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Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes.

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Extrachromosomal circular elements targeted by CRISPR-Cas in Dehalococcoides mccartyi are linked to mobilization of reductive dehalogenase genes.

ISME J. 2018 Aug 13;:

Authors: Molenda O, Tang S, Lomheim L, Gautam VK, Lemak S, Yakunin AF, Maxwell KL, Edwards EA

Abstract
Dehalococcoides mccartyi are obligate organohalide-respiring bacteria that play an important detoxifying role in the environment. They have small genomes (~1.4 Mb) with a core region interrupted by two high plasticity regions (HPRs) containing dozens of genes encoding reductive dehalogenases involved in organohalide respiration. The genomes of eight new strains of D. mccartyi were closed from metagenomic data from a related set of enrichment cultures, bringing the total number of genomes to 24. Two of the newly sequenced strains and three previously sequenced strains contain CRISPR-Cas systems. These D. mccartyi CRISPR-Cas systems were found to primarily target prophages and genomic islands. The genomic islands were identified either as integrated into D. mccartyi genomes or as circular extrachromosomal elements. We observed active circularization of the integrated genomic island containing vcrABC operon encoding the dehalogenase (VcrA) responsible for the transformation of vinyl chloride to non-toxic ethene. We interrogated archived DNA from established enrichment cultures and found that the CRISPR array acquired three new spacers in 11 years. These data provide a glimpse into dynamic processes operating on the genomes distinct to D. mccartyi strains found in enrichment cultures and provide the first insights into possible mechanisms of lateral DNA exchange in D. mccartyi.

PMID: 30104577 [PubMed - as supplied by publisher]



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Stem cell bioengineering: building from stem cell biology.

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Stem cell bioengineering: building from stem cell biology.

Nat Rev Genet. 2018 Aug 08;:

Authors: Tewary M, Shakiba N, Zandstra PW

Abstract
New fundamental discoveries in stem cell biology have yielded potentially transformative regenerative therapeutics. However, widespread implementation of stem-cell-derived therapeutics remains sporadic. Barriers that impede the development of these therapeutics can be linked to our incomplete understanding of how the regulatory networks that encode stem cell fate govern the development of the complex tissues and organs that are ultimately required for restorative function. Bioengineering tools, strategies and design principles represent core components of the stem cell bioengineering toolbox. Applied to the different layers of complexity present in stem-cell-derived systems - from gene regulatory networks in single stem cells to the systemic interactions of stem-cell-derived organs and tissues - stem cell bioengineering can address existing challenges and advance regenerative medicine and cellular therapies.

PMID: 30089805 [PubMed - as supplied by publisher]



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Multiplexed assays of variant effects contribute to a growing genotype-phenotype atlas.

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Multiplexed assays of variant effects contribute to a growing genotype-phenotype atlas.

Hum Genet. 2018 Sep;137(9):665-678

Authors: Weile J, Roth FP

Abstract
Given the constantly improving cost and speed of genome sequencing, it is reasonable to expect that personal genomes will soon be known for many millions of humans. This stands in stark contrast with our limited ability to interpret the sequence variants which we find. Although it is, perhaps, easiest to interpret variants in coding regions, knowledge of functional impact is unknown for the vast majority of missense variants. While many computational approaches can predict the impact of coding variants, they are given a little weight in the current guidelines for interpreting clinical variants. Laboratory assays produce comparatively more trustworthy results, but until recently did not scale to the space of all possible mutations. The development of deep mutational scanning and other multiplexed assays of variant effect has now brought feasibility of this endeavour within view. Here, we review progress in this field over the last decade, break down the different approaches into their components, and compare methodological differences.

PMID: 30073413 [PubMed - indexed for MEDLINE]



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Acute Effects of Drugs on Caenorhabditis elegans Movement Reveal Complex Responses and Plasticity.

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Acute Effects of Drugs on Caenorhabditis elegans Movement Reveal Complex Responses and Plasticity.

