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Cell Biology
[Hypoxia epigenetically bestows astrocytic differentiation potential on human pluripotent cell-derived neural stem/precursor cells].
Feb 12, 2019   Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica
Yasui T, Nakashima K
[Hypoxia epigenetically bestows astrocytic differentiation potential on human pluripotent cell-derived neural stem/precursor cells].
Feb 12, 2019
Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica
The central nervous system (CNS) is composed of three major cell types, neurons, astrocytes, and oligodendrocytes, which differentiate from common multipotent neural stem/precursor cells (NS/PCs). However, NS/PCs do not have this multipotentiality from the beginning: neurons are generated first and astrocytes are later during CNS development. This developmental progression is observed in vitro by using human (h) NS/PCs derived from pluripotent cells, such as embryonic- and induced pluripotent-stem cells (ES/iPSCs), however, in contrast to rodent's pluripotent cells, they require quite long time to obtain astrocytic differentiation potential. Here, we show that hypoxia confers astrocytic differentiation potential on hNS/PCs through epigenetic alteration for gene regulation. Furthermore, we found that these molecular mechanisms can be applied to functional analysis of patient' iPSC-derived astrocytes. In this review, we summarize recent findings that address molecular mechanisms of epigenetic and transcription factor-mediated regulation that specify NS/PC fate and the development of potential therapeutic strategies for treating astrocyte-mediated neurological disorders.
Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells.
Feb 12, 2019   Cancer Cell
Du H, Hirabayashi K, Ahn S, Kren NP, Montgomery SA,   . . . . . .   , Yeh JJ, Liu R, Savoldo B, Ferrone S, Dotti G
Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells.
Feb 12, 2019
Cancer Cell
The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.
Microtubule plus-ends act as physical signaling hubs to activate RhoA during cytokinesis.
Feb 15, 2019   ELife
Verma V, Maresca TJ
Microtubule plus-ends act as physical signaling hubs to activate RhoA during cytokinesis.
Feb 15, 2019
ELife
Microtubules (MTs) are essential for cleavage furrow positioning during cytokinesis, but the mechanisms by which MT-derived signals spatially define regions of cortical contractility are unresolved. In this study cytokinesis regulators visualized in Drosophila melanogaster (Dm) cells were found to localize to and track MT plus-ends during cytokinesis. The RhoA GEF Pebble (Dm ECT2) did not evidently tip-track, but rather localized rapidly to cortical sites contacted by MT plus-tips, resulting in RhoA activation and enrichment of myosin-regulatory light chain. The MT plus-end localization of centralspindlin was compromised following EB1 depletion, which resulted in a higher incidence of cytokinesis failure. Centralspindlin plus-tip localization depended on the C-terminus and a putative EB1-interaction motif (hxxPTxh) in RacGAP50C. We propose that MT plus-end-associated centralspindlin recruits a cortical pool of Dm ECT2 upon physical contact to activate RhoA and to trigger localized contractility.
CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.
Feb 13, 2019   Oncogene
Block I, Müller C, Sdogati D, Pedersen H, List M,   . . . . . .   , Thomassen M, Kruse TA, Karlskov Hansen SW, Kioschis P, Mollenhauer J
CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.
Feb 13, 2019
Oncogene
Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.
The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques.
Feb 13, 2019   Scientific Reports
Antounians L, Tzanetakis A, Pellerito O, Catania VD, Sulistyo A, Montalva L, McVey MJ, Zani A
The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques.
