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The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication  [PDF]
Preeti Malik-Kale, Seth Winfree, Olivia Steele-Mortimer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038732
Abstract: Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens.
Functional Dissection of the TBK1 Molecular Network  [PDF]
Adriana Goncalves, Tilmann Bürckstümmer, Evelyn Dixit, Ruth Scheicher, Maria W. Górna, Evren Karayel, Cristina Sugar, Alexey Stukalov, Tiina Berg, Robert Kralovics, Melanie Planyavsky, Keiryn L. Bennett, Jacques Colinge, Giulio Superti-Furga
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023971
Abstract: TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I:C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.
NEMO Binds Ubiquitinated TANK-Binding Kinase 1 (TBK1) to Regulate Innate Immune Responses to RNA Viruses  [PDF]
Lingyan Wang, Shitao Li, Martin E. Dorf
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043756
Abstract: RIG-I-like receptors (RLR) are intracellular sensors utilized by nearly all cell types for recognition of viral RNA, initiation of antiviral defense, and induction of type I interferons (IFN). TBK1 is a critical kinase implicated in RLR-dependent IFN transcription. Posttranslational modification of TBK1 by K63-linked ubiquitin is required for RLR driven signaling. However, the TBK1 ubiquitin acceptor sites and the function of ubiquitinated TBK1 in the signaling cascade are unknown. We now show that TBK1 is ubiquitinated on residues K69, K154, and K372 in response to infection with RNA virus. The K69 and K154 residues are critical for innate antiviral responses and IFN production. Ubiquitinated TBK1 recruits the downstream adaptor NEMO through ubiquitin binding domains. The assembly of the NEMO/TBK1 complex on the mitochondrial protein MAVS leads to activation of TBK1 kinase activity and phosphorylation of the transcription factor, interferon response factor 3. The combined results refine current views of RLR signaling, define the role of TBK1 polyubiquitination, and detail the mechanisms involved in signalosome assembly.
The Fanconi Anemia Pathway Protects Genome Integrity from R-loops  [PDF]
María L. García-Rubio?,Carmen Pérez-Calero?,Sonia I. Barroso?,Emanuela Tumini?,Emilia Herrera-Moyano?,Iván V. Rosado?,Andrés Aguilera
PLOS Genetics , 2015, DOI: 10.1371/journal.pgen.1005674
Abstract: Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.
Quercetin Protects Saccharomyces cerevisiae against Oxidative Stress by Inducing Trehalose Biosynthesis and the Cell Wall Integrity Pathway  [PDF]
Rita Vila?a, Vanda Mendes, Marta Vaz Mendes, Laura Carreto, Maria Amélia Amorim, Victor de Freitas, Pedro Moradas-Ferreira, Nuno Mateus, Vítor Costa
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045494
Abstract: Background Quercetin is a naturally occurring flavonol with antioxidant, anticancer and anti-ageing properties. In this study we aimed to identify genes differentially expressed in yeast cells treated with quercetin and its role in oxidative stress protection. Methods A microarray analysis was performed to characterize changes in the transcriptome and the expression of selected genes was validated by RT-qPCR. Biological processes significantly affected were identified by using the FUNSPEC software and their relevance in H2O2 resistance induced by quercetin was assessed. Results Genes associated with RNA metabolism and ribosome biogenesis were down regulated in cells treated with quercetin, whereas genes associated with carbohydrate metabolism, endocytosis and vacuolar proteolysis were up regulated. The induction of genes related to the metabolism of energy reserves, leading to the accumulation of the stress protectant disaccharide trehalose, and the activation of the cell wall integrity pathway play a key role in oxidative stress resistance induced by quercetin. Conclusions These results suggest that quercetin may act as a modulator of cell signaling pathways related to carbohydrate metabolism and cell integrity to exert its protective effects against oxidative stress.
Antibody to Ricin A Chain Hinders Intracellular Routing of Toxin and Protects Cells Even after Toxin Has Been Internalized  [PDF]
Kejing Song, R. Ranney Mize, Luis Marrero, Miriam Corti, Jason M. Kirk, Seth H. Pincus
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062417
Abstract: Background Mechanisms of antibody-mediated neutralization are of much interest. For plant and bacterial A-B toxins, A chain mediates toxicity and B chain binds target cells. It is generally accepted and taught that antibody (Ab) neutralizes by preventing toxin binding to cells. Yet for some toxins, ricin included, anti-A chain Abs afford greater protection than anti-B. The mechanism(s) whereby Abs to the A chain neutralize toxins are not understood. Methodology/Principal Findings We use quantitative confocal imaging, neutralization assays, and other techniques to study how anti-A chain Abs function to protect cells. Without Ab, ricin enters cells and penetrates to the endoplasmic reticulum within 15 min. Within 45–60 min, ricin entering and being expelled from cells reaches equilibrium. These results are consistent with previous observations, and support the validity of our novel methodology. The addition of neutralizing Ab causes ricin accumulation at the cell surface, delays internalization, and postpones retrograde transport of ricin. Ab binds ricin for >6hr as they traffic together through the cell. Ab protects cells even when administered hours after exposure. Conclusions/Key Findings We demonstrate the dynamic nature of the interaction between the host cell and toxin, and how Ab can alter the balance in favor of the cell. Ab blocks ricin’s entry into cells, hinders its intracellular routing, and can protect even after ricin is present in the target organelle, providing evidence that the major site of neutralization is intracellular. These data add toxins to the list of pathogenic agents that can be neutralized intracellularly and explain the in vivo efficacy of delayed administration of anti-toxin Abs. The results encourage the use of post-exposure passive Ab therapy, and show the importance of the A chain as a target of Abs.
