RESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.
Asunto(s)
Músculo Esquelético , Distrofia Muscular de Duchenne , Animales , Ratones , Utrofina/genética , Utrofina/metabolismo , Músculo Esquelético/metabolismo , Sistema de Señalización de MAP Quinasas , Distrofia Muscular de Duchenne/genética , Expresión GénicaRESUMEN
A single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic biological samples is presented. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio, but the sequential acquisition of projection images typically requires samples to be immobilized during the acquisition. A proof-of-principle system capable of single-shot tomography of a ~1 mm3 volume is presented, demonstrating camera-limited rates of up to 62.5 volumes/s, which has been applied to 3D imaging of a freely swimming zebrafish embryo. This is achieved by recording eight projection views simultaneously on four low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including high throughput flow cytometry applied to model organisms and fluid dynamics studies.
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Imagenología Tridimensional , Tomografía Óptica , Animales , Imagenología Tridimensional/métodos , Pez Cebra , Tomografía Óptica/métodos , Embrión de MamíferosRESUMEN
Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.
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Epigénesis Genética , Impresión Genómica , Animales , Variación Biológica Poblacional , Metilación de ADN , Dieta Alta en Grasa , Femenino , Mamíferos , Ratones , EmbarazoRESUMEN
The transcriptional effector SMAD4 is a core component of the TGF-ß family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-ß family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-ß family ligands, which has implications for diseases where Smad4 is mutated or deleted.
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Proteínas Morfogenéticas Óseas/metabolismo , Proteína Nodal/metabolismo , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Desarrollo Embrionario , Endodermo/metabolismo , Técnicas de Inactivación de Genes , Mesodermo/metabolismo , Morfogénesis , Transducción de Señal , Proteína Smad4/deficiencia , Proteína Smad4/genética , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.
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Calcio/metabolismo , Mitocondrias/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Glucosa/metabolismo , Secreción de Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm-to-cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts-reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN-based reconstructions of OPT data of other species including live zebrafish.
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Procesamiento de Imagen Asistido por Computador/métodos , Redes Neurales de la Computación , Tomografía Óptica , Animales , Pulmón/diagnóstico por imagen , Ratones , Páncreas/diagnóstico por imagen , Pez CebraRESUMEN
Of current obesity treatments, bariatric surgery induces the most weight loss. Given the marked increase in the number of bariatric surgeries performed, elucidating the mechanisms of action is a key research goal. We compared whole brain activation in response to high-energy dense (HED) vs. low-energy dense (LED) visual and auditory food cues before and approximately 4â¯months after Roux-en-Y Gastric Bypass (RYGB) (nâ¯=â¯16) and Sleeve Gastrectomy (SG) (nâ¯=â¯9). We included two control groups: a low-calorie diet weight loss group (WL) (nâ¯=â¯14) and a non-treatment group (NT) (nâ¯=â¯16). Relative to the control groups, the surgery groups showed increased dorsolateral prefrontal cortex (dlPFC) and decreased parahippocampal/fusiform gyrus (PHG/fusiform) activation in response to HED vs. LED, suggesting greater cognitive dietary inhibition and decreased rewarding effects and attention related to HED foods. dlPFC activation was significantly more increased in RYGB vs. SG. We also found that postprandial increases in GLP-1 concentrations (pre to postsurgery) correlated with postsurgical decreases in RYGB brain activity in the inferior temporal gyrus and the right middle occipital gyrus in addition to increases in the right medial prefrontal gyrus/paracingulate for HEDâ¯>â¯LED stimuli, suggesting involvement of these attention and inhibitory regions in satiety signaling postsurgery.
