Nanoparticle tethered antioxidant response element as a biosensor for oxygen induced toxicity in retinal endothelial cells

Purpose: A novel system, based on biosensor DNA tethered to a nanoparticle, was developed for the treatment of retinopathy of prematurity. Methods: The construction of a five-layered nanoparticle was visualized with gel electrophoresis. Transcriptionally active PCR products (TAP) containing the biosensor sequence, were bioconjugated to the surface of magnetic nanoparticles yielding biosensor tethered magnetic nanoparticles (MNP). The biosensor was based on an enhanced green fluorescent protein (EGFP) reporter gene driven by an enhanced antioxidant response element ( ARE). Image analysis and flow cytometry were used to characterize MNP delivery and biosensor activity. Results: The MNP penetrated dividing and migrating cells more often than quiescent endothelial cells in a wound-healing in vitro assay. Prussian blue staining demonstrated that more cells have nanoparticle cores than are transfected. When compared to naked TAP alone, MNP transfected more cells in a dose dependent manner. Both the biosensor alone and MNP induce gene expression in the presence of hyperoxia, greater than 1.5 fold over normoxic controls. These data also show that the MNP had a signal to noise ratio of 0.5 greater than the plasmid form of the biosensor as demonstrated by flow cytometry. Conclusions: This approach has the potential to allow the endothelial cells of the retinal vasculature to prevent or treat themselves after hyperoxic insult, rather than systemic treatment to protect or treat only the retina

Construction, gene delivery, and expression of DNA tethered nanoparticles

PURPOSE: Layered nanoparticles have the potential to deliver any number of substances to cells both in vitro and in vivo. The purpose of this study was to develop and test a relatively simple alternative to custom synthesized nanoparticles for use in multiple biological systems, with special focus on the eye. METHODS: The biotin-labeled transcriptionally active PCR products (TAP) were conjugated to gold, semiconductor nanocrystals, and magnetic nanoparticles (MNP) coated with streptavidin. The process of nanoparticle construction was monitored with gel electrophoresis. Fluorescence microscopy followed by image analysis was used to examine gene expression levels from DNA alone and tethered MNP in human hepatoma derived Huh-7 cells. Adult retinal endothelial cells from both dog (ADREC) and human (HREC) sources were transfected with nanoparticles and reporter gene expression evaluated with confocal and fluorescent microscopy. Transmission electron microscopy was used to quantify the concentration of nanoparticles in a stock solution. Nanoparticles were evaluated for transfection efficiency, determined by fluorescence microscopy cell counts. Cells treated with MNP were evaluated for increased reactive oxygen species (ROS) and necrosis with flow cytometry. RESULTS: Both 5' and 3' biotin-labeled TAP bound equally to MNP and there were no differences in functionality between the two tethering orientations. Free DNA was easily removed by the use of magnetic columns. These particles were also able to deliver genes to a human hepatoma cell line, Huh-7, but transfection efficiency was greater than TAP. The semiconductor nanocrystals and MNP had the highest transfection efficiencies. The MNP did not induce ROS formation or necrosis after 48 h of incubation. CONCLUSIONS: Once transfected, the MNP had reporter gene expression levels equivalent to TAP. The nanoparticles, however, had better transfection efficiencies than TAP. The magnetic nanoparticles were the most easily purified of all the nanoparticles tested. This strategy for bioconjugating TAP to nanoparticles is valuable because nanoparticle composition can be changed and the system optimized quickly. Since endothelial cells take up MNP, this strategy could be used to target neovascularization as occurs in proliferative retinopathies. Multiple cell types were used to test this technology and in each the nanoparticles were capable of transfection. In adult endothelial cells the MNP appeared innocuous, even at the highest doses tested with respect to ROS and necrosis. This technology has the potential to be used as more than just a vector for gene transfer, because each layer has the potential to perform its own unique function and then degrade to expose the next functional layer

