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      Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs

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          Abstract

          Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.

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          Single-pot, solid-phase-enhanced sample preparation for proteomics experiments

          A critical step in proteomics analysis is the optimal extraction and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technology that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-preparation (SP3) technology is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic analysis. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymatic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic analysis. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aqueous conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of solution additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a standard user in ~30 min, including reagent preparation. As a result of these properties, SP3 has successfully been used to facilitate examination of a broad range of sample types spanning simple and complex protein mixtures in large and very small amounts, across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.
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            The MEK/ERK cascade: from signaling specificity to diverse functions.

            The ERK signaling cascade is a central MAPK pathway that plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, learning, survival and, under some conditions, also apoptosis. The ability of this cascade to regulate so many distinct, and even opposing, cellular processes, raises the question of signaling specificity determination by this cascade. Here we describe mechanisms that cooperate to direct MEK-ERK signals to their appropriate downstream destinations. These include duration and strength of the signals, interaction with specific scaffolds, changes in subcellular localization, crosstalk with other signaling pathways, and presence of multiple components with distinct functions in each tier of the cascade. Since many of the mechanisms do not function properly in cancer cells, understanding them may shed light not only on the regulation of normal cell proliferation, but also on mechanisms of oncogenic transformation.
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              Endocytic pathways and endosomal trafficking: a primer.

              This brief overview of endocytic trafficking is written in honor of Renate Fuchs, who retires this year. In the mid-1980s, Renate pioneered studies on the ion-conducting properties of the recently discovered early and late endosomes and the mechanisms governing endosomal acidification. As described in this review, after uptake through one of many mechanistically distinct endocytic pathways, internalized proteins merge into a common early/sorting endosome. From there they again diverge along distinct sorting pathways, back to the cell surface, on to the trans-Golgi network or across polarized cells. Other transmembrane receptors are packaged into intraluminal vesicles of late endosomes/multivesicular bodies that eventually fuse with and deliver their content to lysosomes for degradation. Endosomal acidification, in part, determines sorting along this pathway. We describe other sorting machinery and mechanisms, as well as the rab proteins and phosphatidylinositol lipids that serve to dynamically define membrane compartments along the endocytic pathway.
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                Author and article information

                Journal
                Viruses
                Viruses
                viruses
                Viruses
                MDPI
                1999-4915
                08 September 2020
                September 2020
                : 12
                : 9
                : 999
                Affiliations
                [1 ]Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Robert-von-Ostertag-Straße 7-13, 14163 Berlin, Germany; pavulraj1vet@ 123456zedat.fu-berlin.de (S.P.); m_salah@ 123456cu.edu.eg (M.K.); no.34@ 123456fu-berlin.de (N.O.)
                [2 ]Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, 12211 Cairo, Egypt
                [3 ]Leibniz Institute of Molecular Pharmacology (FMP Berlin), Robert-Rössle-Str. 10, 13125 Berlin, Germany; Stephanowitz@ 123456fmp-berlin.de (H.S.); FLiu@ 123456fmp-berlin.de (F.L.)
                [4 ]Institut für Veterinäranatomie, Freie Universität Berlin, Koserstraße 20, 14195 Berlin, Germany; plendl.johanna@ 123456vetmed.fu-berlin.de
                Author notes
                [* ]Correspondence: walid.azab@ 123456fu-berlin.de ; Tel.: +49-30-838-50087
                Author information
                https://orcid.org/0000-0001-6804-0808
                https://orcid.org/0000-0002-5313-2176
                Article
                viruses-12-00999
                10.3390/v12090999
                7551999
                32911663
                d6c2019b-200d-47c3-ab70-90a1865081b6
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 10 August 2020
                : 05 September 2020
                Categories
                Article

                Microbiology & Virology
                ehv-1,pathogenesis,flow chamber assay,endothelium,equine,pbmc
                Microbiology & Virology
                ehv-1, pathogenesis, flow chamber assay, endothelium, equine, pbmc

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