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Darren E. Higgins, Ph.D.


Temporal Requirement of virulence Determinants During Intracellular Growth of Listeria Monocytogenes
Summer, 06/07/00 - 08/07/00

The research activities and funding listed below are automatically derived from NIH ExPORTER and other sources, which might result in incorrect or missing items. Faculty can login to make corrections and additions.
  1. R01AI146102 (HIGGINS, DARREN E) Jul 26, 2019 - Jun 30, 2023
    Mechanisms regulating vimentin-dependent invasion of the brain by Listeria monocytogenes
    Role: Principal Investigator
  2. R21AI103806 (HIGGINS, DARREN E) Apr 15, 2014 - Sep 30, 2016
    Listeria Monocytogenes Infection of the Brain
    Role: Principal Investigator
  3. U01AI056446 (HIGGINS, DARREN E) Aug 1, 2003 - Jan 31, 2009
    Novel Vaccine Strategy for Listeria monocytogenes
    Role: Principal Investigator
  4. R01AI053669 (HIGGINS, DARREN E) Dec 1, 2002 - May 31, 2013
    Temporal Requirements for Intracellular Pathogenesis
    Role: Principal Investigator

Publications listed below are automatically derived from MEDLINE/PubMed and other sources, which might result in incorrect or missing publications. Faculty can login to make corrections and additions.
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PMC Citations indicate the number of times the publication was cited by articles in PubMed Central, and the Altmetric score represents citations in news articles and social media. (Note that publications are often cited in additional ways that are not shown here.) Fields are based on how the National Library of Medicine (NLM) classifies the publication's journal and might not represent the specific topic of the publication. Translation tags are based on the publication type and the MeSH terms NLM assigns to the publication. Some publications (especially newer ones and publications not in PubMed) might not yet be assigned Field or Translation tags.) Click a Field or Translation tag to filter the publications.
  1. Tan JMJ, Garner ME, Regeimbal JM, Greene CJ, Márquez JDR, Ammendolia DA, McCluggage ARR, Li T, Wu KJ, Cemma M, Ostrowski PP, Raught B, Diamond MS, Grinstein S, Yates RM, Higgins DE, Brumell JH. Listeria exploits IFITM3 to suppress antibacterial activity in phagocytes. Nat Commun. 2021 08 17; 12(1):4999. PMID: 34404769.
    Citations: 1     Fields:    Translation:AnimalsCells
  2. Laborde RJ, Ishimura ME, Abreu-Butin L, Nogueira CV, Grubaugh D, Cruz-Leal Y, Luzardo MC, Fernández A, Mesa C, Pazos F, Álvarez C, Alonso ME, Starnbach MN, Higgins DE, Fernández LE, Longo-Maugéri IM, Lanio ME. Sticholysins, pore-forming proteins from a marine anemone can induce maturation of dendritic cells through a TLR4 dependent-pathway. Mol Immunol. 2021 03; 131:144-154. PMID: 33422341.
    Citations: 1     Fields:    Translation:AnimalsCells
  3. Rojas Márquez JD, Li T, McCluggage ARR, Tan JMJ, Muise A, Higgins DE, Brumell JH. Cutting Edge: NOX2 NADPH Oxidase Controls Infection by an Intracellular Bacterial Pathogen through Limiting the Type 1 IFN Response. J Immunol. 2021 01 15; 206(2):323-328. PMID: 33288542.
    Citations: 2     Fields:    Translation:AnimalsCells
  4. Ghosh P, Zhou Y, Richardson Q, Higgins DE. Characterization of the pathogenesis and immune response to Listeria monocytogenes strains isolated from a sustained national outbreak. Sci Rep. 2019 12 20; 9(1):19587. PMID: 31862952.
    Citations: 2     Fields:    Translation:HumansAnimalsCellsPHPublic Health
  5. Tan JMJ, Mellouk N, Osborne SE, Ammendolia DA, Dyer DN, Li R, Brunen D, van Rijn JM, Huang J, Czuczman MA, Cemma MA, Won AM, Yip CM, Xavier RJ, MacDuff DA, Reggiori F, Debnath J, Yoshimori T, Kim PK, Fairn GD, Coyaud E, Raught B, Muise AM, Higgins DE, Brumell JH. An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread. Nat Microbiol. 2018 12; 3(12):1472-1485. PMID: 30478389.
    Citations: 24     Fields:    Translation:HumansAnimalsCells
  6. Cruz-Leal Y, Grubaugh D, Nogueira CV, Lopetegui-González I, Del Valle A, Escalona F, Laborde RJ, Alvarez C, Fernández LE, Starnbach MN, Higgins DE, Lanio ME. The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes. Front Immunol. 2018; 9:2473. PMID: 30455685.
