Molecular and functional identification of a novel photopigment in Pecten ciliary photoreceptors

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of General Physiology 150 (2018): 401, doi:10.1085/jgp.201711938.The two basic animal photoreceptor types, ciliary and microvillar, use different light-transduction schemes: their photopigments couple to Gt versus Gq proteins, respectively, to either mobilize cyclic nucleotides or trigger a lipid signaling cascade. A third class of photoreceptors has been described in the dual retina of some marine invertebrates; these present a ciliary morphology but operate via radically divergent mechanisms, prompting the suggestion that they comprise a novel lineage of light sensors. In one of these organisms, an uncommon putative opsin was uncovered that was proposed to signal through Go. Orthologues subsequently emerged in diverse phyla, including mollusks, echinoderms, and chordates, but the cells in which they express have not been identified, and no studies corroborated their function as visual pigments or their suggested signaling mode. Conversely, in only one invertebrate species, Pecten irradians, have the ciliary photoreceptors been physiologically characterized, but their photopigment has not been identified molecularly. We used the transcriptome of Pecten retina to guide the cloning by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) extensions of a new member of this group of putative opsins. In situ hybridization shows selective transcription in the distal retina, and specific antibodies identify a single band of the expected molecular mass in Western blots and distinctly label ciliary photoreceptors in retina sections. RNA interference knockdown resulted in a reduction in the early receptor current—the first manifestation of light transduction—and prevented the prolonged aftercurrent, which requires a large buildup of activated rhodopsin. We also obtained a full-length clone of the α-subunit of a Go from Pecten retina complementary DNA and localized it by in situ hybridization to the distal photoreceptors. Small interfering RNA targeting this Go caused a specific depression of the photocurrent. These results establish this novel putative opsin as a bona fide visual pigment that couples to Go to convey the light signal.Supported by Departamento Administrativo de Ciencia, Tecnología e Innovación (Colcencias; grant FP44842-010-2015) and by Fund for Science

The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in “circadian” light detectors of higher vertebrates. Because in amphioxus melanopsin activates a Gq/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na+. In addition to Na+, Ca2+ is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at Vm > ENa; (b) in Na-free solution a small residual inward photocurrent persists at Vm near resting level, provided that Ca is present; and (c) Vrev exhibits a modest shift with [Ca]o manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K+, as [K]o only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La3+ and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates

Melanopsin-expressing amphioxus photoreceptors transduce light via a phospholipase C signaling cascade

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 7 (2012): e29813, doi:10.1371/journal.pone.0029813.Melanopsin, the receptor molecule that underlies light sensitivity in mammalian ‘circadian’ receptors, is homologous to invertebrate rhodopsins and has been proposed to operate via a similar signaling pathway. Its downstream effectors, however, remain elusive. Melanopsin also expresses in two distinct light-sensitive cell types in the neural tube of amphioxus. This organism is the most basal extant chordate and can help outline the evolutionary history of different photoreceptor lineages and their transduction mechanisms; moreover, isolated amphioxus photoreceptors offer unique advantages, because they are unambiguously identifiable and amenable to single-cell physiological assays. In the present study whole-cell patch clamp recording, pharmacological manipulations, and immunodetection were utilized to investigate light transduction in amphioxus photoreceptors. A Gq was identified and selectively localized to the photosensitive microvillar membrane, while the pivotal role of phospholipase C was established pharmacologically. The photocurrent was profoundly depressed by IP3 receptor antagonists, highlighting the importance of IP3 receptors in light signaling. By contrast, surrogates of diacylglycerol (DAG), as well as poly-unsaturated fatty acids failed to activate a membrane conductance or to alter the light response. The results strengthen the notion that calcium released from the ER via IP3-sensitive channels may fulfill a key role in conveying - directly or indirectly - the melanopsin-initiated light signal to the photoconductance; moreover, they challenge the dogma that microvillar photoreceptors and phoshoinositide-based light transduction are a prerogative of invertebrate eyes.This work was supported by the National Science Foundation of the USA (grant 0918930)

