Stability and ligand binding properties of human cone visual pigments

Human color perception is mediated by cone photoreceptor cells which mainly locate on the fovea of the eye. Bright light activates the photosensitive opsin pigments which are embedded in the outer segment membrane discs of cone retinal cells thereby initiating the complex process of photopic vision with a fast response. The cone visual pigments are G-protein coupled receptors which share analogous structure and functional features with rhodopsin, the most thoroughly studied G-protein coupled receptor from rod photoreceptor cells mediating scotopic vision and distributed throughout the retina. These visual pigments modulate spectral tuning of visible light depending on the molecular variance around the protein bound chromophore, 11-cis-retinal, which is a vitamin A derivative acting as an inverse agonist. Various mutations have been clinically identified in the cone opsin genes and associated with visual dysfunction ranging from mild color blindness to severe cone dystrophies. As the crystal structure of the cone pigments is yet to be resolved, identifying key molecular mechanisms that play major roles in optimal functioning of these photoreceptor proteins, should be helpful in providing a deeper understanding of their function and in designing novel therapeutic strategies for congenital retinal cone dysfunction. In order to study such light sensitive, delicate membrane proteins, the human cone opsin genes have been transiently expressed in mammalian cells, regenerated with their natural chromophore and immunopurified in dark conditions. The purified recombinant chromophore-regenerated cone opsins in solution have been characterized in detail by means of biophysical approaches, including spectroscopic, biochemical, and functional analysis, in order to uncover novel properties that help in optimizing the function of these receptors. Site-directed mutagenesis was employed to obtain the clinically identified mutations in cone opsins related to visual disorders and to compare the structure and function of these mutated opsins with the molecular properties of the wild-type cone opsins expressed in the same way. The results of the present study indicate that the cone opsins are less stable in solution and their retinal binding site is more open than rhodopsin. The ligand binding studies using retinal analogs, show that the photoactivated rhodopsin loses its ability to regenerate with its natural chromophore with time but not with an analog, 9-cis-retinal but cone opsin shows regeneration with both retinal analogs under the same experimental conditions. The highly identical red and green cone opsins exhibit different ligand binding modes during regeneration with their natural chromophore, 11-cis-retinal. A secondary retinal uptake, with a slower kinetics, has been also observed with the red and green cone pigments during regeneration with 11-cis-retinal which is altered in the case of blue cone opsin. The role of specific amino acids involved in the regeneration mechanism has also been clarified. Most of the cone opsin mutants studied, associated with visual disorders, fail to regenerate with the ligand due to protein misfolding resulting in aggregation in solution. R330Q green cone opsin mutant show a regeneration ability similar to that of the native pigment but a compromised transducin binding efficiency. Though the N94K deuteranopic mutant apparently aggregates when expressed in mammalian, chromophore binding to opsin would be through an unprotonated Schiff base linkage. Overall, in the present study the molecular properties of cone opsins have been compared among them and with the well-studied rhodopsin. This has led to the proposal of novel molecular mechanisms for cone opsins. The determined structural differences between visual pigments may be linked to their molecular evolution, and the proposal of secondary retinoid binding to visual pigments may function as a regulatory mechanism of dark adaptation.La percepción del color humano se realiza a través de las células fotorreceptoras de los conos, localizados en la fóvea del ojo. La luz brillante activa los pigmentos fotosensibles que se encuentran en los discos de membrana del segmento externo de estas células iniciando con ello el complejo y rápido proceso de la visión fotópica. Los pigmentos visuales conos son receptores acoplados a proteínas G (GPCR) que comparten estructura análoga y características funcionales a la rodopsina, el GPCR más estudiado y localizado en las células fotorreceptoras de los bastones y responsable de la visión escotópica . Estos pigmentos visuales modulan la sintonización espectral de la luz visible, dependiendo de las diferencias moleculares alrededor del cromóforo ligado a la proteína, 11-cis-retinal, que es un derivado de la vitamina A y que actúa como un agonista inverso. Varias mutaciones han sido identificadas clínicamente en los genes de las opsinas de los conos asociadas a disfunciones visuales; desde la leve ceguera al color a la grave distrofia de las células fotorreceptoras. Como la estructura cristalina de los pigmentos de los conos está todavía por resolver, identificar mecanismos moleculares que juegan un papel importante en el funcionamiento óptimo de estas proteínas, ayudará a obtener una comprensión más profunda de su función y al diseño de nuevas estrategias terapéuticas para la disfunción congénita de los conos. Con el fin de estudiar estas proteínas de membrana sensibles a la luz y delicadas, se han expresado transitoriamente en células de mamífero, regenerado con su cromóforo natural, inmunopurificado en oscuridad y se han caracterizado por medio de técnicas biofísicas, bioquímicas, y funcionales, a fin de descubrir nuevas propiedades que ayuden a la optimización de su función. Mutagénesis dirigida se ha utilizado para obtener las mutaciones clínicamente identificados en opsinas de conos relacionados con los trastornos visuales y comparar la estructura y la función de estas opsinas mutadas con las propiedades moleculares de opsina de conos cono salvaje. Los resultados del presente estudio indican que las opsinas de los conos son menos estables y su sitio de unión al retinal es más abierta que en la rodopsina. Estudios de unión al ligando utilizando análogos del retinal, muestran que la rodopsina fotoactivada pierde su capacidad de regenerarse con su cromóforo natural con el tiempo, aunque la mantiene con el análogo 9-cis-retinal, comparado con la regeneración de los conos, que pueden hacerlo con ambos análogos de retinal. Las altamente idénticas opsinas de conos rojo y verde muestran modos de unión diferente al ligando durante la regeneración con su cromóforo natural, 11-cis retinal. También se ha observado una captación secundaria de retinal, con una cinética más lenta, con los pigmentos de los conos rojos y verdes durante la regeneración con 11-cis-retinal que se altera en el caso de los conos azules. También se discute el papel de los aminoácidos específicos implicados en el mecanismo de la regeneración. La mayoría de los mutantes estudiados, no logran regenerar con el ligando debido al mal plegamiento proteico. El cono verde R330Q muestra una capacidad de regeneración similar a la del pigmento nativo, pero con una baja eficacia de unión a la proteína G, transducina. Aunque inicialmente el mutante N94K parece agregado cuando se expresa en células de mamífero, el cromóforo que se une a la opsina a través de una base de Schiff no protonada. En general, este estudio compara las propiedades moleculares de las opsinas de conos entre ellos y con la rodopsina, proponiendo nuevos mecanismos moleculares para las opsinas de conos. Las diferencias estructurales entre determinados pigmentos visuales pueden estar relacionados con su evolución molecular, y la propuesta de la unión secundaria de retinal a estos pigmentos visuales puede funcionar como un mecanismo de regulación de la adaptación a la oscuridad

