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Background and Objectives: Epidemiological data shows that the prevalence of multiple sclerosis (MS) and the female-to-male sex ratio among MS patients are increasing over time. Endocrine disrupting chemicals (EDCs) are ubiquitous and increasingly recognized for effects on estrogen signaling. This study aimed to determine whether there was an association between EDC levels and disease severity in newly diagnosed, female MS patients., Methods: This exploratory observational cohort study enrolled female patients, ages 18-60, via written informed consent from the Brigham MS Center. Enrollment criteria included diagnosis with MS within the past 5 years and completion of a questionnaire about potential EDC exposures. Exclusion criteria were intravenous steroids in the past 30 days. Collection processes and materials were designed to avoid EDC contamination. Urine samples were analyzed by NSF International (Ann Arbor, Michigan). Primary outcome measures were MRI parameters and clinical disease activity, including multivariable analysis adjusting for MS treatment types. Spearman correlation test was used for analysis and between group comparisons were conducted with one-way ANOVA., Results: 68 patients with MS were enrolled. In the phthalates, mEOHP was negatively correlated with T2 lesion volume over time (R value = -0.522, p-value = 0.002, Bonferroni adjusted p = 0.03). For the phenols, triclocarban was negatively associated with cheese consumption (R value = -0.402, p = 0.001, Bonferroni adjusted p = 0.012) There was no association between EDCs and disease activity or demographic factors, nor significant correlation with exposure to household plastics., Conclusion: This exploratory study identified a negative correlation between triclocarban and cheese consumption. Longitudinally, phthalate metabolite mEOHP was negatively correlated with T2 lesion volume over time. Exposure to EDCs may affect the early disease course in MS, and expansion of research efforts is warranted., Competing Interests: Declaration of competing interest Tzu-Ying Chuang, Brenna A. LaBarre, Osman Corbali, Shristi Saxena, Talia B. Feldman, and Eunnindy Sanon report no disclosures. Brian C. Healy has received research support from Analysis Group, Celgene (Bristol-Myers Squibb), Verily Life Sciences, Merck-Serono, Novartis and Genzyme. Taylor J. Saraceno reports compensation for consulting from the Cumming Foundation, outside of the work being presented. Tanuja Chitnis has received compensation for consulting from Banner Life Sciences*, Biogen, Bristol Myers Squibb, Janssen, Genentech, Novartis Pharmaceuticals, Octave Bioscience, Sandoz, Siemens, TG Therapeutics*, UCB Biopharma, and Vida Ventures*. Dr. Chitnis has received compensation for speaking engagements from Prime Education, LLC*. Dr. Chitnis has received research support from the National Institutes of Health, National MS Society, US Department of Defense, Sumaira Foundation, Brainstorm Cell Therapeutics, Bristol Myers Squibb, Genentech, EMD Serono, I-Mab Biopharma, Mallinckrodt ARD, Massachusetts Life Sciences Center, Novartis Pharmaceuticals, Octave Bioscience, Sanofi Genzyme, Tiziana Life Sciences, and Wesley Clover International. All activities and funding have occurred within the past 24 months (*relationship has since ended) and disclosures do not conflict with the work being presented., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Lipofuscin granules accumulate in the retinal pigment epithelium with age, especially in patients with visual diseases, including progressive age-related macular degeneration. Retinoids (bisretinoids and their oxidation products) are major sources of lipofuscin granule fluorescence. The aim of this work was to analyze the radiation-mediated oxidation of retinoids in lipofuscin granules obtained from the human cadaver eye retinal pigment epithelium. Fluorescent and chromatographic analyses of retinoids were performed before and after irradiation of lipofuscin granules with accelerated protons. The fluorescent properties of chloroform extracts from irradiated lipofuscin granules exhibited an increase in fluorescence intensity in the short-wavelength region of 555 nm. This change is associated with an increase in the quantity of retinoid oxidation cytotoxic products after accelerated proton exposure. The radiation-induced oxidation of retinoids caused a noticeable change in its fluorescent properties allows us to consider this phenomenon as a potential opportunity for non-invasively assessment of the degree of radiation exposure and its relative biological effect in humans. Thus, this research proposes a new strategy for assessing the extent of radiation exposure to humans, which evaluates the effects of ionizing radiation on human eye tissues. This approach is based on the principles of the modern non-invasive method of fundus autofluorescence used in ophthalmology for the diagnosis of the retina and retinal pigment epithelium degenerative diseases., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Photodamage to the outer segments of photoreceptor cells and their impaired utilization by retinal pigment epithelium (RPE) cells contribute to the development of age-related macular degeneration (AMD) leading to blindness. Degeneration of photoreceptor cells and RPE cells is triggered by reactive oxygen species (ROS) produced by photochemical reactions involving bisretinoids, by-products of the visual cycle, which accumulate in photoreceptor discs and lipofuscin granules of RPE. Carotenoids, natural antioxidants with high potential efficacy against a wide range of ROS, may protect against the cytotoxic properties of lipofuscin. To solve the problem of high hydrophobicity of carotenoids and increase their bioaccessibility, specialized proteins can ensure their targeted delivery to the affected tissues. In this study, we present new capabilities of the recombinant water-soluble protein AstaP from Coelastrella astaxanthina Ki-4 (Scenedesmaceae) for protein-mediated carotenoid delivery and demonstrate how zeaxanthin delivery suppresses oxidative stress in a lipofuscin-enriched model of photoreceptor and pigment epithelium cells. AstaP in complex with zeaxanthin can effectively scavenge various ROS (singlet oxygen, free radical cations, hydrogen peroxide) previously reported to be generated in AMD. In addition, we explore the potential of optimizing the structure of AstaP to enhance its thermal stability and resistance to proteolytic activity in the ocular media. This optimization aims to maximize the prevention of retinal degenerative changes in AMD., (© 2024 Federation of European Biochemical Societies.)