    Citations: 4     Fields:    Translation:AnimalsCells
  7. Ghosh P, Halvorsen EM, Ammendolia DA, Mor-Vaknin N, O'Riordan MXD, Brumell JH, Markovitz DM, Higgins DE. Invasion of the Brain by Listeria monocytogenes Is Mediated by InlF and Host Cell Vimentin. mBio. 2018 02 27; 9(1). PMID: 29487235.
    Citations: 28     Fields:    Translation:AnimalsCells
  8. Grubaugh D, Regeimbal JM, Ghosh P, Zhou Y, Lauer P, Dubensky TW, Higgins DE. The VirAB ABC Transporter Is Required for VirR Regulation of Listeria monocytogenes Virulence and Resistance to Nisin. Infect Immun. 2018 03; 86(3). PMID: 29263107.
    Citations: 17     Fields:    Translation:HumansAnimalsCells
  9. Zhang T, Abel S, Abel Zur Wiesch P, Sasabe J, Davis BM, Higgins DE, Waldor MK. Deciphering the landscape of host barriers to Listeria monocytogenes infection. Proc Natl Acad Sci U S A. 2017 06 13; 114(24):6334-6339. PMID: 28559314.
    Citations: 27     Fields:    Translation:AnimalsCells
  10. Laborde RJ, Sanchez-Ferras O, Luzardo MC, Cruz-Leal Y, Fernández A, Mesa C, Oliver L, Canet L, Abreu-Butin L, Nogueira CV, Tejuca M, Pazos F, Álvarez C, Alonso ME, Longo-Maugéri IM, Starnbach MN, Higgins DE, Fernández LE, Lanio ME. Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response. J Immunol. 2017 04 01; 198(7):2772-2784. PMID: 28258198.
    Citations: 8     Fields:    Translation:AnimalsCells
  11. Osborne SE, Sit B, Shaker A, Currie E, Tan JM, van Rijn J, Higgins DE, Brumell JH. Type I interferon promotes cell-to-cell spread of Listeria monocytogenes. Cell Microbiol. 2017 03; 19(3). PMID: 27582004.
    Citations: 13     Fields:    Translation:AnimalsCells
  12. Picard MD, Bodmer JL, Gierahn TM, Lee A, Price J, Cohane K, Clemens V, DeVault VL, Gurok G, Kohberger R, Higgins DE, Siber GR, Flechtner JB, Geisler WM. Resolution of Chlamydia trachomatis Infection Is Associated with a Distinct T Cell Response Profile. Clin Vaccine Immunol. 2015 Nov; 22(11):1206-18. PMID: 26446421.
    Citations: 10     Fields:    Translation:HumansCells
  13. Cemma M, Lam GY, Stöckli M, Higgins DE, Brumell JH. Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells. PLoS One. 2015; 10(5):e0125856. PMID: 25970638.
    Citations: 7     Fields:    Translation:AnimalsCells
  14. Alonso AN, Perry KJ, Regeimbal JM, Regan PM, Higgins DE. Identification of Listeria monocytogenes determinants required for biofilm formation. PLoS One. 2014; 9(12):e113696. PMID: 25517120.
    Citations: 18     Fields:    Translation:Cells
  15. Fattouh R, Kwon H, Czuczman MA, Copeland JW, Pelletier L, Quinlan ME, Muise AM, Higgins DE, Brumell JH. The diaphanous-related formins promote protrusion formation and cell-to-cell spread of Listeria monocytogenes. J Infect Dis. 2015 Apr 01; 211(7):1185-95. PMID: 25281757.
    Citations: 19     Fields:    Translation:HumansCells
  16. Czuczman MA, Fattouh R, van Rijn JM, Canadien V, Osborne S, Muise AM, Kuchroo VK, Higgins DE, Brumell JH. Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread. Nature. 2014 May 08; 509(7499):230-4. PMID: 24739967.
    Citations: 61     Fields:    Translation:HumansAnimalsCells
  17. Grubaugh D, Flechtner JB, Higgins DE. Proteins as T cell antigens: methods for high-throughput identification. Vaccine. 2013 Aug 20; 31(37):3805-10. PMID: 23806245.
    Citations: 14     Fields:    Translation:HumansAnimalsCells
  18. Perry KJ, Higgins DE. A differential fluorescence-based genetic screen identifies Listeria monocytogenes determinants required for intracellular replication. J Bacteriol. 2013 Aug; 195(15):3331-40. PMID: 23687268.
