The single-cell landscape of alternative transcription start sites of diabetic retina.

More than half of mammalian protein-coding genes have multiple transcription start sites. Alternative transcription start site (TSS) modulate mRNA stability, localization, and translation efficiency on post-transcription level, and even generate novel protein isoforms. However, differential TSS usage among cell types in healthy and diabetic retina remains poorly characterized. In this study, by using 5'-tag-based single-cell RNA sequencing, we identified cell type-specific alternative TSS events and key transcription factors for each of retinal cell types. We observed that lengthening of 5'- UTRs in retinal cell types are enriched for multiple RNA binding protein binding sites, including splicing regulators Rbfox1/2/3 and Nova1. Furthermore, by comparing TSS expression between healthy and diabetic retina, we identified elevated apoptosis signal in Müller glia and microglia, which can be served as a putative early sign of diabetic retinopathy. By measuring 5'UTR isoforms in retinal single-cell dataset, our work provides a comprehensive panorama of alternative TSS and its potential consequence related to post-transcriptional regulation. We anticipate our assay can not only provide insights into cellular heterogeneity driven by transcriptional initiation, but also open up the perspectives for identification of novel diagnostic indexes for diabetic retinopathy.

A three-step approach to close refractory persistent macular holes: a releasing-closing-tapping approach.

PURPOSE: The aim of this study was to assess the efficacy and safety of a novel releasing-closing-tapping approach in the treatment of persistent macular holes (PMHs) after initial surgery with internal limiting membrane (ILM) peeling. METHODS: We retrospectively analyzed patients with PMHs after initial surgery with ILM peeling who were treated with a novel releasing-closing-tapping approach. After repeated pars plana vitrectomy (PPV), the surgeon effectively released the adhesion between the edges and retinal pigment epithelium (RPE) by gently scraping the retinal neuroepithelium. Then, the hole was converted into a transverse slit, and the edges were gently tapped flat so that they attached to the RPE, and no space was left under the edges. Finally, air tamponade was carried out. The primary outcome measures included MH closure and the change in best-corrected visual acuity (BCVA) from preoperatively to postoperatively. RESULTS: The study included 11 PMH patients with a mean age of 63.82 ± 3.31 years. The mean minimum linear diameter of PMHs was 666.3 ± 208.1 µm, and the mean basal diameter was 1547.2 ± 351.8 µm. MH closure was achieved in 90.9% (10/11) of eyes, with significant improvement of visual acuity from 1.19 ± 0.30 logMAR to 0.65 ± 0.29 logMAR postoperatively. CONCLUSION: The releasing-closing-tapping approach with repeated PPV is a simple, effective, and safe surgical procedure for refractory PMHs after initial surgery with ILM peeling that can significantly improve the visual outcome and achieve a high surgical success rate.

Comprehensive Analysis of the Immune Cell Infiltration Landscape and Immune-Related Methylation in Retinoblastoma.

Retinoblastoma is a common pediatric intraocular cancer, originating from cone precursors. The development of immunotherapies can help eradicate the tumor without vision loss, which would largely improve the quality of life of patients with retinoblastoma. Investigation of the tumor immune microenvironment provides knowledge for developing novel immunotherapies in cancer. However, the immune cell infiltrative landscape of retinoblastoma is unknown. Here, we compared the relative expression of immune gene signatures among 59 patients with retinoblastoma. The patients were divided into two subgroups according to the 28 types of immune cell infiltration (ICI) scores. We found that a subgroup with high ICI scores had increased expression levels of late cone markers, while the other subgroup exhibited larger tumor size and metastasis propensity. Furthermore, hypermethylated genes in the high-ICI subgroup were associated with immune regulation in the tumor microenvironment, suggesting that DNA methylation may play a vital regulatory role in retinoblastoma immunity. Our study provides a comprehensive framework for the systemic analysis of the influences of epigenetic events on the tumor immune microenvironment. We anticipate that our assay can not only provide insights into tumor immune regulation but also open up the perspectives for the identification of novel immunotherapy targets for retinoblastoma.

Single-Cell Transcriptomic Profiling of Human Retinal Organoids Revealed a Role of IGF1-PHLDA1 Axis in Photoreceptor Precursor Specification.

Purpose: Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. Methods: To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches. Results: Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development. Conclusions: Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.

Somatostatin signalling promotes the differentiation of rod photoreceptors in human pluripotent stem cell-derived retinal organoid.

OBJECTIVES: Stem cell-derived photoreceptor replacement therapy is a promising strategy for the treatment of retinal degenerative disease. The development of 3D retinal organoids has permitted the production of photoreceptors. However, there is no strategy to enrich a specific photoreceptor subtype due to inadequate knowledge of the molecular mechanism underlying the photoreceptor fate determination. Hence, our aim is to explore the uncharacterized function of somatostatin signalling in human pluripotent stem cell-derived photoreceptor differentiation. MATERIALS AND METHODS: 3D retinal organoids were achieved from human embryonic stem cell. The published single-cell RNA-sequencing datasets of human retinal development were utilized to further investigate the transcriptional regulation of photoreceptor differentiation. The assays of immunofluorescence staining, lentivirus transfection, real-time quantitative polymerase chain reaction and western blotting were performed. RESULTS: We identified that the somatostatin receptor 2 (SSTR2)-mediated signalling was essential for rod photoreceptor differentiation at the precursor stage. The addition of the cognate ligand somatostatin in human 3D retinal organoids promoted rod photoreceptor differentiation and inhibited cone photoreceptor production. Furthermore, we found that the genesis of rod photoreceptors was modulated by endogenous somatostatin specifically secreted by developing retinal ganglion cells. CONCLUSIONS: Our study identified SSTR2 signalling as a novel extrinsic regulator for rod photoreceptor fate determination in photoreceptor precursors, which expands the repertoire of functional signalling pathways in photoreceptor development and sheds light on the optimization of the photoreceptor enrichment strategy.

Long noncoding RNA BLACAT1 indicates a poor prognosis of colorectal cancer and affects cell proliferation by epigenetically silencing of p15.

Recently, a novel class of transcripts, long noncoding RNAs (lncRNAs), is being identified at a rapid pace. These RNAs have critical roles in diverse biological processes, including tumorigenesis. One of them, BLACAT1, a cancer-associated long noncoding RNA, exerts regulatory functions in various biological processes in cancer cells, however, the role of BLACAT1 in colon cancer remains unclear. Our experiments showed that increased BLACAT1 was an independent unfavorable prognostic indicator for colorectal cancer, and revealed that BLACAT1 knockdown significantly repressed proliferation, both in vitro and in vivo. Mechanistic investigations demonstrated that BLACAT1 had a key role in G1/G0 arrest, and showed that BLACAT1 can repress p15 expression by binding to EZH2, thus contributing to the regulation of CRC cell cycle and proliferation. Our results suggest that BLACAT1, as a cell cycle regulator, may serve as a potential target for colon cancer prevention and treatment in human CRC.