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Identification and Characterization of USP7 Targets in Cancer Cells.

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Identification and Characterization of USP7 Targets in Cancer Cells.

Sci Rep. 2018 Oct 26;8(1):15833

Authors: Georges A, Marcon E, Greenblatt J, Frappier L

Abstract
The ubiquitin specific protease, USP7, regulates multiple cellular pathways relevant for cancer through its ability to bind and sometimes stabilize specific target proteins through deubiquitylation. To gain a more complete profile of USP7 interactions in cancer cells, we performed affinity purification coupled to mass spectrometry to identify USP7 binding targets in gastric carcinoma cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 E3 ubiquitin ligase as well as identifying novel interactions with two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. Using USP7 binding pocket mutants, we show that USP11, PPM1G, TRIP12 and DDX24 bind USP7 through its TRAF domain binding pocket, while DHX40 interacts with USP7 through a distinct binding pocket in the Ubl2 domain. P/A/ExxS motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity in multiple cells lines showed that USP7 consistently stabilizes DDX24, DHX40 and TRIP12 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Our study better defines the mechanisms of USP7 interaction with known targets and identifies DDX24 and DHX40 as new targets that are specifically bound and regulated by USP7.

PMID: 30367141 [PubMed - in process]



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Mutations in the Guanylate Cyclase gcy-28 Neuronally Dissociate Naïve Attraction and Memory Retrieval.

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Mutations in the Guanylate Cyclase gcy-28 Neuronally Dissociate Naïve Attraction and Memory Retrieval.

Eur J Neurosci. 2018 Oct 25;:

Authors: Li N, van der Kooy D

Abstract
The molecules and mechanisms that are involved in the acquisition, storage, and retrieval of memories in many organisms are unclear. To investigate these processes, we use the nematode worm Caenorhabditis elegans, which is attracted naïvely to the odorant benzaldehyde but learns to avoid it after paired exposure with starvation. Mutations in the receptor-like guanylate cyclase GCY-28 have previously been thought to result in a behavioral switch in the primary chemosensory neuron AWCON , from an attractive state to an aversive (already-learned) state. Here, we offer a different interpretation and show that GCY-28 functions in distinct neurons to modulate two independent processes: naïve attraction to AWCON -sensed odors in the AWCON neuron, and associative learning of benzaldehyde and starvation in the AIA interneurons. Consequently, mutants that lack gcy-28 do not approach AWCON -sensed odors and cannot associate benzaldehyde with starvation. We further show that this learning deficit lies in memory retrieval, not in its acquisition or storage, and that GCY-28 is required in AIA for sensory integration only when both AWC neurons (ON and OFF) are activated by chemical stimuli. Our results reveal a novel role of GCY-28 in the retrieval of associative memories and may have wide implications for the neural machineries of learning and memory in general. This article is protected by copyright. All rights reserved.

PMID: 30362188 [PubMed - as supplied by publisher]



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Bone marrow-derived macrophages enhance vessel stability in modular engineered tissues.

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Bone marrow-derived macrophages enhance vessel stability in modular engineered tissues.

Tissue Eng Part A. 2018 Oct 23;:

Authors: West MED, Sefton EJB, Sefton MV

Abstract
Macrophages play a central role in the host response to tissue engineered constructs and their resultant vascularization. With a diversity of phenotypes, it is important to control the macrophage response to maximize vessel formation. We use a bottom-up tissue fabrication system ("modular tissue engineering") consisting of mesenchymal stromal cells embedded in injectable collagen gel modules that are coated with endothelial cells. Here we characterize the macrophage response to subcutaneously injected HUVEC (human umbilical vein endothelial cell) coated modules with and without embedded human adipose-derived mesenchymal stromal cells (MSC) or additional bone marrow-derived macrophages (BMDM) in SCID Beige mice. Clodronate depletion obviated vessel formation indicating that macrophages are essential to vessel formation. With MSC, macrophage infiltration (at day 3) was lessened relative to the HUVEC only control, while they preferentially adopted a mixed M1/M2 phenotype, characterized by expression of both CD206 and MHCII, which was similar with or without MSC. Vessel numbers were higher with MSC as expected at day 7, but adding BMDM reduced the regression seen by day 14. Pretreating the BMDM with IL-4 or IFNγ had no effect on vessel number or maturity (smooth muscle actin expression) or the inflammatory markers. Co-culturing HUVEC with IFNγ-treated or untreated BMDM altered their expression of polarization markers, reducing the impact of the pretreatment. A similar phenomenon likely occurred in vivo reducing the expected impact of pretreatment. While there is much to learn about the role of macrophages in module vascularization, the versatility of this bottom-up approach, afforded in part by enabling mixtures containing different components, is a useful means to alter the remodeling that occurs when constructs are deployed in vivo.

PMID: 30351235 [PubMed - as supplied by publisher]



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Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.

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Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.

