Project description:We did the RNA-seq analysis to examine the global impact of Nicotinamide (NAM) on hiPSC-derived RPE transcriptome in order to better understand the mechanism of action of NAM. NAM inhibited the expression of Age related Macular degeneration (AMD) associated protein transcripts in hiPSC-derived RPE.
Project description:Nicotinamide (NAM) inhibited the expression of Age related macular degeneration (AMD) associated proteins in hiPSC-derived retinal pigment epithelium (RPE). We did the microarray analysis to examine the global impact of NAM on hiPSC-derived RPE transcriptome in order to better understand the mechanism of action of NAM.
Project description:We generated hiPSCs from patients fibloblast with retinitis pigmentosa (RP) using retrovirus and Sendai virus vectors, which we differentiated into hiPSC derived retinal pigment epithelium using two different methods (SDIA and SFEB methods). We investigated whether these hiPSC-RPE colonies, which were differentiated from various cell lines and methods, showed similar gene expression patterns to those of native RPE. We classified hiPSC-RPE, hiPSCs, and fibroblasts from RP patients, hRPE (commercially available human fetal RPE, Lonza) , ARPE19 (a human RPE cell line), and other human tissues from 54,675 probe sets using microarray data.
Project description:To investigate the efficacy of nicotinamide treatment using our ex-vivo primary lymphocyte model, we performed high-throughput RNA sequencing on libraries generated from untreated and nicotinamide treated samples. PBMC isolated from FRDA affected individuals were cultured to prepare the primary lymphocyte cell lines. The primary cultured cells were either treated with 10mM nicotinamide or without the addition of drug during the 3-days treatment. RNA was extracted after the treatment and then RNA-seq libraries were generated by standard protocols.
Project description:The goals of this study are to generate inflammation-sensitive astrocytes from human induced pluripotent stem cells. We examine the transcriptomic inflammatory signature of generated astrocytes following Il1Beta exposure. Primary human cerebellar astrocytes, human induced pluripotent stem cells (hiPSC)-derived neural precursor cells (NPCs) and hiPSC-derived astrocytes were treated with Il1beta and compared to vehicle treated controls. Results: hiPSC-derived can be differentiated to astrocytes that are inflammation sensitive.
Project description:Microarray analysis was performed to obtain the transcriptomes of GNE7915-, amioarone- and vehicle control-treated hiPSC-derived SFTPC+ cells.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
