553 results on '"mammalian-cells"'
Search Results
2. A novel small molecule RAD51 inactivator overcomes imatinib-resistance in chronic myeloid leukaemia
- Author
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Zhu, Jiewen, Zhou, Longen, Wu, Guikai, Konig, Heiko, Lin, Xiaoqin, Li, Guideng, Qiu, Xiao-Long, Chen, Chi-Fen, Hu, Chun-Mei, Goldblatt, Erin, Bhatia, Ravi, Chamberlin, A. Richard, Chen, Phang-Lang, and Lee, Wen-Hwa
- Subjects
Cancer ,Cml ,Inhibitor ,Rad51 ,Small MoleculeDna-Damage Response ,Homologous Recombination ,Synaptonemal Complexes ,Mammalian-Cells ,Tumor-Cells ,Brc Repeats ,Bcr-Abl ,Protein ,Radiation ,Repair - Published
- 2013
3. CtIP Is Required to Initiate Replication-Dependent Interstrand Crosslink Repair
- Author
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Duquette, Michelle L, Zhu, Qingyuan, Taylor, Ewan R, Tsay, Angela J, Shi, Linda Z, Berns, Michael W, McGowan, Clare H, and Ford, James M
- Subjects
fanconi-anemia pathway ,double-strand breaks ,dna-end resection ,cell-cycle ,homologous recombination ,mammalian-cells ,s-phase ,monoubiquitinated fancd2 ,translesion synthesis ,genome instability - Published
- 2012
4. Condensin I Recruitment to Base Damage-Enriched DNA Lesions Is Modulated by PARP1
- Author
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Kong, Xiangduo, Stephens, Jared, Ball, Alexander R., Heale, Jason T., Newkirk, Daniel A., Berns, Michael W., and Yokomori, Kyoko
- Subjects
sister-chromatid cohesion ,strand break repair ,mammalian-cells ,chromosome condensation ,vertebrate cells ,complex ,activation ,responses ,sites - Abstract
Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I.
- Published
- 2011
5. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation
- Author
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Gomez-Godinez, Veronica, Wu, Tao, Sherman, Adria J., Lee, Christopher S., Liaw, Lih-Huei, Zhongsheng, You, Yokomori, Kyoko, and Berns, Michael W.
- Subjects
histone h2ax phosphorylation ,optical tweezers ,living cells ,nuclear foci ,potorous-tridactylis ,nucleolar organizer ,ubiquitin ligase ,mammalian-cells ,damage response ,mre11 complex - Abstract
In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1–2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ∼34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.
- Published
- 2010
6. Integration of the Beta-Catenin-Dependent Wnt Pathway with Integrin Signaling through the Adaptor Molecule Grb2
- Author
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Crampton, Steve P., Wu, Beibei, Park, Edward J., Kim, Jai-Hyun, Solomon, Candice, Waterman, Marian L., and Hughes, Christopher W.
- Subjects
focal-adhesion-kinase ,c-jun ,mammalian-cells ,linked kinase ,target genes ,protein ,activation ,cancer ,proliferation ,invasion - Abstract
BackgroundThe complexity of wnt signaling likely stems from two sources: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.Methodology/Principal FindingsHere we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of β-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-β-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of β-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.Conclusions/SignificanceThese data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.
- Published
- 2009
7. Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid
- Author
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Murphy, Eric A., Waring, Alan J., Murphy, Jason C., Willson, Richard C., and Longmuir, Kenneth J.
- Subjects
mouse peritoneal-macrophages ,receptor-mediated endocytosis ,nuclear import ,in-vivo ,linear polyethylenimine ,nondividing cells ,intralysosomal ph ,inhibit transfer ,mammalian-cells ,matrix protein - Abstract
We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/µg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 µg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.
- Published
- 2001
8. Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay
- Author
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Congying Zheng, Andrew Collins, Gunnar Brunborg, Frederik-Jan van Schooten, Anne Lene Nordengen, Sergey Shaposhnikov, Roger Godschalk, Farmacologie en Toxicologie, and RS: NUTRIM - R3 - Respiratory & Age-related Health
- Subjects
Antioxidant status ,Health, Toxicology and Mutagenesis ,Cellular repair ,Cell Biology ,CANCER-PATIENTS ,IN-VITRO ,Toxicology ,DOUBLE-STRAND BREAKS ,LYMPHOCYTES ,VALIDATION ,Fpg ,OXIDATIVE DNA-DAMAGE ,SINGLE-STRAND ,MAMMALIAN-CELLS ,DNA repair capacity ,POTASSIUM BROMATE ,RADIOSENSITIVITY ,Comet assay - Abstract
DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0–480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO3). The initial amount of damage induced by Ro 19–8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO3 varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T0 to an equal level can be achieved by the use of KBrO3, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure. Graphical Abstract
- Published
- 2023
9. Hetero-pentamerization determines mobility and conductance of Glycine receptor alpha 3 splice variants
- Author
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Veerle Lemmens, Bart Thevelein, Yana Vella, Svenja Kankowski, Julia Leonhard, Hideaki Mizuno, Susana Rocha, Bert Brône, Jochen C. Meier, and Jelle Hendrix
- Subjects
DYNAMICS ,Biochemistry & Molecular Biology ,Glycine receptors ,Ligand gated ion channels ,Subunit counting ,COLOCALIZATION ,GEPHYRIN ,Ligands ,Synaptic Transmission ,Cellular and Molecular Neuroscience ,Receptors, Glycine ,MAMMALIAN-CELLS ,Image correlation spectroscopy ,Pearson's correlation coefficient ,SUBUNIT STOICHIOMETRY ,SINGLE-MOLECULE ,BETA-SUBUNIT ,Molecular Biology ,Pharmacology ,Science & Technology ,MEMBRANE-PROTEINS ,Single-molecule fluorescence ,Cell Biology ,Protein co-assembly ,Stoichiometry ,DIFFUSION ,Electrophysiology ,Alternative Splicing ,Mutation ,Molecular Medicine ,Patch clamp ,Life Sciences & Biomedicine - Abstract
Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases. ispartof: CELLULAR AND MOLECULAR LIFE SCIENCES vol:79 issue:11 ispartof: location:Switzerland status: published
- Published
- 2022
10. Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy
- Author
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Felix Braun, Richard Wombacher, Frank Rominger, Aleksandra Radenovic, Marvin Busch, Philipp Werther, Edward A. Lemke, Antoni J. Gralak, Klaus Yserentant, Weijie Chi, Michael J. Ziegler, Zhibin Zhang, Dirk-Peter Herten, Christoph Mayer, Xiaogang Liu, Kristin S. Grußmayer, Vytautas Navikas, Miao Yu, and Tiago Buckup
- Subjects
fluorophore ,Fluorescence-lifetime imaging microscopy ,Fluorophore ,Quenching (fluorescence) ,Chemistry ,Super-resolution microscopy ,General Chemical Engineering ,mammalian-cells ,STED microscopy ,Context (language use) ,General Chemistry ,Combinatorial chemistry ,chemistry.chemical_compound ,Tetrazine ,tetrazine probes ,Fluorescence microscope ,strategy ,QD1-999 ,Research Article - Abstract
Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine–dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine–dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size., This work presents probes that turn fluorescent upon specific chemical reactions in biological environments, which are highly beneficial for signal improvement in fluorescence microscopy.
- Published
- 2021
11. Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies
- Subjects
MAMMALIAN-CELLS ,GEL-ELECTROPHORESIS ,OXIDATIVE DAMAGE ,WHOLE-BLOOD ,IN-VITRO ,BREAST-CANCER PATIENTS ,PERIPHERAL LYMPHOCYTES ,SEASONAL-VARIATIONS ,VALIDATION ,CRYOPRESERVED LYMPHOCYTES - Abstract
DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.
- Published
- 2021
12. The cellular response to plasma membrane disruption for nanomaterial delivery
- Author
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Gaëlle Houthaeve, Stefaan C. De Smedt, Kevin Braeckmans, and Winnok H. De Vos
- Subjects
Technology ,Science ,QC1-999 ,TP1-1185 ,Review ,Plasma membrane disruption ,LATERAL MOBILITY ,MAMMALIAN-CELLS ,Medicine and Health Sciences ,Nanotechnology ,General Materials Science ,OXIDATIVE STRESS ,IN-VIVO ,REPAIR ,Chemical technology ,Physics ,Pharmacology. Therapy ,General Engineering ,HAMSTER OVARY CELLS ,Intracellular delivery ,Chemistry ,DNA-DAMAGE ,Cellular homeostasis ,PORE-FORMING TOXINS ,LONG-TERM POTENTIATION ,Engineering sciences. Technology ,TP248.13-248.65 ,EFFICIENT INTRACELLULAR DELIVERY ,Biotechnology - Abstract
Delivery of nanomaterials into cells is of interest for fundamental cell biological research as well as for therapeutic and diagnostic purposes. One way of doing so is by physically disrupting the plasma membrane (PM). Several methods that exploit electrical, mechanical or optical cues have been conceived to temporarily disrupt the PM for intracellular delivery, with variable effects on cell viability. However, apart from acute cytotoxicity, subtler effects on cell physiology may occur as well. Their nature and timing vary with the severity of the insult and the efficiency of repair, but some may provoke permanent phenotypic alterations. With the growing palette of nanoscale delivery methods and applications, comes a need for an in-depth understanding of this cellular response. In this review, we summarize current knowledge about the chronology of cellular events that take place upon PM injury inflicted by different delivery methods. We also elaborate on their significance for cell homeostasis and cell fate. Based on the crucial nodes that govern cell fitness and functionality, we give directions for fine-tuning nano-delivery conditions.