Feb 13, 2019
Scientific Reports
Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) mediate anti-apoptotic, pro-angiogenic, and immune-modulatory effects in multiple disease models, such as skeletal muscle atrophy and Alport syndrome. A source of potential variability in EV biological functions is how EV are isolated from parent cells. Currently, a comparative study of different EV isolation strategies using conditioned medium from AFSCs is lacking. Herein, we examined different isolation strategies for AFSC-EVs, using common techniques based on differential sedimentation (ultracentrifugation), solubility (ExoQuick, Total Exosome Isolation Reagent, Exo-PREP), or size-exclusion chromatography (qEV). All techniques isolated AFSC-EVs with typical EV morphology and protein markers. In contrast, AFSC-EV size, protein content, and yield varied depending on the method of isolation. When equal volumes of the different AFSC-EV preparations were used as treatment in a model of lung epithelial injury, we observed a significant variation in how AFSC-EVs were able to protect against cell death. AFSC-EV enhancement of cell survival appeared to be dose dependent, and largely uninfluenced by variation in EV-size distributions, relative EV-purity, or their total protein content. The variation in EV-mediated cell survival obtained with different isolation strategies emphasizes the importance of testing alternative isolation techniques in order to maximize EV regenerative capacity.
Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.
Feb 13, 2019   Scientific Reports
Ko J, Myeong J, Shin YC, So I
Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.
Feb 13, 2019
Scientific Reports
Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4β, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4β, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4β < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.
KIAA1199 is a secreted molecule that enhances osteoblastic stem cell migration and recruitment.
Feb 16, 2019   Cell Death & Disease
Chen L, Shi K, Andersen TL, Qiu W, Kassem M
KIAA1199 is a secreted molecule that enhances osteoblastic stem cell migration and recruitment.
Feb 16, 2019
Cell Death & Disease
Factors mediating mobilization of osteoblastic stem and progenitor cells from their bone marrow niche to be recruited to bone formation sites during bone remodeling are poorly known. We have studied secreted factors present in the bone marrow microenvironment and identified KIAA1199 (also known as CEMIP, cell migration inducing hyaluronan binding protein) in human bone biopsies as highly expressed in osteoprogenitor reversal cells (Rv.C) recruited to the eroded surfaces (ES), which are the future bone formation sites. In vitro, KIAA1199 did not affect the proliferation of human osteoblastic stem cells (also known as human bone marrow skeletal or stromal stem cells, hMSCs); but it enhanced cell migration as determined by scratch assay and trans-well migration assay. KIAA1199 deficient hMSCs (KIAA1199down) exhibited significant changes in cell size, cell length, ratio of cell width to length and cell roundness, together with reduction of polymerization actin (F-actin) and changes in phos-CFL1 (cofflin1), phos-LIMK1 (LIM domain kinase 1) and DSTN (destrin), key factors regulating actin cytoskeletal dynamics and cell motility. Moreover, KIAA1199down hMSC exhibited impaired Wnt signaling in TCF-reporter assay and decreased expression of Wnt target genes and these effects were rescued by KIAA1199 treatment. Finally, KIAA1199 regulated the activation of P38 kinase and its associated changes in Wnt-signaling. Thus, KIAA1199 is a mobilizing factor that interacts with P38 and Wnt signaling, and induces changes in actin cytoskeleton, as a mechanism mediating recruitment of hMSC to bone formation sites.
PILS6 is a temperature-sensitive regulator of nuclear auxin input and organ growth in Arabidopsis thaliana.
Feb 13, 2019   Proceedings Of The National Academy Of Sciences Of The United States Of America
Feraru E, Feraru MI, Barbez E, Waidmann S, Sun L, Gaidora A, Kleine-Vehn J
PILS6 is a temperature-sensitive regulator of nuclear auxin input and organ growth in Arabidopsis thaliana.
Feb 13, 2019
Proceedings Of The National Academy Of Sciences Of The United States Of America
Temperature modulates growth and development throughout the entire lifecycle of a plant. High temperature (HT) triggers the auxin biosynthesis-dependent growth in aerial tissues. On the other hand, the contribution of auxin to HT-induced root growth is currently under debate. Here we show that the putative intracellular auxin carrier PIN-LIKES 6 (PILS6) is a negative regulator of organ growth and that its abundance is highly sensitive to HT. PILS6 localizes to the endoplasmic reticulum and limits the nuclear availability of auxin, consequently reducing the auxin signaling output. HT represses the PILS6 protein abundance, which impacts on PILS6-dependent auxin signaling in roots and root expansion. Accordingly, we hypothesize that PILS6 is part of an alternative mechanism linking HT to auxin responses in roots.
Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.
Feb 13, 2019   The Journal Of Biological Chemistry
Tang TC, Kienlen-Campard P, Hu Y, Perrin F, Opsomer R, Octave JN, Constantinescu SN, Smith SO
Influence of the familial Alzheimer's disease-associated T43I mutation on the transmembrane structure and γ-secretase processing of the C99 peptide.
Feb 13, 2019
The Journal Of Biological Chemistry
Extracellular deposition of amyloid-β (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). Upon β-secretase-mediated cleavage of the β C-terminal fragment (β-CTF) from the Aβ precursor protein, the γ-secretase complex produces the amyloid β (Aβ) peptides associated with AD. The familial T43I mutation within the transmembrane domain of the β-CTF (also referred to as C99) increases the ratio between the Aβ42 and Aβ40 peptides largely due to a decrease in Aβ40 formation. Aβ42 is the principal component of amyloid deposits within the brain parenchyma, and an increase in the Aβ42:Aβ40 ratio is correlated with early-onset AD. Using NMR and FTIR spectroscopy, here we addressed how the T43I substitution influences the structure of C55, the minimal sequence containing the entire extracellular and transmembrane (TM) domains of C99 needed for γ-secretase processing. 13C NMR chemical shifts indicated that the T43I substitution increases helical structure within the TM domain of C55. These structural changes were associated with a shift of the C55 dimer to the monomer and an increase in the tilt of the TM helix relative to the membrane normal in the T43I mutant compared with that of wild-type C55. The A21G (Flemish) mutation was previously found to increase secreted Aβ40 levels; here, we combined this mutation in the extracellular domain of C99 with T43I and observed that the T43I/A21G double mutant decreases Aβ40 formation. We discuss how the observed structural changes in the T43I mutant may decrease Aβ40 formation and increase the Aβ42:Aβ40 ratio.
Gαs-coupled receptor signaling and sleep regulate integrin activation of human antigen-specific T cells.
Feb 13, 2019   The Journal Of Experimental Medicine
Dimitrov S, Lange T, Gouttefangeas C, Jensen ATR, Szczepanski M, Lehnnolz J, Soekadar S, Rammensee HG, Born J, Besedovsky L
Gαs-coupled receptor signaling and sleep regulate integrin activation of human antigen-specific T cells.
Feb 13, 2019
The Journal Of Experimental Medicine
Efficient T cell responses require the firm adhesion of T cells to their targets, e.g., virus-infected cells, which depends on T cell receptor (TCR)-mediated activation of β2-integrins. Gαs-coupled receptor agonists are known to have immunosuppressive effects, but their impact on TCR-mediated integrin activation is unknown. Using multimers of peptide major histocompatibility complex molecules (pMHC) and of ICAM-1-the ligand of β2-integrins-we show that the Gαs-coupled receptor agonists isoproterenol, epinephrine, norepinephrine, prostaglandin (PG) E2, PGD2, and adenosine strongly inhibit integrin activation on human CMV- and EBV-specific CD8+ T cells in a dose-dependent manner. In contrast, sleep, a natural condition of low levels of Gαs-coupled receptor agonists, up-regulates integrin activation compared with nocturnal wakefulness, a mechanism possibly underlying some of the immune-supportive effects of sleep. The findings are also relevant for several pathologies associated with increased levels of Gαs-coupled receptor agonists (e.g., tumor growth, malaria, hypoxia, stress, and sleep disturbances).
Drug Sensitivity Screening and Targeted Pathway Analysis Reveal a Multi-Driver Proliferative Mechanism and Suggest a Strategy of Combination Targeted Therapy for Colorectal Cancer Cells.
