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Despite recent advances in robotic technology, sewer pipe inspection is still limited to conventional approaches that use cable-tethered robots. Such commercially available tethered robots lack autonomy, and their operation must be manually controlled via their tethered cables. Consequently, they can only travel to a certain distance in pipe, cannot access small-diameter pipes, and their deployment incurs high costs for highly skilled operators. In this paper, we introduce a miniaturised mobile robot for pipe inspection. We present an autonomous control strategy for this robot that is effective, stable, and requires only low-computational resources. The robots used here can access pipes as small as 75 mm in diameter. Due to their small size, low carrying capacity, and limited battery supply, our robots can only carry simple sensors, a small processor, and miniature wheel-legs for locomotion. Yet, our control method is able to compensate for these limitations. We demonstrate fully autonomous robot mobility in a sewer pipe network, without any visual aid or power-hungry image processing. The control algorithm allows the robot to correctly recognise each local network configuration, and to make appropriate decisions accordingly. The control strategy was tested using the physical micro robot in a laboratory pipe network. In both simulation and experiment, the robot autonomously and exhaustively explored an unknown pipe network without missing any pipe section while avoiding obstacles. This is a significant advance towards fully autonomous inspection robot systems for sewer pipe networks.
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Human teeth are mechanically robust through a complex structural composite organisation of materials and morphology. Efforts to replicate mechanical function in artificial teeth (typodont teeth), such as in dental training applications, attempt to replicate the structure and morphology of real teeth but lack tactile similarities during mechanical cutting of the teeth. In this study, biomimetic typodont teeth, with morphology derived from X-ray microtomography scans of extracted teeth, were 3D printed using an approach to develop novel composites. These composites with a range of glass, hydroxyapatite and porcelain reinforcements within a methacrylate-based photopolymer resin were compared to six commercial artificial typodont teeth. Mechanical performance of the extracted human teeth and 3D printed typodont teeth were evaluated using a haptic approach of measuring applied cutting forces. Results indicate 3D printed typodont teeth replicating enamel and dentine can be mechanically comparable to extracted human teeth despite the material compositions differing from the materials found in human teeth. A multiple parameter variable of material elastic modulus and hardness is shown to describe the haptic response when cutting through both human and biomimetic, highlighting a critical dependence between the ratio of material mechanical properties and not absolute material properties in determining tooth mechanical performance under the action of cutting forces.
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Biomimética , Diente , Resinas Compuestas/química , Durapatita , Humanos , Ensayo de Materiales , Impresión Tridimensional , Diente/diagnóstico por imagen , Microtomografía por Rayos XRESUMEN
Additive Manufacturing (AM) often produces complex engineered structures by precisely distributing materials in a layer-by-layer fashion. Multimaterial AM is a particularly flexible technique able to combine a range of hard and soft materials to produce designed composites. Critically, the design of AM multimaterial structures requires the development of precise three-dimensional (3D) computed aided design (CAD) files. While such digital design is heavily used, techniques able to validate the physically manufactured composite against the digital design from which it is generated are lacking for AM, especially as any evaluations must be able to distinguish material variation across the 3D space. Nowadays, there is a growing interest in volumetric tools that can provide topological information hidden by the surface of shaped materials. So far, technologies such as Optical microscopy (OM), Scanning Electron Microscopy (SEM), and Coordinate Measuring Machine (CMM) have paved the way into the metrology field to measure the external geometry of physical objects. Currently, alongside conventional metrology tools, X-ray computed tomography (XCT) is emerging to measure the subsurface of the objects but maintaining the integrity of the probed samples. Thereby, the volumetric nature of the XCT investigations and its associated imaging techniques, ensure 3D quantitative measurements comparable to the output data from 2D metrology tools, but above all, supply the missing subsurface description for an exhaustive metrology study. The reward associated with XCT applied to multimaterial AM is a map reflecting the fabricated distribution of materials following CAD, with the benefits of better understanding the mechanical interplay within phases, hence, describing the hidden processes as well as the changes in phases due to a range of mechanical or chemical phenomena. In this study, a nondestructive approach using