- Published
- 2022
13. Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis
- Author
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Yehudi Bloch, Sandra Kozak, Savvas N. Savvides, Steven De Munck, Aleksandra Mikula, Rob Meijers, and Isabel Bento
- Subjects
0301 basic medicine ,Glycosylation ,crystallization ,receptors ,Crystallography, X-Ray ,AXON GUIDANCE ,chemistry.chemical_compound ,0302 clinical medicine ,MAMMALIAN-CELLS ,CSF-1 ,Structural Biology ,Medicine and Health Sciences ,glycoproteins ,chemistry.chemical_classification ,REFINEMENT ,biology ,Research Papers ,Biochemistry ,Protein folding ,STRUCTURAL BASIS ,EXPRESSION ,Glycan ,glycosylation ,DSCAM ,cell‐ ,Protein–protein interaction ,03 medical and health sciences ,Polysaccharides ,Cell surface receptor ,REVEALS ,surface ,Humans ,ddc:530 ,COMPLEX ,RECEPTOR ,Cryoelectron Microscopy ,HEK 293 cells ,Biology and Life Sciences ,GlycoDelete cell line ,Sialic acid ,carbohydrates (lipids) ,cell-surface receptors ,HEK293 Cells ,030104 developmental biology ,chemistry ,biology.protein ,synthetic biology ,Glycoprotein ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery - Abstract
Acta crystallographica / Section D 76(12), 1244 - 1255 (2020). doi:10.1107/S2059798320013753, Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy., Published by Wiley, Bognor Regis
- Published
- 2020
14. Limited evidence for protein products of non-coding transcripts in the HEK293T cellular cytosol
- Author
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Annelies Bogaert, Daria Fijalkowska, An Staes, Tessa Van de Steene, Hans Demol, and Kris Gevaert
- Subjects
TRANSLATION INITIATION LANDSCAPE ,RNA, Untranslated ,IDENTIFICATION ,Biology and Life Sciences ,PEPTIDES ,N-TERMINAL PROTEOMICS ,Biochemistry ,SMALL ORFS ,ANNOTATION ,Analytical Chemistry ,RNAS ,Open Reading Frames ,Cytosol ,HEK293 Cells ,MAMMALIAN-CELLS ,Protein Biosynthesis ,DISCOVERY ,REVEALS ,Medicine and Health Sciences ,Humans ,Peptides ,Ribosomes ,Molecular Biology - Abstract
Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from non -coding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.
- Published
- 2022
15. Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies
- Author
-
Møller, Peter, Bankoglu, Ezgi Eyluel, Stopper, Helga, Giovannelli, Lisa, Ladeira, Carina, Koppen, Gudrun, Gajski, Goran, Collins, Andrew, Valdiglesias, Vanessa, Laffon, Blanca, Boutet-Robinet, Elisa, Perdry, Herve, Del Bo', Cristian, Langie, Sabine A. S., Dusinska, Maria, Azqueta, Amaya, Møller, Peter, Bankoglu, Ezgi Eyluel, Stopper, Helga, Giovannelli, Lisa, Ladeira, Carina, Koppen, Gudrun, Gajski, Goran, Collins, Andrew, Valdiglesias, Vanessa, Laffon, Blanca, Boutet-Robinet, Elisa, Perdry, Herve, Del Bo', Cristian, Langie, Sabine A. S., Dusinska, Maria, and Azqueta, Amaya
- Abstract
DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.
- Published
- 2021
16. Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies
- Author
-
Goran Gajski, Blanca Laffon, Vanessa Valdiglesias, Peter Møller, Carina Ladeira, Amaya Azqueta, Elisa Boutet-Robinet, Gudrun Koppen, Hervé Perdry, Helga Stopper, Cristian Del Bo, Andrew Collins, Lisa Giovannelli, Maria Dusinska, Sabine A. S. Langie, Ezgi Eyluel Bankoglu, University of Copenhagen = Københavns Universitet (KU), University of Würzburg, NEUROFARBA Department [Firenze, Italy], Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI), Escola Nacional de Saúde Pública (ENSP-NOVA), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Flemish Institute for Technological Research (VITO), Institute for Medical Research and Occupational Health, Institute of Basic Medical Sciences [Oslo], Faculty of Medicine [Oslo], University of Oslo (UiO)-University of Oslo (UiO), Centro de Investigacións Científicas Avanzadas (CICA), Faculty of Sciences, University of A Coruña (UDC), Contaminants & Stress Cellulaire (ToxAlim-COMICS), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Ecole d'Ingénieurs de Purpan (INPT - EI Purpan), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre de recherche en épidémiologie et santé des populations (CESP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Department of Food Environmental and Nutritional Sciences, (DeFENS), University of Milan, School of Nutrition and Translational Research in Metabolism [Maastricht] (NUTRIM), Maastricht University [Maastricht], Norwegian Institute for Air Research (NILU), Universidad de Navarra [Pamplona] (UNAV), and hCOMET project (COST Action, CA 15132)
- Subjects
DNA Repair ,DNA damage ,Epidemiology ,Health, Toxicology and Mutagenesis ,[SDV]Life Sciences [q-bio] ,GEL-ELECTROPHORESIS ,Buffy coat ,Biology ,Toxicology ,Peripheral blood mononuclear cell ,Cryopreservation ,VALIDATION ,Andrology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,MAMMALIAN-CELLS ,Genetics ,WHOLE-BLOOD ,Humans ,DNA damage, DNA repair, comet assay ,Leucocytes ,Comet assay ,Genetics (clinical) ,030304 developmental biology ,Whole blood ,0303 health sciences ,Blood Specimen Collection ,Molecular ,IN-VITRO ,DNA ,PERIPHERAL LYMPHOCYTES ,SEASONAL-VARIATIONS ,In vitro ,CRYOPRESERVED LYMPHOCYTES ,chemistry ,Blood Preservation ,030220 oncology & carcinogenesis ,OXIDATIVE DAMAGE ,Leukocytes, Mononuclear ,Comet Assay ,BREAST-CANCER PATIENTS ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology ,DNA Damage - Abstract
DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors’ experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.
- Published
- 2021
17. SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites
- Author
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Paakinaho, Ville, Lempiäinen, Joanna K, Sigismondo, Gianluca, Niskanen, Einari A, Malinen, Marjo, Jääskeläinen, Tiina, Varjosalo, Markku, Krijgsveld, Jeroen, Palvimo, Jorma J, Institute of Biotechnology, Biosciences, and Molecular Systems Biology
- Subjects
DYNAMICS ,Binding Sites ,IDENTIFICATION ,AcademicSubjects/SCI00010 ,Gene regulation, Chromatin and Epigenetics ,ANDROGEN RECEPTOR ,SUMO MODIFICATIONS ,COREGULATOR ,Sumoylation ,Chromatin ,UBIQUITIN ,STEROID-RECEPTOR ,TRANSCRIPTION FACTORS ,HEK293 Cells ,Nuclear Receptor Coactivator 1 ,Receptors, Glucocorticoid ,Gene Expression Regulation ,MAMMALIAN-CELLS ,TARGET GENE ,Protein Interaction Mapping ,Small Ubiquitin-Related Modifier Proteins ,Humans ,Nuclear Receptor Co-Repressor 1 ,1182 Biochemistry, cell and molecular biology - Abstract
Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
- Published
- 2021
18. Plasmodium translocon component EXP2 facilitates hepatocyte invasion
- Author
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Mello-Vieira, J, Enguita, FJ, De Koning-Ward, T, Zuzarte-Luís, V, Mota, MM, Mello-Vieira, J, Enguita, FJ, De Koning-Ward, T, Zuzarte-Luís, V, and Mota, MM
- Abstract
Plasmodium parasites possess a translocon that exports parasite proteins into the infected erythrocyte. Although the translocon components are also expressed during the mosquito and liver stage of infection, their function remains unexplored. Here, using a combination of genetic and chemical assays, we show that the translocon component Exported Protein 2 (EXP2) is critical for invasion of hepatocytes. EXP2 is a pore-forming protein that is secreted from the sporozoite upon contact with the host cell milieu. EXP2-deficient sporozoites are impaired in invasion, which can be rescued by the exogenous administration of recombinant EXP2 and alpha-hemolysin (an S. aureus pore-forming protein), as well as by acid sphingomyelinase. The latter, together with the negative impact of chemical and genetic inhibition of acid sphingomyelinase on invasion, reveals that EXP2 pore-forming activity induces hepatocyte membrane repair, which plays a key role in parasite invasion. Overall, our findings establish a novel and critical function for EXP2 that leads to an active participation of the host cell in Plasmodium sporozoite invasion, challenging the current view of the establishment of liver stage infection.
- Published
- 2020
19. Phloretin and phloridzin guard against cisplatin-induced nephrotoxicity in mice through inhibiting oxidative stress and inflammation
- Author
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Elif Cadirci, Rustem Anil Ugan, Muhammet Ali Gurbuz, Gokce Kaya, Yasin Bayir, Zekai Halici, Harun Un, Aysenur Kahramanlar, and Belirlenecek
- Subjects
0301 basic medicine ,Antioxidant ,Efficacy ,Phloretin ,medicine.medical_treatment ,Assay ,Injury ,Antineoplastic Agents ,Pharmacology ,Kidney ,medicine.disease_cause ,Kidney Function Tests ,030226 pharmacology & pharmacy ,General Biochemistry, Genetics and Molecular Biology ,Nephrotoxicity ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Heat-Shock-Protein-70 ,medicine ,Phloridzin ,Animals ,General Pharmacology, Toxicology and Pharmaceutics ,Cisplatin ,Inflammation ,Mice, Inbred BALB C ,Tissue ,Toxicity ,General Medicine ,Glutathione ,Increases ,Mammalian-Cells ,Oxidative Stress ,030104 developmental biology ,medicine.anatomical_structure ,Phlorhizin ,chemistry ,Gene Expression Regulation ,Female ,Kidney Diseases ,Oxidative stress ,medicine.drug - Abstract
Aim: Cisplatin (Cis) is widely used chemotherapeutic and has some serious side effects as nephrotoxicity. Phloretin (PH) and Phloridzin (PZ) are known their anti-oxidant anti-inflammatory effects. We aimed to examine the protective effects of PH and PZ on cisplatin-induced nephrotoxicity. Main methods: Totally, 48 Balb/C female mice were separated into eight groups (n = 6). First day, single dose of cisplatin (20 mg/kg intraperitoneal) was administered to induce toxicity. PH and PZ were given (50 and 100 mg/kg orally) to treatment groups during 3 days. After the experimental procedures serum renal function enzymes (BUN and Creatinine), oxidative parameters (SOD, GSH and MDA), nuclear agent NFK beta, inflammatory cytokines (Tnf-alpha and IL1 beta) and HSP70 expressions and histopathological assessments were analyzed. Key findings: Serum enzymes, tissue cytokines and oxidative stress were increased after the Cis treatment. PH and PZ treatments normalized all parameters compared to Cis administrated group. After the treatments, SOD activities and GSH levels were increased while MDA levels were decreased. PH and PZ treatments decreased Tnf-alpha, IL1 beta and NFK beta mRNA expressions. Cis significantly increased the HSP70 expression while PH and PZ administrations significantly decreased. Similar the biochemical and molecular results, PH and PZ showed positive effects on tissue pathological parameters. Cisplatin cause a lot of abnormal structures as tubular and glomeruli damages on the kidney. Significance: PH and PZ play important physiological roles in the prevention of nephrotoxicity. Antioxidant and anti-inflammatory effects of PH and PZ demonstrated visible protective effects in the cisplatin-induced nephrotoxicity model.