Feb 13, 2019   Molecules (Basel, Switzerland)
Shen J, Li L, Yang T, Cheng N, Sun G
Drug Sensitivity Screening and Targeted Pathway Analysis Reveal a Multi-Driver Proliferative Mechanism and Suggest a Strategy of Combination Targeted Therapy for Colorectal Cancer Cells.
Feb 13, 2019
Molecules (Basel, Switzerland)
Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC50's between 10 and 84 nM. These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.
Developmental Erk Signaling Illuminated.
Feb 12, 2019   Developmental Cell
Gagliardi PA, Pertz O
Developmental Erk Signaling Illuminated.
Feb 12, 2019
Developmental Cell
How a small number of signaling pathways can be re-used in distinct embryonic contexts to control different fates remains unclear. In this issue of Developmental Cell, Johnson and Toettcher (2019) use optogenetic approaches to explore how different dynamic ERK signaling states control specific developmental fates in the Drosophila embryo.
SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing.
Feb 12, 2019   PloS One
Mezzadra R, de Bruijn M, Jae LT, Gomez-Eerland R, Duursma A, Scheeren FA, Brummelkamp TR, Schumacher TN
SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing.
Feb 12, 2019
PloS One
Experimental and clinical observations have highlighted the role of cytotoxic T cells in human tumor control. However, the parameters that control tumor cell sensitivity to T cell attack remain incompletely understood. To identify modulators of tumor cell sensitivity to T cell effector mechanisms, we performed a whole genome haploid screen in HAP1 cells. Selection of tumor cells by exposure to tumor-specific T cells identified components of the interferon-γ (IFN-γ) receptor (IFNGR) signaling pathway, and tumor cell killing by cytotoxic T cells was shown to be in large part mediated by the pro-apoptotic effects of IFN-γ. Notably, we identified schlafen 11 (SLFN11), a known modulator of DNA damage toxicity, as a regulator of tumor cell sensitivity to T cell-secreted IFN-γ. SLFN11 does not influence IFNGR signaling, but couples IFNGR signaling to the induction of the DNA damage response (DDR) in a context dependent fashion. In line with this role of SLFN11, loss of SLFN11 can reduce IFN-γ mediated toxicity. Collectively, our data indicate that SLFN11 can couple IFN-γ exposure of tumor cells to DDR and cellular apoptosis. Future work should reveal the mechanistic basis for the link between IFNGR signaling and DNA damage response, and identify tumor cell types in which SLFN11 contributes to the anti-tumor activity of T cells.
Haematopoietic stem cell activity and interactions with the niche.
Feb 12, 2019   Nature Reviews. Molecular Cell Biology
Pinho S, Frenette PS
Haematopoietic stem cell activity and interactions with the niche.
Feb 12, 2019
Nature Reviews. Molecular Cell Biology
The haematopoietic stem cell (HSC) microenvironment in the bone marrow, termed the niche, ensures haematopoietic homeostasis by controlling the proliferation, self-renewal, differentiation and migration of HSCs and progenitor cells at steady state and in response to emergencies and injury. Improved methods for HSC isolation, driven by advances in single-cell and molecular technologies, have led to a better understanding of their behaviour, heterogeneity and lineage fate and of the niche cells and signals that regulate their function. Niche regulatory signals can be in the form of cell-bound or secreted factors and other local physical cues. A combination of technological advances in bone marrow imaging and genetic manipulation of crucial regulatory factors has enabled the identification of several candidate cell types regulating the niche, including both non-haematopoietic (for example, perivascular mesenchymal stem and endothelial cells) and HSC-derived (for example, megakaryocytes, macrophages and regulatory T cells), with better topographical understanding of HSC localization in the bone marrow. Here, we review advances in our understanding of HSC regulation by niches during homeostasis, ageing and cancer, and we discuss their implications for the development of therapies to rejuvenate aged HSCs or niches or to disrupt self-reinforcing malignant niches.