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Encéfalo/diagnóstico por imagen , Gastrectomía , Derivación Gástrica , Obesidad Mórbida/cirugía , Estimulación Acústica , Adulto , Femenino , Alimentos , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Obesidad Mórbida/diagnóstico por imagen , Estimulación Luminosa , Periodo Posoperatorio , Adulto JovenRESUMEN
OBJECTIVE: Hepatic insulin clearance is a significant regulator of glucose homestasis. We hypothesized that the improvement in insulin clearance rates (ICRs) under fasting conditions and in response to oral and intravenous (IV) glucose would improve similarly after Roux-en-Y gastric bypass (RYGB) and adjustable gastric banding (AGB) as a function of weight loss; the difference in ICR after oral and IV glucose stimulation will be enhanced after RYGB compared with AGB, an effect mediated by glucagon-like peptide 1 (GLP-1). RESEARCH DESIGN AND METHODS: In study 1, the ICR was calculated under fasting condition (F-ICR), after oral glucose (O-ICR), and after an isoglycemic IV glucose clamp (IV-ICR) in individuals from an established cohort with type 2 diabetes mellitus (T2DM) before, after 10% matched weight loss, and 1 year after either RYGB (n = 22) or AGB (n = 12). In study 2, O-ICR was studied in a separate cohort of individuals with T2DM (n = 22), before and 3 months after RYGB, with and without exendin(9-39) infusion. RESULTS: In study 1, age, BMI, T2DM duration and control, and ICR did not differ between RYGB and AGB preintervention. Weight loss at 1 year was two times greater after RYGB than after AGB (31.6 ± 5.9% vs. 16.6 ± 9.8%; P < 0.05). RYGB and AGB both significantly increased F-ICR, O-ICR, and IV-ICR at 1 year. ICR was inversely associated with insulinemia. The difference between IV-ICR and O-ICR was significantly greater after RYGB versus AGB. GLP-1 antagonism with exendin(9-39) led to an increase in O-ICR in subjects post-RYGB. CONCLUSIONS: Weight loss increased ICR, an effect more pronounced after RYGB compared with AGB. Our data support a potential role for endogenous GLP-1 in the control of postprandial ICR after RYGB.
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Diabetes Mellitus Tipo 2/cirugía , Derivación Gástrica , Gastroplastia , Glucosa/administración & dosificación , Insulina/metabolismo , Pérdida de Peso/fisiología , Administración Intravenosa , Administración Oral , Adulto , Cirugía Bariátrica/rehabilitación , Estudios de Cohortes , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Derivación Gástrica/rehabilitación , Gastroplastia/rehabilitación , Prueba de Tolerancia a la Glucosa , Humanos , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Estudios Longitudinales , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Periodo PosprandialRESUMEN
In order to study physical relationships within tissue volumes or even organism-level systems, the spatial distribution of multiple fluorescent markers needs to be resolved efficiently in three dimensions. Here, rather than acquiring discrete spectral images sequentially using multiple emission filters, a hyperspectral scanning laser optical tomography system is developed to obtain hyperspectral volumetric data sets with 2-nm spectral resolution of optically transparent mesoscopic (millimeter-centimeter) specimens. This is achieved by acquiring a series of point-scanning hyperspectral extended depth of field images at different angles and subsequently tomographically reconstructing the 3D intensity distribution for each wavelength. This technique is demonstrated to provide robust measurements via the comparison of spectral and intensity profiles of fluorescent bead phantoms. Due to its enhanced spectral resolving ability, this technique is also demonstrated to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual-labeled mouse thymus gland sample and the ability to distinguish tumorous and normal tissues of an unlabeled mouse intestine sample.