    Citations: 8     Fields:    Translation:AnimalsCells
  19. Lam GY, Cemma M, Muise AM, Higgins DE, Brumell JH. Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection. Autophagy. 2013 Jul; 9(7):985-95. PMID: 23584039.
    Citations: 57     Fields:    Translation:AnimalsCells
  20. Chen L, Chen Z, Baker K, Halvorsen EM, da Cunha AP, Flak MB, Gerber G, Huang YH, Hosomi S, Arthur JC, Dery KJ, Nagaishi T, Beauchemin N, Holmes KV, Ho JW, Shively JE, Jobin C, Onderdonk AB, Bry L, Weiner HL, Higgins DE, Blumberg RS. The short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction. Immunity. 2012 Nov 16; 37(5):930-46. PMID: 23123061.
    Citations: 25     Fields:    Translation:AnimalsCells
  21. Picard MD, Cohane KP, Gierahn TM, Higgins DE, Flechtner JB. High-throughput proteomic screening identifies Chlamydia trachomatis antigens that are capable of eliciting T cell and antibody responses that provide protection against vaginal challenge. Vaccine. 2012 Jun 19; 30(29):4387-93. PMID: 22682294.
    Citations: 14     Fields:    Translation:AnimalsCells
  22. Lam GY, Czuczman MA, Higgins DE, Brumell JH. Interactions of Listeria monocytogenes with the autophagy system of host cells. Adv Immunol. 2012; 113:7-18. PMID: 22244576.
    Citations: 13     Fields:    Translation:HumansAnimalsCells
  23. Lam GY, Fattouh R, Muise AM, Grinstein S, Higgins DE, Brumell JH. Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection. Cell Host Microbe. 2011 Dec 15; 10(6):627-34. PMID: 22177565.
    Citations: 28     Fields:    Translation:HumansAnimalsCells
  24. Kamp HD, Higgins DE. A protein thermometer controls temperature-dependent transcription of flagellar motility genes in Listeria monocytogenes. PLoS Pathog. 2011 Aug; 7(8):e1002153. PMID: 21829361.
    Citations: 32     Fields:    Translation:Cells
  25. Moffitt KL, Gierahn TM, Lu YJ, Gouveia P, Alderson M, Flechtner JB, Higgins DE, Malley R. T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization. Cell Host Microbe. 2011 Feb 17; 9(2):158-65. PMID: 21320698.
    Citations: 91     Fields:    Translation:HumansAnimalsCellsPHPublic Health
  26. Lieberman LA, Higgins DE. Inhibition of Listeria monocytogenes infection by neurological drugs. Int J Antimicrob Agents. 2010 Mar; 35(3):292-6. PMID: 20031379.
    Citations: 11     Fields:    Translation:HumansAnimalsCells
  27. Kamp HD, Higgins DE. Transcriptional and post-transcriptional regulation of the GmaR antirepressor governs temperature-dependent control of flagellar motility in Listeria monocytogenes. Mol Microbiol. 2009 Oct; 74(2):421-35. PMID: 19796338.
    Citations: 19     Fields:    Translation:Cells
  28. Burrack LS, Harper JW, Higgins DE. Perturbation of vacuolar maturation promotes listeriolysin O-independent vacuolar escape during Listeria monocytogenes infection of human cells. Cell Microbiol. 2009 Sep; 11(9):1382-98. PMID: 19500109.
    Citations: 14     Fields:    Translation:HumansCells
  29. Shen A, Higgins DE, Panne D. Recognition of AT-rich DNA binding sites by the MogR repressor. Structure. 2009 May 13; 17(5):769-77. PMID: 19446532.
    Citations: 21     Fields:    Translation:Cells
  30. Lieberman LA, Higgins DE. A small-molecule screen identifies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection. Antimicrob Agents Chemother. 2009 Feb; 53(2):756-64. PMID: 19015342.
    Citations: 23     Fields:    Translation:AnimalsCells
  31. Kaser A, Lee AH, Franke A, Glickman JN, Zeissig S, Tilg H, Nieuwenhuis EE, Higgins DE, Schreiber S, Glimcher LH, Blumberg RS. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell. 2008 Sep 05; 134(5):743-56. PMID: 18775308.
    Citations: 628     Fields:    Translation:HumansAnimalsCells
  32. Kirchner M, Higgins DE. Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes. Mol Microbiol. 2008 May; 68(3):749-67. PMID: 18331468.