X-ray computed tomography (XCT) is used to fully evaluate the 3D distribution of multimaterials from an AM process. Specifically, two diverse hard and soft materials are alternatively produced in the form of a fibre embedded in a matrix via ink-jet printing. XCT coupled with imaging evaluation were able to distinguish between the differing materials and, importantly, to demonstrate a reduction in the expected fabricated volumes when compared to the respective CAD designs. LAY DESCRIPTION: Additive Manufacturing (AM) has recently become important in producing complex engineered structures. Using 3D CAD files and/or reconstructed data sets from imaging, hard and soft materials are manufactured independently or in combination, according to geometrical features and shapes in the input data. However, the evaluation of the resultant manufactured parts in comparison with the original 3D drawing is currently lacking. In this sense, X-ray computed tomography (XCT) provides an important metrology tool for mono and multimaterial AM. In this work a volumetric metrology investigation is proposed using higher resolution XCT to provide 3D information comparable to that of the 3D CAD drawings. A commercial high-resolution multijetting material printer (ProJet 5500X, 3D Systems, USA) is used to manufacture single fibre composites, through a complementary deposition of photo sensible polymers. Hard and soft plastics are produced using a UV curable step, resulting in materials of similar attenuation under an X-ray probe. A critical aim of the evaluations is the potential for XCT to distinguish between different UV curable 3D printing materials.
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Impresión Tridimensional , Tomografía Computarizada por Rayos X , Microscopía Electrónica de Rastreo , Polímeros , Tomografía Computarizada por Rayos X/métodosAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos , Agentes Inmunomoduladores/uso terapéutico , Mieloma Múltiple/patología , Mutación , Ubiquitina-Proteína Ligasas/genética , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genéticaAsunto(s)
Atención Odontológica/normas , Fluoruros Tópicos/administración & dosificación , Adhesión a Directriz , Neoplasias de Cabeza y Cuello/terapia , Guías de Práctica Clínica como Asunto , Radiografía Panorámica , Auditoría Clínica , Documentación , Humanos , Estudios Prospectivos , Estudios Retrospectivos , Reino UnidoRESUMEN
A micromechanical characterization of biomaterials for bone tissue engineering is essential to understand the quality of the newly regenerated bone, enabling the improvement of tissue regeneration strategies. A combination of microcomputed tomography in conjunction with in situ mechanical testing and digital volume correlation (DVC) has become a powerful technique to investigate the internal deformation of bone structure at a range of dimensional scales. However, in order to obtain accurate three-dimensional strain measurement at tissue level, high-resolution images must be acquired, and displacement/strain measurement uncertainties evaluated. The aim of this study was to optimize imaging parameters, image postprocessing and DVC settings to enhance computation based on 'zero-strain' repeated high-resolution synchrotron microCT scans of trabecular bone and bone-biomaterial systems. Low exposures to SR X-ray radiation were required to minimize irradiation-induced tissue damage, resulting in the need of advanced three-dimensional filters on the reconstructed images to reduce DVC-measured strain errors. Furthermore, the computation of strain values only in the hard phase (i.e. bone, biomaterial) allowed the exclusion of large artefacts localized in the bone marrow. This study demonstrated the suitability of a local DVC approach based on synchrotron microCT images to investigate the micromechanics of trabecular bone and bone-biomaterial composites at tissue level with a standard deviation of the errors in the region of 100 microstrain after a thorough optimization of DVC computation. LAY DESCRIPTION: Understanding the quality of newly regenerated bone after implantation of novel biomaterials is essential to improve bone tissue engineering strategies and formulation of biomaterials. The relationship between microstructure and mechanics of bone has been previously addressed combining microcomputed tomography with in situ mechanical testing. The addition of an image-based experimental technique such as digital volume correlation (DVC) allows to characterize the deformation of materials in a three-dimensional manner. However, in order to obtain accurate information at the micro-scale, high-resolution images, obtained for example by using synchrotron radiation microcomputed tomography, as well as optimization of the DVC computation are needed. This study presents the effect of different imaging parameters, image postprocessing and DVC settings for as accurate investigation of trabecular bone structure and bone-biomaterial interfaces. The results showed that when appropriate image postprocessing and DVC settings are used DVC computation results in very low strain errors. This is of vital importance for a correct understanding of the deformation in bone-biomaterial systems and the ability of such biomaterials in producing new bone comparable with the native tissue they are meant to replace.