Nat Commun. 2018 Oct 22;9(1):4383

Authors: MacParland SA, Liu JC, Ma XZ, Innes BT, Bartczak AM, Gage BK, Manuel J, Khuu N, Echeverri J, Linares I, Gupta R, Cheng ML, Liu LY, Camat D, Chung SW, Seliga RK, Shao Z, Lee E, Ogawa S, Ogawa M, Wilson MD, Fish JE, Selzner M, Ghanekar A, Grant D, Greig P, Sapisochin G, Selzner N, Winegarden N, Adeyi O, Keller G, Bader GD, McGilvray ID

Abstract
The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment.

PMID: 30348985 [PubMed - in process]



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Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

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Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

Methods Mol Biol. 2019;1869:169-188

Authors: Aregger M, Chandrashekhar M, Tong AHY, Chan K, Moffat J

Abstract
CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

PMID: 30324523 [PubMed - in process]



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EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

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EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

Methods Mol Biol. 2019;1869:155-168

Authors: Hu AX, Adams JJ, Vora P, Qazi M, Singh SK, Moffat J, Sidhu SS

Abstract
The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.

PMID: 30324522 [PubMed - in process]



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Patterned Optoelectronic Tweezers: A New Scheme for Selecting, Moving, and Storing Dielectric Particles and Cells.

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Patterned Optoelectronic Tweezers: A New Scheme for Selecting, Moving, and Storing Dielectric Particles and Cells.

Small. 2018 Oct 11;:e1803342

Authors: Zhang S, Shakiba N, Chen Y, Zhang Y, Tian P, Singh J, Chamberlain MD, Satkauskas M, Flood AG, Kherani NP, Yu S, Zandstra PW, Wheeler AR

Abstract
Optical micromanipulation has become popular for a wide range of applications. In this work, a new type of optical micromanipulation platform, patterned optoelectronic tweezers (p-OET), is introduced. In p-OET devices, the photoconductive layer (that is continuous in a conventional OET device) is patterned, forming regions in which the electrode layer is locally exposed. It is demonstrated that micropatterns in the photoconductive layer are useful for repelling unwanted particles/cells, and also for keeping selected particles/cells in place after turning off the light source, minimizing light-induced heating. To clarify the physical mechanism behind these effects, systematic simulations are carried out, which indicate the existence of strong nonuniform electric fields at the boundary of micropatterns. The simulations are consistent with experimental observations, which are explored for a wide variety of geometries and conditions. It is proposed that the new technique may be useful for myriad applications in the rapidly growing area of optical micromanipulation.

PMID: 30307718 [PubMed - as supplied by publisher]



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Predicting the Effect of Mutations on Protein Folding and Protein-Protein Interactions.

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Predicting the Effect of Mutations on Protein Folding and Protein-Protein Interactions.

Methods Mol Biol. 2019;1851:1-17

Authors: Strokach A, Corbi-Verge C, Teyra J, Kim PM

Abstract
The function of a protein is largely determined by its three-dimensional structure and its interactions with other proteins. Changes to a protein's amino acid sequence can alter its function by perturbing the energy landscapes of protein folding and binding. Many tools have been developed to predict the energetic effect of amino acid changes, utilizing features describing the sequence of a protein, the structure of a protein, or both. Those tools can have many applications, such as distinguishing between deleterious and benign mutations and designing proteins and peptides with attractive properties. In this chapter, we describe how to use one of such tools, ELASPIC, to predict the effect of mutations on the stability of proteins and the affinity between proteins, in the context of a human protein-protein interaction network. ELASPIC uses a wide range of sequential and structural features to predict the change in the Gibbs free energy for protein folding and protein-protein interactions. It can be used both through a web server and as a stand-alone application. Since ELASPIC was trained using homology models and not crystal structures, it can be applied to a much broader range of proteins than traditional methods. It can leverage precalculated sequence alignments, homology models, and other features, in order to drastically lower the amount of time required to evaluate individual mutations and make tractable the analysis of millions of mutations affecting the majority of proteins in a genome.

PMID: 30298389 [PubMed - in process]



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Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

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Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

Methods Mol Biol. 2019;1871:445-454

Authors: Pourhaghighi R, Emili A

Abstract
A high-resolution two-dimensional (2-D) proteomic fractionation technique for the systematic purification and subsequent mass spectrometry-based identification of endogenous protein macromolecular complexes is described. The method hyphenates preparative isoelectric focusing (IEF) with mixed-bed ion exchange chromatography (IEX) to efficiently separate cell- or tissue- derived soluble protein mixtures, allowing for more effective and less biased physiochemical characterization of stable multiprotein assemblies. After comprehensive 2D fractionation of cell-free lysates, each fraction is subjected to quantitative tandem mass spectrometry (MS/MS) and subsequent computational analysis to map high-confidence protein-protein interactions (PPIs). Herein, the experimental component (workflow protocols) for this global "interactome" network mapping platform is described.

PMID: 30276754 [PubMed - in process]



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A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.

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A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.

J Vis Exp. 2018 Sep 13;(139):

Authors: Lakshman N, Xu W, Morshead CM

Abstract
Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site - demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

PMID: 30272658 [PubMed - in process]



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