Effect of Posterior Keratometry on the Accuracy of 10 Intraocular Lens Calculation Formulas: Standard Keratometry versus Total Keratometry.

PURPOSE: To investigate the effect of posterior keratometry (PK) on the accuracy of 10 intraocular lens (IOL) power calculation formulas using standard keratometry (K) and total keratometry (TK). METHODS: This is a retrospective consecutive case-series study. The IOL power was calculated using K and TK measured by IOLMaster 700 in 6 new-generation formulas (Barrett Universal II, Emmetropia Verifying Optical (EVO) 2.0, RBF Calculator 3.0, Hoffer QST, Kane, and Ladas Super Formula) and 4 traditional formulas (Haigis, Hoffer Q, Holladay 1, and SRK/T). The arithmetic prediction error (PE) and mean absolute PE (MAE) were evaluated. The locally-weighted scatterplot smoothing was performed to assess the relationship between PE and PK. RESULTS: A total of 576 patients (576 eyes) who underwent cataract surgery were included. Compared with using K, all formulas using TK showed a hyperopic shift in the whole group. Specifically, for eyes with PK exceeding -5.90 D, all formulas using TK exhibited a hyperopic shift (all P < 0.001), while eyes with PK less than -5.90 D showed a myopic shift (all P < 0.001). The MAE of new-generation formulas calculated with TK and K showed no statistical differences, while the MAE of traditional formulas with TK was larger (TK: 0.34 ~ 0.43 D; K: 0.33 ~ 0.42 D, all P < 0.05). CONCLUSIONS: The prediction bias of formulas with TK increased as PK deviated from -5.90 D. TK did not improve the prediction accuracy of new-generation formulas, and even performed worse in traditional formulas.

RNA fusion in human retinal development.

Chimeric RNAs have been found in both cancerous and healthy human cells. They have regulatory effects on human stem/progenitor cell differentiation, stemness maintenance, and central nervous system development. However, whether they are present in human retinal cells and their physiological functions in the retinal development remain unknown. Based on the human embryonic stem cell-derived retinal organoids (ROs) spanning from days 0 to 120, we present the expression atlas of chimeric RNAs throughout the developing ROs. We confirmed the existence of some common chimeric RNAs and also discovered many novel chimeric RNAs during retinal development. We focused on CTNNBIP1-CLSTN1 (CTCL) whose downregulation caused precocious neuronal differentiation and a marked reduction of neural progenitors in human cerebral organoids. CTCL is universally present in human retinas, ROs, and retinal cell lines, and its loss-of-function biases the progenitor cells toward retinal pigment epithelial cell fate at the expense of retinal cells. Together, this work provides a landscape of chimeric RNAs and reveals evidence for their critical role in human retinal development.

Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSCs.

RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing. RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation, RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.

Genetic detection of two novel LRP5 pathogenic variants in patients with familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a genetic eye disorder that leads to abnormal development of retinal blood vessels, resulting in vision impairment. This study aims to identify pathogenic variants by targeted exome sequencing in 9 independent pedigrees with FEVR and characterize the novel pathogenic variants by molecular dynamics simulation. METHODS: Clinical data were collected from 9 families with FEVR. The causative genes were screened by targeted next-generation sequencing (TGS) and verified by Sanger sequencing. In silico analyses (SIFT, Polyphen2, Revel, MutationTaster, and GERP + +) were carried out to evaluate the pathogenicity of the variants. Molecular dynamics was simulated to predict protein conformation and flexibility transformation alterations on pathogenesis. Furthermore, molecular docking techniques were employed to explore the interactions and binding properties between LRP5 and DKK1 proteins relevant to the disease. RESULTS: A 44% overall detection rate was achieved with four variants including c.4289delC: p.Pro1431Argfs*8, c.2073G > T: p.Trp691Cys, c.1801G > A: p.Gly601Arg in LRP5 and c.633 T > A: p.Tyr211* in TSPAN12 in 4 unrelated probands. Based on in silico analysis and ACMG standard, two of them, c.4289delC: p.Pro1431Argfs*8 and c.2073G > T: p.Trp691Cys of LRP5 were identified as novel pathogenic variants. Based on computational predictions using molecular dynamics simulations and molecular docking, there are indications that these two variants might lead to alterations in the secondary structure and spatial conformation of the protein, potentially impacting its rigidity and flexibility. Furthermore, these pathogenic variants are speculated to potentially influence hydrogen bonding interactions and could result in an increased binding affinity with the DKK1 protein. CONCLUSIONS: Two novel genetic variants of the LRP5 gene were identified, expanding the range of mutations associated with FEVR. Through molecular dynamics simulations and molecular docking, the potential impact of these variants on protein structure and their interactions with the DKK1 protein has been explored. These findings provide further support for the involvement of these variants in the pathogenesis of the disease.