    Citations: 18     Fields:    Translation:HumansAnimalsCells
  33. Smithey MJ, Brandt S, Freitag NE, Higgins DE, Bouwer HG. Stimulation of enhanced CD8 T cell responses following immunization with a hyper-antigen secreting intracytosolic bacterial pathogen. J Immunol. 2008 Mar 01; 180(5):3406-16. PMID: 18292567.
    Citations: 5     Fields:    Translation:AnimalsCells
  34. Birmingham CL, Higgins DE, Brumell JH. Avoiding death by autophagy: interactions of Listeria monocytogenes with the macrophage autophagy system. Autophagy. 2008 Apr; 4(3):368-71. PMID: 18216493.
    Citations: 13     Fields:    Translation:AnimalsCells
  35. Birmingham CL, Canadien V, Kaniuk NA, Steinberg BE, Higgins DE, Brumell JH. Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles. Nature. 2008 Jan 17; 451(7176):350-4. PMID: 18202661.
    Citations: 121     Fields:    Translation:AnimalsCells
  36. Birmingham CL, Canadien V, Gouin E, Troy EB, Yoshimori T, Cossart P, Higgins DE, Brumell JH. Listeria monocytogenes evades killing by autophagy during colonization of host cells. Autophagy. 2007 Sep-Oct; 3(5):442-51. PMID: 17568179.
    Citations: 107     Fields:    Translation:AnimalsCells
  37. Lemon KP, Higgins DE, Kolter R. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol. 2007 Jun; 189(12):4418-24. PMID: 17416647.
    Citations: 121     Fields:    Translation:Cells
  38. Alberti-Segui C, Goeden KR, Higgins DE. Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread. Cell Microbiol. 2007 Jan; 9(1):179-95. PMID: 17222191.
    Citations: 42     Fields:    Translation:HumansCells
  39. Burrack LS, Higgins DE. Genomic approaches to understanding bacterial virulence. Curr Opin Microbiol. 2007 Feb; 10(1):4-9. PMID: 17161645.
    Citations: 11     Fields:    Translation:Cells
  40. Shen A, Kamp HD, Gründling A, Higgins DE. A bifunctional O-GlcNAc transferase governs flagellar motility through anti-repression. Genes Dev. 2006 Dec 01; 20(23):3283-95. PMID: 17158746.
    Citations: 51     Fields:    Translation:Cells
  41. Yoshida M, Kobayashi K, Kuo TT, Bry L, Glickman JN, Claypool SM, Kaser A, Nagaishi T, Higgins DE, Mizoguchi E, Wakatsuki Y, Roopenian DC, Mizoguchi A, Lencer WI, Blumberg RS. Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria. J Clin Invest. 2006 Aug; 116(8):2142-2151. PMID: 16841095.
    Citations: 110     Fields:    Translation:Animals
  42. Roan NR, Gierahn TM, Higgins DE, Starnbach MN. Monitoring the T cell response to genital tract infection. Proc Natl Acad Sci U S A. 2006 Aug 08; 103(32):12069-74. PMID: 16880389.
    Citations: 32     Fields:    Translation:AnimalsCells
  43. Shen A, Higgins DE. The MogR transcriptional repressor regulates nonhierarchal expression of flagellar motility genes and virulence in Listeria monocytogenes. PLoS Pathog. 2006 Apr; 2(4):e30. PMID: 16617375.
    Citations: 52     Fields:    Translation:AnimalsCells
  44. Bouwer HG, Alberti-Segui C, Montfort MJ, Berkowitz ND, Higgins DE. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen. Proc Natl Acad Sci U S A. 2006 Mar 28; 103(13):5102-7. PMID: 16549792.
    Citations: 6     Fields:    Translation:AnimalsCells
  45. Henry R, Shaughnessy L, Loessner MJ, Alberti-Segui C, Higgins DE, Swanson JA. Cytolysin-dependent delay of vacuole maturation in macrophages infected with Listeria monocytogenes. Cell Microbiol. 2006 Jan; 8(1):107-19. PMID: 16367870.
    Citations: 59     Fields:    Translation:AnimalsCells
  46. Shen A, Higgins DE. The 5' untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol Microbiol. 2005 Sep; 57(5):1460-73. PMID: 16102013.
    Citations: 55     Fields:    Translation:AnimalsCells
  47. Li Z, Zhao X, Higgins DE, Frankel FR. Conditional lethality yields a new vaccine strain of Listeria monocytogenes for the induction of cell-mediated immunity. Infect Immun. 2005 Aug; 73(8):5065-73. PMID: 16041022.
