Biology subjects

Su, X.

Publications and source records attributed to Su, X..

149 records · Page 9Linked to original sources

bioRxiv2017

Fundamentals of injection molding for microfluidic cell-based assays

Microscale cell-based assays have demonstrated unique capabilities in reproducing important cellular behaviors for diagnostics and basic biological research. As these assays move beyond the prototyping stage and into biological and clinical research environments, there is a need to produce microscale culture platforms more rapidly, cost-effectively, and reproducibly. Rapid injection molding is poised to meet this need as it enables some of the benefits of traditional high volume injection molding at a fraction of the cost. However, rapid injection molding has limitations due to the material and methods used for mold fabrication. Here, we characterize advantages and limitations of rapid injection molding for microfluidic device fabrication through measurement of key features for cell culture applications including channel geometry, feature consistency, floor thickness, and surface polishing. We demonstrate phase contrast and fluorescence imaging of cells grown in rapid injection molded devices and provide design recommendations to successfully utilize rapid injection molding methods for microscale cell-based assay development in academic laboratory settings.

bioengineering↗

bioRxiv2017

Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development

TRIM family members have been implicated in a variety of biological processes such as differentiation and development. We here found that Trim59 plays a critical role in early embryo development from blastocyst stage to gastrula. There existed delayed development and empty yolk sacs from embryonic day (E) 8.5 in Trim59 -/- embryos. No viable Trim59 -/- embryos were observed beyond E9.5. Trim59 deficiency affected primary germ layer formation at the beginning of gastrulation. In Trim59 -/- embryos at E6.5 and E7.5, the expression of primary germ layer formation associated genes including Brachyury, lefty2, Cer1, Otx2, Wnt3 and BMP4 was reduced. Homozygous mutant embryonic epiblast was contracted and the mesoderm was absent. Trim59 could interact with actin and myosin associated proteins. Trim59 deficiency disturbed F-actin polymerization during inner cell mass differentiation. Trim59 mediated polymerization of F-actin was via WASH K63-linked ubiquitination. Thus, Trim59 may be a critical regulator for early embryo development from blastocyst stage to gastrula through modulating F-actin assembly.

developmental biology↗

bioRxiv2017

Zebrafish models for human ALA-dehydratase-deficient porphyria (ADP) and hereditary coproporphyria (HCP) generated with TALEN and CRISPR-Cas9

Defects in the enzymes involved in heme biosynthesis result in a group of human metabolic genetic disorders known as porphyrias. Using a zebrafish model for human hepatoerythropoietic porphyria (HEP), caused by defective uroporphyrinogen decarboxylase (Urod), the fifth enzyme in the heme biosynthesis pathway, we recently have found a novel aspect of porphyria pathogenesis. However, no hereditable zebrafish models with genetic mutations of alad and cpox, encoding the second enzyme delta-aminolevulinate dehydratase (Alad) and the sixth enzyme coproporphyrinogen oxidase (Cpox), have been established to date. Here we employed site-specific genome-editing tools transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate zebrafish mutants for alad and cpox. These zebrafish mutants display phenotypes of heme deficiency, hypochromia, abnormal erythrocytic maturation and accumulation of heme precursor intermediates, reminiscent of human ALA-dehydratase-deficient porphyria (ADP) and hereditary coproporphyrian (HCP), respectively. Further, we observed altered expression of genes involved in heme biosynthesis and degradation and particularly down-regulation of exocrine pancreatic zymogens in ADP (alad-/-) and HCP (cpox-/-) fishes. These two zebrafish porphyria models can survive at least 7 days and thus provide invaluable resources for elucidating novel pathological aspects of porphyrias, evaluating mutated forms of human ALAD and CPOX, discovering new therapeutic targets and developing effective drugs for these complex genetic diseases. Our studies also highlight generation of zebrafish models for human diseases with two versatile genome-editing tools.

genetics↗

bioRxiv2016

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

T cell signaling initiates upon binding of peptide-major histocompatibility complex (pMHC) on an antigen-presenting cell (APC) to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally-regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (~50%) than the smaller R0 isoform (~20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of [≤]15 M were needed to exclude CD45. We further show that the co-receptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.\n\nSIGNIFICANCE STATEMENTThe interface between a T cell and an antigen-presenting cell (APC) results in the formation of biochemically distinct plasma membrane domains that initiate signaling cascades. Here, using biochemical reconstitution and microscopy, we show that the binding energies of the TCRpMHC and PD-1-PD-L1 complexes are sufficient to create spatial organization at a model membrane-membrane interface. We show that spatial organization depends upon receptor-ligand binding affinity and the relative sizes of the extracellular domains. These biophysical parameters may be used to fine-tune signaling cascades in T cells.

immunology↗

bioRxiv2016

T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition

Programmed death-1 (PD-1) is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). Here, by titrating the strength of PD-1 signaling in both biochemical reconstitution systems and in T cells, we demonstrate that the coreceptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1- recruited Shp2 phosphatase. We also show that PD-1 colocalizes with the costimulatory receptor CD28 in plasma membrane microclusters but partially segregates from the TCR. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways may play unexpected roles in regulating effector T cell function and therapeutic responses to anti-PD-L1/PD-1.

biochemistry↗