Otx2 enhances transdifferentiation of Müller cells-derived retinal stem cells into photoreceptor-like cells.

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal-specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells-derived retinal stem cells (MC-RSCs) into photoreceptor-like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC-RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor-like cells among MC-RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre-treatment with Crx siRNA, Nrl siRNA, or GSK-3 inhibitor SB-216763 reduced the positive rate of photoreceptor-like cells among MC-RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N-methyl-N-nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC-RSCs into photoreceptor-like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.

[Clinical features and genetic analysis of a pedigree affected with non-muscle myosin heavy chain 9 gene related disease].

OBJECTIVE: To identify the mutation type of non-muscle myosin heavy chain 9 (MYH9) gene and investigate the clinical features of a pedigree affected with MYH9 gene-related disease. METHODS: Peripheral blood samples of the proband and his family members were collected. Routine blood tests were performed, which included platelet counting and Wright's staining to observe the granulocyte inclusions and giant platelets. PCR was used to amplify exons 2, 17, 27, 31, 39 and 41 of the MYH9 gene, and the mutation site was determined by Sanger sequencing. RESULTS: All patients from the pedigree presented a typical triad of thrombocytopenia, giant platelets, and inclusion bodies in leukocytes. In addition, two patients had nephritis and cataract. All affected members carried a heterozygous missense mutation of c.5521G>A (p.glu1841Lys) in exon 39 of the MYH9 gene. The same mutation was not found among healthy members of the pedigree and the controls. CONCLUSION: The c.5521G>A (p.Glu1841Lys) mutation in the MYH9 gene probably underlies the MYH9-related disease in this pedigree.

N-methyl-N-nitrosourea induces retinal degeneration in the rat via the inhibition of NF-κB activation.

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N-methyl-N-nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF-κB) in MNU-induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal-pigmented epithelial cells in MNU-treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral-domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF-κB p65 decreased significantly in the retinas of MNU-treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF-κB activation.

Anion Binding Interaction Enhances the Robustness of Iodide for High-Performance Perovskite Solar Cells.

Owing to the ionic bond nature of the Pb-I bond, the iodide at the interface of perovskite polycrystalline films was easily lost during the preparation process, resulting in the formation of a large number of iodine vacancy defects. The presence of iodine vacancy defects can cause nonradiative recombination, provide a pathway for iodide migration, and be harmful to the power conversion efficiency (PCE) and stability of organic-inorganic hybrid perovskite solar cells (HPSCs). Here, in order to increase the robustness of iodides at the interface, a strategy to introduce anion binding effects was developed to stabilize the perovskite films. It was demonstrated that the N,N'-diphenylurea (DPU), characterized by high anionic binding constants and a Y-shaped structure, provides a relatively strong hydrogen bond donor site to effectively reduce the iodine loss during film preparation and inhibits iodide migration in the device working condition. As expected, the reduced iodine loss considerably improves the quality of the perovskite films and suppresses nonradiative recombination. The performance of the device after DPU modification was significantly increased, with the PCE rising from 23.65 to 25.01% with huge stability enhancement as well.

Did you choose appropriate tracer for retrograde tracing of retinal ganglion cells? The differences between cholera toxin subunit B and Fluorogold.

Cholera toxin subunit B (CTB) and Fluorogold(FG) are two widely utilized retrograde tracers to assess the number and function of retinal ganglion cells (RGCs). However, the relative advantages and disadvantages of these tracers remain unclear, which may lead to their inappropriate application. In this study, we compared these tracers by separately injecting the tracer into the superior Colliculi (SC) in rats, one or 2 weeks later, the rats were sacrificed, and their retinas, brains, and optic nerves were collected. From the first to second week, FG displayed a greater number of labeled RGCs and a larger diffusion area in the SC than CTB; The number of CTB labeled RGCs and the diffusion area of CTB in the SC increased significantly, but there was no distinction between FG; Furthermore, CTB exhibited more labeled RGC neurites and longer neurites than FG, but no difference was evident between the same trace; The optic nerves labeled using CTB were much clearer than those labeled using FG. In conclusion, both CTB and FG can be used for the retrograde labeling of RGCs in rats at 1 or 2 weeks. FG achieves retrograde labeling of a greater number of RGCs than CTB, whereas CTB better delineates the morphology of RGCs. Furthermore, CTB seems more suitable for retrograde labeling of some small, non-image forming nuclei in the brain to which certain RGC subtypes project their axons.

Which has more stem-cell characteristics: Müller cells or Müller cells derived from in vivo culture in neurospheres?

