of new diagnostics. Further, we believe this Account can help push the field of mobile diagnostics toward increased productivity, leading to more approved devices and ultimately helping curb the burden of disease worldwide.

PMID: 31430118 [PubMed - as supplied by publisher]



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Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

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Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

Crit Rev Biochem Mol Biol. 2019 06;54(3):301-332

Authors: Gallo D, Brown GW

Abstract
The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

PMID: 31429594 [PubMed - indexed for MEDLINE]



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A 3D Printed Device for Low Cost Neural Stimulation in Mice.

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A 3D Printed Device for Low Cost Neural Stimulation in Mice.

Front Neurosci. 2019;13:784

Authors: Morrison TJ, Sefton E, Marquez-Chin M, Popovic MR, Morshead CM, Naguib HE

Abstract
Electrical stimulation of the brain through the implantation of electrodes is an effective treatment for certain diseases and the focus of a large body of research investigating new cell mechanisms, neurological phenomena, and treatments. Electrode devices developed for stimulation in rodents vary widely in size, cost, and functionality, with the majority of recent studies presenting complex, multi-functional designs. While some experiments require these added features, others are in greater need of reliable, low cost, and readily available devices that will allow surgeries to be scheduled and completed without delay. In this work, we utilize 3D printing and common electrical hardware to produce an effective 2-channel stimulation device that meets these requirements. Our stimulation electrode has not failed in over 60 consecutive surgeries, costs less than $1 USD, and can be assembled in less than 20 min. 3D printing minimizes the amount of material used in manufacturing the device and enables one to match the curvature of the connector's base with the curvature of the mouse skull, producing an ultra-lightweight, low size device with improved adhesion to the mouse skull. The range of the stimulation parameters used with the proposed device was: pulse amplitude 1-200 μA, pulse duration 50-500 μs and pulse frequency 1-285 Hz.

PMID: 31417347 [PubMed]



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