The diversity of the retinal-containing proteins (rhodopsins) in nature is extremely large. Fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of the rhodopsin of types I and II is carried out. The results of experimental studies of the forward and reverse photoreactions of the bacteriorhodopsin (type I) and visual rhodopsin (type II) rhodopsins in the femto- and picosecond time scale, photo-reversible reaction of the octopus rhodopsin (type II), photovoltaic reactions, as well as quantum chemical calculations of the forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The issue of probable convergent evolution of type I and type II rhodopsins is discussed.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Lipofuscin granules (LGs) are accumulated in the retinal pigment epithelium (RPE) cells. The progressive LG accumulation can somehow lead to pathology and accelerate the aging process. The review examines composition, spectral properties and photoactivity of LGs isolated from the human cadaver eyes. By use of atomic force microscopy and near-field microscopy, we have revealed the fluorescent heterogeneity of LGs. We have discovered the generation of reactive oxygen species by LGs, and found that LGs and melanolipofuscin granules are capable of photoinduced oxidation of lipids. It was shown that A2E, as the main fluorophore (bisretinoid) of LGs, is much less active as an oxidation photosensitizer than other fluorophores (bisretinoids) of LGs. Photooxidized products of bisretinoids pose a much greater danger to the cell than non-oxidized one. Our studies of the fluorescent properties of LGs and their fluorophores (bisretinoids) showed for the first time that their spectral characteristics change (shift to the short-wavelength region) in pathology and after exposure to ionizing radiation. By recording the fluorescence spectra and fluorescence decay kinetics of oxidized products of LG fluorophores, it is possible to improve the methods of early diagnosis of degenerative diseases. Lipofuscin ("aging pigment") is not an inert "slag". The photoactivity of LGs can pose a significant danger to the RPE cells. Fluorescence characteristics of LGs are a tool to detect early stages of degeneration in the retina and RPE., Competing Interests: Conflict of interestThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