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Materiales Biocompatibles , Huesos/diagnóstico por imagen , Sincrotrones , Microtomografía por Rayos X/métodos , Humanos , Imagenología Tridimensional , Programas InformáticosRESUMEN
Artificial teeth have several advantages in preclinical training. The aim of this study is to three-dimensionally (3D) print accurate artificial teeth using scans from X-ray microtomography (XMT). Extracted and artificial teeth were imaged at 90 kV and 40 kV, respectively, to create detailed high contrast scans. The dataset was visualised to produce internal and external meshes subsequently exported to 3D modelling software for modification before finally sending to a slicing program for printing. After appropriate parameter setting, the printer deposited material in specific locations layer by layer, to create a 3D physical model. Scans were manipulated to ensure a clean model was imported into the slicing software, where layer height replicated the high spatial resolution that was observed in the XMT scans. The model was then printed in two different materials (polylactic acid and thermoplastic elastomer). A multimaterial print was created to show the different physical characteristics between enamel and dentine. LAY DESCRIPTION: Objectives Trainee dentists practice procedures using artificial teeth that are far from real teeth. Using x-rays and 3D printing technology the project will recreate a real tooth, artificially. Methods X-rays produce a 3D image that can be printed out as a physical replica, after several conversions of files. Different settings can be used to allow the printed model, to be as accurate as possible. Data were collected on the forces from a dental drill on a tooth's surface, to measure hardness and resistance. Results Multiple teeth replicas were printed with a high accuracy. The materials printed did not mimic actual tooth properties, but using the data from real teeth, materials can be tested in future.
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Imagenología Tridimensional , Impresión Tridimensional , Diente Artificial , Microtomografía por Rayos X , Humanos , Programas InformáticosRESUMEN
Molar incisor hypomineralization is a developmental defect of dental enamel associated with rapid caries progression. In order to discover whether molar incisor hypomineralization predisposes to dental caries, a cross-sectional cohort study was conducted in a sample of 414 children aged between eight and nine years. It was found that 24.2% of the children presented molar incisor hypomineralization. Of these, 72% had a mild form and 28% a severe form. Caries prevalence was greater among the children with severe form (60.7%) than in those with mild form (43.1%) or no molar incisor hypomineralization (45.5%). The caries indices were higher in out molar incisor hypomineralization (1.18) or with mild form (1.08). The tooth-surface caries ratio was significantly higher in surfaces with severe hypomineralization than in those with no hypomineralization or mild hypomineralization. A linear regression model showed that cariogenic food intake and the presence of severe molar incisor hypomineralization were significantly associated with DMFS. Consequently, an association was found to exist between dental caries and the presence of surfaces affected by severe molar incisor hypomineralization, which should be considered a risk factor within the multifactorial etiology of caries.
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Caries Dental/epidemiología , Hipoplasia del Esmalte Dental/epidemiología , Niño , Caries Dental/patología , Hipoplasia del Esmalte Dental/patología , Femenino , Humanos , MasculinoRESUMEN
The unexpectedly high flux of cosmic-ray positrons detected at Earth may originate from nearby astrophysical sources, dark matter, or unknown processes of cosmic-ray secondary production. We report the detection, using the High-Altitude Water Cherenkov Observatory (HAWC), of extended tera-electron volt gamma-ray emission coincident with the locations of two nearby middle-aged pulsars (Geminga and PSR B0656+14). The HAWC observations demonstrate that these pulsars are indeed local sources of accelerated leptons, but the measured tera-electron volt emission profile constrains the diffusion of particles away from these sources to be much slower than previously assumed. We demonstrate that the leptons emitted by these objects are therefore unlikely to be the origin of the excess positrons, which may have a more exotic origin.
