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Adaptive testing has a long but largely unrecognized history. The advent of computer-based testing has created new opportunities to incorporate adaptive testing into conventional programmes of study. Relatively recently software has been developed that can automate the delivery of summative assessments that adapt by difficulty or content. Both types of adaptive testing require a large item bank that has been suitably quality assured. Adaptive testing by difficulty enables more reliable evaluation of individual candidate performance, although at the expense of transparency in decision making, and requiring unidirectional navigation. Adaptive testing by content enables reduction in compensation and targeted individual support to enable assurance of performance in all the required outcomes, although at the expense of discovery learning. With both types of adaptive testing, candidates are presented a different set of items to each other, and there is the potential for that to be perceived as unfair. However, when candidates of different abilities receive the same items, they may receive too many they can answer with ease, or too many that are too difficult to answer. Both situations may be considered unfair as neither provides the opportunity to demonstrate what they know. Adapting by difficulty addresses this. Similarly, when everyone is presented with the same items, but answer different items incorrectly, not providing individualized support and opportunity to demonstrate performance in all the required outcomes by revisiting content previously answered incorrectly could also be considered unfair; a point addressed when adapting by content. We review the educational rationale behind the evolution of adaptive testing and consider its inherent strengths and limitations. We explore the continuous pursuit of improvement of examination methodology and how software can facilitate personalized assessment. We highlight how this can serve as a catalyst for learning and refinement of curricula; fostering engagement of learner and educator alike.
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PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.
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Hipopituitarismo , Insuficiencia Ovárica Primaria , Animales , Femenino , Humanos , Hipopituitarismo/genética , Masculino , Ratones , Proteínas Nucleares/genética , Linaje , Fenotipo , Insuficiencia Ovárica Primaria/genética , Prolactina/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The positive regulatory (PR) domain containing 13 (PRDM13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis, and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia and normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the cooccurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.
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Cerebelo/anomalías , N-Metiltransferasa de Histona-Lisina , Hipogonadismo , Hipotálamo/enzimología , Mutación , Malformaciones del Sistema Nervioso , Factores de Transcripción , Animales , Cerebelo/enzimología , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Modelos Animales de Enfermedad , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Hipogonadismo/enzimología , Hipogonadismo/genética , Ratones , Ratones Mutantes , Malformaciones del Sistema Nervioso/enzimología , Malformaciones del Sistema Nervioso/genética , Neuronas/enzimología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.
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Mutación con Ganancia de Función , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófisis/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Niño , Preescolar , Corticotrofos/citología , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Facies , Insuficiencia de Crecimiento/genética , Células HEK293 , Cardiopatías Congénitas/genética , Humanos , Lactante , Sistema de Señalización de MAP Quinasas/genética , Melanotrofos/citología , Melanotrofos/metabolismo , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Secuenciación del Exoma/métodosRESUMEN
A miniature thermal infrared laser heterodyne spectro-radiometer based on hybrid optical integration is demonstrated. A quantum cascade laser emitting at 953â cm-1 (10.5 µm) is used as the local oscillator. Integration is achieved using hollow waveguides inscribed in a copper substrate, with slot-encapsulated optical components positioned to maintain fundamental hybrid mode coupling. The demonstrator performances are studied in the laboratory and show a noise level within 1.6 times of the ideal case. Atmospheric high-resolution transmittance spectroscopy of carbon dioxide and water vapor in solar occultation is demonstrated. The total column concentrations are derived as well as measurement uncertainties, 399.5 ± 2.2â ppm for CO2 and 1066 ± 62â ppm for H2O. The miniature laser heterodyne spectro-radiometer demonstration opens the prospect for nanosatellite-based high spectral resolution thermal infrared atmospheric sounding.