The present study evaluated the effects of proton and gamma-ray ionizing radiation on the mouse eye. The aim of this work was to analyze radiation-mediated retinoid oxidation in the retina and retinal pigment epithelium (RPE). The findings from this analysis can be used to develop a noninvasive method for rapid assessment of the effects of ionizing radiation. Comparative fluorescence and chromatographic analyses of retinoids before and after irradiations were performed. The fluorescent properties of chloroform extracts from irradiated mouse retina and RPE exhibited an increase in fluorescence intensity in the short-wave region of the spectrum (λ < 550 nm). This change is due to increased retinal and RPE retinoid oxidation and degradation products after radiation exposure. Comparative analyses of radiation effects demonstrated that the effect of proton exposure on the retina and RPE was higher than that of gamma-ray exposure. The present study revealed a new approach to assessing the level of radiation exposure in ocular tissues., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

The sex-bias of disease susceptibility has remained a puzzling aspect of several autoimmune conditions, including post-infection viral autoimmunity. In the last half of the twentieth century, the incidence rate of female-biased autoimmunity has steadily increased independent of medical advances. This has suggested a role for environmental factors, such as endocrine disrupting chemicals, which have been described to interfere with endocrine signaling. Endocrine involvement in the proper function of innate and adaptive immunity has also been defined, however, these two areas have rarely been reviewed in correlation. In addition, studies addressing the effects of endocrine disruptors have reported findings resulting from a broad range of exposure doses, schedules and models. This experimental heterogeneity adds confusion and may mislead the translation of findings to human health. Our work will normalize results across experiments and provide a necessary summary relevant to human exposure. Through a novel approach, we describe how different categories of ubiquitously used environmental endocrine disruptors interfere with immune relevant endocrine signaling and contribute to autoimmunity. We hope this review will guide identification of mechanisms and concentration-dependent EDC effects important not only for the sex-bias of autoimmunity, but also for other conditions of immune dysfunction, including post-infection autoreactivity such as may arise following severe acute respiratory syndrome coronavirus 2, Epstein-Barr virus, Herpes Simplex virus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2021 Popescu, Feldman and Chitnis.)

The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bR PM ) and in recombinant form (bR rec ). The primary intermediates of the ESR photocycle were similar to intermediates I , J , and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J , and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bR PM and 0.74 ps in bR rec . The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530-580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.

The objective of this study was screening of ommochromes from the compound eyes of insects and comparison of their antioxidant properties. Ommochromes were isolated in preparative quantities from insects of five different families: Stratiomyidae, Sphingidae, Blaberidae, Acrididae, and Tenebrionidae. The yield of ommochromes (dry pigment weight) was 0.9-5.4% of tissue wet weight depending on the insect species. Isolated pigments were analyzed by high-performance liquid chromatography and represented a mixture of several ommochromes of the ommatin series. The isolated ommochromes displayed a pronounced fluorescence with the emission maxima at 435-450 nm and 520-535 nm; furthermore, the emission intensity increased significantly upon ommochrome oxidation with hydrogen peroxide. The ommochromes produced a stable EPR signal consisting of a singlet line with g = 2.0045-2.0048, width of 1.20-1.27 mT, and high concentration of paramagnetic centers (> 10 17 spin/g dry weight). All the investigated ommochromes demonstrated high antiradical activity measured from the degree of chemiluminescence quenching in a model system containing luminol, hemoglobin, and hydrogen peroxide. The ommochromes strongly inhibited peroxidation of the photoreceptor cell outer segments induced by visible light in the presence of lipofuscin granules from the human retinal pigment epithelium, as well as suppressed iron/ascorbate-mediated lipid peroxidation. The obtained results are important for understanding the biological functions of ommochromes in invertebrates and identifying invertebrate species that could be used as efficient sources of ommochromes for pharmacological preparations to prevent and treat pathologies associated with the oxidative stress development.

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE)., Methods: RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method., Results: Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes., Conclusion: Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.