Process evaluation of the project standardization strategy as a public management tool in Colombia in the years 2016 to 2018

El presente documento tiene por objetivo evaluar el proceso de implementación de la estrategia de estandarización de proyectos como herramienta de gestión pública a escala nacional, teniendo en cuenta los lineamientos establecidos en el Consejo Nacional de Política Económica y Social - CONPES 3856 de 2016 así como sus objetivos y resultados esperados. De acuerdo con el estudio realizado, el uso de Proyectos Tipo del Departamento Nacional de Planeación fue el esperado, debido a que se cumplió con los principios básicos de la estrategia: reducción de costos y tiempos en el proceso de estructuración de los proyectos ante los Órganos Colegiados de Administración y Decisión – OCAD, así como la implementación de estos en todo el territorio nacional. Sin embargo, los requisitos requeridos en el proceso de presentación de los proyectos, así como la singularidad de estructuración de cada proyecto, hace que la estrategia no esté del todo optimizada. A partir de estos hallazgos se dan recomendaciones al proceso de implementación, así como a la coyuntura de ejecución del programa.The purpose of this paper is to evaluate the implementation of the project standardization strategy process as a public management tool at the national scale, considering the guidelines established in the National Council of Economic and Social Policy - CONPES 3856 of 2016 as well as its objectives and expected results. According to the study, the use of projects of the National Planning Department was expected because it met the basic principles of the strategy: reducing costs and time in the process of structuring projects to the Organs Administration and Decision Associates - OCAD, as well as their implementation throughout the national territory. However, the requirements required in the project presentation process, as well as the unique structuring of each project, make the strategy not fully optimized. Based on these findings, recommendations are given to the implementation process, as well as to the program execution situation.Magíster en Gobierno del Territorio y Gestión PúblicaMaestrí

Development of a Web Prototype that Supports the Implementation of the Model Cmmi-Dev Level 3 Using a NoSQL Persistence Model

El número de empresas colombianas certificadas en CMMI-DEV es relativamente bajo, comparado con otros países como China, Estados Unidos, India o Brasil, según las estadísticas del CMMI Institute Published Appraisal(Published Appraisal Results, 2017), no solamente por la interpretación de las prácticas del modelo, sino que para muchas empresas resulta dispendiosa la gestión de la documentación que se debe generar como soporte a los procesos de desarrollo en cada una de las áreas definidas por el modelo, lo cual se traduce en un esfuerzo adicional, tanto en tiempo como en costos, que las empresas no siempre están dispuestas a asumir. Es por esto que en este trabajo se propone el desarrollo de un prototipo de software que soporte la gestión, tanto del proceso, como de la documentación generada en la implementación del modelo CMMI-DEV nivel 3, utilizando una base de datos NoSQL para el manejo de la persistencia que permita la escalabilidad del sistema y se adapte fácilmente a los cambios en los documentos que soportan el modelo.The number of Colombian organizations certified on CMMI-DEV is relatively low compared to countries like China, The United States, India or Brazil, according to statistics from the CMMI Institute Published Appraisal(Published Appraisal Results, 2017). Not only because the model's interpretation are complex and the resulting practices hard to follow but because the handling of large amount of documentation encouraged by the model is cumbersome and expensive, so most organizations don't see the benefit for the big investment. That's the main reason behind this project, to propose a software prototype that supports the handling of both the process and the documents generated in organizations that plan or are using the CMMI-DEV model in Level 3 using a NoSQL database as storage. This should prove easily adaptive and scalable to the vast amount of documentation that supports the CMMI-DEV model

