SciCombinator

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Concept: Cytosol

170

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

Concepts: Cell, Bacteria, Cytosol, Cell membrane, Endoplasmic reticulum, Intestinal parasite, Digenea, Fasciola hepatica

150

Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca(2+)-activated-K(+)-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca(2+) and eventually an activation of hIK channels.

Concepts: DNA, Cell nucleus, Cancer, Ionizing radiation, X-ray, Cytosol, Cytoplasm, Electromagnetic spectrum

147

B7-H4, one of the costimulatory molecules of B7 family, has been found to be widely expressed in many kinds of tumor tissues and to play an important part in tumor progression and poor prognosis. However, the role of B7-H4 in esophageal squamous cell carcinoma (ESCC) cells has not been elucidated. In this study, we found that, compared with normal esophageal tissue, B7-H4 was highly expressed in three ESCC cell lines, Eca109, TE1 and TE13. Besides, B7-H4 silence suppressed cells proliferation and colony formation. Additionally, compared with control cells, B7-H4 silence cells showed higher apoptosis rate, Bcl-2 and Survivin upregulation as well as BAX downregulation. Further study demonstrated that B7-H4 silence cells also exhibited reduction of IL-6 secretion, STAT3 activation and p-STAT3 translocation from cytoplasm to nucleus. Moreover, B7-H4 depletion inhibited the IL-6 secretion of control cells but not JAK2/STAT3 inhibitor FLLL32 treated cells. IL-6 receptor antagonist Tocilizumab didn’t block the p-JAK2 and p-STAT3 downregulation induced by B7-H4 silence. It was suggested that B7-H4 silence suppressed IL-6 secretion through JAK2/STAT3 inactivation. Furthermore, cells proliferation and colony formation were downregulated by Tocilizumab in control cells but not B7-H4 silence cells demonstrating that IL-6 upregulation induced by B7-H4 was necessary for cells growth. On the other hand, B7-H4 expression was downregulated by Tocilizumab. In all, our study provided the first evidence that B7-H4 facilitated ESCC cells proliferation through promoting IL-6/STAT3 positive loopback pathway activation. This article is protected by copyright. All rights reserved.

Concepts: Cell nucleus, Gene expression, Cell, Cancer, Cytosol, Cell biology, Squamous cell carcinoma, Downregulation and upregulation

46

The uptake of cytoplasmic calcium into mitochondria is critical for a variety of physiological processes, including calcium buffering, metabolism, and cell survival. Here, we demonstrate that inhibiting the mitochondrial calcium uniporter in the Drosophila mushroom body neurons (MBn)-a brain region critical for olfactory memory formation-causes memory impairment without altering the capacity to learn. Inhibiting uniporter activity only during pupation impaired adult memory, whereas the same inhibition during adulthood was without effect. The behavioral impairment was associated with structural defects in MBn, including a decrease in synaptic vesicles and an increased length in the axons of the αβ MBn. Our results reveal an in vivo developmental role for the mitochondrial uniporter complex in establishing the necessary structural and functional neuronal substrates for normal memory formation in the adult organism.

Concepts: Alzheimer's disease, Neuron, Cell, Bacteria, Eukaryote, Mitochondrion, Cytosol, Action potential

45

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer.

Concepts: Immune system, DNA, Cell nucleus, Cell, Cancer, Bacteria, Cytosol, Cell biology

34

There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

Concepts: Cell, Cytosol, Cell membrane, Heart, Cytoplasm, Cardiac muscle, Cellular differentiation, Cell type

34

Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated.

Concepts: Cell, Signal transduction, Cytosol, Cell membrane, Regulation, Endocytosis, Administrative law, Development approvals

33

By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.

Concepts: DNA, Cell, Eukaryote, Mitochondrion, Cytosol, Organelle, Cell division, Cell biology

31

BACKGROUND:Erythrocyte cell membranes undergo morphologic changes during storage, but it is unclear whether these changes are reversible. We assessed erythrocyte cell membrane deformability in patients before and after transfusion to determine the effects of storage duration and whether changes in deformability are reversible after transfusion.METHODS:Sixteen patients undergoing posterior spinal fusion surgery were studied. Erythrocyte deformability was compared between those who required moderate transfusion (≥5 units erythrocytes) and those who received minimal transfusion (0-4 units erythrocytes). Deformability was measured in samples drawn directly from the blood storage bags before transfusion and in samples drawn from patients before and after transfusion (over 3 postoperative days). In samples taken from the blood storage bags, we compared deformability of erythrocytes stored for a long duration (≥21 days), those stored for a shorter duration (<21 days), and cell-salvaged erythrocytes. Deformability was assessed quantitatively using the elongation index (EI) measured by ektacytometry, a method that determines the ability for the cell to elongate when exposed to shear stress.RESULTS:Erythrocyte deformability was significantly decreased from the preoperative baseline in patients after moderate transfusion (EI decreased by 12% ± 4% to 20% ± 6%; P = 0.03) but not after minimal transfusion (EI decreased by 3% ± 1% to 4% ± 1%; P = 0.68). These changes did not reverse over 3 postoperative days. Deformability was significantly less in erythrocytes stored for ≥21 days (EI = 0.28 ± 0.02) than in those stored for <21 days (EI = 0.33 ± 0.02; P = 0.001) or those drawn from patients preoperatively (EI = 0.33 ± 0.02; P = 0.001). Cell-salvaged erythrocytes had intermediate deformability (EI = 0.30 ± 0.03) that was greater than that of erythrocytes stored ≥21 days (P = 0.047), but less than that of erythrocytes stored <21 days (P = 0.03).CONCLUSIONS:The findings demonstrate that increased duration of erythrocyte storage is associated with decreased cell membrane deformability and that these changes are not readily reversible after transfusion.

Concepts: Protein, Gene, Cell nucleus, Cell, Cytosol, Cell membrane, Red blood cell, Spinal fusion

28

The transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.

Concepts: Protein, Cell, Cytosol, Cell membrane, Endoplasmic reticulum, Low-density lipoprotein, Apolipoprotein B, Protein targeting