On the influence of cannabinoids on cell morphology and motility of glioblastoma cells.
Feb 12, 2019   PloS One
Hohmann T, Feese K, Ghadban C, Dehghani F, Grabiec U
On the influence of cannabinoids on cell morphology and motility of glioblastoma cells.
Feb 12, 2019
PloS One
The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways.
Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila.
Feb 12, 2019   PloS One
Castells-Nobau A, Eidhof I, Fenckova M, Brenman-Suttner DB, Scheffer-de Gooyert JM,   . . . . . .   , Fisher SE, Kramer JM, Sigrist SJ, Simon AF, Schenck A
Conserved regulation of neurodevelopmental processes and behavior by FoxP in Drosophila.
Feb 12, 2019
PloS One
FOXP proteins form a subfamily of evolutionarily conserved transcription factors involved in the development and functioning of several tissues, including the central nervous system. In humans, mutations in FOXP1 and FOXP2 have been implicated in cognitive deficits including intellectual disability and speech disorders. Drosophila exhibits a single ortholog, called FoxP, but due to a lack of characterized mutants, our understanding of the gene remains poor. Here we show that the dimerization property required for mammalian FOXP function is conserved in Drosophila. In flies, FoxP is enriched in the adult brain, showing strong expression in ~1000 neurons of cholinergic, glutamatergic and GABAergic nature. We generate Drosophila loss-of-function mutants and UAS-FoxP transgenic lines for ectopic expression, and use them to characterize FoxP function in the nervous system. At the cellular level, we demonstrate that Drosophila FoxP is required in larvae for synaptic morphogenesis at axonal terminals of the neuromuscular junction and for dendrite development of dorsal multidendritic sensory neurons. In the developing brain, we find that FoxP plays important roles in α-lobe mushroom body formation. Finally, at a behavioral level, we show that Drosophila FoxP is important for locomotion, habituation learning and social space behavior of adult flies. Our work shows that Drosophila FoxP is important for regulating several neurodevelopmental processes and behaviors that are related to human disease or vertebrate disease model phenotypes. This suggests a high degree of functional conservation with vertebrate FOXP orthologues and established flies as a model system for understanding FOXP related pathologies.
Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii.
Feb 16, 2019   ELife
Tosetti N, Dos Santos Pacheco N, Soldati-Favre D, Jacot D
Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii.
Feb 16, 2019
ELife
Toxoplasma gondii possesses a limited set of actin-regulatory proteins and relies on only three formins (FRMs) to nucleate and polymerize actin. We combined filamentous actin (F-actin) chromobodies with gene disruption to assign specific populations of actin filaments to individual formins. FRM2 localizes to the apical juxtanuclear region and participates in apicoplast inheritance. Restricted to the residual body, FRM3 maintains the intravacuolar cell-cell communication. Conoidal FRM1 initiates a flux of F-actin crucial for motility, invasion and egress. This flux depends on myosins A and H and is controlled by phosphorylation via PKG (protein kinase G) and CDPK1 (calcium-dependent protein kinase 1) and by methylation via AKMT (apical lysine methyltransferase). This flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC). This study offers a coherent model of the key players controlling actin polymerization, stressing the importance of well-timed post-translational modifications to power parasite motility.
Monitoring Phenotypic Switching in Candida albicans and the Use of Next-Gen Fluorescence Reporters.
Feb 12, 2019   Current Protocols In Microbiology
Frazer C, Hernday AD, Bennett RJ
Monitoring Phenotypic Switching in Candida albicans and the Use of Next-Gen Fluorescence Reporters.
Feb 12, 2019
Current Protocols In Microbiology
Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.
CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.
