Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.Subject terms: Translational research, Stem-cell research, Retina     

Structural Coupling of 11‐cis‐7‐Methyl‐retinal and Amino Acids at the Ligand Binding Pocket of Rhodopsin†

It was previously shown that opsin can be regenerated with the newly synthesized 11‐cis‐7‐methyl‐retinal forming an artificial visual pigment. We now extend this study to include mutants at positions close to the retinal to further dissect the interactions of native and artificial chromophores with opsin. Several mutants at M207, W265 and Y268 have been obtained and regenerated with 11‐cis‐retinal and the 7‐methyl analog. M207 is the site of the point mutation M207R associated with the retinal degenerative disease retinitis pigmentosa. All the studied mutants regenerated with 11‐cis‐retinal except for M207C which proved to be completely misfolded. The naturally occurring M207R mutant formed a pigment with an unprotonated Schiff base linkage, altered photobleaching and low MetarhodopsinII stability. Mutants regenerated with the 7‐methyl analog showed altered photobleaching reflecting a structural perturbation in the vicinity of M207. The newly obtained mutants at M207 also showed reduced levels of transducin activation with M207R showing essentially no transducin activation. Our results highlight the tight coupling of the vicinity of C7 of retinal and M207 and support the involvement of this amino acid residue in the conformational changes associated with rhodopsin photoactivation.     

Retinal Photodamage Mediated by All‐trans‐retinal†

Accumulation of all‐trans‐retinal (all‐trans‐RAL), reactive vitamin A aldehyde, is one of the key factors in initiating retinal photodamage. This photodamage is characterized by progressive retinal cell death evoked by light exposure in both an acute and chronic fashion. Photoactivated rhodopsin releases all‐trans‐RAL, which is subsequently transported by ATP‐binding cassette transporter 4 and reduced to all‐trans‐retinol by all‐trans‐retinol dehydrogenases located in photoreceptor cells. Any interruptions in the clearing of all‐trans‐RAL in the photoreceptors can cause an accumulation of this reactive aldehyde and its toxic condensation products. This accumulation may result in the manifestation of retinal dystrophy including human retinal degenerative diseases such as Stargardt’s disease and age‐related macular degeneration. Herein, we discuss the mechanisms of all‐trans‐RAL clearance in photoreceptor cells by sequential enzymatic reactions, the visual (retinoid) cycle, and potential molecular pathways of retinal photodamage. We also review recent imaging technologies to monitor retinal health status as well as novel therapeutic strategies preventing all‐trans‐RAL‐associated retinal photodamage.     

Effect of visible light on normal and P23H-3 transgenic rat retinas: characterization of a novel retinoic acid derivative present in the P23H-3 retina

Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.     

Cherry antioxidants: from farm to table

The dietary consumption of fruits and vegetables is associated with a lower incidence of degenerative diseases such as cardiovascular disease and certain types of cancers. Most recent interest has focused on the bioactive phenolic compounds found in vegetable products. Sweet and sour cherries contain several antioxidants and polyphenols that possess many biological activities, such as antioxidant, anticancer and anti-inflammation properties. The review describes the effect of environment and other factors (such as production, handling and storage) on the nutritional properties of cherries, with particular attention to polyphenol compounds. Moreover the health benefits of cherries and their polyphenols against human diseases such as heart disease, cancers, diabetes are reviewed.     

Gq-coupled rhodopsin subfamily composed of invertebrate visual pigment and melanopsin

Rhodopsins (rhodopsins and their related photopigments) are phylogenetically classified into at least seven subfamilies, which are also roughly discriminated by molecular function. The Gq-coupled rhodopsin subfamily, members of which activate the Gq type G protein upon light absorption, contains pigments which underlie both visual and nonvisual physiologic functions. Gq-coupled visual pigments have been found in the rhabdomeric photoreceptor cells of varied protostomes, and those of molluskans and arthropods have been extensively investigated. Recently, a novel photopigment, melanopsin, and its homologs have been identified in varied vertebrates. In mammals, melanopsin is localized in retinal ganglion cells and is involved in nonvisual systems, including circadian entrainment and pupillary light responses. More recently, we discovered a melanopsin homolog in amphioxus, the closest living invertebrate to vertebrates. Amphioxus melanopsin is localized in putative nonvisual photoreceptor cells with rhabdomeric morphology and exhibits molecular properties almost identical to those of invertebrate Gq-coupled visual pigments. The localization and properties of amphioxus melanopsin bridged the functional and evolutionary gap between invertebrate Gq-coupled visual pigments and vertebrate circadian photopigment melanopsins. Research into the Gq-coupled rhodopsin subfamily, especially invertebrate melanopsins, will provide an opportunity to investigate the evolution of various physiologic functions, based on orthologous genes, during animal evolution.     