REG1A protects retinal photoreceptors from blue light damage.

With the increased use of artificial light and the prolonged use of optoelectronic products, light damage (LD) to the human retina has been identified as a global vision-threatening problem. While there is evidence of a significant correlation between light-induced retinal damage and age-related vision impairment in age-related macular degeneration, it is unclear how light-induced retinal degeneration manifests itself and whether there are agents capable of preventing the development of LD in the retina. This study investigated a mechanism by which blue light leads to photoreceptor death. By observing blue light exposure in retinal organoids and photoreceptor cells, we concluded that there could be significant apoptosis of the photoreceptors. We demonstrate that regenerating islet-derived 1 alpha (REG1A) prevents photoreceptors from undergoing this LD-induced apoptosis by increasing expression of the anti-apoptotic gene Bcl2 and downregulating expression of the pro-apoptotic gene Bax, resulting in reduced mitochondrial damage and improved aerobic capacity in photoreceptor cells. For the first time, REG1A has been shown to restore mitochondrial function and cell apoptosis after LD-induced damage, suggesting its potential application in the prevention and treatment of retinal vision loss.

Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory.

Retinoblastoma (Rb) is a type of malignant tumor due to abnormal retinogenesis with biallelic mutations of the RB1 gene. Its pathogenesis has been proposed as a "two-mutation hypothesis" by Knudson since 1971; however, there remain some debates on disease onset sufficiency of the biallelic RB1 mutations. To obtain straightforward evidence for this hypothesis, we investigated whether two-hit mutations of the RB1 gene drive tumorigenesis in patient-induced pluripotent stem cell (hiPSC)-derived human retinal organoids (hROs) and whether single allelic mutation hiPSC-derived hROs exhibit molecular and cellular defects. We generated hiPSCs with a heterozygous germline mutation (RB1m1/ wt ) from a Rb patient. A second-allele RB1 gene mutation was knocked in to produce compound heterozygous mutations (RB1m1/m2 ) in the hiPSCs. These two hiPSC lines were independently developed into hROs through a stepwise differentiation. The hiPSC-RB1m1/m2 derived organoids demonstrated tumorigenesis in dishes, consistent with Rb profiles in spatiotemporal transcriptomes, in which developmentally photoreceptor fate-determining markers, CRX and OTX2, were highly expressed in hiPSC-RB1m1/m2 derived hROs. Additionally, ARR3+ maturing cone precursors were co-labeled with proliferative markers Ki67 or PCNA, in agreement with the consensus that human Rb is originated from maturing cone precursors. Finally, we demonstrated that retinal cells of hROs with monoallelic RB1 mutation were abnormal in molecular aspects due to its haploinsufficiency. In conclusion, this study provides straightforward supporting evidence in a way of reverse genetics for "two-hit hypothesis" in the Rb tumorigenesis and opens new avenues for development of early intervention and treatment of Rb.

Retinal Organoid Technology: Where Are We Now?

It is difficult to regenerate mammalian retinal cells once the adult retina is damaged, and current clinical approaches to retinal damages are very limited. The introduction of the retinal organoid technique empowers researchers to study the molecular mechanisms controlling retinal development, explore the pathogenesis of retinal diseases, develop novel treatment options, and pursue cell/tissue transplantation under a certain genetic background. Here, we revisit the historical background of retinal organoid technology, categorize current methods of organoid induction, and outline the obstacles and potential solutions to next-generation retinal organoids. Meanwhile, we recapitulate recent research progress in cell/tissue transplantation to treat retinal diseases, and discuss the pros and cons of transplanting single-cell suspension versus retinal organoid sheet for cell therapies.