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Cortical bone is an example of a mineralized tissue containing a compositional distribution of hard and soft phases in 3-dimensional space for mechanical function. X-ray computed tomography (XCT) is able to describe this compositional and morphological complexity but methods to provide a physical output with comparable mechanical function is lacking. A workflow is presented here to establish a method of using high contrast XCT to establish a virtual model of cortical bone that is manufactured using a multiple material capable 3D printer. Resultant 3D printed structures were produced based on more and less remodelled bone designs exhibiting a range of secondary osteon density. Variation in resultant mechanical properties of the 3D printed composite structures for each bone design was achieved using a combination of material components and reasonable prediction of elastic modulus provided using a Hashin-Shtrikman approach. The ability to 3D print composite structures using high contrast XCT to distinguish between compositional phases in a biological structure promises improved anatomical models as well as next-generation mechano-mimetic implants.
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Molar incisor hypomineralization (MIH) is a developmental defect of dental enamel that shares features with hypomineralized second primary molars (HSPM). Prior to permanent tooth eruption, second primary molars could have predictive value for permanent molar and incisor hypomineralization. To assess this possible relationship, a cross-sectional study was conducted in a sample of 414 children aged 8 and 9 years from the INMA cohort in Valencia (Spain). A calibrated examiner (linear-weighted Kappa 0.83) performed the intraoral examinations at the University of Valencia between November 2013 and 2014, applying the diagnostic criteria for MIH and HSPM adopted by the European Academy of Paediatric Dentistry. 100 children (24.2%) presented MIH and 60 (14.5%) presented HSPM. Co-occurrence of the two defects was observed in 11.1% of the children examined. The positive predictive value was 76.7% (63.9-86.6) and the negative predictive value 84.7% (80.6-88.3). The positive likelihood ratio (S/1-E) was 10.3 (5.9-17.9) and the negative likelihood ratio (1-S/E) 0.57 (0.47-0.68). The odds ratio was 18.2 (9.39-35.48). It was concluded that while the presence of HSPM can be considered a predictor of MIH, indicating the need for monitoring and control, the absence of this defect in primary dentition does not rule out the appearance of MIH.
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Hipoplasia del Esmalte Dental/diagnóstico , Incisivo/metabolismo , Diente Molar/metabolismo , Niño , Estudios Transversales , Hipoplasia del Esmalte Dental/epidemiología , Hipoplasia del Esmalte Dental/patología , Femenino , Humanos , Incisivo/patología , Masculino , Diente Molar/patología , Oportunidad Relativa , Valor Predictivo de las Pruebas , Prevalencia , Índice de Severidad de la EnfermedadRESUMEN
AIM: Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines with a key role in glucose and lipid metabolism. In the current investigation, we examined the in vivo and in vitro effects of CT-1 treatment on intestinal sugar absorption in different experimental models. METHODS: rCT-1 effects on α-Methyl-D-glucoside uptake were assessed in everted intestinal rings from wild-type and CT-1(-/-) mice and in Caco-2 cells. rCT-1 actions on SGLT-1 expression in brush border membrane vesicles and the identification of the potential signalling pathways involved were determined by Western blot. RESULTS: In vivo administration (0.2 mg kg(-1) ) of rCT-1 caused a significant decrease on α-Methyl-D-glucoside uptake in everted intestinal rings from wild-type and CT-1(-/-) mice after short-term and long-term treatments. Similarly, in vitro treatment (1-50 ng mL(-1) ) with rCT-1 reduced α-Methyl-D-glucoside uptake in everted intestinal rings. In Caco-2 cells, rCT-1 treatment (20 ng mL(-1) , 1 and 24 h) lowered apical uptake of α-Methyl-D-glucoside in parallel with a decrease on SGLT-1 protein expression. rCT-1 promoted the phosphorylation of STAT-3 after 5 and 15 min treatment, but inhibited the activation by phosphorylation of AMPK after 30 and 60 min. Interestingly, pre-treatment with the JAK/STAT inhibitor (AG490) and with the AMPK activator (AICAR) reversed the inhibitory effects of rCT-1 on α-Methyl-D-glucoside uptake. AICAR also prevented the inhibition of SGLT-1 observed in rCT-1-treated cells. CONCLUSIONS: CT-1 inhibits intestinal sugar absorption by the reduction of SGLT-1 levels through the AMPK pathway, which could also contribute to explain the hypoglycaemic and anti-obesity properties of CT-1.
