Prolonged survival of the phosphorylated form of rhodopsin during dark adaptation of Royal College Surgeons rat

AbstractTo study rhodopsin (Rho) phosphorylation and dephosphorylation in Royal College of Surgeons (RCS) rat retina, specific antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser, were prepared by immunization of authentic phosphorylated peptides in rabbit. Enzyme-linked immunosorbent assay identified that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immunofluorescence labeling, both antibodies recognized photoreceptor outer segments in light-adapted retinas from Sprague–Dawley (SD), Brown–Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward phosphorylated 338Ser and 334Ser sites were diminished within several hours (0.2–2 h) in SD and BN rat retinas. However, those toward phosphorylated 338Ser and 334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly affected than the phosphorylation reaction. Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat

Review : Clinical and Pathological Aspects of Cancer-Associated Retinopathy (CAR)

Cancer-associated retinopathy (CAR) is an ocular manifestation of para-neoplastic syndrome and is further classified into two distinct diseases, CAR and MAR (melanoma-associated retinopathy) . CAR is associated with epithe-lial cancers, mostly lung small cell carcinoma, and is characterized by retini-tis pigmentosa-like retinal degeneration, such as ring scotoma, night blind-ness, and lowering of a and b-waves in electroretinogram (ERG) . Usually CAR can be found before an underlying primary cancer is diagnosed. In con-trast, MAR is associated with cutaneous malignant melanoma and is characte-rized by the relatively sudden onset of photophobia, nyctalopia, and lowering of b-wave in ERG. Histopathological study has revealed that photoreceptor and bipolar degeneration are primarily involved in CAR and MAR, respec-tively. Both CAR and MAR are believed to result from an autoimmune basis. In CAR, a calcium binding protein called recoverin, heat shock cog-nate protein 70 (hsc 70) , and other proteins are identified as retinal autoanti-gens, while the retinal antigens in MAR have not yet been identified. In this text, we review up-to-date information about the clinical and pathological aspects of these diseases

Identification of Antigenic Site Within hsc 70 by Serum Autoantibody in Patients with Cancer-associated Retinopathy

In our previous studies, both recoverin and heat shock cognate protein 70 (hsc 70) were found as autoantigens recognized by sera from four patients with cancer-associated retinopathy (CAR) . In the present study on the mole-cular mechanism of antibody generation in CAR, we identified the antigenic site within hsc 70 by the patients\u27 sera using deletion mutants of hsc 73. We expressed a series of deletion mutants of hsc 73 proteins and subjected then to western blot analysis. In western blot analysis, CAR patient\u27s serum reacted with wild type hsp 70, but not with the C-terminal truncated mutants. This data demonstrated that the antigenic site was located within the C-terminus region of hsc 73 as identified by CAR patient\u27s serum

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin’s stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration

Interferon-α/β and Anti-Fibroblast Growth Factor Receptor 1 Monoclonal Antibody Suppress Hepatic Cancer Cells In Vitro and In Vivo

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC..Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

Trends of Stem Cell Therapies in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a highly prevalent irreversible impairment in the elderly population worldwide. Stem cell therapies have been considered potentially viable for treating AMD through the direct replacement of degenerated cells or secretion of trophic factors that facilitate the survival of existing cells. Among them, the safety of pluripotent stem cell-derived retinal pigment epithelial (RPE) cell transplantation against AMD, and some hereditary retinal degenerative diseases, has been discussed to a certain extent in clinical studies of RPE cell transplantation. Preparations are in progress for its clinical application. On the other hand, clinical trials using somatic stem cells are also being conducted, though these had controversial outcomes. Retinal regenerative medicine using stem cells is expected to make steady progress toward practical use while new technologies are incorporated from various fields, thereby making the role of ophthalmologists in this field increasingly important