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Rayos Láser , Tomografía Óptica/métodos , Animales , Imagenología Tridimensional , Intestinos/diagnóstico por imagen , Ratones , Fantasmas de ImagenRESUMEN
To improve the imaging performance of optical projection tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
RESUMEN
The present study was a 4-week randomized trial to assess the efficacy and safety of sitagliptin, a dipeptidyl-peptidase-4 inhibitor, in persistent or recurring type 2 diabetes after Roux-en-Y gastric bypass surgery (RYGB). Participants (n = 32) completed a mixed meal test (MMT) and self-monitoring of plasma glucose (SMPG) before and 4 weeks after randomization to either sitagliptin 100 mg daily or placebo daily. Questionnaires were administered to assess gastrointestinal discomfort. Outcome variables were glucose, active glucagon-like peptide-1 and ß-cell function during the MMT, and glucose levels during SMPG. Age (56.3 ± 8.2 years), body mass index (34.4 ± 6.7 kg/m2 ), glycated haemoglobin (7.21 ± 0.77%), diabetes duration (12.9 ± 10.0 years), years since RYGB (5.6 ± 3.3 years) and ß-cell function did not differ between the placebo and sitagliptin groups at pre-intervention. Sitagliptin was well tolerated, decreased postprandial glucose levels during the MMT (from 8.31 ± 1.92 mmol/L to 7.67 ± 1.59 mmol/L, P = 0.03) and mean SMPG levels, but had no effect on ß-cell function. In patients with diabetes and mild hyperglycemia after RYGB, a short course of sitagliptin provided a small but significant glucose-lowering effect, with no identified improvement in ß-cell function.
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Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/cirugía , Derivación Gástrica , Fosfato de Sitagliptina/farmacología , Adulto , Anciano , Glucemia/metabolismo , Terapia Combinada , Diabetes Mellitus Tipo 2/sangre , Femenino , Hemoglobina Glucada/efectos de los fármacos , Hemoglobina Glucada/metabolismo , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fosfato de Sitagliptina/uso terapéuticoRESUMEN
BACKGROUND: Roux-en-Y gastric bypass (RYGB) accelerates gastric pouch emptying, enhances postprandial glucagon-like peptide 1 (GLP-1) and insulin, and lowers glucose concentrations. To prevent discomfort and reactive hypoglycemia, it is recommended that post-RYGB patients eat small, frequent meals and avoid caloric drinks. However, the effect of meal size and texture on GLP-1 and metabolic response has not been studied. OBJECTIVES: To demonstrate that frequent minimeals and solid meals (S) elicit less GLP-1 and insulin release and less reactive hypoglycemia and are better tolerated compared with a single isocaloric liquid meal (L). SETTING: A university hospital. METHODS: In this prospective study, 32 RYGB candidates were enrolled and randomized to L or S groups before gastric bypass. Each subject received an L or S 600-kcal single meal (SM) administered at hour 0 or three 200-kcal minimeals administered at hours 0, 2, and 4 on 2 separate days. Twenty-one patients were retested 1 year after RYGB. Blood and visual analogue scale measurements were collected up to 6 hours postprandially. Outcome measures included gastric pouch emptying, glucose, insulin, and GLP-1; hunger, fullness, and stomach discomfort were measured by visual analogue scale. RESULTS: Twenty-one were patients retested after RYGB (L: n = 12; S: n = 9). Meal texture had a significant effect on peak GLP-1 (L-SM: 106.1 ± 67.2 versus S-SM: 45.3 ± 25.2 pM, P = .004), peak insulin, and postprandial glucose. Hypoglycemia was more frequent after the L-SM meal compared with the S-SM. Gastric pouch emptying was 2.4 times faster after RYGB but was not affected by texture. CONCLUSIONS: Meal texture and size have significant impact on tolerance and metabolic response after RYGB.
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Dieta , Derivación Gástrica , Vaciamiento Gástrico/fisiología , Péptido 1 Similar al Glucagón/metabolismo , Obesidad Mórbida/metabolismo , Obesidad Mórbida/cirugía , Adulto , Glucemia , Femenino , Humanos , Insulina/sangre , Masculino , Comidas , Obesidad Mórbida/fisiopatología , Estudios Prospectivos , Adulto JovenRESUMEN
Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing - with the latter typically requiring weeks for cm scale tissue samples. To address this challenge, we present a new, convenient, aqueous optical clearing agent, termed UbasM: Urea-Based Amino-Sugar Mixture, that rapidly renders fixed tissue samples highly transparent and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues. UbasM is simple, inexpensive, reproducible and compatible with all labeling methods that we have encountered. It can enable convenient, volumetric imaging of tissue up to the scale of whole adult mouse organs and should be useful for a wide range of light microscopy and tomography techniques applied to biomedical research, especially the study on organism-level systems biology at multiple levels.