- Published
- 2020
20. Comb-Like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes
- Author
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Liwei Wu, Rongjun Chen, Victoria L. Bemmer, Jie Ren, Richard Zajicek, Siyuan Chen, and GlaxoSmithKline Services Unlimited
- Subjects
MECHANISM ,Technology ,STRESS ,Erythrocytes ,Polymers ,02 engineering and technology ,030226 pharmacology & pharmacy ,Cryopreservation ,09 Engineering ,chemistry.chemical_compound ,pseudopeptide ,RED-BLOOD-CELLS ,0302 clinical medicine ,Cryoprotective Agents ,MAMMALIAN-CELLS ,Side chain ,General Materials Science ,pH-responsive ,PH-RESPONSIVE POLYMERS ,Temperature ,Hydrogen-Ion Concentration ,021001 nanoscience & nanotechnology ,Membrane curvature ,SURVIVAL ,Science & Technology - Other Topics ,0210 nano-technology ,03 Chemical Sciences ,Hydrophobic and Hydrophilic Interactions ,Intracellular ,Materials science ,Cryoprotectant ,MEMBRANE CURVATURE ,PROTEINS ,Materials Science ,pH-sensitive polymers ,comb-like polymer ,Materials Science, Multidisciplinary ,Hemolysis ,03 medical and health sciences ,blood ,Extracellular ,Nanoscience & Nanotechnology ,Science & Technology ,Trehalose ,DESICCATION ,chemistry ,Biophysics ,CRYOSURVIVAL - Abstract
Cell cryopreservation plays a key role in the development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing- and desiccation-tolerant organisms in nature has been sought as an attractive nontoxic cryoprotectant. Herein, we report a coincubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution, and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature, and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation but also other membrane-impermeable cargos.
- Published
- 2020
- Full Text
- View/download PDF
21. Cx43 channels and signaling via IP3/Ca2+, ATP, and ROS/NO propagate radiation-induced DNA damage to non-irradiated brain microvascular endothelial cells
- Author
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Christian Vanhove, Valérie Van Haver, Benedicte Descamps, Luc Leybaert, Geert Bultynck, Dmitri V. Krysko, Maarten De Smet, Tinneke Delvaeye, Delphine Hoorelbeke, Elke Decrock, and Marijke De Bock
- Subjects
Cancer Research ,DNA damage ,Immunology ,Connexin ,DOUBLE-STRAND BREAKS ,medicine.disease_cause ,CONNEXIN-43 HEMICHANNELS ,Nitric oxide ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Paracrine signalling ,MAMMALIAN-CELLS ,INTERCELLULAR CA2+ WAVES ,Medicine and Health Sciences ,Bystander effect ,medicine ,Patch clamp ,lcsh:QH573-671 ,OXIDATIVE STRESS ,Science & Technology ,NITRIC-OXIDE ,lcsh:Cytology ,Biology and Life Sciences ,Cell Biology ,SISTER-CHROMATID EXCHANGES ,GAP-JUNCTION HEMICHANNELS ,Cell biology ,chemistry ,BYSTANDER RESPONSES ,IONIZING-RADIATION ,Radiation Induced DNA Damage ,Life Sciences & Biomedicine ,Oxidative stress - Abstract
Radiotherapeutic treatment consists of targeted application of radiation beams to a tumor but exposure of surrounding healthy tissue is inevitable. In the brain, ionizing radiation induces breakdown of the blood–brain barrier by effects on brain microvascular endothelial cells. Damage from directly irradiated cells can be transferred to surrounding non-exposed bystander cells, known as the radiation-induced bystander effect. We investigated involvement of connexin channels and paracrine signaling in radiation-induced bystander DNA damage in brain microvascular endothelial cells exposed to focused X-rays. Irradiation caused DNA damage in the directly exposed area, which propagated over several millimeters in the bystander area. DNA damage was significantly reduced by the connexin channel-targeting peptide Gap26 and the Cx43 hemichannel blocker TAT-Gap19. ATP release, dye uptake, and patch clamp experiments showed that hemichannels opened within 5 min post irradiation in both irradiated and bystander areas. Bystander signaling involved cellular Ca2+ dynamics and IP3, ATP, ROS, and NO signaling, with Ca2+, IP3, and ROS as crucial propagators of DNA damage. We conclude that bystander effects are communicated by a concerted cascade involving connexin channels, and IP3/Ca2+, ATP, ROS, and NO as major contributors of regenerative signal expansion.
- Published
- 2020
22. Microcarriers for Upscaling Cultured Meat Production
- Author
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Vincent Bodiou, Panagiota Moutsatsou, and Mark J. Post
- Subjects
0301 basic medicine ,Computer science ,Endocrinology, Diabetes and Metabolism ,SKELETAL-MUSCLE CELLS ,030209 endocrinology & metabolism ,lcsh:TX341-641 ,Review ,03 medical and health sciences ,Global population ,Cultured meat ,0302 clinical medicine ,cultivated meat ,MAMMALIAN-CELLS ,EXTRACELLULAR-MATRIX ,Production (economics) ,bovine myoblasts ,Bioprocess ,LARGE-SCALE PRODUCTION ,Nutrition ,satellite cells ,030109 nutrition & dietetics ,Nutrition and Dietetics ,CURRENT TECHNOLOGIES ,SHEAR-STRESS ,bioprocessing ,Final product ,Microcarrier ,HYDROXYBUTYL CHITOSAN ,ENDOTHELIAL-CELLS ,Skeletal muscle cell proliferation ,clean meat ,microbeads ,Biochemical engineering ,Sustainable production ,EMBRYONIC STEM-CELLS ,lcsh:Nutrition. Foods and food supply ,cell expansion ,SATELLITE CELL ,Food Science - Abstract
Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.
- Published
- 2020
23. Microcarriers for Upscaling Cultured Meat Production
- Subjects
satellite cells ,CURRENT TECHNOLOGIES ,SHEAR-STRESS ,bioprocessing ,SKELETAL-MUSCLE CELLS ,HYDROXYBUTYL CHITOSAN ,ENDOTHELIAL-CELLS ,cultivated meat ,clean meat ,MAMMALIAN-CELLS ,EXTRACELLULAR-MATRIX ,bovine myoblasts ,microbeads ,LARGE-SCALE PRODUCTION ,EMBRYONIC STEM-CELLS ,cell expansion ,SATELLITE CELL - Abstract
Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.
- Published
- 2020
24. Extracellular citrate fuels cancer cell metabolism and growth
- Author
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Sebastian, Haferkamp, Konstantin, Drexler, Marianne, Federlin, Hans J, Schlitt, Mark, Berneburg, Jerzy, Adamski, Andreas, Gaumann, Edward K, Geissler, Vadivel, Ganapathy, E Kenneth, Parkinson, Maria E, Mycielska, and Publica
- Subjects
Cell and Developmental Biology ,ddc:610 ,cancer associated fibroblast (CAF) ,transporter ,610 Medizin ,cancer ,senescent fibroblasts ,Cancer ,Cancer Associated Fibroblast (caf) ,Metabolism ,Senescent Fibroblasts ,Transporter ,Review ,metabolism ,GENOMIC ORGANIZATION ,FUNCTIONAL FEATURES ,TUMOR METABOLISM ,MAMMALIAN-CELLS ,PROSTATE-CANCER ,DOWN-REGULATION ,LIVER-CANCER ,TRANSPORTER ,FIBROBLASTS ,IRON - Abstract
Cancer cells need excess energy and essential nutrients/metabolites not only to divide and proliferate but also to migrate and invade distant organs for metastasis. Fatty acid and cholesterol synthesis, considered a hallmark of cancer for anabolism and membrane biogenesis, requires citrate. We review here potential pathways in which citrate is synthesized and/or supplied to cancer cells and the impact of extracellular citrate on cancer cell metabolism and growth. Cancer cells employ different mechanisms to support mitochondrial activity and citrate synthesis when some of the necessary substrates are missing in the extracellular space. We also discuss the different transport mechanisms available for the entry of extracellular citrate into cancer cells and how citrate as a master metabolite enhances ATP production and fuels anabolic pathways. The available literature suggests that cancer cells show an increased metabolic flexibility with which they tackle changing environmental conditions, a phenomenon crucial for cancer cell proliferation and metastasis.
- Published
- 2020
25. Development of MTL-CEBPA: Small Activating RNA Drug for Hepatocellular Carcinoma
- Author
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Nagy A. Habib, Helen L. Lightfoot, Ryan L. Setten, John J. Rossi, and Mina Therapeutics Ltd
- Subjects
0301 basic medicine ,Druggability ,Pharmaceutical Science ,MAMMALIAN-CELLS ,Transcription (biology) ,CEBPA ,Genes, Tumor Suppressor ,Pharmacology & Pharmacy ,RNA, Small Interfering ,LEUCINE-ZIPPER ,Regulation of gene expression ,CEBPα ,Liver Neoplasms ,hepatocellular carcinoma ,Drug development ,1115 Pharmacology and Pharmaceutical Sciences ,Life Sciences & Biomedicine ,MTL-CEBPA ,Biotechnology ,MiNA therapeutics ,Biochemistry & Molecular Biology ,Carcinoma, Hepatocellular ,ARGONAUTE PROTEINS ,DNA-BINDING ,RNA activation ,ACUTE MYELOID-LEUKEMIA ,Biology ,liver ,ANTISENSE OLIGONUCLEOTIDES ,Article ,MESENCHYMAL STEM-CELLS ,CCAAT/ENHANCER-BINDING-PROTEIN ,03 medical and health sciences ,Animals ,Humans ,Transcription factor ,RNA, Double-Stranded ,Science & Technology ,1004 Medical Biotechnology ,RNA therapeutics ,C/EBP-ALPHA GENE ,saRNA ,030104 developmental biology ,INDUCE TRANSCRIPTIONAL ACTIVATION ,Gene Expression Regulation ,CEBPa ,CCAAT-Enhancer-Binding Proteins ,Cancer research ,RNA, Small Untranslated ,Transcription Factors - Abstract
BACKGROUND: Oligonucleotide drug development has revolutionised the drug discovery field allowing the notoriously "undruggable" genome to potentially become "druggable". Within this field, 'small' or 'short' activating RNAs (saRNA) are a more recently discovered category of short double stranded RNA with clinical potential. SaRNAs promote endogenous transcription from target loci, a phenomenon widely observed in mammals known as RNA activation (RNAa). The ability to target a particular gene is dependent on the sequence of the saRNA. Hence, the potential clinical application of saRNA is to increase target gene expression in a sequence specific manner. SaRNA based oligonucleotide therapeutics present great promise in expanding the "druggable" genome with particular areas of interest including transcription factor activation and haploinsufficency. Review and Conclusion: In this mini-review, we describe the pre-clinical development of the first saRNA drug to enter the clinic. This saRNA, referred to as MTL-CEBPA, induces transcription of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα), a tumour suppressor and critical regulator of hepatocyte function. MTL-CEBPA is presently in Phase I clinical trials for hepatocellular carcinoma (HCC). The clinical development of MTL-CEBPA will demonstrate "proof of concept", showing that saRNAs can provide the basis for drugs which enhance targeted gene expression and consequently improve disease outcome in patients.
