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The mammalian cell cycle is regulated by a well-studied but complex biochemical reaction system. Computational models provide a particularly systematic and systemic description of the mechanisms governing mammalian cell cycle control. By combining both state-of-the-art multiplexed experimental methods and powerful computational tools, this work aims at improving on these models along four dimensions: model structure, validation data, validation methodology and model reusability. We developed a comprehensive model structure of the full cell cycle that qualitatively explains the behaviour of human retinal pigment epithelial-1 cells. To estimate the model parameters, time courses of eight cell cycle regulators in two compartments were reconstructed from single cell snapshot measurements. After optimisation with a parallel global optimisation metaheuristic we obtained excellent agreements between simulations and measurements. The PEtab specification of the optimisation problem facilitates reuse of model, data and/or optimisation results. Future perturbation experiments will improve parameter identifiability and allow for testing model predictive power. Such a predictive model may aid in drug discovery for cell cycle-related disorders.
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Descubrimiento de Drogas , Neuronas , Humanos , Animales , División Celular , Ciclo Celular , Proyectos de Investigación , MamíferosRESUMEN
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation.
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Proteína Quinasa CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Mitosis , Proteína Fosfatasa 2/metabolismo , Proteína Quinasa CDC2/genética , Línea Celular , Ciclina A/genética , Ciclina B/genética , Humanos , Proteína Fosfatasa 2/genéticaRESUMEN
Elite endurance athletes possess a high capacity for whole-body maximal fat oxidation (MFO). The aim was to investigate the determinants of a high MFO in endurance athletes. The hypotheses were that augmented MFO in endurance athletes is related to concomitantly increments of skeletal muscle mitochondrial volume density (MitoVD ) and mitochondrial fatty acid oxidation (FAOp ), that is, quantitative mitochondrial adaptations as well as intrinsic FAOp per mitochondria, that is, qualitative adaptations. Eight competitive male cross-country skiers and eight untrained controls were compared in the study. A graded exercise test was performed to determine MFO, the intensity where MFO occurs (FatMax ), and V Ë O 2 Max . Skeletal muscle biopsies were obtained to determine MitoVD (electron microscopy), FAOp , and OXPHOSp (high-resolution respirometry). The following were higher (P < 0.05) in endurance athletes compared to controls: MFO (mean [95% confidence intervals]) (0.60 g/min [0.50-0.70] vs 0.32 [0.24-0.39]), FatMax (46% V Ë O 2 Max [44-47] vs 35 [34-37]), V Ë O 2 Max (71 mL/min/kg [69-72] vs 48 [47-49]), MitoVD (7.8% [7.2-8.5] vs 6.0 [5.3-6.8]), FAOp (34 pmol/s/mg muscle ww [27-40] vs 21 [17-25]), and OXPHOSp (108 pmol/s/mg muscle ww [104-112] vs 69 [68-71]). Intrinsic FAOp (4.0 pmol/s/mg muscle w.w/MitoVD [2.7-5.3] vs 3.3 [2.7-3.9]) and OXPHOSp (14 pmol/s/mg muscle ww/MitoVD [13-15] vs 11 [10-13]) were, however, similar in the endurance athletes and untrained controls. MFO and MitoVD correlated (r2 = 0.504, P < 0.05) in the endurance athletes. A strong correlation between MitoVD and MFO suggests that expansion of MitoVD might be rate-limiting for MFO in the endurance athletes. In contrast, intrinsic mitochondrial changes were not associated with augmented MFO.