Virus del papiloma humano y cáncer bucal: Análisis Bibliométrico

Objetivo: identificar en la literatura las investigaciones que relacionen el Virus de Papiloma Humano (VPH) y cáncer bucal. Metodología: se realizó un análisis bibliométrico para lo cual se consultaron las bases de datos Embase, Pubmed, Web of Science y Scopus. El análisis bibliométrico valoró la dinámica científica para la obtención del conocimiento y el impacto de una investigación, identificó las tendencias y el incremento del conocimiento en el tema. Resultados: la tendencia de investigación está liderada por las revistas British Journal of Cancer, Head and Neck, International Journal of Cancer y Journal of Clinical. Se encontraron estudios transversales en un 42% de la muestra, seguido de casos y controles que representaría un 22%. Se observó que el país con mayor colaboración es Estados Unidos con 39 publicaciones en red, así mismo lideró las investigaciones realizadas en esta temática, seguido de Italia y España. También se encontró que el VPH tipo 16 se menciona en 99 publicaciones de los artículos relacionados con cáncer bucal, así mismo, se observó un menor número de publicaciones del VPH sub tipo 18. Conclusiones: en el análisis de las bases de datos se identificaron 919 documentos que fueron filtrados por año, repeticiones y pertinencia dejando al final 151 documentos susceptibles de analizar, de los cuales 42 artículos correspondieron a estudios transversales, 22 a casos y controles, 21 a estudios longitudinales, nueve a ensayos clínicos, tres a estudios de cohortes y tres reportes de caso

Lack of effect of diacylglycerol analogs on membrane conductance and light-evoked current.

<p>(A) Control Hesse cell dialyzed with standard internal solution. (<i>Left</i>) Recording of membrane current in the dark immediately after attaining the whole-cell configuration; (<i>right</i>) currents evoked by flashes of increasing intensity. (B,C) Similar experiments conducted in Hesse cells dialyzed with SAG (1-Stearoyl-2-arachidonoyl-<i>sn</i>-glycerol; 10 µM) and OAG (1-Oleoyl-2-acetyl-sn-glycerol; 10 µM), respectively. Neither the holding current nor the photocurrents were affected. Membrane potential was clamped at −50 mV throughout.</p

Amphioxus melanopsin groups with G_q-coupled rhodopsins.

<p>(<i>A</i>) The translated sequence of <i>Branchiostoma</i> was subjected to a BLAST search, and subsequently aligned with the highest-ranking hits among <i>non</i>-melanopsin photopigments. All belong to microvillar photoreceptors from eyes of invertebrates. The Clustal W alignment shows the core region of the proteins (omitting the carboxy-terminus, and part of the amino-terminus, which are generally divergent); black shading indicates identity, whereas grey shading indicates conservative aminoacid substitutions. Accession numbers: amphioxus (<i>Branchiostoma</i>) Q4R1I4; scallop (<i>Mizuhopecten</i>), O15973.1, octopus (<i>Octopus</i>). P09241.1; horseshoe crab (<i>Limulus</i>), ACO05013.1; and squid (<i>Loligo</i>) P24603.1. (<i>B</i>) Western blot with anti-melanopsin antibodies. A single band of ≈71 kDa was detected, closely approaching the size expected from the predicted aminoacid sequence. This confirms that the melanopsin protein expresses in the neural tube of <i>B. floridae</i>. (<i>C</i>) Phylogenetic tree illustrating the grouping of the main classes of photopigments found in the animal kingdom. Amphioxus melanopsin is closely related to the G_q-coupled rhodopsin of mollusks and arthropods.</p

G_q expresses in the microvillar membrane of Hesse cells.

<p>(<i>A</i>) The sequence of the immunogenic peptide used to raise anti-G_q antibodies was aligned with the predicted C-terminal region of G_q of amphioxus, and those of other organisms. (<i>B</i>) Western blot of neural tube using anti G_q, showing the detection of a single band of ≈42 kDa. (<i>C</i>) Morphological characteristics of the Hesse cell: the accessory pigmented cell engulfs the microvilli-covered region of the clear sensory cell; the position of the villous membrane within the occluded region is drawn in red. (<i>D</i>) <i>Left</i>: Nomarski micrograph of a 10 µm section of fixed neural tube containing two Hesse cells. <i>Right</i>: fluorescence image of the same section incubated with anti-G_q antibodies and Alexa Fluo 488-conjugated secondary antibodies. The immunostaining is confined to the region of the microvilli.</p