- Published
- 2018
26. Molecular signalling towards mitochondrial breakdown is enhanced in skeletal muscle of patients with chronic obstructive pulmonary disease (COPD)
- Author
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Marco C. J. M. Kelders, Annemie M. W. J. Schols, C. C. de Theije, Anita Kneppers, Mitja Lainscak, Harry R. Gosker, Pieter A. Leermakers, Promovendi NTM, Pulmonologie, RS: NUTRIM - R3 - Respiratory & Age-related Health, and Ondersteunend personeel NTM
- Subjects
0301 basic medicine ,Male ,RECEPTOR-MEDIATED MITOPHAGY ,PROTEIN ,lcsh:Medicine ,Systemic inflammation ,Pulmonary Disease, Chronic Obstructive ,MAMMALIAN-CELLS ,Mitophagy ,lcsh:Science ,COPD ,Multidisciplinary ,Anemia, Iron-Deficiency ,Iron deficiency ,Middle Aged ,CHRONIC HEART-FAILURE ,Mitochondria ,medicine.anatomical_structure ,C-Reactive Protein ,Female ,LOCOMOTOR MUSCLE ,medicine.symptom ,Signal Transduction ,FUNDC1 ,REHABILITATION ,medicine.medical_specialty ,Mitochondrial DNA ,Anemia ,Ubiquitin-Protein Ligases ,METABOLISM ,DNA, Mitochondrial ,Article ,Mitochondrial Proteins ,03 medical and health sciences ,Internal medicine ,Proto-Oncogene Proteins ,medicine ,Autophagy ,Humans ,Muscle, Skeletal ,Aged ,Inflammation ,business.industry ,Tumor Suppressor Proteins ,lcsh:R ,Skeletal muscle ,Membrane Proteins ,medicine.disease ,DYSFUNCTION ,030104 developmental biology ,Endocrinology ,Gene Expression Regulation ,lcsh:Q ,business - Abstract
Loss of skeletal muscle mitochondrial oxidative capacity is well-established in patients with COPD, but the role of mitochondrial breakdown herein is largely unexplored. Currently, we studied if mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients and associates with the loss of mitochondrial content, and whether it is affected in patients with iron deficiency (ID) or systemic inflammation. Therefore, mitophagy, autophagy, mitochondrial dynamics and content markers were analysed in vastus lateralis biopsies of COPD patients (N = 95, FEV1% predicted: 39.0 [31.0–53.6]) and healthy controls (N = 15, FEV1% predicted: 112.8 [107.5–125.5]). Sub-analyses were performed on patients stratified by ID or C-reactive protein (CRP). Compared with controls, COPD patients had lower muscle mitochondrial content, higher BNIP3L and lower FUNDC1 protein, and higher Parkin protein and gene-expression. BNIP3L and Parkin protein levels inversely correlated with mtDNA/gDNA ratio and FEV1% predicted. ID-COPD patients had lower BNIP3L protein and higher BNIP3 gene-expression, while high CRP patients had higher BNIP3 and autophagy-related protein levels. In conclusion, our data indicates that mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients, and is related to disease severity and loss of mitochondrial content. Moreover, systemic inflammation is associated with higher BNIP3 and autophagy-related protein levels.
- Published
- 2018
27. Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20
- Author
-
F. Gisou van der Goot, Laurence Abrami, Michael Veit, and Mohamed Rasheed Gadalla
- Subjects
chemistry.chemical_classification ,Zinc finger ,binding ,biology ,hef ,mammalian-cells ,Hemagglutinin (influenza) ,m2 protein ,fatty-acids ,site-specific attachment ,Transmembrane protein ,Acylation ,fusion pore formation ,cytoplasmic tail ,chemistry ,Biochemistry ,s-acylation ,biology.protein ,palmitoylation ,Influenza C Virus ,Glycoprotein ,DHHC domain ,Cysteine - Abstract
Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton-channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by acyl-RAC and confirmed by 3H-palmitate labelling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co- localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the hemagglutinin-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs.
- Published
- 2019
- Full Text
- View/download PDF
28. Acute depletion of diacylglycerol from the cis-Golgi affects localized nuclear envelope morphology during mitosis
- Author
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Chung, Gary Hong Chun, Domart, Marie-Charlotte, Peddie, Christopher, Mantell, Judith, Mclaverty, Kieran, Arabiotorre, Angela, Hodgson, Lorna, Byrne, Richard D., Verkade, Paul, Arkill, Kenton, Collinson, Lucy M., and Larijani, Banafshé
- Subjects
fusion ,vesicles ,phosphatidylinositol ,binding ,education ,mammalian-cells ,endoplasmic-reticulum ,dynamics ,QD415-436 ,in-vitro ,Biochemistry ,serial block face scanning electron microscopy ,correlative light electron microscopy ,membrane curvature ,lipids (amino acids, peptides, and proteins) ,phospholipid-bilayer membranes ,health care economics and organizations - Abstract
Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis is a highly regulated process, but the detailed mechanism still remains incompletely understood. Previous studies have found that isolated diacylglycerol (DAG)-containing vesicles are essential for completing the fusion of the NE in nonsomatic cells. We investigated the impact of DAG depletion from the cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a mini singlet oxygen generator-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the reforming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly. This work was supported by the British Heart Foundation [Grant number PG/15/37/31438 (K.A.)]; the Medical Research Council [Grant number MR/P003214/1 (K.A.), FC001999 (Francis Crick Institute), MR/K01580X/1 (L.M.C.)]; Cancer Research UK (Grant number FC001999); the Wellcome Trust (Grant number FC001999); the Biotechnology and Biological Sciences Research Council [Grant number BB/L014181/1 (P.V.)]; MR/K01580X/1 (L.M.C.)]; the Engineering and Physical Sciences Research Council [grant number MR/K01580X/1 (L.M.C.)]; the Ministerio de Economia y Competitividad Grant BFU2015-65625-P; and Bizkaia Talent Fellowship Grant AYD-000-256 (K.A.).
- Published
- 2018
29. Interferon priming is essential for human CD34+ cell-derived plasmacytoid dendritic cell maturation and function
- Author
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Matt Porteus, Niels Uldbjerg, Nadia R. Roan, L Kjær, Satish K. Pillai, Anders Laustsen, J H Egedahl, Paul W. Denton, Christian Krapp, Hai Q Tang, Martin R. Jakobsen, Rasmus O. Bak, Charlotte Christie Petersen, and Mette Nyegaard
- Subjects
0301 basic medicine ,ADJUVANT ACTIVITY ,General Physics and Astronomy ,Priming (immunology) ,Antigens, CD34 ,Receptor, Interferon alpha-beta ,Plasmacytoid dendritic cell ,Polymerase Chain Reaction ,0302 clinical medicine ,MAMMALIAN-CELLS ,Interferon ,Receptors, Immunologic ,lcsh:Science ,Cells, Cultured ,Gene Editing ,Multidisciplinary ,Cell Differentiation ,hemic and immune systems ,Nucleotidyltransferases ,Cell biology ,I-INTERFERON ,Haematopoiesis ,030220 oncology & carcinogenesis ,SYNTHETIC SIRNA ,Interferon Type I ,DEAD Box Protein 58 ,medicine.drug ,BONE-MARROW ,FLT3 LIGAND ,Science ,Enzyme-Linked Immunosorbent Assay ,macromolecular substances ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Immune system ,medicine ,Humans ,HEMATOPOIETIC STEM-CELLS ,Progenitor cell ,Interleukin-6 ,IFN-ALPHA ,TLR9 ,DIABETES-MELLITUS ,Dendritic Cells ,General Chemistry ,TLR7 ,030104 developmental biology ,PROGENITOR CELLS ,Toll-Like Receptor 7 ,Toll-Like Receptor 9 ,lcsh:Q ,CRISPR-Cas Systems - Abstract
Plasmacytoid dendritic cells (pDC) are essential for immune competence. Here we show that pDC precursor differentiated from human CD34+ hematopoietic stem and progenitor cells (HSPC) has low surface expression of pDC markers, and has limited induction of type I interferon (IFN) and IL-6 upon TLR7 and TLR9 agonists treatment; by contrast, cGAS or RIG-I agonists-mediated activation is not altered. Importantly, after priming with type I and II IFN, these precursor pDCs attain a phenotype and functional activity similar to that of peripheral blood-derived pDCs. Data from CRISPR/Cas9-mediated genome editing of HSPCs further show that HSPC-pDCs with genetic modifications can be obtained, and that expression of the IFN-α receptor is essential for the optimal function, but dispensable for the differentiation, of HSPC-pDC percursor. Our results thus demonstrate the biological effects of IFNs for regulating pDC function, and provide the means of generating of gene-modified human pDCs., Plasmacytoid dendritic cells (pDC) are an important regulator of immune responses. Here the authors show that pDC precursors, similar to peripheral blood-derived pDCs, can be differentiated from human CD34+ hematopoietic stem and progenitor cells, with type I/II IFN priming being required for their functional maturation and differentiation.