OBJECTIVE: Müller cells can be acquired from in vitro culture or a neurosphere culture system. Both culture methods yield cells with progenitor-cell characteristics that can differentiate into mature nervous cells. We compared the progenitor-cell traits of Müller cells acquired from each method. METHODS: Primary murine Müller cells were isolated in serum culture media and used to generate Müller cells derived from neurospheres in serum-free culture conditions. Gene expression of neural progenitor cell markers was examined by Q-PCR in the two groups. Expression of rhodopsin and the cone-rod homeobox protein CRX were assessed after induction with 1 µM all-trans retinoic acid (RA) for 7 days. RESULTS: After more than four passages, many cells were large, flattened, and difficult to passage. A spontaneously immortalized Müller cell line was not established. Three-passage neurospheres yielded few new spheres. Genes coding for Nestin, Sox2, Chx10, and Vimentin were downregulated in cells derived from neurospheres compared to the cells from standard culture, while Pax6 was upregulated. Müller cells from both culture methods were induced into rod photoreceptors, but expression of rhodopsin and CRX was greater in the Müller cells from the standard culture. CONCLUSION: Both culture methods yielded cells with stem-cell characteristics that can be induced into rod photoreceptor neurons by RA. Serum had no influence on the "stemness" of the cells. Cells from standard culture had greater "stemness" than cells derived from neurospheres. The standard Müller cells would seem to be the best choice for transplantation in cell replacement therapy for photoreceptor degeneration.

MicroRNA-28 potentially regulates the photoreceptor lineage commitment of Müller glia-derived progenitors.

Retinal degenerative diseases ultimately result into irreversible photoreceptor death or loss. At present, the most promising treatment for these diseases is cell replacement therapy. Müller glia are the major glia in the retina, displaying cardinal features of retinal progenitor cells, and can be candidate of seed cells for retinal degenerative diseases. Here, mouse retinal Müller glia dissociated and cultured in vitro amplified and were dedifferentiated into Müller glia-derived progenitors (MGDPs), demonstrating expression of stem/progenitor cell markers Nestin, Sox2 and self-renewal capacity. MicroRNAs (miRNAs) play unique roles in the retinogenesis, so we hypothesized miRNAs would contribute to photoreceptor lineage commitment of MGDPs. By TargetScan, Miranda, and Pictar bioinformatics, gain/loss-of-function models, dual luciferase assay, we identified and validated that miR-28 targeted the photoreceptor-specific CRX transcription factor. Anti-miR-28 could induce MGDPs to differentiate into neurons strongly expressing CRX and Rhodopsin, while miR-28 mimic suppressed CRX and Rhodopsin expression. Knockdown of CRX by siRNA blocked the expression of CRX and Rhodospin upregulated by anti-miR-28, indicating that anti-miR-28 potentially induced photoreceptor commitment of MGDPs by targeting CRX, but more experiments are necessary to confirm their role in differentiation.

[Expression and significance of DNA-dependent protein kinase in human laryngeal squamous cell carcinoma].

OBJECTIVE: To study the expression of DNA-dependent protein kinase (DNA-PK) in human laryngeal squamous cell carcinoma (LSCC) and normal laryngeal mucosa (NLM), and to analysize the relationship between the expression and the clinicopathologic parameters of LSCC. METHOD: Immunohistochemical technique (Envision) was used to detect the expression of DNA-PK in 64 cases of LSCC and 15 cases of NLM. To investigate an investigation was conducted on the relationship between the expression and clinico-pathological features of LSCC. RESULT: DNA-PK was lowly expressed in NLM and highly expressed in LSCC,the positive rate of DNA-PK expression was 26.67% (4/15), 78.13% (50/64), respectively, and there was significant different difference between the two groups (P < 0.05). Its expression was correlated with the level of histodifferentiation (P < 0.05), but not with TNM stages and neck lymph node metastasis (P > 0.05). CONCLUSION: DNA-PK may be involved in disease development of LSCC.

Dynamic changes of tear fluid matrix metralloproteinase-9 within 1 year after laser in situ keratomileusis.

OBJECTIVE: To investigate the dynamic changes of matrix metalloproteinase-9 (MMP-9) level in tear fluid within 12 months after laser in situ keratomileusis (LASIK). METHODS: Twenty-two myopic patients undergoing uneventful LASIK were enrolled in this study. Tear fluid samples were collected from the patients for measurements of MMP-9 level using Western blotting preoperatively, at 7 and 14 days, and at 1, 2, 3, 6, and 12 months after the surgery. RESULTS: MMP-9 concentrations in the tear fluid of post-LASIK patients showed a time-dependent variation pattern. MMP-9 reached its peak level in the tear fluid at 14 days postoperatively, which was 2.70 times the preoperative level; it gradually decreased thereafter but was still 1.38 times the preoperative level at 12 months after the surgery. CONCLUSIONS: MMP-9 concentrations in the tear fluid of post-LASIK patients show a time-dependent variation pattern and remains higher than the preoperative level even at 12 months after the surgery, suggesting that corneal wound healing after LASIK lasts for more than 12 months.