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Metabolismo de los Lípidos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/fisiología , Esquí/fisiología , Tejido Adiposo/metabolismo , Atletas , Prueba de Esfuerzo , Humanos , Masculino , Oligopéptidos , Oxidación-Reducción , Consumo de Oxígeno , Adulto JovenRESUMEN
This study aims to identify risk factors associated with complications in retinoblastoma patients following primary and secondary enucleations with porous implant placement. A retrospective case-control study was performed between 2010 and 2015. Data pertaining to subjects' demographics, medical history, clinical, and pathological findings, implant characteristics and complications were collected. The analysis included 103 eyes of 101 patients age 27.8 ± 21.9 months undergoing enucleation for retinoblastoma. Postoperatively, 19/103 (18%) eyes developed exposure, extrusion, or hematoma requiring subsequent surgery. Exposure was the most common postoperative complication (12/19, 63%). Age at enucleation 24 months or younger, Hispanic ethnicity, female gender, and intravenous chemotherapy prior to enucleation were associated with increased odds of implant complications. In contrast, patients who were given intravitreal melphalan (IM), subtenons carboplatin (SC), or external beam radiation therapy (EBRT) did not demonstrate an increased risk of complications. In this cohort of retinoblastoma patients undergoing primary or secondary enucleation with porous implants, implant exposure was the most common postoperative complication. Our findings suggest that female gender, Hispanic ethnicity, age at enucleation 24 months or younger, and intravenous chemotherapy prior to enucleation may increase the risk of complications.
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Enucleación del Ojo , Implantes Orbitales , Neoplasias de la Retina/cirugía , Retinoblastoma/cirugía , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Estudios de Casos y Controles , Preescolar , Femenino , Humanos , Lactante , Complicaciones Intraoperatorias , Masculino , Polietilenos , Porosidad , Complicaciones Posoperatorias , Implantación de Prótesis , Radioterapia , Neoplasias de la Retina/patología , Retinoblastoma/patología , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Julia is a general purpose programming language that was designed for simplifying and accelerating numerical analysis and computational science. In particular the Scientific Machine Learning (SciML) ecosystem of Julia packages includes frameworks for high-performance symbolic-numeric computations. It allows users to automatically enhance high-level descriptions of their models with symbolic preprocessing and automatic sparsification and parallelization of computations. This enables performant solution of differential equations, efficient parameter estimation and methodologies for automated model discovery with neural differential equations and sparse identification of nonlinear dynamics. To give the systems biology community easy access to SciML, we developed SBMLToolkit.jl. SBMLToolkit.jl imports dynamic SBML models into the SciML ecosystem to accelerate model simulation and fitting of kinetic parameters. By providing computational systems biologists with easy access to the open-source Julia ecosystevnm, we hope to catalyze the development of further Julia tools in this domain and the growth of the Julia bioscience community. SBMLToolkit.jl is freely available under the MIT license. The source code is available at https://github.com/SciML/SBMLToolkit.jl.
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Lenguajes de Programación , Programas Informáticos , Aprendizaje Automático , Biología de Sistemas , Modelos Biológicos , Simulación por ComputadorRESUMEN
Previous data have suggested an antiviral effect of teriflunomide, including against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent underlying the ongoing COVID-19 pandemic. We undertook an in vitro investigation to evaluate the inhibitory activity of teriflunomide against SARS-CoV-2 in a cell-based assay. Teriflunomide was added to Vero (kidney epithelial) cells that had been infected with SARS-CoV-2. A nucleocapsid immunofluorescence assay was performed to examine viral inhibition with teriflunomide and any potential cytotoxic effect. The 50% effective concentration (EC50) for teriflunomide against SARS-CoV-2 was 15.22 µM. No cytotoxicity was evident for teriflunomide in the Vero cells (i.e., the 50% cytotoxic concentration [CC50] was greater than the highest test concentration of 100 µM). The data were supported by additional experiments using other coronaviruses and human cell lines. In the SARS-CoV-2-infected Vero cells, the prodrug leflunomide had an EC50 of 16.49 µM and a CC50 of 54.80 µM. Our finding of teriflunomide-mediated inhibition of SARS-CoV-2 infection at double-digit micromolar potency adds to a growing body of evidence for a broad-ranging antiviral effect of teriflunomide.
