Druse-Induced Morphology Evolution in Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a key site of pathogenesis for many retina diseases. The formation of drusen in the retina is characteristic of retinal degeneration. We investigate morphological changes in the RPE in the presence of soft drusen using an integrated experimental and modeling approach. We collect RPE flat mount images from donated human eyes and develop 1) statistical tools to quantify the images and 2) a cell-based model to simulate the morphology evolution. We compare three different mechanisms of RPE repair evolution, cell apoptosis, cell fusion, and expansion, and Simulations of our RPE morphogenesis model quantitatively reproduce deformations of human RPE morphology due to drusen, suggesting that a purse-string mechanism is sufficient to explain how RPE heals cell loss caused by drusen-damage. We found that drusen beneath tissue promote cell death in a number that far exceeds the cell numbers covering the drusen. Tissue deformations are studied using area distributions, Voronoi domains and a texture tensor.Fil: Mazzitello, Karina Irma. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Zhang, Qing. University of Emory; Estados UnidosFil: Chrenek, M. A.. University of Emory; Estados UnidosFil: Family, F.. University of Emory; Estados UnidosFil: Grossniklaus, H. E.. University of Emory; Estados UnidosFil: Nickerson, J. M.. University of Emory; Estados UnidosFil: Jiang, Y.. Georgia State University; Estados Unido

Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation

The RAD21/REC8 gene family has been implicated in sister chromatid cohesion and DNA repair in several organisms. Unlike most eukaryotes, Arabidopsis thaliana has three RAD21 gene homologues, and their cloning and characterization are reported here. All three genes, AtRAD21.1, AtRAD21.2, and AtRAD21.3, are expressed in tissues rich in cells undergoing cell division, and AtRAD21.3 shows the highest relative level of expression. An increase in steady-state levels of AtRAD21.1 transcript was also observed, specifically after the induction of DNA damage. Phenotypic analysis of the atrad21.1 and atrad21.3 mutants revealed that neither of the single mutants was lethal, probably due to the redundancy in function of the AtRAD21 genes. However, AtRAD21.1 plays a critical role in recovery from DNA damage during seed imbibition, prior to germination, as atrad21.1 mutant seeds are hypersensitive to radiation damag

Evaluation of variants in the selectin genes in age-related macular degeneration

<p>Abstract</p> <p>Background</p> <p>Age-related macular degeneration (AMD) is a common disease of the elderly that leads to loss of the central visual field due to atrophic or neovascular events. Evidence from human eyes and animal models suggests an important role for macrophages and endothelial cell activation in the pathogenesis of AMD. We sought to determine whether common ancestral variants in genes encoding the selectin family of proteins are associated with AMD.</p> <p>Methods</p> <p>Expression of E-selectin, L-selectin and P-selectin was examined in choroid and retina by quantitative PCR and immunofluorescence. Samples from patients with AMD (n = 341) and controls (n = 400) were genotyped at a total of 34 SNPs in the <it>SELE</it>, <it>SELL </it>and <it>SELP </it>genes. Allele and genotype frequencies at these SNPs were compared between AMD patients and controls as well as between subtypes of AMD (dry, geographic atrophy, and wet) and controls.</p> <p>Results</p> <p>High expression of all three selectin genes was observed in the choroid as compared to the retina. Some selectin labeling of retinal microglia, drusen cores and the choroidal vasculature was observed. In the genetic screen of AMD versus controls, no positive associations were observed for <it>SELE </it>or <it>SELL</it>. One SNP in <it>SELP </it>(rs3917751) produced p-values < 0.05 (uncorrected for multiple measures). In the subtype analyses, 6 SNPs (one in <it>SELE</it>, two in <it>SELL</it>, and three in <it>SELP</it>) produced p-values < 0.05. However, when adjusted for multiple measures with a Bonferroni correction, only one SNP in <it>SELP </it>(rs3917751) produced a statistically significant p-value (p = 0.0029).</p> <p>Conclusions</p> <p>This genetic screen did not detect any SNPs that were highly associated with AMD affection status overall. However, subtype analysis showed that a single SNP located within an intron of <it>SELP </it>(rs3917751) is statistically associated with dry AMD in our cohort. Future studies with additional cohorts and functional assays will clarify the biological significance of this discovery. Based on our findings, it is unlikely that common ancestral variants in the other selectin genes (<it>SELE </it>and <it>SELL</it>) are risk factors for AMD. Finally, it remains possible that sporadic or rare mutations in <it>SELE</it>, <it>SELL</it>, or <it>SELP </it>have a role in the pathogenesis of AMD.</p