- Published
- 2018
30. Structure of a mitochondrial fission dynamin in the closed conformation
- Author
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Harry H. Low, Olga Bohuszewicz, and Wellcome Trust
- Subjects
0301 basic medicine ,Dynamins ,Biochemistry & Molecular Biology ,DIMERIZATION ,Conformational change ,Biophysics ,Molecular Conformation ,PROTEIN ,macromolecular substances ,GTPase ,Mitochondrion ,PEROXISOMAL FISSION ,Crystallography, X-Ray ,Mitochondrial Dynamics ,Article ,Catalysis ,Protein filament ,03 medical and health sciences ,0302 clinical medicine ,DOMAIN ,Membrane fission ,MAMMALIAN-CELLS ,Structural Biology ,CRYSTAL-STRUCTURE ,CONSTRICTION ,Molecular Biology ,Dynamin ,Science & Technology ,biology ,MEMBRANE FISSION ,Chemistry ,Reproducibility of Results ,Cell Biology ,11 Medical And Health Sciences ,06 Biological Sciences ,biology.organism_classification ,030104 developmental biology ,Cyanidioschyzon merolae ,STALK REGION ,Mitochondrial fission ,03 Chemical Sciences ,Life Sciences & Biomedicine ,GTPASE ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Dynamin 1-like proteins (DNM1-L) are mechanochemical GTPases that induce membrane fission in mitochondria and peroxisomes. Their mechanism depends on conformational changes driven by nucleotide and lipid cycling. Here we show the crystal structure of a mitochondrial fission dynamin (CmDnm1) from the algae Cyanidioschyzon merolae. Contrary to other eukaryotic dynamin structures, CmDnm1 is in a hinge 1 closed conformation with the GTPase domain compacted against the stalk. Within the crystal, CmDnm1 packs as a diamond shaped tetramer that is consistent with an inactive off-membrane state. Cross-linking, photoinduced electron transfer (PET) assays, and electron microscopy verify these structures. In vitro, CmDnm1 forms concentration dependent rings and protein-lipid tubes reminiscent of DNM1-L and classical dynamin with hinge 1 open. Our data provides a mechanism for filament collapse and membrane release that may extend to other dynamin family members. Additionally, hinge 1 closing may represent a key conformational change that contributes to membrane fission.
- Published
- 2018
31. Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences
- Author
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Rizwan Ali, Sivaramakrishnan Ramadurai, Heinz-Peter Nasheuer, Frank Barry, Science Foundation Ireland, and Else Kröner‐Fresenius‐Stiftung
- Subjects
green fluorescent protein ,0301 basic medicine ,mammalian-cells ,Negative regulatory element ,promoter sequences ,in-vivo ,General Biochemistry, Genetics and Molecular Biology ,Green fluorescent protein ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,fluorescence (cross-)correlation spectroscopy ,quantitative fluorescence microscopy ,icp4 ,Chemistry ,Promoter ,gene-expression ,Molecular biology ,Fusion protein ,mobility ,mcherry ,cross-correlation spectroscopy ,030104 developmental biology ,Thymidine kinase ,030220 oncology & carcinogenesis ,Ectopic expression ,single-molecule ,transcription ,mCherry ,living cells ,fusion proteins - Abstract
The modulation of expression levels of fluorescent fusion proteins (FFPs) is central for recombinant DNA technologies in modern biology as overexpression of proteins contributes to artifacts in biological experiments. In addition, some microscopy techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule-based techniques are very sensitive to high expression levels of FFPs. To reduce the levels of recombinant protein expression in comparison with the commonly used, very strong CMV promoter, the herpes simplex virus thymidine kinase (TK) gene promoter, and mutants thereof were analyzed. Deletion mutants of the TK promoter were constructed and introduced into the Gateway (R) system for ectopic expression of enhanced green fluorescent protein (eGFP), monomeric cherry (mCherry), and FFPs containing these FPs. Two promoter constructs, TK2ST and TKTSC, were established, which have optimal low expression levels suitable for FCS studies in U2OS, HeLa CCL2, NIH 3T3, and BALB/c cells. Interestingly, when tested in these four cell lines, promoter constructs having a deletion within TK gene 5'-UTR showed significantly higher protein expression levels than the equivalent constructs lacking this deletion. This suggests that a negative regulatory element is localized within the TK gene 5'-UTR. Science Foundation Ireland. Grant Number: 06/CE/B1129 Else Kröner‐Fresenius‐Stiftung. Grant Number: 2013_A215 peer-reviewed
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- 2018
32. Engineering a Clinically Translatable Bioartificial Pancreas to Treat Type I Diabetes
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BETA-CELLS ,MAMMALIAN-CELLS ,GLYCEMIC CONTROL ,HUMAN ISLETS ,INFLAMMATORY RESPONSES ,ALGINATE-PLL CAPSULES ,ENCAPSULATION ,PLURIPOTENT STEM-CELLS ,L-LYSINE MICROCAPSULES ,BIOCOMPATIBILITY - Abstract
Encapsulating, or immunoisolating, insulin-secreting cells within implantable, semipermeable membranes is an emerging treatment for type 1 diabetes. This approach can eliminate the need for immunosuppressive drug treatments to prevent transplant rejection and overcome the shortage of donor tissues by utilizing cells derived from allogeneic or xenogeneic sources. Encapsulation device designs are being optimized alongside the development of clinically viable, replenishable, insulin-producing stem cells, for the first time creating the possibility of widespread therapeutic use of this technology. Here, we highlight the status of the most advanced and widely explored implementations of cell encapsulation with an eye toward translating the potential of this technological approach to medical reality.
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- 2018
33. An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability
- Author
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Mónica Álvarez-Fernández, Marcos Malumbres, Aintzane Apraiz, Asier Fullaondo, Ana M. Zubiaga, Jone Mitxelena, Iraia García-Santisteban, Jon Vallejo-Rodríguez, Ministerio de Ciencia e Innovación (España), Basque Government (España), University of the Basque Country (España), and Worldwide Cancer Research
- Subjects
nucleotide excision-repair ,0301 basic medicine ,DNA Repair ,Transcription, Genetic ,DNA repair ,DNA damage ,melphalan resistance ,mammalian-cells ,RAD51 ,homologous recombination ,Genome Integrity, Repair and Replication ,fanconi-anemia pathway ,Biology ,Genomic Instability ,Cell Line ,03 medical and health sciences ,E2F7 Transcription Factor ,E2F ,sites ,Genetics ,Transcriptional regulation ,Humans ,Promoter Regions, Genetic ,cross-link repair ,double-strand breaks ,Cell Cycle ,OMOLOGOUS RECOMBINATION ,Recombinational DNA Repair ,cell-cycle genes ,Chromosome Breakage ,Cell cycle ,Cell biology ,030104 developmental biology ,Gene Expression Regulation ,Tumor Suppressor Protein p53 ,Corrigendum ,Transcriptome ,Homologous recombination ,DNA Damage ,Nucleotide excision repair - Abstract
Corrigendum published on 03 July 2019 Nucleic Acids Research 47 (14) : 7716–7717 (2019) https://doi.org/10.1093/nar/gkz587 The cellular response to DNA damage is essential for maintaining the integrity of the genome. Recent evidence has identified E2F7 as a key player in DNA damage-dependent transcriptional regulation of cell-cycle genes. However, the contribution of E2F7 to cellular responses upon genotoxic damage is still poorly defined. Here we show that E2F7 represses the expression of genes involved in the maintenance of genomic stability, both throughout the cell cycle and upon induction of DNA lesions that interfere with replication fork progression. Knockdown of E2F7 leads to a reduction in 53BP1 and FANCD2 foci and to fewer chromosomal aberrations following treatment with agents that cause interstrand crosslink (ICL) lesions but not upon ionizing radiation. Accordingly, E2F7-depleted cells exhibit enhanced cell-cycle re-entry and clonogenic survival after exposure to ICL-inducing agents. We further report that expression and functional activity of E2F7 are p53-independent in this context. Using a cell-based assay, we show that E2F7 restricts homologous recombination through the transcriptional repression of RAD51. Finally, we present evidence that downregulation of E2F7 confers an increased resistance to chemotherapy in recombination-deficient cells. Taken together, our results reveal an E2F7-dependent transcriptional program that contributes to the regulation of DNA repair and genomic integrity. This work was supported by grants from the Spanish Ministry [SAF2012-33551 and SAF2015-67562-R, co-financed by FEDER funds, and SAF2014-57791-REDC], the Basque Government [IT634-13 and KK-2015/89], and the University of the Basque Country UPV/EHU [UFI11/20] to AMZ; and grants from the Spanish Ministry [SAF2015-69920-R], and Worldwide Cancer Research [15-0278] to MM. JM was recipient of a Basque Government fellowship for graduate studies and JVR is recipient of a UPV/EHU fellowship for graduate studies. M.A.F. was supported by a young investigator grant from MINECO [SAF2014-60442-JIN; co-financed by FEDER funds]. Funding for open access charge: Spanish Ministry [SAF2015-67562-R, co-financed by FEDER funds]; Basque Government [IT634-13].
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- 2018
34. No Effects of Hyperosmolar Culture Medium on Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells Despite Upregulation Extracellular Matrix Genes
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Ruud A. Bank, Jelena Popov-Celeketic, Saskia G M Plomp, Anita Krouwels, F. Cumhur Oner, Wouter J.A. Dhert, Laura B. Creemers, Restoring Organ Function by Means of Regenerative Medicine (REGENERATE), and Groningen Kidney Center (GKC)
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Male ,0301 basic medicine ,osmolyte ,PROTEIN EXPRESSION ,INTERVERTEBRAL DISC CELLS ,OSMOLARITY ,Intervertebral Disc Degeneration ,Extracellular matrix ,DNA ,0302 clinical medicine ,MAMMALIAN-CELLS ,Gene expression ,Orthopedics and Sports Medicine ,Aggrecans ,Cells, Cultured ,Glycosaminoglycans ,GAG ,collagen II ,nucleus pulposus ,CHONDROCYTES ,hyperosmotic ,sucrose ,Middle Aged ,musculoskeletal system ,Extracellular Matrix ,Up-Regulation ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,Osmolyte ,sodium chloride ,Female ,Proteoglycans ,Amino Acid Oxidoreductases ,aggrecan ,MESSENGER-RNA ,Adult ,musculoskeletal diseases ,urea ,METABOLISM ,Biology ,03 medical and health sciences ,Downregulation and upregulation ,CARTILAGE ,medicine ,Humans ,Regeneration ,osmolality ,GLYCOSAMINOGLYCAN PRODUCTION ,Aggrecan ,Aged ,030203 arthritis & rheumatology ,Cartilage ,Regeneration (biology) ,COLLAGEN ,Culture Media ,030104 developmental biology ,intervertebral disc ,Neurology (clinical) ,Nucleus - Abstract
Study Design. An in vitro study using human degenerated nucleus pulposus cells.Objective. To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production.Summary of Background Data. Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration.Methods. In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured.Results. Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points.Conclusion. Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system.