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Synthetic biology relies on rapid and efficient methods to stably integrate DNA payloads encoding for synthetic biological systems into the genome of living cells. The size of designed biological systems increases with their complexity, and novel methods are needed that enable efficient and simultaneous integration of multiple payloads into single cells. By assembling natural and synthetic protein-protein dimerization domains, we have engineered a set of multipartite transcription factors for driving heterologous target gene expression. With the distribution of single parts of multipartite transcription factors on piggyback transposon-based donor plasmids, we have created a logic genome integration control (LOGIC) system that allows for efficient one-step selection of stable mammalian cell lines with up to three plasmids. LOGIC significantly enhances the efficiency of multiplexed payload integration in mammalian cells compared to traditional cotransfection and may advance cell line engineering in synthetic biology and biotechnology.
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Genoma/genética , Mamíferos/genética , Factores de Transcripción/genética , Animales , Biotecnología/métodos , Línea Celular , Expresión Génica/genética , Ingeniería Genética/métodos , Lógica , Plásmidos/genética , Dominios Proteicos/genética , Biología Sintética/métodosRESUMEN
Non-canonical residues, caps, crosslinks, and nicks are important to many functions of DNAs, RNAs, proteins, and complexes. However, we do not fully understand how networks of such non-canonical macromolecules generate behavior. One barrier is our limited formats for describing macromolecules. To overcome this barrier, we develop BpForms and BcForms, a toolkit for representing the primary structure of macromolecules as combinations of residues, caps, crosslinks, and nicks. The toolkit can help omics researchers perform quality control and exchange information about macromolecules, help systems biologists assemble global models of cells that encompass processes such as post-translational modification, and help bioengineers design cells.
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Sustancias Macromoleculares/normas , Estructura Molecular , Programas Informáticos , Sustancias Macromoleculares/química , Proteómica , Biología Sintética , Biología de SistemasRESUMEN
Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.
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Diseño de Fármacos , Microscopía Confocal , Microscopía Fluorescente , Animales , Biomarcadores , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Pruebas de MicronúcleosRESUMEN
All organisms evolved defense mechanisms to counteract oxidative stress and buildup of reactive oxygen species (ROS). To test whether a potentially conserved mechanism exists for the rapid response, we investigated immediate metabolic dynamics of Escherichia coli, yeast, and human dermal fibroblasts to oxidative stress that we found to be conserved between species. To elucidate the regulatory mechanisms that implement this metabolic response, we developed mechanistic kinetic models for each organism's central metabolism and systematically tested activation and inactivation of each irreversible reaction by each metabolite. This ensemble modeling predicts in vivo relevant metabolite-enzyme interactions based on their ability to quantitatively describe metabolite dynamics. All three species appear to inhibit their oxidative pentose phosphate pathway during normal growth by the redox cofactor NADPH and relieve this inhibition to increase the pathway flux for detoxification of ROS during stress, with the sole exception of yeast when exposed to high levels of stress.