Long-term remission of myopic choroidal neovascular membrane after treatment with ranibizumab: a case report

<p>Abstract</p> <p>Introduction</p> <p>Myopia has become a big public health problem in certain parts of the world. Sight-threatening complications like choroidal neovascularisation membranes occur in up to 10% of pathological myopia, and natural history studies show a trend towards progressive visual loss. There are long-term financial and quality-of-life implications in this group of patients, and treatment strategies should aim for long-term preservation of vision.</p> <p>Case presentation</p> <p>A 56-year-old Caucasian woman presented with a best-corrected visual acuity of 6/6-1 in her right eye and 6/24 in her left. Fundal examination revealed pathological myopia in both eyes and an elevated lesion associated with pre-retinal haemorrhage in the left macula. Ocular coherence tomography and fundus fluorescein angiogram confirmed a subfoveal classic choroidal neovascularisation membrane. The patient decided to proceed with intravitreal ranibizumab (0.5 mg) therapy. One month after treatment, best-corrected visual acuity improved to 6/12 in her left eye, with complete resolution subretinal fluid on ocular coherence tomography. After three months, best-corrected visual acuity further improved to 6/9, which was maintained up to 16 months post-treatment.</p> <p>Conclusion</p> <p>We suggest intravitreal ranibizumab as an alternative treatment for long-term remission of myopic choroidal neovascular membrane. It also suggests that myopic choroidal neovascularisation membranes may require fewer treatments to achieve sustained remission. Furthermore, this could serve as a feasible long-term management option if used in conjunction with ocular coherence tomography.</p

Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344

PURPOSE: To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). RESULTS: The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1⁻/⁻ mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001). CONCLUSIONS: INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration

Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate

Adhesion Failures Determine the Pattern of Choroidal Neovascularization in the Eye: A Computer Simulation Study

Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation

The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. Methods: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. Results: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. Conclusions: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses

A Multicenter Analysis of Nucleic Acid Quantification Using Aqueous Humor Liquid Biopsy in Retinoblastoma: Implications for Clinical Testing

PURPOSE: Retinoblastoma (RB) is most often diagnosed with clinical features and not diagnosed with tumor biopsy. This study describes tumor-derived analyte concentrations from aqueous humor (AH) liquid biopsy and its use in clinical assays. DESIGN: Case series study. PARTICIPANTS: Sixty-two RB eyes from 55 children and 14 control eyes from 12 children from 4 medical centers. METHODS: This study included 128 RB AH samples including: diagnostic (DX) samples, samples from eyes undergoing treatment (TX), samples after completing treatment (END), and during bevacizumab injection for radiation therapy after completing RB treatment (BEV). Fourteen-control AH were analyzed for unprocessed analytes (double-stranded DNA [dsDNA], single-stranded DNA [ssDNA], micro-RNA [miRNA], RNA, and protein) with Qubit fluorescence assays. Double-stranded DNA from 2 RB AH samples underwent low-pass whole-genome sequencing to detect somatic copy number alterations. Logistic regression was used to predict disease burden given analyte concentrations. MAIN OUTCOME MEASURES: Unprocessed analyte (dsDNA, ssDNA, miRNA, RNA and protein) concentrations. RESULTS: Results revealed dsDNA, ssDNA, miRNA, and proteins, but not RNA, were quantifiable in most samples (up to 98%) with Qubit fluorescence assays. Median dsDNA concentration was significantly higher in DX (3.08 ng/μl) compared to TX (0.18 ng/μl; CONCLUSIONS: Aqueous humor liquid biopsy in RB is a high-yield source of dsDNA, ssDNA, miRNA, and protein. Diagnostic samples are most useful for RB 1 gene mutational analyses. Genomic analysis may be more informative of tumor activity status than quantification alone and can be performed even with smaller analyte concentrations obtained from TX samples. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found after the references