The presence of carotenoids in the vitreous body, retina, lens, retinal pigment epithelium together with choroid (hereinafter RPE), and ciliary body and iris together with choroidal stroma (hereinafter CBI) was studied throughout the second trimester of prenatal development of the human eye. It has been found that the vitreous body, retina, and RPE contain lutein and its oxidized forms. Zeaxanthin was not found in the tissues studied. The presence of lutein in the vitreous body is transient and no longer detected after 28 weeks of gestation. Lutein was not detected in the lens and CBI, but its oxidized forms were found. The presence of carotenoids in different tissues of the eye in the course of normal eye development and the antioxidant role of carotenoids are discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A comparative analysis of fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology was carried out. Measurements of fluorescence lifetimes of bis-retinoids and their photooxidation and photodegradation products were carried out using the method of counting time-correlated photons. Comparative analysis showed that, in the case of visualized pathology, the contribution of photooxidation and photodegradation products of bis-retinoids to the total fluorescence of the retinal pigment epithelium cell suspension increases in comparison with the norm.

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bR rec ) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bR rec was produced in an Escherichia coli expression system. Since bR rec was prepared in a DMPC-CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bR trim and bR mon , respectively) was carried out. We found that bR rec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bR trim and bR mon . Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bR rec , occurred more slowly compared to bR trim , but similarly to bR mon . The lifetime of intermediate I, judging from the signal of ΔA ESA (470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bR rec , 0.52 ps (73%) and 1.7 ps (27%) for bR mon , and 0.45 ps (90%) and 1.75 ps (10%) for bR trim . The formation time of intermediate K, judging from the signal of ΔA GSA (625-635 nm), was 13.5 ps for bR rec , 9.8 ps for bR mon , and 4.3 ps for bR trim . In addition, there was a decrease in the photoreaction efficiency of bR rec and bR mon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bR rec are similar to those of the monomeric form of the native protein, bR rec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE-fundus autofluorescence (FAF).

A quantum-classical model of photoisomerization of the visual pigment rhodopsin chromophore is proposed. At certain (and more realistic) parameter value combinations, the model is shown to accurately reproduce a number of independent experimental data on the photoreaction dynamics: the quantum yield, the time to reach the point of conical intersection of potential energy surfaces, the termination time of the evolution of quantum subsystem, as well as the characteristic low frequencies of retinal molecular lattice fluctuations during photoisomerization. In addition, the model behavior is in good accordance with experimental data about coherence and local character of quantum transition.

Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20-25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3-5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Anion-selective opsins slow ChloC and ACR2 were expressed in rat brain cortical neurons by electroporation in utero. It is shown that the light-activated channel ACR2 has pronounced advantages in terms of both the inactivation kinetics and the neuron inhibition intensity, which is associated with a more negative value of the light-activated current reversal potential compared to the slow ChloC channel. The identified properties of opsin ACR2 indicate that it can be used for strictly controlled suppression of neuronal activity in optogenetic experiments, including the expression in the retinal ganglionic cells for reconstituting the OFF-component of their receptive field, which is essential for optogenetic prosthetics of degenerative retina.

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF-SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF-SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.

Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.

Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

Supramolecular organization of rhodopsin in the photoreceptor membrane was investigated by small-angle neutron scattering method. The experiments, which were performed with mixtures of heavy/light water as solvent (contrast variation method), were aimed at obtaining information about the lipid and protein components of the photoreceptor disc membrane separately. It was shown that the packaging density of the rhodopsin molecules in the photoreceptor membrane was unusually high: the distance between the centers of the molecules was approximately 56 Å. The probability of the monomeric state of rhodopsin molecules in the photoreceptor membrane, according to the data obtained, is rather high.

The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.

We have reproduced the model system containing A2-rhodopsin, NR-PE, A2-PE, and ATR-dimer-PE in order to study photosensitized damage of rhodopsin within photoreceptor membranes of rod outer segments. We have demonstrated that irradiation of such a system with visible light (400-700 nm) distorts the most important functional property of native visual pigment--its ability to regenerate after addition of 11-cis-retinal in the dark. We have also shown that all-trans-retinal bound to membrane phospholipids and rhodopsin has less photosensitizing activity that free all-trans-retinal.