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PURPOSE: To compare the relative 12-month corneal crosslinking (CXL) functional outcomes using standard protocol and accelerated protocols in patients with progressive keratoconus. METHODS: CXL was performed using 3 epithelium-off protocols: standard [3 mW/cm for 30 minutes, 5.4 J/cm (S3/30-CXL)], accelerated with equivalent total irradiance [9 mW/cm for 10 minutes, 5.4 J/cm (A9/10-CXL)], and accelerated with increased total irradiance [30 mW/cm for 4 minutes, 7.2 J/cm (A30/4-CXL)]. Efficacy measurements were evaluated 12 months after treatment with Scheimpflug imaging (Pentacam HR) and included change in maximum keratometry (K Max), corrected distance visual acuity (CDVA), other keratometric variables, pachymetry, keratoconus indices, astigmatism, asphericity, manifest refraction, and higher order aberrations. RESULTS: Ninety-three eyes (67 patients) were evaluated: 35 eyes (26 patients) with S3/30-CXL, 29 eyes (19 patients) with A9/10-CXL, and 29 eyes (22 patients) with A30/4-CXL. Mean [INCREMENT]K Max was -1.53 ± 2.1 diopter (D) for S3/30-CXL, -0.71 ± 1.3 D for A9/10-CXL, and -0.70 ± 2.3 D for A30/4-CXL (P = 0.37). Mean [INCREMENT]CDVA(logMAR) was -0.18 ± 0.2 for S3/30-CXL, -0.13 ± 0.2 for A9/10-CXL, and -0.18 ± 0.2 for A30/4-CXL (P = 0.79). [INCREMENT]K Mean (r = -0.29 to -0.46), anterior asphericity (r = -0.34 to -0.40), and central keratoconus index (r = -0.18 to -0.38) best correlated with [INCREMENT]CDVA. S3/30-CXL had greater changes in index of surface variance, index of vertical asymmetry, keratoconus index, and regularization index compared to A9/10-CXL and A30/4-CXL. There were no other differences between protocols. CONCLUSIONS: All 3 protocols showed improvements in K Max, CDVA, and other variables, with similar functional outcomes for each despite greater change in keratoconus indices after S3/30-CXL. Correlations between change in measured variables and CDVA were poor overall; however, K Mean, central keratoconus index, and anterior asphericity were better correlated with CDVA than K Max.
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Colágeno/metabolismo , Reactivos de Enlaces Cruzados/uso terapéutico , Queratocono/tratamiento farmacológico , Fotoquimioterapia/métodos , Adulto , Paquimetría Corneal , Sustancia Propia/metabolismo , Topografía de la Córnea , Femenino , Humanos , Queratocono/metabolismo , Queratocono/fisiopatología , Masculino , Persona de Mediana Edad , Fármacos Fotosensibilizantes/uso terapéutico , Refracción Ocular/fisiología , Estudios Retrospectivos , Riboflavina/uso terapéutico , Rayos Ultravioleta , Agudeza Visual/fisiología , Adulto JovenRESUMEN
PURPOSE: To evaluate the correlation between anterior axial curvature difference maps following corneal cross-linking (CXL) for progressive keratoconus obtained from Scheimpflug-based tomography and Placido-based topography. DESIGN: Between-devices reliability analysis of randomized clinical trial data. METHODS: Corneal imaging was collected at a single-center institution preoperatively and at 3, 6, and 12 months postoperatively using Scheimpflug-based tomography (Pentacam; Oculus Inc, Lynnwood, Washington, USA) and scanning-slit, Placido-based topography (Orbscan II; Bausch & Lomb, Rochester, New York, USA) in patients with progressive keratoconus receiving standard protocol CXL (3 mW/cm2 for 30 minutes). Regularization index (RI), absolute maximum keratometry (K Max), and change in K Max (ΔK Max) were compared between the 2 devices at each time point. RESULTS: Fifty-one eyes from 36 patients were evaluated at all time points. Values were significantly different at all time points (56.01 ± 5.3 diopters [D] Scheimpflug vs 55.04 ± 5.1 D scanning-slit preoperatively [P = .003]; 54.58 ± 5.3 D Scheimpflug vs 53.12 ± 4.9 D scanning-slit at 12 months [P < .0001]) but strongly correlated between devices (r = 0.90-0.93) at all time points. The devices were not significantly different at any time point for either ΔK Max or RI but were poorly correlated at all time points (r = 0.41-0.53 for ΔK Max, r = 0.29-0.48 for RI). At 12 months, 95% limits of agreement were 7.51 D for absolute K Max, 8.61 D for ΔK Max, and 19.86 D for RI. CONCLUSIONS: Measurements using Scheimpflug and scanning-slit Placido-based technology are correlated but not interchangeable. Both devices appear reasonable for separately monitoring the cornea's response to CXL; however, caution should be used when comparing results obtained with one measuring technology to the other.