G3 (Bethesda). 2018 08 30;8(9):2941-2952

Authors: Spensley M, Del Borrello S, Pajkic D, Fraser AG

Abstract
Many drugs act very rapidly - they can turn on or off their targets within minutes in a whole animal. What are the acute effects of drug treatment and how does an animal respond to these? We developed a simple assay to measure the acute effects of drugs on C. elegans movement and examined the effects of a range of compounds including neuroactive drugs, toxins, environmental stresses and novel compounds on worm movement over a time period of 3 hr. We found a wide variety of acute responses. Many compounds cause rapid paralysis which may be permanent or followed by one or more recovery phases. The recoveries are not the result of some generic stress response but are specific to the drug e.g., recovery from paralysis due to a neuroactive drug requires neurotransmitter pathways whereas recovery from a metabolic inhibitor requires metabolic changes. Finally, we also find that acute responses can vary greatly across development and that there is extensive natural variation in acute responses. In summary, acute responses are sensitive probes of the ability of biological networks to respond to drug treatment and these responses can reveal the action of unexplored pathways.

PMID: 30061375 [PubMed - indexed for MEDLINE]



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mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β.

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mTOR complex 1 controls the nuclear localization and function of glycogen synthase kinase 3β.

J Biol Chem. 2018 Jul 30;:

Authors: Bautista SJ, Boras I, Vissa A, Mecica N, Yip CM, Kim PK, Antonescu CN

Abstract
Glycogen synthase kinase 3β (GSK3β) phosphorylates and thereby regulates a wide range of protein substrates involved in diverse cellular functions. Some GSK3β substrates, such as c-myc and snail, are nuclear transcription factors, suggesting the possibility that GSK3β function is controlled through its nuclear localization. Here, using ARPE-19 and MDA-MB-231 human cell lines, we found that inhibition of mTOR complex 1 (mTORC1) leads to partial redistribution of GSK3β from the cytosol to the nucleus and to a GSK3β-dependent reduction of the levels of both c-myc and snail. mTORC1 is known to be controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance, and we observed here that AMPK activation or amino acid deprivation promotes GSK3β nuclear localization in an mTORC1-dependent manner. GSK3β was detected on several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through perturbation of RAB7, member RAS oncogene family 7 (Rab7) resulted in loss of GSK3β from lysosomes and in enhanced GSK3β nuclear localization as well as GSK3β-dependent reduction of c-myc levels. These findings indicate that the nuclear localization and function of GSK3β is suppressed by mTORC1 and suggest a link between metabolic conditions sensed by mTORC1 and GSK3β-dependent regulation of transcriptional networks controlling cellular biomass production.

PMID: 30061153 [PubMed - as supplied by publisher]



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Skin-derived precursor cells undergo substrate-dependent galvanotaxis that can be modified by neighbouring cells.

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Skin-derived precursor cells undergo substrate-dependent galvanotaxis that can be modified by neighbouring cells.

Stem Cell Res. 2018 Jul 19;31:95-101

Authors: Iwasa SN, Popovic MR, Morshead CM

Abstract
Many cell types respond to electric fields (EFs) through cell migration, a process termed galvanotaxis. The galvanotactic response is critical for development and wound healing. Here we investigate whether skin-derived precursor cells (SKPs), which have the potential to differentiate into mesodermal and peripheral neural cell types, undergo directed migration in the presence of an EF. We found that EF application promotes SKP migration towards the anode. The migratory response is substrate-dependent as SKPs undergo directed migration on laminin and Matrigel, but not collagen. The majority of SKPs express the undifferentiated cell markers nestin, fibronectin and Sox2, after both EF application and in sister cultures with no EF application, suggesting that EFs do not promote cell differentiation. Co-cultures of SKPs and brain-derived neural precursor cells (NPCs), a population of cells that undergo rapid, cathode-directed migration, reveal that in the presence of NPCs an increased percentage of SKPs undergo galvanotaxis, providing evidence that cells can provide cues to modify the galvanotactic response. We propose that a better understanding of SKP migration in the presence of EFs may provide insight into improved strategies for wound repair.