Yacon Product (PBY) Modulates Intestinal Constipation and Protects the Integrity of Crypts in Wistar Rats  [PDF]
M?nica de Souza Lima Sant’Anna, Vivian Carolina Rodrigues, Tatiane Ferreira Araújo, Tania Toledo de Oliveira, Maria do Carmo Gouveia Pelúzio, Célia Lúcia de Luces Fortes Ferreira
Food and Nutrition Sciences (FNS) , 2018, DOI: 10.4236/fns.2018.912101
Abstract: This study investigated the use of a product based on yacon (PBY) in microbiological, physical-chemical and intestinal characteristics of Wistar rats artificially constipated with Loperamide®. Thirty-two rats were divided into four groups: Control (C), Constipated Control (CC), PBY (not constipated) and Constipated PBY (PBYC). The dosage of 0.14 g of FOS+ inulin/kg was tested. Microbiota, pH and faeces characteristics of faeces and caecal contents were evaluated. Caecal weight, morphometry of caecal villi and the concentration of short-chain fatty acids were determined. Higher caecal weight was identified in the PBYC animals as well as higher width, height and depth of cripts. The PBY group showed the highest (p < 0.05) concentration of butyrate (93.2 ± 65.5 mmol/L). The supplementation with PBY positively altered the intestine epithelial tissue in constipated animals, keeping the integrity of the caecum crypts.
Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites  [PDF]
Sébastien Besteiro ,Carrie F. Brooks,Boris Striepen,Jean-Fran?ois Dubremetz
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002416
Abstract: Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
L-arginine Supplementation Protects Exercise Performance and Structural Integrity of Muscle Fibers after a Single Bout of Eccentric Exercise in Rats  [PDF]
Yulia N. Lomonosova, Boris S. Shenkman, Grigorii R. Kalamkarov, Tatiana Y. Kostrominova, Tatyana L. Nemirovskaya
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094448
Abstract: Eccentric exercise is known to disrupt sarcolemmal integrity and induce damage of skeletal muscle fibers. We hypothesized that L-arginine (L-Arg; nitric oxide synthase (NOS) substrate) supplementation prior to a single bout of eccentric exercise would diminish exercise-induced damage. In addition, we used N-nitro-L-arginine methyl ester hydrochloride (L-NAME; NOS inhibitor) to clarify the role of native NOS activity in the development of exercise-induced muscle damage. Rats were divided into four groups: non-treated control (C), downhill running with (RA) or without (R) L-Arg supplementation and downhill running with L-NAME supplementation (RN). Twenty four hours following eccentric exercise seven rats in each group were sacrificed and soleus muscles were dissected and frozen for further analysis. The remaining seven rats in each group were subjected to the exercise performance test. Our experiments showed that L-Arg supplementation prior to a single bout of eccentric exercise improved subsequent exercise performance capacity tests in RA rats when compared with R, RN and C rats by 37%, 27% and 13%, respectively. This outcome is mediated by L-Arg protection against post-exercise damage of sarcolemma (2.26- and 0.87-fold less than R and RN groups, respectively), reduced numbers of damaged muscle fibers indicated by the reduced loss of desmin content in the muscle (15% and 25% less than R and RN groups, respectively), and diminished μ-calpain mRNA up-regulation (42% and 30% less than R and RN groups, respectively). In conclusion, our study indicates that L-Arg supplementation prior to a single bout of eccentric exercise alleviates muscle fiber damage and preserves exercise performance capacity.
The Pivotal Role of TBK1 in Inflammatory Responses Mediated by Macrophages  [PDF]
Tao Yu,Young-Su Yi,Yanyan Yang,Jueun Oh,Deok Jeong,Jae Youl Cho
Mediators of Inflammation , 2012, DOI: 10.1155/2012/979105
Abstract: Inflammation is a complex biological response of tissues to harmful stimuli such as pathogens, cell damage, or irritants. Inflammation is considered to be a major cause of most chronic diseases, especially in more than 100 types of inflammatory diseases which include Alzheimer's disease, rheumatoid arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, hepatitis, and Parkinson's disease. Recently, an increasing number of studies have focused on inflammatory diseases. TBK1 is a serine/threonine-protein kinase which regulates antiviral defense, host-virus interaction, and immunity. It is ubiquitously expressed in mouse stomach, colon, thymus, and liver. Interestingly, high levels of active TBK1 have also been found to be associated with inflammatory diseases, indicating that TBK1 is closely related to inflammatory responses. Even though relatively few studies have addressed the functional roles of TBK1 relating to inflammation, this paper discusses some recent findings that support the critical role of TBK1 in inflammatory diseases and underlie the necessity of trials to develop useful remedies or therapeutics that target TBK1 for the treatment of inflammatory diseases. 1. Introduction Inflammation is the immune response of tissues to pathogens, cell damage, or irritants [1]. It is a protective mechanism used by organisms to remove injurious stimuli. In the process, several symptoms appear, which include redness, swelling, and pain, which are general responses to infection. Inflammation is classified as either acute or chronic. Acute inflammation is the initial response of the organism to harmful stimuli and is induced by the increased movement of plasma and leukocytes from the blood into the injured sites. Chronic inflammation leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and generation of the tissues from the inflammatory process. Inflammation is considered to be the main cause of most chronic diseases including not only inflammatory diseases, such as heart disease, diabetes, Alzheimer’s disease, and arthritis, but also cancers [2–5]. Therefore, the study of inflammation should be considered a priority. The inflammation that occurs during innate immune responses is largely regulated by macrophages [6, 7]. This inflammation is driven by immunopathological events such as the overproduction of various proinflammatory cytokines, including tumor necrosis factor (TNF-α), interleukin (IL-1β), interferon (IFN-β), and several types of inflammatory
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