All Publications


  • N6-Methyladenosine Modification Controls Circular RNA Immunity. Molecular cell Chen, Y. G., Chen, R., Ahmad, S., Verma, R., Kasturi, S. P., Amaya, L., Broughton, J. P., Kim, J., Cadena, C., Pulendran, B., Hur, S., Chang, H. Y. 2019

    Abstract

    Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific Tcell activation, antibody production, and anti-tumor immunity invivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

    View details for DOI 10.1016/j.molcel.2019.07.016

    View details for PubMedID 31474572

  • Optical Control of Fast and Processive Engineered Myosins In Vitro and in Living Cells Ruijgrok, P. V., Ghosh, R. P., Nakamura, M., Zemsky, S., Chen, R., Vachharajani, V., Liphardt, J. T., Bryant, Z. CELL PRESS. 2019: 259A
  • Optical Control of Fast and Processive Engineered Myosins: Optimization and Characterization in Vitro and in Living Cells Ruijgrok, P. V., Ghosh, R. P., Nakamura, M., Chen, R., Vachharajani, V., Liphardt, J., Bryant, Z. CELL PRESS. 2018: 318A
  • A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy ACS SYNTHETIC BIOLOGY Chen, R., Rishi, H. S., Potapov, V., Yamada, M. R., Yeh, V. J., Chow, T., Cheung, C. L., Jones, A. T., Johnson, T. D., Keating, A. E., DeLoache, W. C., Dueber, J. E. 2015; 4 (11): 1205–16

    Abstract

    Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

    View details for DOI 10.1021/acssynbio.5b00060

    View details for Web of Science ID 000365461200006

    View details for PubMedID 26155738

    View details for PubMedCentralID PMC4654675

  • Effective Treatment of Mouse Sepsis With an Inhibitory Antibody Targeting Integrin alpha v beta 5 CRITICAL CARE MEDICINE Su, G., Atakilit, A., Li, J. T., Wu, N., Luong, J., Chen, R., Bhattacharya, M., Sheppard, D. 2013; 41 (2): 546-553

    Abstract

    Integrin αvβ5 has been identified as a regulator of vascular leak and endothelial permeability. We hypothesized that targeting αvβ5 could represent a viable treatment strategy for sepsis.Integrin β5 subunit knockout and wild-type 129/svJae mice and wild-type mice treated with αvβ5 blocking or control antibodies were tested in models of intraperitoneal lipopolysaccharide and cecal ligation and puncture. Human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers were treated with αvβ5 antibodies to assess for effects on lipopolysaccharide-induced changes in transendothelial resistance and on patterns of cytoskeletal reorganization.Laboratory-based research.Mice and endothelial cell monolayers.Measurements taken after intraperitoneal lipopolysaccharide and/or cecal ligation and puncture included mortality, vascular leak, hematocrit, quantification of a panel of serum cytokines/chemokines, and assessment of thioglyccolate-induced leukocyte migration. β5 knockout mice had decreased mortality after intraperitoneal lipopolysaccharide and cecal ligation and puncture and decreased vascular leak, as measured by extravasation of an I-labeled intravascular tracer. Treating clinically ill mice with αvβ5 antibodies, up to 20 hrs after intraperitoneal lipopolysaccharide and cecal ligation and puncture, also resulted in decreased mortality. αvβ5 antibodies attenuated lipopolysaccharide-induced transendothelial resistance changes and cytoskeletal stress fiber formation in both human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers. αvβ5 antibodies had no effect on cytokine/chemokine serum levels after cecal ligation and puncture. β5 knockout mice and wild-type controls did not exhibit differences in thioglyccolate-induced leukocyte migration.Our studies suggest that αvβ5 is an important regulator of the vascular endothelial leak response in sepsis and that αvβ5 blockade may provide a novel approach to treating this devastating disease syndrome.

    View details for DOI 10.1097/CCM.0b013e3182711b1e

    View details for Web of Science ID 000314106000039

    View details for PubMedID 23263571

  • Absence of Integrin alpha v beta 3 Enhances Vascular Leak in Mice by Inhibiting Endothelial Cortical Actin Formation AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Su, G., Atakilit, A., Li, J. T., Wu, N., Bhattacharya, M., Zhu, J., Shieh, J. E., Li, E., Chen, R., Sun, S., Su, C. P., Sheppard, D. 2012; 185 (1): 58-66

    Abstract

    Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms.We hypothesized that integrin αvβ3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI.β3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of β3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvβ3 antibodies were evaluated for sphingosine-1 phosphate (S1P)–mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization.β3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvβ3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvβ3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvβ3 localization after S1P was decreased by αvβ3 antibodies, suggesting that ligation of the αvβ3 with its extracellular matrix ligands is required to stabilize cortical αvβ3 focal adhesions.Our studies identify a novel mechanism by which αvβ3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvβ3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.

    View details for DOI 10.1164/rccm.201108-1381OC

    View details for Web of Science ID 000298806600015

    View details for PubMedID 21980034

    View details for PubMedCentralID PMC3262039