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Técnicas Histológicas/métodos , Imagen Óptica/métodos , Amino Azúcares/química , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/patología , Soluciones , Urea/químicaRESUMEN
We describe the implementation of an OPT plate to perform optical projection tomography (OPT) on a commercial wide-field inverted microscope, using our open-source hardware and software. The OPT plate includes a tilt adjustment for alignment and a stepper motor for sample rotation as required by standard projection tomography. Depending on magnification requirements, three methods of performing OPT are detailed using this adaptor plate: a conventional direct OPT method requiring only the addition of a limiting aperture behind the objective lens; an external optical-relay method allowing conventional OPT to be performed at magnifications >4x; a remote focal scanning and region-of-interest method for improved spatial resolution OPT (up to ~1.6 µm). All three methods use the microscope's existing incoherent light source (i.e. arc-lamp) and all of its inherent functionality is maintained for day-to-day use. OPT acquisitions are performed on in vivo zebrafish embryos to demonstrate the implementations' viability.
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Microscopía/métodos , Programas Informáticos , Tomografía Óptica/métodos , Algoritmos , Animales , Pez CebraRESUMEN
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Animales , Técnicas Biosensibles , Células COS , Chlorocebus aethiops , Humanos , Imagen Óptica , Programas Informáticos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
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Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Impresión Genómica/fisiología , Alelos , Animales , Cromatina/fisiología , Metilación de ADN/fisiología , Epigénesis Genética/fisiología , RatonesRESUMEN
OBJECTIVE: The superior effect of Roux-en-Y gastric bypass (RYGB) on glucose control compared with laparoscopic adjustable gastric banding (LAGB) is confounded by the greater weight loss after RYGB. We therefore examined the effect of these two surgeries on metabolic parameters matched on small and large amounts of weight loss. RESEARCH DESIGN AND METHODS: Severely obese individuals with type 2 diabetes were tested for glucose metabolism, ß-cell function, and insulin sensitivity after oral and intravenous glucose stimuli, before and 1 year after RYGB and LAGB, and at 10% and 20% weight loss after each surgery. RESULTS: RYGB resulted in greater glucagon-like peptide 1 release and incretin effect, compared with LAGB, at any level of weight loss. RYGB decreased glucose levels (120 min and area under the curve for glucose) more than LAGB at 10% weight loss. However, the improvement in glucose metabolism, the rate of diabetes remission and use of diabetes medications, insulin sensitivity, and ß-cell function were similar after the two types of surgery after 20% equivalent weight loss. CONCLUSIONS: Although RYGB retained its unique effect on incretins, the superiority of the effect of RYGB over that of LAGB on glucose metabolism, which is apparent after 10% weight loss, was attenuated after larger weight loss.
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Cirugía Bariátrica/métodos , Diabetes Mellitus Tipo 2/metabolismo , Derivación Gástrica/métodos , Glucosa/metabolismo , Obesidad/cirugía , Edulcorantes/metabolismo , Adulto , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/cirugía , Femenino , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/administración & dosificación , Humanos , Incretinas/metabolismo , Resistencia a la Insulina/fisiología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/metabolismo , Periodo Posoperatorio , Estudios Prospectivos , Edulcorantes/administración & dosificación , Pérdida de Peso/fisiologíaRESUMEN
Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering.
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Cartílago/embriología , Miembro Posterior/embriología , Morfogénesis/fisiología , Movimiento/fisiología , Animales , Cartílago/fisiología , Embrión de Pollo , Miembro Posterior/fisiología , Estimulación FísicaRESUMEN
We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This "mesoscopic" imaging method bridges a gap between established ~µm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.