Extremely Complex Populations of Small RNAs in the Mouse Retina and RPE/Choroid

Purpose: MicroRNAs (miRNAs) are small non-coding RNAs of ~18-22 nucleotides in length that regulate gene expression. They are widely expressed in the retina, being both required for its normal development and perturbed in disease. The aim of this study was to apply new high-throughput sequencing techniques to more fully characterise the microRNAs and other small RNAs expressed in the retina and retinal pigment epithelium (RPE)/choroid of the mouse.

Methods: Retina and RPE/choroid were dissected from eyes of 3 month-old C57BL/6J mice. Small RNA libraries were prepared and deep sequencing performed on a Genome Analyzer (Illumina). Reads were annotated by alignment to miRBase, other non-coding RNA databases and the mouse genome.

Results: Annotation of 9 million reads to 320 microRNAs in retina and 340 in RPE/choroid provides the most comprehensive profiling of microRNAs to date. Two novel microRNAs were identified in retina. Members of the sensory organ specific miR-183,-182,-96 cluster were amongst the most highly expressed, retina-enriched microRNAs. Remarkably, microRNA 'isomiRs', which vary slightly in length and are differentially detected by Taqman RT-PCR assays, existed for all the microRNAs identified in both tissues. More variation occurred at the 3' ends, including non-templated additions of T and A. Drosha-independent mirtron microRNAs and other small RNAs derived from snoRNAs were also detected.

Conclusions: Deep sequencing has revealed the complexity of small RNA expression in the mouse retina and RPE/choroid. This knowledge will improve the design and interpretation of future functional studies of the role of microRNAs and other small RNAs in retinal disease.

Comparison of the mouse spleen cell assay and a radioimmunoassay for the measurement of serum erythropoietin

The mouse spleen cell assay (MSCA) has been compared with a radioimmunoassay for the measurement of serum erythropoietin (Ep). In 20 normal subjects the serum values ranged from 15 to 73 mU/ml for the MSCA compared with 5-30 mU/ml for the RIA. For normal sera there was no correlation between the results of the two assays. In 37 patients with anaemias of differing aetiologies and at various stages of treatment values ranged from 10 to 3645 mU/ml for the MSCA and 13-10,000 mU/ml for the RIA. Although patient values from the two assays were highly correlated (r = 0.98, P less than 0.001), the MSCA results were generally lower. These discrepancies can be largely accounted for by two factors. Firstly the MSCA is sensitive to non-specific matrix effects. Secondly, heat inactivation of serum, a prerequisite for the MSCA, but not for the RIA, destroys a variable and unpredictable proportion of the Ep in the test sera leading to an underestimation of Ep in the MSCA. We conclude that the RIA is more reliable than the MSCA which, in its present form, cannot be recommended for the accurate measurement of serum erythropoietin.

Secreted Proteins from the Helminth Fasciola hepatica Inhibit the Initiation of Autoreactive T Cell Responses and Prevent Diabetes in the NOD Mouse

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

Intravitreal Injection of Normal Saline Induces Retinal Degeneration in the C57BL/6J Mouse

Purpose: To investigate the adverse effect of intravitreal injection of normal saline (NS) and phosphate buffered saline (PBS) in mouse eyes.

Methods: NS or PBS was injected intravitreally into C57BL/6J mouse eyes. Retinal lesions were monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), and histological investigations. Retinal immune gene expression was determined by real-time polymerase chain reaction (PCR). The toxic effect of NS and PBS or retinal protein from NS- or PBS-injected eyes on retinal pigment epithelium (RPE) was tested in B6-RPE-07 mouse RPE cell cultures.

Results: Intravitreal injection of NS dose-dependently induced localized retinal lesion in mice. Histological investigations revealed multiple vacuoles in photoreceptor outer segments and RPE cells. The lesions recovered over time and by 3 weeks post injection the majority of lesions vanished in eyes receiving 1 μl NS. Inflammatory genes, including TNF-α, IL-1β, IL-6, iNOS, and VEGF were upregulated in NS injected eyes. Intravitreal injection of PBS did not cause any pathology. The treatment of B6-RPE07 cells with 30% PBS or 30% NS did not affect RPE viability. However, incubation of 1-μg/ml retinal protein from NS-injected eyes, but not PBS-injected eyes induced RPE cell death.

Conclusion: NS is toxic to the C57BL/6J mouse retina and should not be used as a vehicle for intraocular injection. PBS is not toxic to the retina and is a preferred vehicle.

Translational Relevance: NS is not a physiological solution for intraocular injection in the C57BL/6J mice and questions its suitability for intraocular injection in other species, including human.