Feb 16, 2019   ELife
Deneault E, Faheem M, White SH, Rodrigues DC, Sun S,   . . . . . .   , Roth FP, Yuen RK, Singh KK, Ellis J, Scherer SW
CNTN5-/+or EHMT2-/+human iPSC-derived neurons from individuals with autism develop hyperactive neuronal networks.
Feb 16, 2019
ELife
Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, 'isogenic' iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.
Feb 14, 2019   Science Advances
Oshima T, Niwa Y, Kuwata K, Srivastava A, Hyoda T,   . . . . . .   , Ikeda M, Kinoshita T, Kay SA, Itami K, Hirota T
Cell-based screen identifies a new potent and highly selective CK2 inhibitor for modulation of circadian rhythms and cancer cell growth.
Feb 14, 2019
Science Advances
Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.
Celastrol Inhibits the Growth of Ovarian Cancer Cells in vitro and in vivo.
Feb 14, 2019   Frontiers In Oncology
Xu LN, Zhao N, Chen JY, Ye PP, Nan XW,   . . . . . .   , Xing ZH, Wei MN, Li Y, Shi Z, Yan XJ
Celastrol Inhibits the Growth of Ovarian Cancer Cells in vitro and in vivo.
Feb 14, 2019
Frontiers In Oncology
Celastrol is a natural triterpene isolated from the Chinese plant Thunder God Vine with potent antitumor activity. However, the effect of celastrol on the growth of ovarian cancer cells in vitro and in vivo is still unclear. In this study, we found that celastrol induced cell growth inhibition, cell cycle arrest in G2/M phase and apoptosis with the increased intracellular reactive oxygen species (ROS) accumulation in ovarian cancer cells. Pretreatment with ROS scavenger N-acetyl-cysteine totally blocked the apoptosis induced by celastrol. Additionally, celastrol inhibited the growth of ovarian cancer xenografts in nude mice. Altogether, these findings suggest celastrol is a potential therapeutic agent for treating ovarian cancer.
Inhibition of Heme Oxygenase-1 enhances hyperthermia-induced autophagy and antiviral effect.
Feb 14, 2019   International Journal Of Biological Sciences
Yang Y, Wang HX, Zhang L, Huo W, Li XD, Qi RQ, Song XY, Wei S, Gao XH, Han S, Cao L
Inhibition of Heme Oxygenase-1 enhances hyperthermia-induced autophagy and antiviral effect.
Feb 14, 2019
International Journal Of Biological Sciences
Hyperthermia has been clinically utilized as an adjuvant therapy in the treatment of cervical carcinoma. However, thermotolerance induced by heme oxygenase-1 (HO-1), a stress-inducible cytoprotective protein, limits the efficacy of hyperthermic therapy, for which the exact mechanism remains unknown. In the present study, we found that heat treatment induced HO-1 expression and decreased copy number of HPV16 in cervical cancer cells and tissues from cervical cancer and precursor lesions. Knockdown of HO-1 stimulated autophagy accompanied by downregulation of X-linked inhibitor of apoptosis protein. Furthermore, silencing of HO-1 led to cell intolerance to hyperthermia, as manifested by inhibition of cell viability and induction of autophagic apoptosis. Moreover, HO-1 modulated hyperthermia-induced, autophagy-dependent antiviral effect. Thus, the findings indicate that blockade of HO-1 enhances hyperthermia-induced autophagy, an event resulting in apoptosis of cervical cancer cells through an antiviral mechanism. These observations imply the potential clinical utility of hyperthermia in combination with HO-1 inhibition in the treatment of cervical cancer.
Cyclin G2 Inhibits the Warburg Effect and Tumour Progression by Suppressing LDHA Phosphorylation in Glioma.
Feb 14, 2019   International Journal Of Biological Sciences
Li S, Gao J, Zhuang X, Zhao C, Hou X, Xing X, Chen C, Liu Q, Liu S, Luo Y
Cyclin G2 Inhibits the Warburg Effect and Tumour Progression by Suppressing LDHA Phosphorylation in Glioma.