    Citations: 13     Fields:    Translation:AnimalsCells
  48. Agaisse H, Burrack LS, Philips JA, Rubin EJ, Perrimon N, Higgins DE. Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science. 2005 Aug 19; 309(5738):1248-51. PMID: 16020693.
    Citations: 131     Fields:    Translation:AnimalsCells
  49. Pan H, Yan BS, Rojas M, Shebzukhov YV, Zhou H, Kobzik L, Higgins DE, Daly MJ, Bloom BR, Kramnik I. Ipr1 gene mediates innate immunity to tuberculosis. Nature. 2005 Apr 07; 434(7034):767-72. PMID: 15815631.
    Citations: 199     Fields:    Translation:HumansAnimalsCells
  50. Boyartchuk V, Rojas M, Yan BS, Jobe O, Hurt N, Dorfman DM, Higgins DE, Dietrich WF, Kramnik I. The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice. J Immunol. 2004 Oct 15; 173(8):5112-20. PMID: 15470055.
    Citations: 18     Fields:    Translation:AnimalsCells
  51. Gründling A, Burrack LS, Bouwer HG, Higgins DE. Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. Proc Natl Acad Sci U S A. 2004 Aug 17; 101(33):12318-23. PMID: 15302931.
    Citations: 88     Fields:    Translation:AnimalsCells
  52. Starks H, Bruhn KW, Shen H, Barry RA, Dubensky TW, Brockstedt D, Hinrichs DJ, Higgins DE, Miller JF, Giedlin M, Bouwer HG. Listeria monocytogenes as a vaccine vector: virulence attenuation or existing antivector immunity does not diminish therapeutic efficacy. J Immunol. 2004 Jul 01; 173(1):420-7. PMID: 15210801.
    Citations: 37     Fields:    Translation:AnimalsCells
  53. Hu PQ, Tuma-Warrino RJ, Bryan MA, Mitchell KG, Higgins DE, Watkins SC, Salter RD. Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I-restricted CD8+ T cells following uptake by human APC. J Immunol. 2004 Feb 01; 172(3):1595-601. PMID: 14734740.
    Citations: 16     Fields:    Translation:HumansCells
  54. Gründling A, Gonzalez MD, Higgins DE. Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. J Bacteriol. 2003 Nov; 185(21):6295-307. PMID: 14563864.
    Citations: 53     Fields:    Translation:HumansCells
  55. Radford KJ, Higgins DE, Pasquini S, Cheadle EJ, Carta L, Jackson AM, Lemoine NR, Vassaux G. A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: application to cancer immunotherapy. Gene Ther. 2002 Nov; 9(21):1455-63. PMID: 12378408.
    Citations: 19     Fields:    Translation:AnimalsCells
  56. Dancz CE, Haraga A, Portnoy DA, Higgins DE. Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection. J Bacteriol. 2002 Nov; 184(21):5935-45. PMID: 12374827.
    Citations: 28     Fields:    Translation:AnimalsCells
  57. Parker JS, Broering TJ, Kim J, Higgins DE, Nibert ML. Reovirus core protein mu2 determines the filamentous morphology of viral inclusion bodies by interacting with and stabilizing microtubules. J Virol. 2002 May; 76(9):4483-96. PMID: 11932414.
    Citations: 104     Fields:    Translation:HumansAnimalsCells
  58. Gedde MM, Higgins DE, Tilney LG, Portnoy DA. Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes. Infect Immun. 2000 Feb; 68(2):999-1003. PMID: 10639481.
    Citations: 83     Fields:    Translation:AnimalsCells
  59. Higgins DE, Shastri N, Portnoy DA. Delivery of protein to the cytosol of macrophages using Escherichia coli K-12. Mol Microbiol. 1999 Mar; 31(6):1631-41. PMID: 10209738.
    Citations: 21     Fields:    Translation:AnimalsCells
  60. Higgins DE, Portnoy DA. Bacterial delivery of DNA evolves. Nat Biotechnol. 1998 Feb; 16(2):138-9. PMID: 9487514.
    Citations:    Fields:    Translation:AnimalsCells
  61. Higgins DE, DiRita VJ. Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA. J Bacteriol. 1996 Feb; 178(4):1080-7. PMID: 8576041.
    Citations: 15     Fields:    Translation:Cells
  62. Higgins DE, DiRita VJ. Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae. Mol Microbiol. 1994 Oct; 14(1):17-29. PMID: 7830555.
    Citations: 70     Fields:    Translation:Cells
  63. Higgins DE, Nazareno E, DiRita VJ. The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. J Bacteriol. 1992 Nov; 174(21):6974-80. PMID: 1400247.
    Citations: 91     Fields:    Translation:Cells
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Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.