Ligand binding mechanisms in human cone visual pigments

Vertebrate vision starts with light absorption by visual pigments in rod and cone photoreceptor cells of the retina. Rhodopsin, in rod cells, responds to dim light, whereas three types of cone opsins (red, green, and blue) function under bright light and mediate color vision. Cone opsins regenerate with retinal much faster than rhodopsin, but the molecular mechanism of regeneration is still unclear. Recent advances in the area pinpoint transient intermediate opsin conformations, and a possible secondary retinal-binding site, as determinant factors for regeneration. In this Review, we compile previous and recent findings to discuss possible mechanisms of ligand entry in cone opsins, involving a secondary binding site, which may have relevant functional and evolutionary implicationsPeer ReviewedPostprint (author's final draft

Performance evaluation of multi-stage reverse osmosis process with permeate and retentate recycling strategy for the removal of chlorophenol from wastewater

YesReverse Osmosis (RO) is one of the most widely used technologies for wastewater treatment for the removal of toxic impurities, such as phenol and phenolic compounds from industrial effluents. In this research, performance of multi-stage RO wastewater treatment system is evaluated for the removal of chlorophenol from wastewater using model-based techniques. A number of alternative configurations with recycling of permeate, retentate, and permeate-retentate streams are considered. The performance is measured in terms of total recovery rate, permeate product concentration, overall chlorophenol rejection and energy consumption and the effect of a number of operating parameters on the overall performance of the alternative configurations are evaluated. The results clearly show that the permeate recycling scheme at fixed plant feed flow rate can remarkably improve the final chlorophenol concentration of the product despite a reduction in the total recovery rate

Human blue cone opsin regeneration involves secondary retinal binding with analog specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigmentsPeer ReviewedPostprint (author's final draft

Occurrence of Oratosquilla oratoria (De Haan, 1844) and Oratosquillina gravieri (Manning, 1978) from the trawl bycatches of Nagapattinam region, Tamil Nadu