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Citocinas/farmacología , Hipoglucemiantes/farmacología , Absorción Intestinal/efectos de los fármacos , Azúcares/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Células CACO-2 , Citocinas/genética , Citocinas/metabolismo , Activación Enzimática , Humanos , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metilglucósidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Ribonucleótidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transportador 1 de Sodio-Glucosa/biosíntesis , Transportador 1 de Sodio-Glucosa/genética , Tirfostinos/farmacologíaRESUMEN
PURPOSE: Functionally relevant alterations in resting state fMRI (rs-fMRI) connectivity have been identified in adults with traumatic brain injury (TBI). We evaluated rs-fMRI connectivity in children with TBI and explored the relationship between altered connectivity and measures of neurological function. METHODS: Rs-fMRI was obtained in 14 children after TBI and 14 controls matched for age, sex, and handedness. Whole-brain connectivity was evaluated separately for the default mode network (DMN) and dorsal attention network (DAN); Between-group contrasts identified regions with altered connectivity between TBI and control cohorts. In children with TBI, the relationships between regions of altered connectivity and performance on relevant functional measures were examined. RESULTS: Compared to controls, children with TBI showed significantly greater connectivity between DMN and right dorsal premotor cortex (RdPM) and between DAN and bilateral sensorimotor cortex (SM1). In children with TBI, greater DMN-RdPM connectivity was associated with worse motor performance whereas greater DAN-LSM1 connectivity was associated with better motor performance; furthermore, DMN-RdPM and DAN-LSM1 connectivity were negatively correlated. CONCLUSION: Rs-fMRI reveals significant altered connectivity in children with TBI compared to controls. After TBI in children, patterns of altered connectivity appear divergent, with increased DMN-motor network connectivity associated with worse motor control whereas increased DAN-motor network connectivity appears compensatory.
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Lesiones Encefálicas/fisiopatología , Adolescente , Niño , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Imagen por Resonancia Magnética , Masculino , Red Nerviosa/fisiopatologíaRESUMEN
Diabetic retinopathy is one of the most common complications of diabetes. A plethora of literature indicates that oxidative stress may play a central role in the pathogenesis of diabetic retinopathy. One could thus hypothesise that antioxidant therapies may be protective for diabetic retinopathy. Anthocyanins are important natural bioactive pigments responsible for red-blue colour of fruits, leaves, seeds, stems and flowers in a variety of plant species. Apart from their colours, anthocyanins are known to be health-promoting phytochemicals with potential properties useful to protect against oxidative stress in some degenerative diseases. They also have a variety of biological properties including anti-inflammatory, antibacterial, anticancer, and cardio-protective properties. Some reports further suggest a therapeutic role of anthocyanins to prevent and/or protect against ocular diseases but more studies are needed to examine their potential as alternative therapy to diabetic retinopathy. The present article reviews the available literature concerning the beneficial role of anthocyanins in diabetic retinopathy.
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Antocianinas/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Antocianinas/química , Antocianinas/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Ensayos Clínicos como Asunto , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Humanos , Metaloproteinasas de la Matriz/metabolismo , Estrés Oxidativo/efectos de los fármacos , Plantas/química , Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The inelastic deformability of the mineralised matrix in bones is critical to their high toughness, but the nanoscale mechanisms are incompletely understood. Antler is a tough bone type, with a nanostructure composed of mineralised collagen fibrils â¼100nm diameter. We track the fibrillar deformation of antler tissue during cyclic loading using in situ synchrotron small-angle X-ray diffraction (SAXD), finding that residual strain remains in the fibrils after the load was removed. During repeated unloading/reloading cycles, the fibril strain shows minimal hysteresis when plotted as a function of tissue strain, indicating that permanent plastic strain accumulates inside the fibril. We model the tensile response of the mineralised collagen fibril by a two - level staggered model - including both elastic - and inelastic regimes - with debonding between mineral and collagen within fibrils triggering macroscopic inelasticity. In the model, the subsequent frictional sliding at intrafibrillar mineral/collagen interfaces accounts for subsequent inelastic deformation of the tissue in tension. The model is compared to experimental measurements of fibrillar and mineral platelet strain during tensile deformation, measured by in situ synchrotron SAXD and wide-angle X-ray diffraction (WAXD) respectively, as well as macroscopic tissue stress and strain. By fitting the model predictions to experimentally observed parameters like the yield point, elastic modulus and post-yield slope, extremely good agreement is found between the model and experimental data at both the macro- and at the nanoscale. Our results provide strong evidence that intrafibrillar sliding between mineral and collagen leads to permanent plastic strain at both the fibril and the tissue level, and that the energy thus dissipated is a significant factor behind the high toughness of antler bone.