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- 2018
35. Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing
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Jean-Jacques Vasseur, Françoise Debart, Christelle Dupouy, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
stimuli-responsive nucleic acids ,PROOLIGONUCLEOTIDE APPROACH ,oligonucleotide prodrugs ,Stimuli responsive ,Review ,SOLID-PHASE SYNTHESIS ,ACTIVATED RNA INTERFERENCE ,[CHIM.THER]Chemical Sciences/Medicinal Chemistry ,light-responsive group ,Computational biology ,Stimulus (physiology) ,010402 general chemistry ,01 natural sciences ,lcsh:QD241-441 ,PROTECTING GROUPS ,Solid-phase synthesis ,lcsh:Organic chemistry ,MAMMALIAN-CELLS ,[CHIM.ANAL]Chemical Sciences/Analytical chemistry ,In vivo ,Gene expression ,[CHIM]Chemical Sciences ,Gene silencing ,lcsh:Science ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,010405 organic chemistry ,Chemistry ,Oligonucleotide ,Organic Chemistry ,CELLULAR UPTAKE PROPERTIES ,DNA OLIGONUCLEOTIDES ,REDUCING ENVIRONMENT ,Prodrug ,0104 chemical sciences ,enzymolabile group ,CAGED SIRNAS ,thermolytic prodrugs ,FLIGHT MASS-SPECTROMETRY ,lcsh:Q ,reduction-responsive - Abstract
Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought.This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules.
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- 2018
36. Visualization of human karyopherin beta-1/importin beta-1 interactions with protein partners in mitotic cells by co-immunoprecipitation and proximity ligation assays
- Author
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Laura Di Francesco, Annalisa Verrico, Patrizia Lavia, Giulia Guarguaglini, Maria Eugenia Schininà, Italia Anna Asteriti, Paola Rovella, and Pietro Cirigliano
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0301 basic medicine ,import receptor kpn-beta-1 ,chromosome alignment ,nuclear transporter ,mammalian-cells ,rangtp gradient ,messenger-rna ,spindle poles ,up-regulation ,mitosis ,cancer ,Interactome ,Mitosis ,lcsh:Medicine ,Importin ,Proximity ligation assay ,Biology ,Polymerase Chain Reaction ,environment and public health ,Article ,03 medical and health sciences ,Nuclear transport receptors ,Image Processing, Computer-Assisted ,Humans ,Immunoprecipitation ,Protein interaction networks ,Protein Interaction Domains and Motifs ,lcsh:Science ,Karyopherin ,chemistry.chemical_classification ,Multidisciplinary ,lcsh:R ,Cell cycle ,beta Karyopherins ,Cell biology ,030104 developmental biology ,chemistry ,Ran ,embryonic structures ,Biological Assay ,Beta Karyopherins ,lcsh:Q ,Nuclear transport ,HeLa Cells - Abstract
Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected (e.g., RAN and SUMO pathway factors) and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized in situ in intact mitotic cells. Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.
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- 2018
37. Surface Disinfections: Present and Future
- Author
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Maurizio Bossù, Gabriele Di Carlo, Daniela Uccelletti, Erika Bruni, Matteo Saccucci, Antonella Polimeni, Maria Sabrina Sarto, and Agnese Bregnocchi
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multidrug-resistant bacteria ,near-infrared irradiation ,walled carbon nanotubes ,drinking-water sources ,graphene oxide ,antibacterial activity ,clostridium-difficile ,staphylococcus-aureus ,mammalian-cells ,zno-nanorods ,Materials science ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Antibiotic resistance ,lcsh:Technology (General) ,General Materials Science ,Health related ,021001 nanoscience & nanotechnology ,Antimicrobial ,0104 chemical sciences ,Risk analysis (engineering) ,lcsh:T1-995 ,0210 nano-technology ,Countermeasure (computer) - Abstract
The propagation of antibiotic resistance increases the chances of major infections for patients during hospitalization and the spread of health related diseases. Therefore finding new and effective solutions to prevent the proliferation of pathogenic microorganisms is critical, in order to protect hospital environment, such as the surfaces of biomedical devices. Modern nanotechnology has proven to be an effective countermeasure to tackle the threat of infections. On this note, recent scientific breakthroughs have demonstrated that antimicrobial nanomaterials are effective in preventing pathogens from developing resistance. Despite the ability to destroy a great deal of bacteria and control the outbreak of infections, nanomaterials present many other advantages. Moreover, it is unlikely for nanomaterials to develop resistance due to their multiple and simultaneous bactericidal mechanisms. In recent years, science has explored more complex antimicrobial coatings and nanomaterials based on graphene have shown great potential in antibacterial treatment. The purpose of this article is to deepen the discussion on the threat of infections related to surface disinfection and to assess the state of the art and potential solutions, with specific focus on disinfection procedures using nanomaterials.
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- 2018
38. Shielding and activation of a viral membrane fusion protein
- Author
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Halldorsson, Steinar, Li, Sai, Li, Mengqiu, Harlos, Karl, Bowden, Thomas A., Huiskonen, Juha T., Helsinki Institute of Life Science HiLIFE, Laboratory of Structural Biology, and Molecular and Integrative Biosciences Research Programme
- Subjects
Protein Folding ,Science ,viruses ,SEMLIKI-FOREST-VIRUS ,CRYO-EM ,Crystallography, X-Ray ,Article ,MAMMALIAN-CELLS ,Humans ,lcsh:Science ,ELECTRON-MICROSCOPY ,Cryoelectron Microscopy ,UUKUNIEMI PHLEBOVIRUS ,Virion ,ENVELOPE PROTEINS ,Virus Internalization ,Rift Valley fever virus ,HEK293 Cells ,X-RAY CRYSTALLOGRAPHY ,MOLECULAR-DYNAMICS ,Multiprotein Complexes ,1182 Biochemistry, cell and molecular biology ,lcsh:Q ,VALLEY FEVER VIRUS ,Hydrophobic and Hydrophilic Interactions ,Viral Fusion Proteins - Abstract
Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families., Viral fusion proteins undergo extensive conformational changes during entry but intermediate conformations often remain unknown. Here, the authors show how Gn of Rift Valley fever virus fusion protein shields hydrophobic fusion loops of Gc and how these loops embed in the target membrane at acidic conditions.
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- 2018
39. eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs
- Author
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Elvis Ndah, Gerben Menschaert, Daria Fijalkowska, Veronique Jonckheere, Steven Verbruggen, and Petra Van Damme
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0301 basic medicine ,Five prime untranslated region ,START CODON SELECTION ,OPEN READING FRAMES ,ISOFORM EXPRESSION ,Codon, Initiator ,Gene Expression ,Nerve Tissue Proteins ,Leaky scanning ,Biology ,UPSTREAM ORFS ,Cell Line ,Open Reading Frames ,03 medical and health sciences ,Eukaryotic translation ,Stress, Physiological ,MAMMALIAN-CELLS ,Eukaryotic initiation factor ,Upstream open reading frame ,Genetics ,Humans ,Initiation factor ,RNA, Messenger ,Eukaryotic Initiation Factors ,Peptide Chain Initiation, Translational ,EUKARYOTIC RIBOSOMES ,Biology and Life Sciences ,QUANTIFICATION ,HCT116 Cells ,Eukaryotic translation initiation factor 4 gamma ,Neoplasm Proteins ,Cell biology ,EIF1 ,030104 developmental biology ,MESSENGER-RNA TRANSLATION ,Gene Knockdown Techniques ,DISCOVERY ,Nucleic Acid Conformation ,RNA ,DEEP PROTEOME COVERAGE ,Energy Metabolism ,Ribosomes - Abstract
Alternative translation initiation mechanisms such as leaky scanning and reinitiation potentiate the polycistronic nature of human transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4000 proteins and 10 000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. Here, the stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with suboptimal start. Interestingly, genes affected by eIF1 deprivation were implicated in energy production and sensing of metabolic stress.
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- 2017
40. Pathways of Unconventional Protein Secretion
- Author
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Catherine Rabouille and Hubrecht Institute for Developmental Biology and Stem Cell Research
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0301 basic medicine ,Signal peptide ,GROWTH-FACTOR 2 ,translocation ,HEPARAN-SULFATE PROTEOGLYCANS ,Review ,Biology ,Golgi bypass ,MECHANISMS ,FIBROBLAST-GROWTH-FACTOR-2 ,03 medical and health sciences ,symbols.namesake ,stress ,MAMMALIAN-CELLS ,GRASP ,Journal Article ,Animals ,Humans ,Secretion ,TRAFFICKING ,HIV-1 TAT ,Secretory pathway ,Unconventional protein secretion ,Secretory Pathway ,Endoplasmic reticulum ,Cell Membrane ,Proteins ,STIM1 ,Cell Biology ,Golgi apparatus ,Endoplasmic Reticulum Stress ,Cell biology ,membrane-bound organelle ,Protein Transport ,DROSOPHILA ,030104 developmental biology ,Secretory protein ,unconventional protein secretion ,symbols ,AUTOPHAGY ,pore - Abstract
Secretory proteins are conventionally transported through the endoplasmic reticulum to the Golgi and then to the plasma membrane where they are released into the extracellular space. However, numerous substrates also reach these destinations using unconventional pathways. Unconventional protein secretion (UPS) is complex and comprises cargos without a signal peptide or a transmembrane domain that can translocate across the plasma membrane, and cargos that reach the plasma membrane by bypassing the Golgi despite entering the endoplasmic reticulum (ER). With a few exceptions, unconventional secretion is largely triggered by stress. Here I review new results and concepts that are beginning to define these pathways.
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- 2017
41. Pathways of Unconventional Protein Secretion
- Subjects
GROWTH-FACTOR 2 ,DROSOPHILA ,MAMMALIAN-CELLS ,GRASP ,HEPARAN-SULFATE PROTEOGLYCANS ,AUTOPHAGY ,TRAFFICKING ,HIV-1 TAT ,MECHANISMS ,FIBROBLAST-GROWTH-FACTOR-2 - Abstract
Secretory proteins are conventionally transported through the endoplasmic reticulum to the Golgi and then to the plasma membrane where they are released into the extracellular space. However, numerous substrates also reach these destinations using unconventional pathways. Unconventional protein secretion (UPS) is complex and comprises cargos without a signal peptide or a transmembrane domain that can translocate across the plasma membrane, and cargos that reach the plasma membrane by bypassing the Golgi despite entering the endoplasmic reticulum (ER). With a few exceptions, unconventional secretion is largely triggered by stress. Here I review new results and concepts that are beginning to define these pathways.
