Cloning and characterization of a glycosyltransferase from Catharanthus roseus for glycosylation of cardiotonic steroids and phenolic compounds.

OBJECTIVES: To characterize a glycosyltransferase (UGT74AN3) from Catharanthus roseus and investigate its specificity toward cardiotonic steroids and phenolic compounds. RESULTS: UGT74AN3, a novel permissive GT from C. roseus, displayed average high conversion rate (> 90%) toward eight structurally different cardiotonic steroids. Among them, resibufogenin, digitoxigenin, and uzarigenin gave 100% yield. Based on LC-MS, 1H-NMR and 13C-NMR analysis, structure elucidation of eight glycosides was consistent with 3-O-ß-D-glucosides. We further confirmed UGT74AN3 was permissive enough to glycosylate curcumin, resveratrol, and phloretin. The cDNA sequence of UGT74AN3 contained an ORF of 1,425 nucleotides encoding 474 amino acids. UGT74AN3 performed the maximum catalytic activity at 40 °C, pH 8.0, and was divalent cation-independent. Km values of UGT74AN3 toward resibufogenin, digitoxigenin, and uzarigenin were 7.0 µM, 12.3 µM, and 17.4 µM, respectively. CONCLUSIONS: UGT74AN3, a glycosyltransferase from a noncardenolide-producing plant, displayed catalytic efficiency toward cardiotonic steroids and phenolic compounds, which would make it feasible for glycosylation of bioactive molecules.

Modeling retinitis pigmentosa through patient-derived retinal organoids.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into well-structured retinal organoids. In this protocol, we successfully established 3D retinae from patient-derived hiPSCs and built the retinitis pigmentosa model in vitro. Moreover, mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene was corrected by CRISPR-Cas9 gene editing, which rescued the structure and function of the 3D retinae. For complete details on the use and execution of this protocol, please refer to Deng et al. (2018).

An ionic molecular glass as electron injection layer for efficient polymer light-emitting diode.

An ionic molecular glass based on a dendronized monoammonium salt has been facilely synthesized and utilized as an interfacial electron-injection layer in a light-emitting diode (LED). The characterization of a yellow-green LED that involves an Al cathode and a thin layer of the new compound spin cast from a methanol solution has shown device performances comparable to those obtained with a Ba/Al cathode. Photovoltaic measurements under white light irradiation reveal that a thin layer of the new compound can significantly increase the built-in potential and thus facilitate electron injection from an Al cathode. Furthermore, it is interesting to observe that the new ionic salt could undergo reorganization on the emissive conjugated polymer layer, which leads to the formation of nearly uniform nanoaggregates.

Stemming retinal regeneration with pluripotent stem cells.

Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases, such as age-related macular degeneration (AMD), Stargardt's disease, retinitis pigmentosa (RP) and glaucoma. These diseases are all characterized by the degeneration of one or two retinal cell types that cannot regenerate spontaneously in humans. Aberrant retinal pigment epithelial (RPE) cells can be observed through optical coherence tomography (OCT) in AMD patients. In RP patients, the morphological and functional abnormalities of RPE and photoreceptor layers are caused by a genetic abnormality. Stargardt's disease or juvenile macular degeneration, which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age. Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it. Transplantation-based approaches have been proposed as a universal therapy to target patients with various concomitant diseases. Both the replacement of dead cells and neuroprotection are strategies used to rescue visual function in animal models of retinal degeneration. Diverse retinal cell types derived from pluripotent stem cells, including RPE cells, photoreceptors, RGCs and even retinal organoids with a layered structure, provide unlimited cell sources for transplantation. In addition, mesenchymal stem cells (MSCs) are multifunctional and protect degenerating retinal cells. The aim of this review is to summarize current findings from preclinical and clinical studies. We begin with a brief introduction to retinal degenerative diseases and cell death in diverse diseases, followed by methods for retinal cell generation. Preclinical and clinical studies are discussed, and future concerns about efficacy, safety and immunorejection are also addressed.

Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.

Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.

Toll-Like Receptor 3 Activation Initiates Photoreceptor Cell Death In Vivo and In Vitro.

Purpose: Accumulating evidence has demonstrated that excessive immunoreaction plays a prominent role in the pathogenesis of dry AMD. Toll-like receptor 3 (TLR3) can be activated by double-stranded (ds)RNA in retinal pigment epithelia and trigger an innate immunity-mediated inflammatory response. However, its role in photoreceptor cells, the effectors of AMD geographic atrophy, remains unclear. Methods: The expression of TLR3 was examined in mouse retina and in a murine photoreceptor cell line (661W). Retinal structure, function, and cell death in the polyinosine-polycytidylic acid (poly I:C)-treated retina were investigated by optical coherence tomography, electroretinography (ERG), and immunostaining. Cytokine and chemokine expression as well as cell death were measured in poly I:C-exposed 661W cells and explant retinas. By comparing the RNA sequencing (seq) data of 661W cells and murine retina, we comprehensively investigated the contribution of photoreceptor in poly I:C-induced retinal immune response. Results: Toll-like receptor 3 was highly expressed in the inner segment of the photoreceptor and in 661W cells. We found poly I:C induced significant retinal structural damages and impairment of ERG responses. Focal ERG demonstrated that injected and parainjected zones were functionally damaged by poly I:C. In addition, poly I:C acted on cultured photoreceptor cells directly and evoked an inflammatory response that exhibited similarities with the immune response in mouse retina. Moreover, TLR3 activation initiated cell death in murine photoreceptor cells in vivo and in vitro. Additionally, poly I:C initiated immune response in explant retinas. Conclusions: We deciphered the TLR3-mediated inflammatory response in photoreceptor cells. Our findings suggested TLR3-mediated inflammatory response in photoreceptor cells may play an important role in dry AMD, offering new insights of potential treatments targeting photoreceptor immunity.

Molecular genetic analysis of patients with sporadic and X-linked infantile nystagmus.

OBJECTIVES: Infantile nystagmus (IN) is a genetically heterogeneous condition characterised by involuntary rhythmic oscillations of the eyes accompanied by different degrees of vision impairment. Two genes have been identified as mainly causing IN: FRMD7 and GPR143. The aim of our study was to identify the genetic basis of both sporadic IN and X-linked IN. DESIGN: Prospective analysis. PATIENTS: Twenty Chinese patients, including 15 sporadic IN cases and 5 from X-linked IN families, were recruited and underwent molecular genetic analysis. We first performed PCR-based DNA sequencing of the entire coding region and the splice junctions of the FRMD7 and GPR143 genes in participants. Mutational analysis and co-segregation confirmation were then performed. SETTING: All clinical examinations and genetic experiments were performed in the Eye Hospital of Wenzhou Medical University. RESULTS: Two mutations in the FRMD7 gene, including one novel nonsense mutation (c.1090C>T, p.Q364X) and one reported missense mutation (c.781C>G, p.R261G), were identified in two of the five (40%) X-linked IN families. However, none of putative mutations were identified in FRMD7 or GPR143 in any of the sporadic cases. CONCLUSIONS: The results suggest that mutations in FRMD7 appeared to be the major genetic cause of X-linked IN, but not of sporadic IN. Our findings provide further insights into FRMD7 mutations, which could be helpful for future genetic diagnosis and genetic counselling of Chinese patients with nystagmus.