A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye

We have developed a method to label and image myeloid cells infiltrating the mouse retina and choroid in vivo, using a single depot injection of indocyanine green dye (ICG). This was demonstrated using the following ocular models of inflammation and angiogenesis: endotoxin-induced uveitis, experimental autoimmune uveoretinitis and laser-induced choroidal neovascularization model. A near-infrared scanning ophthalmoscope was used for in vivo imaging of the eye, and flow cytometry was used on blood and spleen to assess the number and phenotype of labelled cells. ICG was administered 72 h before the induction of inflammation to ensure clearance from the systemic circulation. We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina. Progression of inflammation in the retina could be traced over a period of 14 days following a single depot injection of ICG. Additionally, bright-field microscopy, spectrophotometry and flow cytometric analysis suggest that the predominant population of cells stained by ICG are circulating myeloid cells. The translation of this approach into clinical practice would enable visualization of immune cells in situ. This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders

Choriocapillaris and Choroidal Microvasculature Imaging with Ultrahigh Speed OCT Angiography

We demonstrate in vivo choriocapillaris and choroidal microvasculature imaging in normal human subjects using optical coherence tomography (OCT). An ultrahigh speed swept source OCT prototype at 1060 nm wavelengths with a 400 kHz A-scan rate is developed for three-dimensional ultrahigh speed imaging of the posterior eye. OCT angiography is used to image three-dimensional vascular structure without the need for exogenous fluorophores by detecting erythrocyte motion contrast between OCT intensity cross-sectional images acquired rapidly and repeatedly from the same location on the retina. En face OCT angiograms of the choriocapillaris and choroidal vasculature are visualized by acquiring cross-sectional OCT angiograms volumetrically via raster scanning and segmenting the three-dimensional angiographic data at multiple depths below the retinal pigment epithelium (RPE). Fine microvasculature of the choriocapillaris, as well as tightly packed networks of feeding arterioles and draining venules, can be visualized at different en face depths. Panoramic ultra-wide field stitched OCT angiograms of the choriocapillaris spanning ~32 mm on the retina show distinct vascular structures at different fundus locations. Isolated smaller fields at the central fovea and ~6 mm nasal to the fovea at the depths of the choriocapillaris and Sattler's layer show vasculature structures consistent with established architectural morphology from histological and electron micrograph corrosion casting studies. Choriocapillaris imaging was performed in eight healthy volunteers with OCT angiograms successfully acquired from all subjects. These results demonstrate the feasibility of ultrahigh speed OCT for in vivo dye-free choriocapillaris and choroidal vasculature imaging, in addition to conventional structural imaging.National Institutes of Health (U.S.) (NIH R01-EY011289-27)National Institutes of Health (U.S.) (NIH R01-EY013178-12)National Institutes of Health (U.S.) (NIH R44-EY022864-01)National Institutes of Health (U.S.) (NIH R01-CA075289-16)United States. Air Force Office of Scientific Research (AFOSR FA9550-10-1-0551)United States. Air Force Office of Scientific Research (AFOSR FA9550-12-1-0499

Desarrollo de aplicaciones móviles

El presente trabajo monográfico está centrado a el desarrollo de una aplicación móvil, partiendo del concepto de que es esta, en cómo funcionan, se tratara de los tipos de aplicaciones móviles; aplicaciones nativas, web e hibridas, sus ventajas y desventajas de cada una de ellas. Sobre lo más destacado de cuál de es la mejor. En la siguiente sección veremos la importancia de una aplicación móvil en una empresa, desarrollando las ventajas y puntos a favor dentro de una empresa. A continuación se verá los sistemas operativos en la cual funciona un dispositivo portátil, las plataformas para desarrollar un programa para móviles; y como no sus ventajas y desventajas para su desarrollo, sus plataformas de distribución para poder adquirir una en caso se requiriera una en especial. Ya en la última sección de este trabajo monográfico teniendo en cuenta los conceptos antes mencionados tendremos ya los conocimientos para empezar a crear una aplicación para móviles, siguiendo los consejos para el desarrollo de la misma, una sección en la cual nos indica los procesos para desarrollar una aplicación y en un apartado (anexos) un manual de cómo crear una aplicación móvil.Trabajo de suficiencia profesiona

Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice

<p>Abstract</p> <p>Background</p> <p>Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in <it>Lama1</it>, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these <it>Lama1 </it>mutants.</p> <p>Results</p> <p>Although astrocytes and blood vessels initially migrate into <it>Lama1 </it>mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy.</p> <p>Conclusions</p> <p><it>Lama1 </it>and a fully intact ILM are required for normal retinal vascular development. Mutations in <it>Lama1 </it>allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The <it>Lama1 </it>mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.</p