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This article was migrated. The article was marked as recommended. Context: We challenge the philosophical acceptability of the Angoff method, and propose an alternative method of standard setting based on how important it is for candidates to know the material each test item assesses, and not how difficult it is for a subgroup of candidates to answer each item. Methods: The practicalities of an alternative method of standard setting are evaluated here, for the first time, with direct comparison to an Angoff method. To negate bias due to any leading effects, a prospective cross-over design was adopted involving two groups of judges (n=7 and n=8), both of which set the standards for the same two 100 item multiple choice question tests, by the two different methods. Results: Overall, we found that the two methods took a similar amount of time to complete. The alternative method produced a higher cut-score (by 12-14%), and had a higher degree of variability between judges' cut-scores (by 5%). When using the alternative method, judges reported a small, but statistically significant, increase in their confidence to decide accurately the standard (by 3%). Conclusion: This is a new approach to standard setting where the quantitative differences are slight, but there are clear qualitative advantages associated with use of the alternative method.
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Using hollow waveguide hybrid optical integration, a miniaturized mid-infrared laser absorption spectrometer for 13CO2/12CO2 isotopologue ratio analysis is presented. The laser analyzer described focuses on applications where samples contain a few percent of CO2, such as breath analysis and characterization of geo-carbon fluxes, where miniaturization facilitates deployment. As part of the spectrometer design, hollow waveguide mode coupling and propagation is analyzed to inform the arrangement of the integrated optical system. The encapsulated optical system of the spectrometer occupies a volume of 158 × 60 × 30 mm3 and requires a low sample volume (56 µL) for analysis, while integrating a quantum cascade laser, coupling lens, hollow waveguide cell and optical detector into a single copper alloy substrate. The isotopic analyzer performance is characterized through robust error propagation analysis, from spectral inversion to calibration errors. The analyzer achieves a precision of 0.2 in 500 s integration. A stability time greater than 500 s was established to allow two-point calibration. The accuracy achieved is 1.5, including a contribution of 0.7 from calibrant gases that can be addressed with improved calibration mixtures.
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BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.
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Factor 2 Eucariótico de Iniciación/genética , Genes Ligados a X , Glucosa/metabolismo , Hipopituitarismo/etiología , Hipopituitarismo/metabolismo , Fenotipo , Sustitución de Aminoácidos , Apoptosis , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Línea Celular , Preescolar , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Hipopituitarismo/diagnóstico , Hibridación in Situ , Lactante , Imagen por Resonancia Magnética , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Biosíntesis de ProteínasRESUMEN
Nutrition plays a critical role in programming and shaping linear growth during early postnatal life through direct action on the development of the neuroendocrine somatotropic (GH/IGF-1) axis. IGF-1 is a key factor in modulating the programming of linear growth during this period. Notably, IGF-1 preferentially stimulates axonal growth of GHRH neurons in the arcuate nucleus of the hypothalamus (Arc), which is crucial for the proliferation of somatotroph progenitors in the pituitary, thus influencing later GH secretory capacity. However, other nutrition-related hormones may also be involved. Among them, insulin shares several structural and functional similarities with IGF-1, as well as downstream signaling effectors. We investigated the role of insulin in the control of Arc axonal growth using an in vitro model of arcuate explants culture and a cell-type specific approach (GHRH-eGFP mice) under both physiological conditions (normally fed pups) and those of dietary restriction (underfed pups). Our data suggest that insulin failed to directly control axonal growth of Arc neurons or influence specific IGF-1-mediated effects on GHRH neurons. Insulin may act on neuronal welfare, which appears to be dependent on neuronal sub-populations and is influenced by the nutritional status of pups in which Arc neurons develop.
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Núcleo Arqueado del Hipotálamo/metabolismo , Axones/metabolismo , Insulina/farmacología , Estado Nutricional , Animales , Animales Recién Nacidos , Núcleo Arqueado del Hipotálamo/citología , Técnicas de Cultivo de Célula , Células Cultivadas , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones TransgénicosRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0170083.].
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Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postnatal period with programming of the growth trajectory.