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- 2017
42. Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells
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Bernard L. Schneider, Daniel Blessing, Maria Rey, Catherine Pythoud, Vivianne Padrun, Nicole Déglon, Gabriel Vachey, and Florian M. Wurm
- Subjects
0301 basic medicine ,lcsh:QH426-470 ,viral vectors ,purification ,Genetic enhancement ,viruses ,mammalian-cells ,serotype 8 ,high-titer ,adeno-associated virus ,medicine.disease_cause ,Virus ,Article ,03 medical and health sciences ,Transduction (genetics) ,gene-transfer ,0302 clinical medicine ,expression ,Genetics ,medicine ,orbitally shaken bioreactors ,lcsh:QH573-671 ,Molecular Biology ,Adeno-associated virus ,CNS transduction ,Chemistry ,lcsh:Cytology ,HEK 293 cells ,Transfection ,serum-free production ,suspension HEK293 cells ,Cell biology ,AAV8 ,AAV9 ,immune affinity chromatography ,transient transfection ,lcsh:Genetics ,030104 developmental biology ,recombinant-adenoassociated-virus ,Cell culture ,030220 oncology & carcinogenesis ,Molecular Medicine ,Nuclear localization sequence - Abstract
Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells. Keywords: adeno-associated virus, AAV8, AAV9, suspension HEK293 cells, transient transfection, orbitally shaken bioreactors, immune affinity chromatography, CNS transduction
- Published
- 2019
43. Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives
- Author
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Jewel Ann Joseph, Simen Akkermans, Philippe Nimmegeers, and Jan F. M. Van Impe
- Subjects
Microbiology (medical) ,FED-BATCH ,Systems biology ,lcsh:QR1-502 ,AMINO-ACID-SEQUENCE ,Review ,Sugar consumption ,Biology ,recombinant proteins ,Microbiology ,lcsh:Microbiology ,law.invention ,Pichia pastoris ,natural sweetener ,03 medical and health sciences ,ANION-EXCHANGE CHROMATOGRAPHY ,MAMMALIAN-CELLS ,law ,ALCOHOL OXIDASE ,030304 developmental biology ,0303 health sciences ,Science & Technology ,030306 microbiology ,HIGH-LEVEL SECRETION ,food and beverages ,systems biology ,biology.organism_classification ,SCALE METABOLIC MODEL ,Bioproduction ,Artificial Sweetener ,Yeast ,Chemistry ,PICHIA-PASTORIS ,Biochemistry ,Thaumatin ,thaumatin ,sweet protein ,Recombinant DNA ,METHYLOTROPHIC YEAST ,Life Sciences & Biomedicine ,Engineering sciences. Technology ,TASTE-MODIFYING PROTEIN - Abstract
There is currently a worldwide trend to reduce sugar consumption. This trend is mostly met by the use of artificial non-nutritive sweeteners. However, these sweeteners have also been proven to have adverse health effects such as dizziness, headaches, gastrointestinal issues, and mood changes for aspartame. One of the solutions lies in the commercialization of sweet proteins, which are not associated with adverse health effects. Of these proteins, thaumatin is one of the most studied and most promising alternatives for sugars and artificial sweeteners. Since the natural production of these proteins is often too expensive, biochemical production methods are currently under investigation. With these methods, recombinant DNA technology is used for the production of sweet proteins in a host organism. The most promising host known today is the methylotrophic yeast, Pichia pastoris. This yeast has a tightly regulated methanol-induced promotor, allowing a good control over the recombinant protein production. Great efforts have been undertaken for improving the yields and purities of thaumatin productions, but a further optimization is still desired. This review focuses on (i) the motivation for using and producing sweet proteins, (ii) the properties and history of thaumatin, (iii) the production of recombinant sweet proteins, and (iv) future possibilities for process optimization based on a systems biology approach. ispartof: FRONTIERS IN MICROBIOLOGY vol:10 ispartof: location:Switzerland status: published
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- 2019
44. Single-neuron level one-photon voltage imaging with sparsely targeted genetically encoded voltage indicators
- Author
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Quicke, Peter, Song, Chenchen, McKimm, Eric J., Milosevic, Milena M., Howe, Carmel L., Neil, Mark, Schultz, Simon R., Antic, Srdjan D., Foust, Amanda J., Knöpfel, Thomas, Biotechnology and Biological Sciences Research Council (BBSRC), Engineering & Physical Science Research Council (E, Royal Academy Of Engineering, National Institutes of Health, Wellcome Trust, and The Royal Society
- Subjects
Science & Technology ,CIRCUIT DYNAMICS ,Neurosciences ,FLUORESCENT ,MICE ,MAMMALIAN-CELLS ,sparse expression ,EXCITATION ,cerebral cortex ,Neurosciences & Neurology ,optogenetics ,Life Sciences & Biomedicine ,Neuroscience ,Original Research ,voltage imaging ,transgenic ,GENE-EXPRESSION - Abstract
Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.
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- 2019
45. High-throughput multiplexed fluorescence-activated droplet sorting
- Author
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M. S. Suryateja Jammalamadaka, Tobias M. Schneider, Jeremy Vrignon, Philippe Nizard, Jean-Christophe Baret, Ouriel Caen, Valérie Taly, Simon Schütz, Médecine Personnalisée, Pharmacogénomique, Optimisation Thérapeutique (MEPPOT - U1147), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Emergent Complexity in Physical Systems Laboratory [Lausanne, Switzerland] (ECPS), Ecole Polytechnique Fédérale de Lausanne (EPFL), Centre de Recherche Paul Pascal (CRPP), Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Ministère de l’Enseignement Supérieur et de la Recherche, the Université Paris-Descartes, the Centre National de la Recherche Scientifique (CNRS) and the Institut National de la Santé et de la Recherche Médicale (INSERM). V. Taly acknowledges the financial support of Agence Nationale de la Recherche (ANR Chipset, no. ANR-15-CE11-0008-04), ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé, National Alliance for Life Sciences and Health) within the framework of the Cancer Plan, the ligue nationale contre le cancer (LNCC, Program 'Equipe labelisée LIGUE' no. EL2016.LNCC/VaT) and HTE Program-HetColi network, INSERM Physicancer program (no. PC201423) and the SIRIC CARPEM for financial support. O. Caen thanks ITMO National Cancer Alliance for Life Sciences and Health (AVIESAN, INSERM, no.HAP_2012001) and the Association pour la Recherche sur le Cancer for fellowships within the Frontiers in Life Science Ph.D. program (FdV).J.-C.B. acknowledges the support of the Institut Universitaire de France, of the ERC (FP7/2007-2013/ERC Starting Grant 306385-SofI), of the French state in the frame of the 'Investments for the future', Programme IdEx Bordeaux, ANR-10-IDEX-03-02 and of the 'Region Aquitaine'. We would like to thank RainDance for kindly providing us the EA-surfactant., ANR-15-CE11-0008,CHIPSeT,Caracterisation Structurale des Couplages entre Chromatine et Homeostasie Protéique par Combinaison d'Analyses de Coevolution et de Perturbation des Interfaces de Complexes a Haut-Debit(2015), ANR-10-IDEX-0003,IDEX BORDEAUX,Initiative d'excellence de l'Université de Bordeaux(2010), European Project: 306385,EC:FP7:ERC,ERC-2012-StG_20111012,SOFI(2013), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Baret, Jean-Christophe, Caracterisation Structurale des Couplages entre Chromatine et Homeostasie Protéique par Combinaison d'Analyses de Coevolution et de Perturbation des Interfaces de Complexes a Haut-Debit - - CHIPSeT2015 - ANR-15-CE11-0008 - AAPG2015 - VALID, Initiative d'excellence de l'Université de Bordeaux - - IDEX BORDEAUX2010 - ANR-10-IDEX-0003 - IDEX - VALID, and SOFt Interfaces: control of interfacial layers for biotechnological applications - SOFI - - EC:FP7:ERC2013-01-01 - 2017-12-31 - 306385 - VALID
- Subjects
0301 basic medicine ,protein-signaling networks ,Computer science ,[SDV]Life Sciences [q-bio] ,Materials Science (miscellaneous) ,mammalian-cells ,Microfluidics ,microfluidics ,selection ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,breakup ,lcsh:Technology ,Multiplexing ,Industrial and Manufacturing Engineering ,directed enzyme evolution ,03 medical and health sciences ,Microsystem ,sort ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Electrical and Electronic Engineering ,[PHYS.COND]Physics [physics]/Condensed Matter [cond-mat] ,Throughput (business) ,lcsh:T ,Sorting ,single-cell ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,sorter ,030104 developmental biology ,lcsh:TA1-2040 ,manipulation ,flow-cytometry ,lcsh:Engineering (General). Civil engineering (General) ,Biological system ,[PHYS.COND] Physics [physics]/Condensed Matter [cond-mat] - Abstract
Bioanalysis: microfluidic-sorters to make better choices Droplet-based microfluidics provides new technologies for cell screening by steering cell-containing microdroplets of interest into different channels at the kilohertz frequency. Until now, in conventional microfluidic sorting, droplets are sorted using electric pulses to move them into one of the two channels. Jean–Christophe Baret from the CNRS and University of Bordeaux in France, Valérie Taly from the CNRS and University of Paris Descartes and Tobias Schneider from EPFL in Switzerland have now boosted dielectrophoretic droplet manipulation to allow for multiple selections in a single device. The teams show how emulsified dye-loaded microdroplets are sorted in five different channels depending on fluorescence intensity: a computer algorithm controls the application of electric pulses to direct the droplets into one of the five channels depending on the fluorescence signals. Numerical models unravel the effect of the dielectrophoretic actuation on the droplet trajectories and are now used to predict the sorting conditions for droplet volumes compatible with single-cell encapsulation.
- Published
- 2018
46. Multicompartmentalized Microreactors Containing Nuclei and Catalase-Loaded Liposomes
- Author
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Brigitte Städler, Rona Chandrawati, Fabian Itel, Chuntao Zhu, and Xiaojun Han
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Polymers and Plastics ,02 engineering and technology ,01 natural sciences ,Oxygen ,chemistry.chemical_compound ,Mice ,Bioreactors ,Tissue engineering ,MAMMALIAN-CELLS ,ARTIFICIAL CELLS ,Materials Chemistry ,Protein biosynthesis ,ENCAPSULATION ,LIVING CELLS ,Hydrogen peroxide ,Cells, Cultured ,Liposome ,biology ,Hydrogels ,Hep G2 Cells ,021001 nanoscience & nanotechnology ,Catalase ,ALPHA-AMANITIN ,MESSENGER-RNA TRANSLATION ,0210 nano-technology ,Cell Survival ,chemistry.chemical_element ,Bioengineering ,010402 general chemistry ,Biomaterials ,HYDROGEN-PEROXIDE ,ALGINATE BEADS ,Bioreactor ,DRUG CARRIERS ,Animals ,Humans ,RNA, Messenger ,Cell Nucleus ,Tissue Engineering ,Macrophages ,Hydrogen Peroxide ,DNA ,0104 chemical sciences ,chemistry ,Liposomes ,Biophysics ,biology.protein ,Microreactor - Abstract
Multicompartmentalized microreactors are considered as cell mimics with hierarchical structures inspired by mammalian cells. We report the successful assembly and encapsulation of purified nuclei from RAW 264.7 cells (pNuc) into alginate-based micro reactors. We demonstrate the preserved function of nuclei within the microreactors for mRNA production. Further, we load catalase-loaded liposomes (Lcat) into the microreactors to break down hydrogen peroxide (H2O2) into oxygen and water. Assemblies containing both natural pNuc and synthetic Lcat show significantly higher mRNA production in the presence of H2O2 compared to microreactors without Lcat or no H2O2 present, suggesting a beneficial effect of the locally enzymatically produced oxygen for transcription. This novel type of microreactors, containing both natural and synthetic compartments, presents a substantial advancement from assemblies equipped with solely synthetic units and offers opportunities as hypoxia models or for cell-free protein synthesis.
- Published
- 2018
47. Listeria monocytogenes: cell biology of invasion and intracellular growth
- Author
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Javier Pizarro-Cerdá, Pascale Cossart, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), 1.Institut Pasteur 2.Institut Pasteur ‘Transversal Research Program’ - PTR5213.National Research Agency (ANR) - grant ANR-15-CE15-0017 ‘StopBugEntry’4.Institut National de la Santé et de la Recherche Médicale - Unité 6045.Institut National de la Recherche Agronomique - Unité Sous Contrat 20206.Fondation Balzan7.Fondation le Roch les Mousquetaires8.Howard Hughes Medical Institute9.Human Frontier Science Program Organization - grant RGP011/201310.European Research Council Advanced - Grant BacCellEpi 67082311.French Government's ‘Investissement d’Avenir’ program, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ - Grant ANR-10-LABX-62-IBEID12.ERANET Infect-ERA - Grant ANR-13-IFEC-0004-02 PROANTILIS, ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-13-IFEC-0004,PROANTILIS,Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes(2013), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Prodinra ZT, Migration, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, ERA-NET Infect-ERA - Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes - - PROANTILIS2013 - ANR-13-IFEC-0004 - IFEC - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, and Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID
- Subjects
0301 basic medicine ,Microbiology (medical) ,rich repeat region ,dependent internalization ,Physiology ,media_common.quotation_subject ,[SDV]Life Sciences [q-bio] ,030106 microbiology ,mammalian-cells ,Vacuole ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Biology ,medicine.disease_cause ,virulence factor ,03 medical and health sciences ,Listeria monocytogenes ,Genetics ,medicine ,Internalization ,Cytoskeleton ,[SDV.BC] Life Sciences [q-bio]/Cellular Biology ,media_common ,030304 developmental biology ,surface protein ,0303 health sciences ,protein-kinase-c ,Innate immune system ,General Immunology and Microbiology ,Ecology ,030306 microbiology ,Listeriolysin O ,Cell Biology ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Cell biology ,[SDV] Life Sciences [q-bio] ,Infectious Diseases ,Cytoplasm ,eukaryotic cells ,range phospholipase-c ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,mediated internalization ,Intracellular ,e-cadherin - Abstract
The Gram-positive pathogen Listeria monocytogenes is able to promote its entry into a diverse range of mammalian host cells by triggering plasma membrane remodeling, leading to bacterial engulfment. Upon cell invasion, L. monocytogenes disrupts its internalization vacuole and translocates to the cytoplasm, where bacterial replication takes place. Subsequently, L. monocytogenes uses an actin-based motility system that allows bacterial cytoplasmic movement and cell-to-cell spread. L. monocytogenes therefore subverts host cell receptors, organelles and the cytoskeleton at different infection steps, manipulating diverse cellular functions that include ion transport, membrane trafficking, post-translational modifications, phosphoinositide production, innate immune responses as well as gene expression and DNA stability.
- Published
- 2018
48. OSBP-related protein-2 (ORP2) : a novel Akt effector that controls cellular energy metabolism
- Author
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Henriikka Kentala, Robert Andrews, Zoltán Pataj, Vesa M. Olkkonen, Eija Jokitalo, Juho Pirhonen, Silke Matysik, Leena Karhinen, Eeva Jääskeläinen, Leena Meriläinen, Shiqian Li, Annika Koponen, Helena Vihinen, You Zhou, Elina Ikonen, Annukka M. Kivelä, Gerhard Liebisch, Institute of Biotechnology, Electron Microscopy, University of Helsinki, Medicum, Department of Anatomy, Faculty of Medicine, Research Services, and Lipid Trafficking Lab
- Subjects
0301 basic medicine ,Receptors, Steroid ,OSBP-related protein ,Chaperonins ,Cell Cycle Proteins ,STEROL ,Gene Knockout Techniques ,GSK-3 ,Cell Movement ,MAMMALIAN-CELLS ,Lipid droplet ,Akt signaling ,CHOLESTEROL-METABOLISM ,biology ,Chemistry ,Hsp90 ,Cell biology ,Actin Cytoskeleton ,OSBPL2 ,Molecular Medicine ,LIPID DROPLETS ,CRISPR-Cas9 ,Glycolysis ,Signal Transduction ,Triacylglycerol ,Cell Line ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Humans ,HSP90 Heat-Shock Proteins ,RNA, Messenger ,Glycogen synthase ,Molecular Biology ,OSBP ,Protein kinase B ,Cell Proliferation ,Pharmacology ,Organelles ,030102 biochemistry & molecular biology ,Endoplasmic reticulum ,GLUCOSE-UPTAKE ,Lipid metabolism ,Biological Transport ,TRIACYLGLYCEROL SYNTHESIS ,Cell Biology ,PHOSPHATIDYLSERINE TRANSPORT ,Lipid Metabolism ,030104 developmental biology ,ER ,PLASMA-MEMBRANE ,biology.protein ,1182 Biochemistry, cell and molecular biology ,3111 Biomedicine ,Energy Metabolism ,Proto-Oncogene Proteins c-akt ,OXYSTEROL-BINDING PROTEINS - Abstract
ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.
- Published
- 2018
49. An Inside Job: Applications of Intracellular Single Domain Antibodies
- Author
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Eline Soetens, Marlies Ballegeer, and Xavier Saelens
- Subjects
NANOBODIES ,0301 basic medicine ,single domain antibody ,MONOCLONAL-ANTIBODY ,medicine.drug_class ,research tool ,lcsh:QR1-502 ,Intracellular Space ,PROTEIN ,Review ,Computational biology ,Monoclonal antibody ,Biochemistry ,lcsh:Microbiology ,Intrabody ,Affinity maturation ,DELIVERY ,03 medical and health sciences ,Drug Delivery Systems ,AFFINITY MATURATION ,0302 clinical medicine ,MAMMALIAN-CELLS ,medicine ,Animals ,Humans ,CELL-PENETRATING PEPTIDES ,Molecular Biology ,therapy ,OCULOPHARYNGEAL MUSCULAR-DYSTROPHY ,biology ,Chemistry ,Biology and Life Sciences ,IN-VITRO ,Single-Domain Antibodies ,In vitro ,intrabody ,030104 developmental biology ,Single-domain antibody ,HCV REPLICATION ,Cytoplasm ,030220 oncology & carcinogenesis ,biology.protein ,Antibody ,delivery methods ,Intracellular - Abstract
Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.
- Published
- 2020
50. An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication
- Author
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Fulvio Reggiori, Jennifer Jung, Sharon A. Tooze, Søren R. Paludan, Mario Mauthe, Bjoern Stork, Cornelis A. M. de Haan, Martijn A. Langereis, Dave Egan, Frank J. M. van Kuppeveld, Alex Jones, Wienand A. Omta, Tero Ahola, Michal Mokry, Xingdong Zhou, Christian Behrends, Microbes in Health and Disease (MHD), Center for Liver, Digestive and Metabolic Diseases (CLDM), dI&I I&I-1, LS Virologie, and Sub Software Production
- Subjects
0301 basic medicine ,Proteome ,Picornavirus ,SELF-DIGESTION ,Proteome/metabolism ,SEMLIKI-FOREST-VIRUS ,Autophagy-Related Proteins ,Virus Replication ,RNA, Small Interfering/metabolism ,Mice ,MAMMALIAN-CELLS ,RNA, Small Interfering ,EUKARYOTIC MESSENGER-RNA ,Research Articles ,IN-VIVO ,biology ,MOUSE MODEL ,Protein-Tyrosine Kinases ,Autophagy-related protein 13 ,VACCINIA VIRUS ,Cell biology ,Interferon Type I/metabolism ,Gene Knockdown Techniques ,Interferon Type I ,Viruses ,endocrine system ,VIRAL REPLICATION ,ENDOPLASMIC-RETICULUM ,Tools ,03 medical and health sciences ,Autophagy ,Journal Article ,Animals ,Humans ,Gene ,Viruses/metabolism ,Sequence Analysis, RNA ,HEK 293 cells ,COXSACKIEVIRUS INFECTION ,Autophagy-Related Proteins/metabolism ,RNA ,Cell Biology ,Virus Internalization ,biology.organism_classification ,Protein-Tyrosine Kinases/metabolism ,030104 developmental biology ,HEK293 Cells ,Viral replication ,HeLa Cells - Abstract
Autophagy-related (ATG) proteins regulate autophagy, but recent work indicates that some also have autophagy-independent roles. Here, Mauthe et al. perform an unbiased siRNA screen to examine the effects of ATG protein depletion on viral replication and demonstrate autophagy-independent functions for ATG13 and FIP200 in the picornaviral life cycle., Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy.
- Published
- 2016
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