Severe hypotonia and developmental delay due to an EBF3 pathogenic variant: Clinical implications of a molecular defect and narrative review

peer reviewedHypotonia Ataxia and Delayed Development Syndrome (HADDS) is a neurodevelopmental syndrome due to missense pathogenic variants of the EBF3 gene, located on chromosome 10q26.3. In most cases, these variants appear de novo and the transmission is autosomal dominant. HADDS would affect about 200 people worldwide and is characterized by a high clinical variability in the expression of these different symptoms : severe hypotonia, failure to thrive, psychomotor delay, digestive and feeding disorders, vesicoureteral anomalies, strabismus, and moderate facial dysmorphia. Although our knowledge is still limited, the significance of these symptoms seems to depend upon the EBF3 expression during embryogenesis. Animal studies suggest that EBF3 plays a critical role in neuronal migration and differentiation and interacts with CDKN1A, NeuroD, and ARX regulation pathways. With respect to diaphragmatic and vesicoureteral dysfunction and hypotonia, EBF3 appears to be involved in myocyte calcium metabolism. In addition, EBF3 has recently been identified as a novel tumor suppressor gene in some cancers. Further research on the EBF3 gene and the associated pathological pathways is needed to improve our understanding of HADDS and to provide appropriate care for such rare diseases

Conocimiento y actitudes en relación al código de ética y deontología en internos de pregrado de Medicina Humana, Lambayeque 2023

El objetivo principal de esta investigación fue evaluar el nivel de conocimiento y actitudes en relación al Código de Ética y Deontología en Internos de Pregrado de Medicina Humana, Lambayeque 2023. Metodológicamente, se trató de una investigación con enfoque cuantitativo, de tipo descriptivo, correlacional, no experimental y transversal. La población de estudio estuvo conformada por 290 Médicos Internos de Pregrado, los cuales ejercieron sus actividades en el periodo enero 2022 – diciembre 2022, de los cuales se obtuvo una muestra de 166. La técnica empleada en esta investigación fue la encuesta y sus instrumentos (Uno para cada variable) fueron construidos con las dimensiones identificadas por Majdor A, Herring J, Wheeler R. en su obra “Medical Ethics and Law” y por Ter, Marieke y Lissenberg-Witte, Birgit en su obra “A Quick Guide on How to Conduct Medical Research”. Los resultados obtenidos muestran que el 92,9% de los Médicos Internos de Pregrado consultados conocen y poseen el Código de Ética y Deontología del Colegio Médico del Perú en alguna de sus formas, (virtual, digital o impresa) y solo un 7.1% declaró ignorar sobre la materia. Asimismo, el 88,2% declaró que la formación ética fue adquirida durante sus cursos regulares proporcionados por sus respectivas casas de estudio. En relación al nivel de conocimientos de los internos de medicina de la Región Lambayeque respecto al Código de Ética y Deontología, un 32.35% reflejan un nivel deficiente; un 28.8% regular, un 27.65% declaran poseer un nivel bueno y solo un 11.18% manifiestan tener un conocimiento muy bueno. Finalmente, se concluye que, si bien se maneja el Código de Ética y Deontología, existe un alto porcentaje que desconoce de su aplicación práctica, lo que representa un serio problema para el adecuado ejercicio de la profesión médica.TesisCiencias de la vida y cuidado de la salud human

Immunohistochemical localization of low density lipoprotein receptor-related protein 1 and α2-Macroglobulin in retinal and choroidal tissue of proliferative retinopathies