EL CVn-type binaries - Discovery of 17 helium white dwarf precursors in bright eclipsing binary star systems

The star 1SWASP J024743.37-251549.2 was recently discovered to be a binary star in which an A-type dwarf star eclipses the remnant of a disrupted red giant star (WASP 0247-25 B). The remnant is in a rarely observed state evolving to higher effective temperatures at nearly constant luminosity prior to becoming a very low mass white dwarf composed almost entirely of helium, i.e. it is a pre-helium white dwarf (pre-He-WD). We have used the photometric database from theWide Angle Search for Planets (WASP) to find 17 eclipsing binary stars with orbital periods P = 0.7-2.2 d with similar light curves to 1SWASP J024743.37-251549.2. The only star in this group previously identified as a variable star is the brightest one, EL CVn, which we adopt as the prototype for this class of eclipsing binary star. The characteristic light curves of EL CVn-type stars show a total eclipse by an A-type dwarf star of a smaller, hotter star and a secondary eclipse of comparable depth to the primary eclipse. We have used new spectroscopic observations for six of these systems to confirm that the companions to the A-type stars in these binaries have very low masses (≈0.2M⊙). This includes the companion to EL CVn which was not previously known to be a pre-He-WD. EL CVn-type binary star systems will enable us to study the formation of very low mass white dwarfs in great detail, particularly in those cases where the pre-He-WD star shows non-radial pulsations similar to those recently discovered in WASP0247-25 B. © 2013 The Authors Published by Oxford University Press on behalf of the Royal Astronomical Society.

A functional role of the sky’s polarization pattern for orientation in the greater mouse-eared bat

Animals can call on a multitude of sensory information to orient and navigate. In some cases they may calibrate these cues against each other to establish the most accurate information available. One such cue is the pattern of polarized light in the sky, which may be used as a geographical reference to calibrate other cues in the compass mechanism. Mammals, however, have not been shown to use this cue, even though they do calibrate a magnetic compass with sunset. In this paper we demonstrate that bats use polarization cues at sunset to calibrate a magnetic compass, subsequently used for orientation during a homing experiment. It is thus the only mammal known so far to make use of the polarization pattern in the sky. This is an intriguing finding as currently there is no clear understanding of how this cue is perceived in this taxon and has general implications for the sensory biology of mammalian vision.

Loss of Synaptic Connectivity, Particularly in Second Order Neurons Is a Key Feature of Diabetic Retinal Neuropathy in the Ins2Akita Mouse

Retinal neurodegeneration is a key component of diabetic retinopathy (DR), although the detailed neuronal damage remains ill-defined. Recent evidence suggests that in addition to amacrine and ganglion cell, diabetes may also impact on other retinal neurons. In this study, we examined retinal degenerative changes in Ins2^Akita diabetic mice. In scotopic electroretinograms (ERG), b-wave and oscillatory potentials were severely impaired in 9-month old Ins2^Akita mice. Despite no obvious pathology in fundoscopic examination, optical coherence tomography (OCT) revealed a progressive thinning of the retina from 3 months onwards. Cone but not rod photoreceptor loss was observed in 3-month-old diabetic mice. Severe impairment of synaptic connectivity at the outer plexiform layer (OPL) was detected in 9-month old Ins2Akita mice. Specifically, photoreceptor presynaptic ribbons were reduced by 25% and postsynaptic boutons by 70%, although the density of horizontal, rod- and cone-bipolar cells remained similar to non-diabetic controls. Significant reductions in GABAergic and glycinergic amacrine cells and Brn3a+ retinal ganglion cells were also observed in 9-month old Ins2^Akita mice. In conclusion, the Ins2^Akita mouse develops cone photoreceptor degeneration and the impairment of synaptic connectivity at the OPL, predominately resulting from the loss of postsynaptic terminal boutons. Our findings suggest that the Ins2^Akita mouse is a good model to study diabetic retinal neuropathy.

SN 2010LP - A type IA supernova from a violent merger of two carbon-oxygen white dwarfs

SN 2010lp is a subluminous Type Ia supernova (SN Ia) with slowly evolving lightcurves. Moreover, it is the only subluminous SN Ia observed so far that shows narrow emission lines of [O I] in late-time spectra, indicating unburned oxygen close to the center of the ejecta. Most explosion models for SNe Ia cannot explain the narrow [O I] emission. Here, we present hydrodynamic explosion and radiative transfer calculations showing that the violent merger of two carbon-oxygen white dwarfs of 0.9 and 0.76 M adequately reproduces the early-time observables of SN 2010lp. Moreover, our model predicts oxygen close to the center of the explosion ejecta, a pre-requisite for narrow [O I] emission in nebular spectra as observed in SN 2010lp. © 2013. The American Astronomical Society. All rights reserved.