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Continued activation of the photocycle of the dim‐light receptor rhodopsin leads to the accumulation of all‐trans‐retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre‐activated truncated bovine visual arrestin (Arr^Tr) with rhodopsin in 1,2‐diheptanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin–arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr^Tr leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all‐trans‐retinal from rhodopsin.     

Origin of the vertebrate visual cycle

In vertebrates, the absorption of light by rhodopsin leads to the isomerization of 11-cis-retinal chromophore to its all-trans form. In the visual cycle, all-trans retinal is converted back to 11-cis retinal. Mammalian visual cycle takes place in photoreceptor cells and retinal pigment epithelial (RPE) cells, while that of cephalopods is completed within a photoreceptor cell. To identify visual cycle system in the primitive chordate ascidians, we studied the localization of the ascidian visual cycle genes and proteins by in situ hybridization and whole-mount immunohistochemistry, respectively. We identified four genes encoding putative visual cycle proteins, homologs of retinal G protein-coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP), beta-carotene 15,15'monooxygenase (Ci-BCO) and RPE-specific 65 kDa protein (Ci-RPE65) in the ascidian, Ciona intestinalis. In contrast to Ci-BCO, which is predominantly localized in ocellus photoreceptor cells of the larva, Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable with that of Ci-opsin3 and Ci-CRALBP. Proteins of Ci-opsin3, Ci-CRALBP and Ci-BCO are localized in photoreceptor cells. These results suggest that the larval visual cycle uses Ci-opsin3 as a photoisomerase, while the visual cycle of the adult photoreceptors is RPE65-dependent. The colocalization of visual cycle proteins in the photoreceptor cells suggest that ascidian visual cycle takes place in a photoreceptor cell as seen in the cephalopod visual cycle.     

Studies on the Stability of the Human Cone Visual Pigments†

The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high‐resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11‐cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site‐directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo‐protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high‐resolution structural studies.     

The Analysis of Phenolic Compounds in Walnut Husk and Pellicle by UPLC-Q-Orbitrap HRMS and HPLC

Husk and pellicle as the agri-food waste in the walnut-product industry are in soaring demand because of their rich polyphenol content. This study investigated the differential compounds related to walnut polyphenol between husk and pellicle during fruit development stage. By using ultra-high performance liquid chromatography-quadrupole-orbitrap (UHPLC-Q-Orbitrap), a total of 110 bioactive components, including hydrolysable tannins, flavonoids, phenolic acids and quinones, were tentatively identified, 33 of which were different between husk and pellicle. The trend of dynamic content of 16 polyphenols was clarified during walnut development stage by high-performance liquid chromatography (HPLC). This is the first time to comprehensive identification of phenolic compounds in walnut husk and pellicle, and our results indicated that the pellicle is a rich resource of polyphenols. The dynamic trend of some polyphenols was consistent with total phenols. The comprehensive characterization of walnut polyphenol and quantification of main phenolic compounds will be beneficial for understanding the potential application value of walnut and for exploiting its metabolism pathway.     

Visual functional effects of constant blue light in a retinal degenerate rat model

Retinal degenerative conditions increase susceptibility to light damage, but rapid retinal degeneration (RD) models show less susceptibility to cyclic dim light. We investigated whether constant blue light (BL) exposure can eliminate the residual visual responses in a comparatively rapid RD rat model. Pigmented rhodopsin mutant S334ter line-3 rat pups (21 days old) were exposed for 5-6 consecutive days to constant BL. Visual behavior was evaluated with an optokinetic head tracking apparatus. Electrophysiological recordings were made from the superior colliculus (SC). S-antigen, red-green opsin and rhodopsin immunoreactive residual photoreceptors were counted. Following BL exposure, head tracking was significantly reduced at 0.25 cycles degree(-1) in 38-day-old line 3 rats. With a 0.125 cycles degree(-1) stimulus, the head tracking performance of 80-day-old BL rats were similar to that of 220-day-old no-BL-treated line-3 rats. SC recordings also revealed a significant decrease in the residual photoreceptor activity. Histological evaluation showed reduction of the rod population in the central area of the light-damaged retina. Exposure to constant BL considerably reduces the residual visual responses in a rapid degenerating RD rat model.     