The immunolocalization of the low density lipoprotein receptor-related protein 1 (LRP1) and its ligand α 2-Macroglobulin (α2M) was examined in tissues from human donor eyes of normal, diabetic and sickle cell disease subjects. Streptavidin alkaline phosphatase immunohistochemistry was performed with a mouse anti-human LRP1 and rabbit anti-human α2M antibodies. Retinal and choroidal blood vessels were labeled with mouse anti-human CD34 antibody in adjacent tissue sections. Mean scores for immunostaining from the pathological and control eyes were statistically compared.LRP1 immunoreactivity was very weak to negative in the neural retina of normal subjects except in scattered astrocytes. LRP1 expression in diabetic eyes was detected in the internal limiting membrane (ILM), astrocytes, inner photoreceptor matrix, choriocapillaris and choroidal stroma. The ligand α2M, however, was limited mainly to blood vessel walls, some areas of the inner nuclear layer (INL), photoreceptors, RPE-Bruch's membrane-choriocapillaris complex, intercapillary septa, and choroidal stroma. In sickle cell eyes, avascular and vascular retina as well as choroidal neovascularization (CNV) were analyzed. In avascular areas, LRP1 immunoreactivity was in innermost retina (presumably ILM, astrocytes, and Muller cells) and INL as well as RPE-Bruch's membrane-choriocapillaris complex and choroidal stroma α2M was very weak in avascular peripheral retina compared to vascularized areas and limited to stroma in choroid. In contrast, in areas with CNV, LRP1 immunoreactivity was significantly decreased in overlying retina and in RPE-Bruch's membrane and choroidal stroma compared to the controls, while α2M was elevated in RPE-Bruch's membrane near CNV compared to normal areas in sickle cell choroid. The mean scores revealed that LRP1 and α2M in neural retina were significantly elevated in astrocytes and ILM in diabetic eyes (p ≤ 0.05), whereas in sickle cell eyes scores were elevated in ILM and INL (p ≤ 0.05). In addition, α2M immunoreactivity was in photoreceptors in both ischemic retinopathies. In choroid, the patterns of LRP1 and α2M expression were different and not coincident.This is the first demonstration of the presence of LRP1 and α2M in human proliferative retinopathies. Elevated LRP1 expression in sickle cell neural retina and diabetic inner retina and choroid suggests that LRP1 plays an important role in ischemic neovascular diseases. © 2010 Elsevier Ltd.Fil: Barcelona, Pablo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Luna, J. D.. Fundación VER. Departamento de Oftalmología; ArgentinaFil: Chiabrando, Gustavo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Juarez, P. C.. Fundación VER. Departamento de Oftalmología; ArgentinaFil: Bhutto IA. University Johns Hopkins; Estados UnidosFil: McLeod, D. S.. University Johns Hopkins; Estados UnidosFil: Sanchez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Lutty, G. A.. University Johns Hopkins; Estados Unido

Feasibility of laser-targeted photoocclusion of the choriocapillary layer in rats

Purpose. A new method, laser-targeted photoocclusion, was developed to occlude choroidal neovascularization while minimizing damage to the overlying retina. The ability to occlude normal choriocapillary layer in rats was evaluated as a first test of die feasibility of treating choroidal neovascularization with this method. Method. A photosensitive agent, aluminum phdialocyanine tetrasulfonate, encapsulated in heat-sensitive liposomes, was administered intravenously along with carboxyfluorescein liposomes. A low-power argon laser (retinal power density of 5.7 W/cm 2 ) locally released a photosensitizer bolus, monitored by the simultaneous release of carboxyfluorescein. A diode laser (operating at 675 nm with a retinal power density of 0.27 W/cm 2 ) activated the photosensitizer with its release. Results. Vessels in the choriocapillary layer were occluded at day 3 after laser treatment and remained unchanged during die 30-day follow-up. Larger choroidal vessels and retinal capillaries remained perfused. Control experiments excluded possible effects of heat or activation of free photosensitizer. Pilot histologic studies showed no damage to the retinal pigment epithelium. Conclusions. Laser-targeted photoocclusion caused selective occlusion of normal choriocapillaries while sparing overlying retinal pigment epithelium and retinal vessels. The method has potential as a treatment of choroidal neovascularization diat may minimize iatrogenic loss of vision. Invest Ophthalmol Vis Sci. 1997;38:2702-2710 Age-related macular degeneration (ARMD) is one of the leading causes of severe loss of vision in people more than 50 years old. &quot; 3 Choroidal neovascularization (CNV), which occurs in ARMD, is often treated by laser photocoagulation. However, the thermal damage and the scarring of large macular areas can caus