Project description:The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of the NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. Therefore we isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44k Agilent microarrays.
Project description:The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of the NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. Therefore we isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44k Agilent microarrays. We performed the microarrays against a common reference sample, namely human RPE/choroid RNA. We performed 7 replicates of PE samples from 7 different donors and 7 NPE replicates from the same 7 different donors.
Project description:Ciliary margin zone cells have differential neurogenic potential in albino and pigmented eyes. We used single cell RNA sequencing (scRNA-seq) of albino and pigmented eyes to determine transcriptomic changes underlying differential RGC genesis.
Project description:The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2,959, 2,867, and 2,755 non-redundant proteins with protein false positive rate <1%. There were 43 unambiguous protein isoforms identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were found in ciliary body. The MS proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001424.
Project description:The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2,959, 2,867, and 2,755 non-redundant proteins with protein false positive rate <1%. There were 43 unambiguous protein isoforms identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were found in ciliary body. The MS proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001424.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
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.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.