PMID: 30059907 [PubMed - as supplied by publisher]



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Integrating genetic and protein-protein interaction networks maps a functional wiring diagram of a cell.

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Integrating genetic and protein-protein interaction networks maps a functional wiring diagram of a cell.

Curr Opin Microbiol. 2018 Jul 27;45:170-179

Authors: VanderSluis B, Costanzo M, Billmann M, Ward HN, Myers CL, Andrews BJ, Boone C

Abstract
Systematic experimental approaches have led to construction of comprehensive genetic and protein-protein interaction networks for the budding yeast, Saccharomyces cerevisiae. Genetic interactions capture functional relationships between genes using phenotypic readouts, while protein-protein interactions identify physical connections between gene products. These complementary, and largely non-overlapping, networks provide a global view of the functional architecture of a cell, revealing general organizing principles, many of which appear to be evolutionarily conserved. Here, we focus on insights derived from the integration of large-scale genetic and protein-protein interaction networks, highlighting principles that apply to both unicellular and more complex systems, including human cells. Network integration reveals fundamental connections involving key functional modules of eukaryotic cells, defining a core network of cellular function, which could be elaborated to explore cell-type specificity in metazoans.

PMID: 30059827 [PubMed - as supplied by publisher]



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Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

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Melatonin receptors limit dopamine reuptake by regulating dopamine transporter cell-surface exposure.

Cell Mol Life Sci. 2018 Dec;75(23):4357-4370

Authors: Benleulmi-Chaachoua A, Hegron A, Le Boulch M, Karamitri A, Wierzbicka M, Wong V, Stagljar I, Delagrange P, Ahmad R, Jockers R

Abstract
Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.

PMID: 30043140 [PubMed - indexed for MEDLINE]



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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

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A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases.

Structure. 2018 Jun 26;:

Authors: Gorelik M, Manczyk N, Pavlenco A, Kurinov I, Sidhu SS, Sicheri F

Abstract
Skp1-Cul1-F-box (SCF) E3 ligases constitute the largest and best-characterized family of the multisubunit E3 ligases with important cellular functions and numerous disease links. The specificity of an SCF E3 ligase is established by one of the 69 human F-box proteins that are recruited to Cul1 through the Skp1 adaptor. We previously reported generation of ubiquitin variants (UbVs) targeting Fbw7 and Fbw11, which inhibit ligase activity by binding at the F-box-Skp1 interface to competitively displace Cul1. In the present study, we employed an optimized engineering strategy to generate specific binding UbVs against 17 additional Skp1-F-box complexes. We validated our design strategy and uncovered the structural basis of binding specificity by crystallographic analyses of representative UbVs bound to Skp1-Fbl10 and Skp1-Fbl11. Our study highlights the power of combining phage display with structure-based design to develop UbVs targeting specific protein surfaces.

PMID: 30033217 [PubMed - as supplied by publisher]



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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

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Single-Cell RNA Sequencing: A New Window into Cell Scale Dynamics.

Biophys J. 2018 Jul 11;:

Authors: Dasgupta S, Bader GD, Goyal S

Abstract
Single-cell genomics has recently emerged as a powerful tool for observing multicellular systems at a much higher level of resolution and depth than previously possible. High-throughput single-cell RNA sequencing techniques are able to simultaneously quantify expression levels of several thousands of genes within individual cells for tens of thousands of cells within a complex tissue. This has led to development of novel computational methods to analyze this high-dimensional data, investigating longstanding and fundamental questions regarding the granularity of cell types, the definition of cell states, and transitions from one cell type to another along developmental trajectories. In this perspective, we outline this emerging field starting from the "input data" (e.g., quantifying transcription levels in single cells), which are analyzed to define "identities" (e.g., cell types, states, and key genes) and to build "interactions" using models that can infer relations and transitions between cells.

PMID: 30033145 [PubMed - as supplied by publisher]



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