Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders

Capacity of Retinal Ganglion Cells Derived from Human Induced Pluripotent Stem Cells to Suppress T-Cells

Retinal ganglion cells (RGCs) are impaired in patients such as those with glaucoma and optic neuritis, resulting in permanent vision loss. To restore visual function, development of RGC transplantation therapy is now underway. Induced pluripotent stem cells (iPSCs) are an important source of RGCs for human allogeneic transplantation. We therefore analyzed the immunological characteristics of iPSC-derived RGCs (iPSC-RGCs) to evaluate the possibility of rejection after RGC transplantation. We first assessed the expression of human leukocyte antigen (HLA) molecules on iPSC-RGCs using immunostaining, and then evaluated the effects of iPSC-RGCs to activate lymphocytes using the mixed lymphocyte reaction (MLR) and iPSC-RGC co-cultures. We observed low expression of HLA class I and no expression of HLA class II molecules on iPSC-RGCs. We also found that iPSC-RGCs strongly suppressed various inflammatory immune cells including activated T-cells in the MLR assay and that transforming growth factor-β2 produced by iPSC-RGCs played a critical role in suppression of inflammatory cells in vitro. Our data suggest that iPSC-RGCs have low immunogenicity, and immunosuppressive capacity on lymphocytes. Our study will contribute to predicting immune attacks after RGC transplantation.This work was supported by the Research Center Network for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) to M.T., and by a Scientific Research Grant (B, 18H02959) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to S.S. A.E. was financially supported from RIKEN by a Junior Research Associate (JRA) program for graduate students

Stereocontrolled spirocyclization of exo-glucal derivatives for stereodivergent synthesis of spiro[5.5]ketals

This Letter describes a stereoselective synthetic approach to spiro[5.5]ketals from the exo-glucals, constructed from 1-(4-hydroxyalkylidene)-1,5-anhydro-d-glucitol, by spirocyclization based on intramolecular α- and β-selective glycosylation. Stereodivergent synthesis of both α- and β-glucoside of spiro[5.5]ketals was attained by combination of chiral d-glucopyranose environment and C-4′ chiral center on the side chain

A New Dyspnea Evaluation System Focusing on Patients’ Perceptions of Dyspnea and Their Living Disabilities: The Linkage between COPD and Frailty

Background: As much as there are unmet needs for brief frailty assessment in patients with chronic obstructive pulmonary disease (COPD), the lack of a simplified and comprehensive dyspnea evaluation system that focuses on the patients’ perceptions of dyspnea and their COPD living disabilities remains a major challenge. We developed patient-reported outcome measures for dyspnea-related behavior and activity limitation (PROMs-D), which consisted of the Activity-limit Dyspnea Scale (ADS) and Self-Limit Dyspnea Scale (SDS), while investigated the usefulness of PROMs-D in identifying frailty. Methods: We administered PROMs-D and frailty status evaluations in 128 outpatients. Results: We classified 30 (23.4%), 50 (39.0%), and 48 (37.5%) patients as robust, prefrail, and frail, respectively. There was a positive correlation between SDS and ADS (ρ = 0.67, p < 0.001), and both ADS and SDS had high accuracies for detecting frailty (AUC, 0.82 and 0.78, respectively). Moreover, a PROMs-D score that consisted of the sum of ADS and SDS was more effective in stratifying frailty (cutoff value, 2; AUC, 0.85; sensitivity, 60%; specificity, 95%). Conclusions: PROMs-D could be used as the first step for frailty screening in patients with COPD, and we propose the importance of capturing the troublesome nature of living behaviors due to dyspnea in daily clinical practice

Enhanced Expression of IGFBP-3 Reduces Radiosensitivity and Is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) modulates various cell functions through IGF-dependent or independent mechanisms. However, its biological roles in the radiosensitivity of oral squamous cell carcinoma (OSCC) remain largely unknown. The purpose of this study was to determine the clinical significance and molecular mechanisms of the association between IGFBP-3 and OSCC radiosensitivity. We performed an immunohistochemical analysis of IGFBP-3 in 52 OSCC specimens from patients treated with preoperative chemoradiotherapy and surgery (phase II study). Associations between IGFBP-3 expression and clinicopathological features were also evaluated. In addition, we examined the effects of IGFBP-3 on post-X-ray irradiation radiosensitivity and DNA damage in vitro. High IGFBP-3 expression was significantly correlated with poor chemoradiotherapy responses and prognosis. With IGFBP-3 knockdown, irradiated OSCC cells exhibited significantly higher radiosensitivity compared with that of control cells. Moreover, IGFBP-3 depletion in OSCC cells reduced phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is required for DNA double-strand break repair during non-homologous end joining. These findings indicate that IGFBP-3 may have a significant role in regulating DNA repair and is be a potential biomarker for predicting clinical response to radiotherapy and prognosis in OSCC