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Cuernos de Venado , Huesos/metabolismo , Colágeno/metabolismo , Fenómenos Mecánicos , Minerales/metabolismo , Animales , Fenómenos Biomecánicos , Ciervos , Módulo de ElasticidadRESUMEN
Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.
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Apoptosis , Caspasas/metabolismo , Animales , Caenorhabditis elegans/enzimología , Línea Celular , Drosophila/enzimología , Humanos , Espectrometría de Masas , Ratones , Transducción de Señal , Especificidad por SustratoRESUMEN
Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.
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Células Fotorreceptoras Retinianas Bastones/fisiología , Células Fotorreceptoras Retinianas Bastones/trasplante , Visión Ocular/fisiología , Animales , Subunidades alfa de la Proteína de Unión al GTP/deficiencia , Subunidades alfa de la Proteína de Unión al GTP/genética , Luz , Aprendizaje por Laberinto , Ratones , Células Bipolares de la Retina/ultraestructura , Células Horizontales de la Retina/ultraestructura , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Transducina/deficiencia , Transducina/genética , Visión Ocular/efectos de la radiación , Corteza Visual/fisiología , Corteza Visual/efectos de la radiaciónRESUMEN
AIM: Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved. METHODS: Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin, and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot. RESULTS: Leptin inhibited 0.1 mm α-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 µm glutamine and 0.1 mm phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na(+) -dependent neutral amino acid transporters ASCT2 and B(0) AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mm proline and ß-alanine uptake in Na(+) medium at pH 6, conditions for optimal activity of the H(+) -dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on ß-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin's regulation of PAT1 activity. CONCLUSION: These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, that is, D-glucose (SGLT1) and amino acids (ASCT2, B(0) AT1 and PAT1).
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Sistemas de Transporte de Aminoácidos/metabolismo , Leptina/farmacología , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Células Cultivadas , Glutamina/metabolismo , Glutamina/farmacología , Humanos , Leptina/metabolismoRESUMEN
Retinal degenerative diseases are a major cause of untreatable blindness. Stem cell therapy to replace lost photoreceptors represents a feasible future treatment. We previously demonstrated that postmitotic photoreceptor precursors expressing an NrlGFP transgene integrate into the diseased retina and restore some light sensitivity. As genetic modification of precursor cells derived from stem cell cultures is not desirable for therapy, we have tested cell selection strategies using fluorochrome-conjugated antibodies recognizing cell surface antigens to sort photoreceptor precursors. Microarray analysis of postnatal NrlGFP-expressing precursors identified four candidate genes encoding cell surface antigens (Nt5e, Prom1, Podxl, and Cd24a). To test the feasibility of using donor cells isolated using cell surface markers for retinal therapy, cells selected from developing retinae by fluorescence-activated cell sorting based on Cd24a expression (using CD24 antibody) and/or Nt5e expression (using CD73 antibody) were transplanted into the wild-type or Crb1(rd8/rd8) or Prph2(rd2/rd2) mouse eye. The CD73/CD24-sorted cells migrated into the outer nuclear layer, acquired the morphology of mature photoreceptors and expressed outer segment markers. They showed an 18-fold higher integration efficiency than that of unsorted cells and 2.3-fold higher than cells sorted based on a single genetic marker, NrlGFP, expression. These proof-of-principle studies show that transplantation competent photoreceptor precursor cells can be efficiently isolated from a heterogeneous mix of cells using cell surface antigens without loss of viability for the purpose of retinal stem cell therapy. Refinement of the selection of donorphotoreceptor precursor cells can increase the number of integrated photoreceptor cells,which is a prerequisite for the restoration of sight.