Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (\u3e105 copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES

Genetically Determined Plasma Lipid Levels and Risk of Diabetic Retinopathy: A Mendelian Randomization Study.

Results from observational studies examining dyslipidemia as a risk factor for diabetic retinopathy (DR) have been inconsistent. We evaluated the causal relationship between plasma lipids and DR using a Mendelian randomization approach. We pooled genome-wide association studies summary statistics from 18 studies for two DR phenotypes: any DR (N = 2,969 case and 4,096 control subjects) and severe DR (N = 1,277 case and 3,980 control subjects). Previously identified lipid-associated single nucleotide polymorphisms served as instrumental variables. Meta-analysis to combine the Mendelian randomization estimates from different cohorts was conducted. There was no statistically significant change in odds ratios of having any DR or severe DR for any of the lipid fractions in the primary analysis that used single nucleotide polymorphisms that did not have a pleiotropic effect on another lipid fraction. Similarly, there was no significant association in the Caucasian and Chinese subgroup analyses. This study did not show evidence of a causal role of the four lipid fractions on DR. However, the study had limited power to detect odds ratios less than 1.23 per SD in genetically induced increase in plasma lipid levels, thus we cannot exclude that causal relationships with more modest effect sizes exist

Posterior eye shape measurement with retinal OCT compared to MRI

PURPOSE. Posterior eye shape assessment by magnetic resonance imaging (MRI) is used to study myopia. We tested the hypothesis that optical coherence tomography (OCT), as an alternative, could measure posterior eye shape similarly to MRI. METHODS. Macular spectral-domain OCT and brain MRI images previously acquired as part of the Singapore Epidemiology of Eye Diseases study were analyzed. The right eye in the MRI and OCT images was automatically segmented. Optical coherence tomography segmentations were corrected for optical and display distortions requiring biometry data. The segmentations were fitted to spheres and ellipsoids to obtain the posterior eye radius of curvature (Rc) and asphericity (Qxz). The differences in Rc and Qxz measured by MRI and OCT were tested using paired t-tests. Categorical assignments of prolateness or oblateness using Qxz were compared. RESULTS. Fifty-two subjects (67.8 ± 5.6 years old) with spherical equivalent refraction from +0.50 to -5.38 were included. The mean paired difference between MRI and original OCT posterior eye Rc was 24.03 ± 46.49 mm (P = 0.0005). For corrected OCT images, the difference in Rc decreased to -0.23 ± 2.47 mm (P = 0.51). The difference between MRI and OCT asphericity, Qxz, was -0.052 ± 0.343 (P = 0.28). However, categorical agreement was only moderate (j = 0.50). CONCLUSIONS. Distortion-corrected OCT measurements of Rc and Qxz were not statistically significantly different from MRI, although the moderate categorical agreement suggests that individual differences remained. This study provides evidence that with distortion correction, noninvasive office-based OCT could potentially be used instead of MRI for the study of posterior eye shape

Rapid expression and purification of the hepatitis delta virus antigen using the methylotropic yeast Pichia pastoris

Objective: Patients with dual hepatitis B (HBV) and hepatitis D (HDV) virus infection are at an increased risk of progression to liver cirrhosis and hepatocellular carcinoma than patients with a single viral infection. Treatment of viral hepatitis due to dual HBV/HDV infection represents a challenge. Currently there is no vaccine against HDV. Recombinant production of HDV antigen (HDAg) is the first step towards a potential vaccine candidate and the development of assays for HDV detection. Results: This study demonstrates the expression of one HDAg isoform, S-HDAg, in Pichia pastoris. A recombinant vector carrying a tagged gene encoding S-HDAg under the control of the methanol-inducible promoter AOX1 was designed and integrated into P. pastoris X33. The protein, which was purified using a Ni2+ affinity column and eluted at 100-150 mM imidazole, has potential as a recombinant antigen for further study

Induced pseudoscalar coupling of the proton weak interaction

The induced pseudoscalar coupling $g_p$ is the least well known of the weak coupling constants of the proton's charged--current interaction. Its size is dictated by chiral symmetry arguments, and its measurement represents an important test of quantum chromodynamics at low energies. During the past decade a large body of new data relevant to the coupling $g_p$ has been accumulated. This data includes measurements of radiative and non radiative muon capture on targets ranging from hydrogen and few--nucleon systems to complex nuclei. Herein the authors review the theoretical underpinnings of $g_p$, the experimental studies of $g_p$, and the procedures and uncertainties in extracting the coupling from data. Current puzzles are highlighted and future opportunities are discussed.Comment: 58 pages, Latex, Revtex4, prepared for Reviews of Modern Physic

Targeted Delivery of Chemotherapy Agents Using a Liver Cancer-Specific Aptamer

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general

Genome-Wide Association Study of Retinopathy in Individuals without Diabetes

10.1371/journal.pone.0054232PLoS ONE82