Feb 14, 2019
International Journal Of Biological Sciences
Cyclin G2 has been identified as a tumour suppressor in several cancers. However, its regulatory roles and underlying mechanisms in tumours are still unknown. In this study, we demonstrated that cyclin G2 was expressed at low levels in glioma, which was as a poor prognostic factor for this disease. We also found that, cyclin G2 could suppress cell proliferation, initiate cell apoptosis and reduce aerobic glycolysis, suggesting that cyclin G2 plays a tumour suppressive role in glioma. Mechanistically, cyclin G2 could negatively regulate tyrosine-10 phosphorylation of a critical glycolytic enzyme, lactate dehydrogenase A, through direct interaction. Taken together, these results indicate that cyclin G2 acts as a tumour suppressor in glioma by repressing glycolysis and tumour progression through its interaction with LDHA.
Autophagy resists EMT process to maintain retinal pigment epithelium homeostasis.
Feb 14, 2019   International Journal Of Biological Sciences
Feng H, Zhao X, Guo Q, Feng Y, Ma M, Guo W, Dong X, Deng C, Li C, Song X, Han S, Cao L
Autophagy resists EMT process to maintain retinal pigment epithelium homeostasis.
Feb 14, 2019
International Journal Of Biological Sciences
Proliferative vitreoretinopathy (PVR) is the most serious fibrous complication that causes vision loss after intraocular surgery, and there is currently no effective treatment in clinical. Autophagy is an important cell biological mechanism in maintaining the homeostasis of tissues and cells, resisting the process of EMT. However, it is still unclear whether autophagy could resist intraocular fibrosis and prevent PVR progression. In this study, we investigated the expression of mesenchymal biomarkers in autophagy deficiency cells and found these proteins were increased. The mesenchymal protein transcription factor Twist can bind to autophagy related protein p62 and promote the degradation of Twist, which reduced the expression of mesenchymal markers. By constructing an EMT model of retinal pigment epithelial (RPE) cells in vitro, we found that autophagy was activated in the EMT process of RPE cells. Moreover, in autophagy deficient RPE cell line via knockdown autophagy related protein 7 (Atg7), the expression of epithelial marker claudin-1 was suppressed and the mesenchymal markers were increased, accompanied by an increase in cell migration and contractility. Importantly, RPE epithelial properties can be maintained by promoting autophagy and effectively reversing TFG-β2-induced RPE fibrosis. These observations reveal that autophagy may be an effective way to treat PVR.
NUP214 deficiency causes severe encephalopathy and microcephaly in humans.
Feb 13, 2019   Human Genetics
Shamseldin HE, Makhseed N, Ibrahim N, Al-Sheddi T, Alobeid E, Abdulwahab F, Alkuraya FS
NUP214 deficiency causes severe encephalopathy and microcephaly in humans.
Feb 13, 2019
Human Genetics
Nuclear pore complex (NPC) is a fundamental component of the nuclear envelope and is key to the nucleocytoplasmic transport. Mutations in several NUP genes that encode individual components of NPC known as nucleoporins have been identified in recent years among patients with static encephalopathies characterized by developmental delay and microcephaly. We describe a multiplex consanguineous family in which four affected members presented with severe neonatal hypotonia, profound global developmental delay, progressive microcephaly and early death. Autozygome and linkage analysis revealed that this phenotype is linked to a founder disease haplotype (chr9:127,113,732-135,288,807) in which whole exome sequencing revealed the presence of a novel homozygous missense variant in NUP214. Functional analysis of patient-derived fibroblasts recapitulated the dysmorphic phenotype of nuclei that was previously described in NUP214 knockdown cells. In addition, the typical rim staining of NUP214 is largely displaced, further supporting the deleterious effect of the variant. Our data expand the list of NUP genes that are mutated in encephalopathy disorders in humans.

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