Six species of mantis shrimps were recorded in the trawl catches of Nagapattinam region during January – December, 2018. The occurrence of two species namely Oratosquilla oratoria (De Haan, 1844) and Oratosquillina gravieri (Manning, 1978) were reported for the first time in the trawl bycatches of Nagapattinam region. The morphometrics of these two species is reported in this paper. Presence of inferodistal spine in the merus of the raptorial claw, rostral plate short and trapezoid to square in shape, 5th abdominal segment without submedian patch, 4th abdominal segment submedian carinae unarmed in Oratosquilla oratoria and rostral plate elongate and rectangle, lateral process of 6th thoracic somite with slender, triangular anterior lobe and dactylus of the raptorial claw with 6 teeth in Oratosquillina gravieri are chief characters to differentiate from other species

Occurrence of Oratosquilla oratoria (De Haan, 1844) and Oratosquillina gravieri (Manning, 1978) from the trawl bycatches of Nagapattinam region, Tamil Nadu

1029-1036Six species of mantis shrimps were recorded in the trawl catches of Nagapattinam region during January – December, 2018. The occurrence of two species namely Oratosquilla oratoria (De Haan, 1844) and Oratosquillina gravieri (Manning, 1978) were reported for the first time in the trawl bycatches of Nagapattinam region. The morphometrics of these two species is reported in this paper. Presence of inferodistal spine in the merus of the raptorial claw, rostral plate short and trapezoid to square in shape, 5th abdominal segment without submedian patch, 4th abdominal segment submedian carinae unarmed in Oratosquilla oratoria and rostral plate elongate and rectangle, lateral process of 6th thoracic somite with slender, triangular anterior lobe and dactylus of the raptorial claw with 6 teeth in Oratosquillina gravieri are chief characters to differentiate from other species

Removal of phenol from wastewater using spiral-wound reverse osmosis process: model development based on experiment and simulation

YesThe removal of the ubiquitous phenol and phenolic compounds in industrial wastes is a critical environmental issue due to their harmful threats to wildlife and potential adverse human health effects. The removal of such compounds is therefore of significant importance in water treatment and reuse. In recent years, reverse osmosis (RO) has been successfully utilised in several industrial processes and wastewater treatment including phenol removal. In this paper, a new model based on a spiral-wound RO process is developed for the removal of phenol from wastewater. A simplified mathematical algorithm using an irreversible thermodynamic approach is developed. This results in a set of non-linear Differential and Algebraic Equations (DAEs), which are solved based on a number of optimised model parameters using a combined methodology of parameter estimation and experimental phenol-water data derived from the literature. The effects of several operational parameters on the performance (in terms of removal of phenol) of the process are explored using the model

Simulation and optimisation of a two-stage/two-pass reverse osmosis system for improved removal of chlorophenol from wastewater

YesReverse osmosis (RO) has become a common method for treating wastewater and removing several harmful organic compounds because of its relative ease of use and reduced costs. Chlorophenol is a toxic compound for humans and can readily be found in the wastewater of a wide range of industries. Previous research in this area of work has already provided promising results in respect of the performance of an individual spiral wound RO process for removing chlorophenol from wastewater, but the associated removal rates have stayed stubbornly low. The literature has so far confirmed that the efficiency of eliminating chlorophenol from wastewater using a pilot-scale of an individual spiral wound RO process is around 83 %, compared to 97 % for dimethylphenol. This paper explores the potential of an alternative configuration of two-stage/two-pass RO process for improving such low chlorophenol rejection rates via simulation and optimisation. The operational optimisation carried out is enhanced by constraining the total recovery rate to a realistic value by varying the system operating parameters according to the allowable limits of the process. The results indicate that the proposed configuration has the potential to increase the rejection of chlorophenol by 12.4 % while achieving 40 % total water recovery at an energy consumption of 1.949 kWh/m³

Simulation of hybrid trickle bed reactor-reverse osmosis process for the removal of phenol from wastewater

YesPhenol and phenolic derivatives found in different industrial effluents are highly toxic and extremely harmful to human and the aquatic ecosystem. In the past, trickle bed reactor (TBR), reverse osmosis (RO) and other processes have been used to remove phenol from wastewater. However, each of these technologies has limitations in terms of the phenol concentration in the feed water and the efficiency of phenol rejection rate. In this work, an integrated hybrid TBR-RO process for removing high concentration phenol from wastewater is suggested and model-based simulation of the process is presented to evaluate the performance of the process. The models for both TBR and RO processes were independently validated against experimental data from the literature before coupling together to make the hybrid process. The results clearly show that the combined process significantly improves the rejection rate of phenol compared to that obtained via the individual processes

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field