Impacting the Remedial Potential of Nano Delivery-Based Flavonoids for Breast Cancer Treatment

Breast cancer persists as a diffuse source of cancer despite persistent detection and treatment. Flavonoids, a type of polyphenol, appear to be a productive option in the treatment of breast cancer, because of their capacity to regulate the tumor related functions of class of compounds. Plant polyphenols are flavonoids that appear to exhibit properties which are beneficial for breast cancer therapy. Numerous epidemiologic studies have been performed on the dynamic effect of plant polyphenols in the prevention of breast cancer. There are also subclasses of flavonoids that have antioxidant and anticarcinogenic activity. These can regulate the scavenging activity of reactive oxygen species (ROS) which help in cell cycle arrest and suppress the uncontrolled division of cancer cells. Numerous studies have also been performed at the population level, one of which reported a connection between cancer risk and intake of dietary flavonoids. Breast cancer appears to show intertumoral heterogeneity with estrogen receptor positive and negative cells. This review describes breast cancer, its various factors, and the function of flavonoids in the prevention and treatment of breast cancer, namely, how flavonoids and their subtypes are used in treatment. This review proposes that cancer risk can be reduced, and that cancer can be even cured by improving dietary intake. A large number of studies also suggested that the intake of fruit and vegetables is associated with reduced breast cancer and paper also includes the role and the use of nanodelivery of flavonoids in the healing of breast cancer. In addition, the therapeutic potential of orally administered phyto-bioactive compounds (PBCs) is narrowed because of poor stability and oral bioavailability of compounds in the gastrointestinal tract (GIT), and solubility also affects bioavailability. In recent years, creative nanotechnology-based approaches have been advised to enhance the activity of PBCs. Nanotechnology also offers the potential to become aware of disease at earlier stages, such as the detection of hidden or unconcealed metastasis colonies in patients diagnosed with lung, colon, prostate, ovarian, and breast cancer. However, nanoformulation-related effects and safety must not be overlooked. This review gives a brief discussion of nanoformulations and the effect of nanotechnology on herbal drugs.     

Predicting how polyphenol antioxidants prevent DNA damage by binding to iron

Prevention of oxidative DNA damage due to hydroxyl radical is important for the prevention and treatment of disease. Because of their widely recognized antioxidant ability, 12 polyphenolic compounds were assayed by gel electrophoresis to directly quantify the inhibition of DNA damage by polyphenols with Fe(2+) and H2O2. All of the polyphenol compounds have IC50 values ranging from 1-59 microM and inhibit 100% of DNA damage at 50-500 microM concentrations. Gel electrophoresis results with iron(II)EDTA and UV-vis spectroscopy experiments confirm that binding of the polyphenol to iron is essential for antioxidant activity. Furthermore, antioxidant potency of polyphenol compounds correlates to the pKa of the first phenolic hydrogen, representing the first predictive model of antioxidant potency based on metal-binding. Understanding this iron-coordination mechanism for polyphenol antioxidant activity will aid in the design of more-potent antioxidants to treat and prevent diseases caused by oxidative stress, and help develop structure-activity relationships for these compounds.     

硅基纳米探针用于眼部疾病的成像检测与治疗

快速精准的诊断和高效的治疗对于减轻眼部疾病造成的危害至关重要. 在过去的几十年里, 由于具有尺寸小、 比表面积大、 表面易修饰及独特的光/电子/机械性能等优点, 纳米材料已被用于构建不同种类的高性能纳米探针. 其中, 基于其良好的生物相容性, 科学家们已经将硅纳米材料设计为可用于不同眼部疾病诊断与治疗的功能化纳米探针. 本综述主要概述了将硅基纳米探针用于检测和治疗不同眼部疾病(如角膜疾病、 视网膜疾病、 青光眼等)的近期研究进展. 首先, 重点介绍了硅基纳米探针的设计制备及在角膜新生血管、 细菌性角膜炎等角膜疾病的成像检测与治疗中的应用; 然后, 介绍了用于成像检测和治疗视网膜疾病(如色素性视网膜炎和视网膜新生血管)的硅基持续性给药系统的研究成果; 随后, 概述了多功能硅基纳米载药系统的构建及在青光眼治疗领域的应用研究进展; 最后, 简要讨论了将硅基纳米探针用于眼部疾病诊治面临的挑战并对未来的发展前景进行了展望.     

