Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique.

This paper presents a novel clustered regularly interspaced short palindromic repeat (CRISPR)-associated HRCA technique (CART). During the entire detection process of CART, the target DNA is first specifically recognized and cleaved by a pair of Cas9/sgRNA complexes; then, the cleaved product is ligated into circular DNA as the template of HRCA, and the circular DNA is efficiently amplified by HRCA. Therefore, CART has the advantages of Cas9/sgRNA (single-base mismatch specificity) and HRCA (isothermal reaction temperature and high sensitivity). This technique has been verified by detecting various human papillomavirus (HPV) genes with numerous subtypes. In summary, this study provides a new and effective method for the detection of nucleic acids.

Up-regulation of VEGF by retinoic acid during hyperoxia prevents retinal neovascularization and retinopathy.

PURPOSE: Retinopathy of prematurity (ROP) is directly associated with abnormal expression of retinal vascular endothelial growth factor (VEGF) in premature neonates. This study was to investigate whether the systemic administration of retinoic acid (RA) regulates retinal VEGF expression and prevents retinal neovascularization and retinopathy in the oxygen-induced retinopathy (OIR) mouse model. METHODS: C57BL/6 mice were subjected to OIR by exposure to 75% oxygen from postnatal day (P) 7 to 12 of age. RA was intraperitoneally injected daily to pups from P6 to P9. Retinal whole mount staining and image analysis, immunostaining, Western blotting, quantitative RT-PCR, TUNEL assay, and electroretinography were performed to evaluate the effects of RA on VEGF expression, retinal neovascularization, and retinal neuron functions. RESULTS: Systemic administration of RA in OIR mice promoted retinal VEGF mRNA and protein expression in phase I; the stabilized level of VEGF in phase I supported retinal vascular development and counteracted vaso-obliteration in OIR mice. Subsequently, the excessive generation of VEGF in phase II was attenuated; the retinal vascular leakage and apoptotic cells were significantly ameliorated. As a result, RA significantly prevented the development of hypoxia-induced retinal neovascularization and retinopathy in OIR mice and improved the functional recovery of retinal neurons downstream of photoreceptor cells as measured by focal electroretinography. CONCLUSIONS: Systemic administration of RA regulates retinal VEGF expression and supports retinal vascular development in OIR mouse model. We propose that systemic administration of RA to extremely low birth weight, preterm infants during oxygen therapy could potentially be an effective therapeutic approach for the prevention of ROP.