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Axones/efectos de los fármacos , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proyección Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Axones/metabolismo , Axones/fisiología , Femenino , Crecimiento y Desarrollo/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
This article was migrated. The article was marked as recommended. The Angoff standard setting method depends fundamentally on the conceptualisation of an anchor statement. The precise wording and consequent interpretation of anchor statements varies in practice. Emphasis is often placed on standard setting judges' perceptions of difficulty for a candidate subgroup. The current review focusses on the meaning of anchor statements and argues that when determining the required standard of performance it is more appropriate to consider: (1) what it is important to achieve, and not how difficult it is to achieve it; (2) what all candidates should achieve, and not what a subgroup of candidates would achieve. In summary, current practice should be refined by using an anchor statement which refers to estimating the 'minimum acceptable performance by every candidate' for each item being tested, and then requiring each judge to score the relevant aspects of importance which could then be combined to derive a cut-score.
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Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.
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Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Drosophila melanogaster/metabolismo , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Modelos Neurológicos , Mutación , Fosfoproteínas/metabolismo , Isoformas de Proteínas , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.
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Potenciales de Acción/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hipotálamo/fisiología , Somatostatina/fisiología , Animales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-ClampRESUMEN
Pancreatic ß-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory ß-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of ß-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping ß-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.
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Permeabilidad Capilar , Células Secretoras de Insulina/fisiología , Insulina/metabolismo , Animales , Velocidad del Flujo Sanguíneo , Proliferación Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Gestacional/metabolismo , Dieta Alta en Grasa/efectos adversos , Femenino , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Microscopía Intravital , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Páncreas/irrigación sanguínea , EmbarazoRESUMEN
The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to ß-catenin accumulation, and pharmacological inhibition of ß-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of ß-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of ß-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent ß-catenin signaling, highlighting ubiquitin homeostasis and ß-catenin as potential therapeutic targets for SMA.
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Atrofia Muscular Espinal/etiología , Atrofia Muscular Espinal/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , beta Catenina/metabolismo , Empalme Alternativo , Animales , Modelos Animales de Enfermedad , Drosophila , Homeostasis , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Médula Espinal/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Pez CebraRESUMEN
Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.
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Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Células Ependimogliales/patología , Hipopituitarismo/etiología , Hipotálamo/patología , Neuronas/patología , Uniones Estrechas/patología , Animales , Núcleo Arqueado del Hipotálamo/inmunología , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/patología , Biomarcadores/metabolismo , Células Ependimogliales/inmunología , Células Ependimogliales/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hipopituitarismo/inmunología , Hipopituitarismo/metabolismo , Hipopituitarismo/patología , Hipotálamo/inmunología , Hipotálamo/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Eminencia Media/inmunología , Eminencia Media/metabolismo , Eminencia Media/patología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/inmunología , Neuronas/metabolismo , Permeabilidad , Proteínas Recombinantes de Fusión/metabolismo , Tercer Ventrículo/inmunología , Tercer Ventrículo/metabolismo , Tercer Ventrículo/patología , Uniones Estrechas/inmunología , Uniones Estrechas/metabolismoRESUMEN
Alcohol dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) ß1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN x C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.
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Consumo de Bebidas Alcohólicas/genética , Receptores de GABA-A/genética , Trastornos Relacionados con Alcohol/genética , Animales , Femenino , Genes Dominantes , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Núcleo Accumbens/fisiología , Mutación Puntual , Receptores de GABA-A/metabolismoRESUMEN
Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional in vivo analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. Here, we investigate the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll(10b), in neurons only, rescued the semi-lethality of spz(2) and DNT1(41), DNT2(e03444) double mutants, indicating retrograde functions in neurons. In spz(2) mutants, DNT1(41), DNT2(e03444) double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in spz(2) mutants and DNT1(41), DNT2(e03444) double mutants, but frequency and amplitude of spontaneous events were reduced in spz(2) mutants suggesting defective neurotransmission. Our data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ.
Asunto(s)
Proteínas de Drosophila/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Neuronas/metabolismo , Animales , Drosophila , Transmisión Sináptica/fisiologíaRESUMEN
Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.