Beneficial Effects of Epigallocatechin-3-O-Gallate,Chlorogenic Acid,Resveratrol, and Curcumin on Neurodegenerative Diseases

Many observational and clinical studies have shown that consumption of diets rich in plant polyphenols have beneficial effects on various diseases such as cancer, obesity, diabetes, cardiovascular diseases, and neurodegenerative diseases (NDDs). Animal and cellular studies have indicated that these polyphenolic compounds contribute to such effects. The representative polyphenols are epigallocatechin-3-O-gallate in tea, chlorogenic acids in coffee, resveratrol in wine, and curcumin in curry. The results of human studies have suggested the beneficial effects of consumption of these foods on NDDs including Alzheimer’s and Parkinson’s diseases, and cellular animal experiments have provided molecular basis to indicate contribution of these representative polyphenols to these effects. This article provides updated information on the effects of these foods and their polyphenols on NDDs with discussions on mechanistic aspects of their actions mainly based on the findings derived from basic experiments.     

Retinal photoisomerase: role in invertebrate visual cells.

In invertebrate visual cells, the rhodopsin content is maintained at a high level by the fast process of photoregeneration during daylight. Rhodopsin is converted by photoabsorption to metarhodopsin, which is reconverted to rhodopsin by light. In addition, rhodopsin is regenerated by a slow process of renewal which takes days to complete and involves the biosynthesis of opsin. It is well known that rhodopsin can be formed from opsin only when 11-cis-retinal is present; this requires the existence of an isomerizing enzyme which is capable of transforming all-trans-retinal, released from the degradation of metarhodopsin, into the 11-cis-retinal isomer. In some invertebrate visual systems, experiments on rhodopsin regeneration have been interpreted by assuming that the isomerization reaction is a light-dependent process involving a retinal-protein complex. Two retinal photoisomerases which have been well characterized, i.e. bee photoisomerase and cephalopod retinochrome, are reviewed here. Their properties are compared in order to determine their physiological role, which is likely to be in the renewal of visual pigment rhodopsin. To conclude, a visual pigment cycle is proposed in which rhodopsin regeneration follows two light-dependent pathways. This greatly simplifies the rhodopsin regeneration scheme for invertebrate visual systems.     

Dietary Plant Polyphenols: Effects of Food Processing on Their Content and Bioavailability

Dietary plant polyphenols are natural bioactive compounds that are increasingly attracting the attention of food scientists and nutritionists because of their nutraceutical properties. In fact, many studies have shown that polyphenol-rich diets have protective effects against most chronic diseases. However, these health benefits are strongly related to both polyphenol content and bioavailability, which in turn depend on their origin, food matrix, processing, digestion, and cellular metabolism. Although most fruits and vegetables are valuable sources of polyphenols, they are not usually consumed raw. Instead, they go through some processing steps, either industrially or domestically (e.g., cooling, heating, drying, fermentation, etc.), that affect their content, bioaccessibility, and bioavailability. This review summarizes the status of knowledge on the possible (positive or negative) effects of commonly used food-processing techniques on phenolic compound content and bioavailability in fruits and vegetables. These effects depend on the plant type and applied processing parameters (type, duration, media, and intensity). This review attempts to shed light on the importance of more comprehensive dietary guidelines that consider the recommendations of processing parameters to take full advantage of phenolic compounds toward healthier foods.     

Flavonoids: A Myth or a Reality for Cancer Therapy?

Nutraceuticals are biologically active molecules present in foods; they can have beneficial effects on health, but they are not available in large enough quantities to perform this function. Plant metabolites, such as polyphenols, are widely diffused in the plant kingdom, where they play fundamental roles in plant development and interactions with the environment. Among these, flavonoids are of particular interest as they have significant effects on human health. In vitro and/or in vivo studies described flavonoids as essential nutrients for preventing several diseases. They display broad and promising bioactivities to fight cancer, inflammation, bacterial infections, as well as to reduce the severity of neurodegenerative and cardiovascular diseases or diabetes. Therefore, it is not surprising that interest in flavonoids has sharply increased in recent years. More than 23,000 scientific publications on flavonoids have described the potential anticancer activity of these natural molecules in the last decade. Studies, in vitro and in vivo, show that flavonoids exhibit anticancer properties, and many epidemiological studies confirm that dietary intake of flavonoids leads to a reduced risk of cancer. This review provides a glimpse of the mechanisms of action of flavonoids on cancer cells.