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OBJECTIVE To identify and characterize myeloid cell populations within the CSF of patients with MS and anti-myelin oligodendrocyte glycoprotein (MOG) disorder by high-resolution single-cell gene expression analysis. METHODS Single-cell RNA sequencing (scRNA-seq) was used to profile individual cells of CSF and blood from 2 subjects with relapsing-remitting MS (RRMS) and one with anti-MOG disorder. Publicly available scRNA-seq data from the blood and CSF of 2 subjects with HIV were also analyzed. An informatics pipeline was used to cluster cell populations by transcriptomic profiling. Based on gene expression by CSF myeloid cells, a flow cytometry panel was devised to examine myeloid cell populations from the CSF of 11 additional subjects, including individuals with RRMS, anti-MOG disorder, and control subjects without inflammatory demyelination. RESULTS Common myeloid populations were identified within the CSF of subjects with RRMS, anti-MOG disorder, and HIV. These included monocytes, conventional and plasmacytoid dendritic cells, and cells with a transcriptomic signature matching microglia. Microglia could be discriminated from other myeloid cell populations in the CSF by flow cytometry. CONCLUSIONS High-resolution single-cell gene expression analysis clearly distinguishes distinct myeloid cell types present within the CSF of subjects with neuroinflammation. A population of microglia exists within the human CSF, which is detectable by surface protein expression. The function of these cells during immunity and disease requires further investigation. Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.OBJECTIVE To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs). METHODS We collected splenocytes and CD4+ T cells from C57BL/6 wild-type and interferon (IFN)-γ-deficient mice. For human PBMCs, venous blood was collected from healthy donors, and PBMCs were collected using the Percoll gradient method. Cells were cultured with anti-CD3/28 in the presence/absence of DF for 3 to 5 days. Cells were stained and analyzed by flow cytometry. Cytokines were measured by ELISA in cell supernatants. For in vivo experiments, EAE was induced by myelin oligodendrocyte glycoprotein35-55 and mice were treated with oral DF or vehicle daily. RESULTS DF acts directly on CD4+ T cells and suppresses GM-CSF-producing Th1 not Th17 or single GM-CSF+ T cells in EAE. In addition, GM-CSF suppression depends on the IFN-γ pathway. We also show that DF specifically suppresses Th1 and GM-CSF-producing Th1 cells in PBMCs from healthy donors. CONCLUSIONS We suggest that DF exclusively suppresses GM-CSF-producing Th1 cells in both animal and human CD4+ T cells through an IFN-γ-dependent pathway. These findings indicate that DF has a better therapeutic effect on patients with Th1-dominant immunophenotype. However, future longitudinal study to validate this finding in MS is needed. Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.The Arabidopsis (Arabidopsis thaliana) root epidermis consists of a position-dependent pattern of root-hair cells and non-hair cells. Underlying this cell-type patterning is a network of transcription factors including a central MYB-bHLH-WD40 complex containing WEREWOLF (WER), GLABRA 3/ENHANCER OF GLABRA 3 (GL3/EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1). In this study, we used a genetic enhancer screen to identify apum23-4, a mutant allele of the ribosome biogenesis factor (RBF) gene PUMILIO 23 (APUM23), which caused prospective root-hair cells to instead adopt the non-hair cell fate. We discovered that this cell fate switch relied on MYB23, a MYB protein encoded by a WER target gene and acting redundantly with WER. In the apum23-4 mutant, MYB23 exhibited ectopic expression that was WER-independent and instead required ANAC082, a recently identified ribosomal stress response mediator. We examined additional RBF mutants that produced ectopic non-hair cells and determined that this cell fate switch is generally linked to defects in ribosome biogenesis. Further, the flagellin peptide flg22 triggers the ANAC082-MYB23-GL2 pathway. Taken together, our study provides a molecular explanation for root epidermal cell fate switch in response to ribosomal defects and, more generally, it demonstrates a novel regulatory connection between stress conditions and cell fate control in plants. © 2020 American Society of Plant Biologists. All rights reserved.How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks is not well understood in plants. The TRAnsport Protein Particle (TRAPP) complexes play key roles in selective delivery of membrane vesicles to various subcellular compartments in yeast and animals, but are not characterized in plants. Here we interrogate TRAPP complexes in Arabidopsis. Affinity purification of a core subunit AtTRS33 followed by quantitative mass spectrometry identified fourteen interacting proteins; including homologues of all thirteen TRAPP components in yeast and mammals and a novel protein we named TRAPP-interacting plant protein (TRIPP), which is conserved in multi-cellular photosynthetic organisms. Proteomic and molecular analyses showed that TRIPP specifically associates with the TRAPPII complex through binary interactions with two TRAPPII-specific subunits. TRIPP co-localizes with a subset of TRS33 compartments and trans-Golgi network markers in a TRS33-dependent manner. Loss-of-function tripp mutation caused dwarfism, sterility, partial photomorphogenesis in the dark, reduced polarity of the auxin transporter PIN2, incomplete cross wall formation and altered localization of a TRAPPII-specific component. Our study demonstrates that plants possess at least two distinct TRAPP complexes similar to metazoans, and identifies TRIPP as a novel plant-specific component of the TRAPPII complex with important functions in trafficking, plant growth and development. 1-Azakenpaullone © 2020 American Society of Plant Biologists. All rights reserved.
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