A Novel Mutation p.S93R in CRYBB1 Associated with Dominant Congenital Cataract and Microphthalmia.

Purpose: To identify the pathogenetic mutations in a four-generation Chinese family with dominant congenital cataracts and microphthalmia.Methods: A four-generation Chinese family with dominant congenital cataracts were recruited. Genomic DNAs were collected from their peripheral blood leukocytes and subjected to whole exome sequencing. The genetic mutations were identified by bioinformatic analyses and verified by Sanger sequencing.Results: Whole exome sequencing revealed a c.279C>G point mutation in the CRYBB1 gene which was further verified by Sanger sequencing. The nucleotide replacement results in a novel mutation p.S93R in a conserved residue of ßB1 crystallin which is predicted to disrupt normal ßB1 structure and function.Conclusions: We identified a novel missense mutation p.S93R in CRYBB1 in a Chinese family with autosomal dominant congenital cataracts and microphthalmia. This serine residue is extremely conserved evolutionarily in more than 50 ßγ-crystallins of many species. These data will be very helpful to further understand the structural and functional features of crystallins.

Coexpression of YY1 Is Required to Elaborate the Effector Functions Controlled by PLZF in NKT Cells.

The promyelocytic leukemia zinc-finger transcription factor (PLZF) is essential for nearly all of the unique, innate-like functions and characteristics of NKT cells. It is not known, however, if the activity of PLZF is regulated by other factors. In this article, we show that the function of PLZF is completely dependent on the transcription factor Yin Yang 1 (YY1). Mouse NKT cells expressing wild-type levels of PLZF, but deficient for YY1, had developmental defects, lost their characteristic "preformed" mRNA for cytokines, and failed to produce cytokine protein upon primary activation. Immunoprecipitation experiments showed that YY1 and PLZF were coassociated. Taken together, these biochemical and genetic data show that the broadly expressed transcription factor, YY1, is required for the cell-specific "master regulator" functions of PLZF.

Necessity and Sufficiency of Ldb1 in the Generation, Differentiation and Maintenance of Non-photoreceptor Cell Types During Retinal Development.

During mammalian retinal development, the multipotent progenitors differentiate into all classes of retinal cells under the delicate control of transcriptional factors. The deficiency of a transcription cofactor, the LIM-domain binding protein Ldb1, has been shown to cause proliferation and developmental defects in multiple tissues including cardiovascular, hematopoietic, and nervous systems; however, it remains unclear whether and how it regulates retinal development. By expression profiling, RNA in situ hybridization and immunostaining, here we show that Ldb1 is expressed in the progenitors during early retinal development, but later its expression gradually shifts to non-photoreceptor cell types including bipolar, amacrine, horizontal, ganglion, and Müller glial cells. Retina-specific ablation of Ldb1 in mice resulted in microphthalmia, optic nerve hypoplasia, retinal thinning and detachment, and profound vision impairment as determined by electroretinography. In the mutant retina, there was precocious differentiation of amacrine and horizontal cells, indicating a requirement of Ldb1 in maintaining the retinal progenitor pool. Additionally, all non-photoreceptor cell types were greatly reduced which appeared to be caused by a generation defect and/or retinal degeneration via excessive cell apoptosis. Furthermore, we showed that misexpressed Ldb1 was sufficient to promote the generation of bipolar, amacrine, horizontal, ganglion, and Müller glial cells at the expense of photoreceptors. Together, these results demonstrate that Ldb1 is not only necessary but also sufficient for the development and/or maintenance of non-photoreceptor cell types, and implicate that the pleiotropic functions of Ldb1 during retinal development are context-dependent and determined by its interaction with diverse LIM-HD (LIM-homeodomain) and LMO (LIM domain-only) binding protein partners.

In vitro explant culture and related protocols for the study of mouse retinal development.

The mouse retina is composed of many cell types and subtypes with distinct morphology and function; how these cells are differentiated from the multipotent progenitors is still largely unknown. Retinal in vitro explant culture has proven to be a useful tool to study the molecular and cellular mechanisms underlying retinal development. Here, we provide detailed descriptions about how to prepare retroviruses, dissect retinal cups, perform in vitro explant culture, and collect explant samples.