a-reversible-silicon-oil-induced-ocular-hypertension-model-in-mice

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Stanford University School of Medicine

Second Xiangya Hospital of Central South University

The aetiology of nasopharyngeal carcinoma (NPC) remains enigmatic in endemic and non-endemic areas. Early-life infection with Epstein-Barr virus (EBV) may predispose to NPC development, whereas delayed primary infection with EBV may cause infectious mononucleosis (IM).

Sibship size, birth order and risk of nasopharyngeal carcinoma and infectious mononucleosis: a nationwide study in Sweden.

Generation of midbrain dopaminergic (mDA) neurons from human pluripotent stem cells provides a platform for inquiry into basic and translational studies of Parkinson's disease (PD). However, heterogeneity in differentiation in vitro makes it difficult to identify mDA neurons in culture or in vivo following transplantation. Here, we report the generation of a human embryonic stem cell (hESC) line with a tyrosine hydroxylase (TH)-RFP (red fluorescent protein) reporter. We validated that RFP faithfully mimicked TH expression during differentiation. Use of this TH-RFP reporter cell line enabled purification of mDA-like neurons from heterogeneous cultures with subsequent characterization of neuron transcriptional and epigenetic programs (global binding profiles of H3K27ac, H3K4me1, and 5-hydroxymethylcytosine [5hmC]) at four different stages of development. We anticipate that the tools and data described here will contribute to the development of mDA neurons for applications in disease modeling and/or drug screening and cell replacement therapies for PD.

A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations.

To prepare and evaluate a new radiotracer F-IRS for molecular imaging mutant EGF Receptors in vitro and vivo. Uptake and efflux of F-IRS were performed with four NSCLC cell lines including HCC827, H1975, H358 and H520. In vivo tumor targeting and pharmacokinetics of the radiotracers were also evaluated in HCC827, H1975, H358 and H520 tumor-bearing nude mice by PET/CT imaging. Ex vivo biodistribution assays were performed to quantify the accumulation of F-IRS in vivo. We also performed F-IRS PET/CT imaging of three patients with NSCLC. We labeled this small molecule with QD620 for flow cytometry and confocal imaging analyses. The uptakes of F-IRS by HCC827 and HCC827 tumors were significantly higher than those of H358, H1975 and H520, and they were reduced by the addition of 100 μM of gefitinib. Biodistribution experiments showed an accumulation of F-IRS in tumors of HCC827 xenografts. Flow cytometry and confocal imaging with QD620-IRS further demonstrated that binding specifically to HCC827 cells. F-IRS accumulation was preferential in the tumor, which was NSCLC with responsive EGFR exon 19 deleted. F-IRS showed high binding stability and specificity to 19 exon deleted EGFR mutation in vitro and vivo.

Development and Evaluation of F-IRS for Molecular Imaging Mutant EGF Receptors in NSCLC.

Tumor heterogeneity and changes in epidermal growth factor receptor (EGFR) mutation status over time challenge the design of effective EGFR tyrosine kinase inhibitor (TKI) treatment strategies for non-small cell lung cancer (NSCLC). Therefore, there is an urgent need to develop techniques for comprehensive tumor EGFR profiling in real time, particularly in lung cancer precision medicine trials. We report a positron emission tomography (PET) tracer, -(3-chloro-4-fluorophenyl)-7-(2-(2-(2-(2-F-fluoroethoxy) ethoxy) ethoxy) ethoxy)-6-methoxyquinazolin-4-amine (F-MPG), with high specificity to activating EGFR mutant kinase. We evaluate the feasibility of using F-MPG PET for noninvasive imaging and quantification of EGFR-activating mutation status in preclinical models of NSCLC and in patients with primary and metastatic NSCLC tumors. F-MPG PET in NSCLC animal models showed a significant correlation ( = 0.9050) between F-MPG uptake and activating EGFR mutation status. In clinical studies with NSCLC patients ( = 75), the concordance between the detection of EGFR activation by F-MPG PET/computed tomography (CT) and tissue biopsy reached 84.29%. There was a greater response to EGFR-TKIs (81.58% versus 6.06%) and longer median progression-free survival (348 days versus 183 days) in NSCLC patients when F-MPG PET/CT SUV (maximum standard uptake value) was ≥2.23 versus <2.23. Our study demonstrates that F-MPG PET/CT is a powerful method for precise quantification of EGFR-activating mutation status in NSCLC patients, and it is a promising strategy for noninvasively identifying patients sensitive to EGFR-TKIs and for monitoring the efficacy of EGFR-TKI therapy.

A PET imaging approach for determining EGFR mutation status for improved lung cancer patient management.

Elevated expression of the c-Met receptor plays a crucial role in cancers. In non-small cell lung cancer (NSCLC), aberrant activation of the c-Met signaling pathway contributes to tumorigenesis and cancer progression and may mediate acquired resistance to epidermal growth factor receptor-targeted therapy. c-Met is therefore emerging as a promising therapeutic target for NSCLC, and methods for noninvasive in vivo assessment of c-Met expression would improve NSCLC treatment and diagnosis.  We developed a new c-Met-binding peptide (cMBP) radiotracer, Tc-hydrazine nicotinamide (HYNIC)-cMBP, for SPECT imaging. Cell uptake assays were performed on 2 NSCLC cell lines with different c-Met expressions: H1993 (high expression) and H1299 (no expression). In vivo tumor specificity was assessed by SPECT imaging in tumor-bearing mice at 0.5, 1, 2, and 4 h after injection of the probe. Blocking assays, biodistribution, and autoradiography were also conducted to determine probe specificity. Tc-HYNIC-cMBP was prepared with high efficiency and showed higher uptake in H1993 cells than in H1299 cells. Biodistribution and autoradiography also showed significantly higher percentages of the injected dose for Tc-HYNIC-cMBP in H1993 tumors than in H1299 tumors at 0.5 h (4.74 ± 1.43%/g and 1.00 ± 0.37%/g, respectively;  < 0.05). H1993 tumors were clearly visualized at 0.5 h in SPECT images, whereas H1299 tumors were not observed at any time. The specificity of Tc-HYNIC-cMBP for c-Met was demonstrated by a competitive block with an excess of nonradiolabeled peptide.  For c-Met-targeted SPECT imaging of NSCLC, we developed Tc-HYNIC-cMBP, a tracer that specifically binds to c-Met with favorable pharmacokinetics in vitro and in vivo.

Development of a SPECT Tracer to Image c-Met Expression in a Xenograft Model of Non-Small Cell Lung Cancer.

Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm.

A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells.

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.

Bi-allelic ADPRHL2 Mutations Cause Neurodegeneration with Developmental Delay, Ataxia, and Axonal Neuropathy.

Understanding the molecular mechanism of glaucoma and development of neuroprotectants is significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here, we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive retinal ganglion cell (RGC) loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.

Fang Fang

Department of Ophthalmology

Stanford University School of Medicine

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice