Project description:The project aimed to profile the cell surface proteins of Nomo-1 (AML cell line) using structural surfaceomics for identification of protein conformation-based cancer antigens thereby expanding the toolkit for cancer target discovery for immunotherapeutic targeting. To achieve the goal, cell surface capture (CSC) was integrated with cross-linking mass spectrometry (XL-MS). DSSO was used as a cross-linker to freeze the structural conformations of protein in three-dimensional space, followed by biotinylation of cell surface proteins to enable enrichment of cell surface proteins to allow focused XL-MS analysis of those enriched proteins. DSSO being MS cleavable cross-linker, allowed higher order MS3 approach for analysis.
Project description:This ArrayExpress experiment contains RNA-seq data from human tissues, generated by Michael Snyder's lab at Stanford University as part of the ENCODE project ( https://www.encodeproject.org/search/?type=Experiment ). Sample information was obtained from the ENCODE data coordination center and protocol information was obtained from the associated publication in PNAS (Lin et al., 2014, PubMed ID 25413365). All samples, except for the ovary, were sequenced with technical replicates. The second technical replicate was performed in May 2015 and was not part of the original Lin et al. 2014 publication. Technical replicates are marked in the âComment[technical replicate group]â column of sample-data-relationship format (SDRF) file for this record.
Project description:Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Numerous clinical trials confirmed safety and efficacy of Treg treatment of for deleterious immune responses. However, Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. In our experiment we demonstrate that mild hypothermia of 33C induces robust proliferation of human Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33C showed stronger immunosuppressive potential and anti-inflammatory phenotype. We show that a simple change in temperature can preserve Treg stability, function and accelerate their proliferation in vitro.
Project description:Because of the extensive data in mice supporting the concept that ST2+ Tregs might have desirable therapeutic properties, including tissue repair function, high suppressive capacity, and enhanced stability, we engineered human blood Tregs to constitutively express ST2 (IL-33R). Here we used RNA sequencing to explore the effects of short-term culture with IL-33 on human ST2-transduced Tregs.
Project description:Establish the microRNA expression profile between normoxic- and hypoxic-culture during differentiation of mouse embryonic stem cells
Project description:Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin)is activated, resulting in binding of the activated transducin α-subunit (Gtα) toPDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal disease. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβin its nonactivated state. Following reconstitution of PDE6 and activated Gtαwith liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of interaction of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6-Tα*complex during visual excitation, thereby enhancing our understanding of the molecular etiology of inherited retinal diseases.
Project description:Transcriptome analysis of murine foetal NSCs (E14) after short-term (48 hours) and long-term (13 days) hypoxic (3% oxygen) culture compared to normoxic culture (21% oxygen) We focused on whole-transcriptome analyses using gene chip microarrays to compare expression profiles of NSCs cultured at hypoxic conditions to those of normoxic cells. Therefore, we used NSCs derived from the mesencephalon and the cortex and cultured them for short- and long-term at hypoxia/normoxia.
Project description:Whether or not human Tregs are susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how a tonic-signaling CAR, which induce Tconv exhaustion, affect human Tregs. We thus sorted naïve Tregs and naïve conventional CD4s and either left them untransduced (UT) or transduced them with a non-TS CAR (CD19) or a TS-CAR (HA). After 12 days in culture, we isolated their RNA and did RNA sequencing.
Project description:To define ncRNA expression in hypoxic endothelial cells, we applied pro-angiogenic hypoxia to cultured endothelial cells. Afterwards total RNA was isolated and underwent genechip analysis. HUVECs were subjected to normoxic or hypoxic (0.1-0.2% O2) cell culture conditions.
Project description:To define ncRNA expression in hypoxic endothelial cells, we applied pro-angiogenic hypoxia to cultured endothelial cells. Afterwards, total RNA was isolated and underwent RNA-seq analysis. HUVECs were subjected to normoxic or hypoxic (0.1-0.2% O2) cell culture conditions.