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Córnea/patología , Paquimetría Corneal/instrumentación , Topografía de la Córnea/instrumentación , Reactivos de Enlaces Cruzados/farmacología , Imagenología Tridimensional , Queratocono/diagnóstico , Fotoquimioterapia/métodos , Adulto , Diseño de Equipo , Femenino , Estudios de Seguimiento , Humanos , Queratocono/tratamiento farmacológico , Masculino , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: To compare posterior corneal features and their discriminating power for differentiating normal corneas from subclinical keratoconus using the Placido dual-Scheimpflug analyzer. METHODS: Patients were retrospectively included in the study. The preoperative normal right eyes of 79 patients imaged with a Placido dual-Scheimpflug system and with a stable postoperative LASIK follow-up of a minimum of 36 months were included in the normal group and were compared to 39 contralateral topographically normal eyes with clinically evident keratoconus in the fellow eye. The posterior surface variables measured were categorized according to the feature of the corneal shape they were characterizing (curvature, elevation, asymmetry, and eccentricity) and compared between the two groups using the Student's two-sample t test. The discriminating ability of the posterior surface variables was compared by receiver operator characteristics curves. RESULTS: Variables that related to asymmetry and elevation of the posterior surface were statistically significantly different between groups (P < .05), whereas eccentricity and curvature-related parameters were not. Receiver operator characteristics curves analysis showed that the maximum posterior elevation over the best-fit toric and aspheric surface reference shape had the highest discriminating ability for distinguishing normal corneas from subclinical keratoconus, with an area under the curve of 0.877, followed by the asphericity asymmetry index, with an area under the curve of 0.871, and posterior inferior-superior value, with an area under the curve of 0.851. CONCLUSIONS: Posterior cornea measured with a dual-Scheimpflug analyzer provides useful parameters for differentiating normal corneas from subclinical keratoconus. Of the posterior surface parameters, asymmetry and elevation seem to be the most sensitive shape modifications for differentiating both populations. [J Refract Surg. 2018;34(10):664-670.].
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Córnea/anatomía & histología , Córnea/patología , Técnicas de Diagnóstico Oftalmológico , Queratocono/diagnóstico , Adulto , Paquimetría Corneal , Topografía de la Córnea , Dilatación Patológica/diagnóstico , Dilatación Patológica/cirugía , Femenino , Humanos , Queratocono/cirugía , Queratomileusis por Láser In Situ , Láseres de Excímeros/uso terapéutico , Masculino , Curva ROC , Estudios Retrospectivos , Sensibilidad y Especificidad , Adulto JovenRESUMEN
PURPOSE: To evaluate changes in regional corneal epithelial thickness after corneal crosslinking (CXL) using higher fluence (7.2 J/cm2) and accelerated treatment time (4 minutes) in eyes with progressive keratoconus using spectral-domain optical coherence tomography (SD-OCT) and to correlate focal epithelial and focal anterior curvature changes. SETTING: Academic medical center in the United States. DESIGN: Prospective case series. METHODS: Patients had anterior segment SD-OCT (RTVue-100) with focal stromal and epithelial measurements and Scheimpflug imaging before and 1, 3, 6, and 12 months after accelerated CXL. RESULTS: Twenty-seven eyes from 20 patients were evaluated. Before the accelerated CXL, the epithelium was thinnest in the inferior inner and outer temporal regions, whereas at 12 months postoperatively, the epithelium was significantly thinned in multiple inferior and nasal regions by 1.1 to 3.2 µm (P < .05, all measurements), with no significant changes from 6 to 12 months. Epithelial thickness standard deviation across the central 6.0 mm was significantly reduced by 3 months (1.4 µm, P = .09) and remained stable at 12 months (P = .009). Change in epithelial thickness was poorly correlated to change in anterior curvature (r = -0.035). CONCLUSIONS: Significant epithelial remodeling occurred after accelerated CXL in eyes with progressive keratoconus, with improved regularity across the central 6.0 mm, by 6 months after treatment. There was poor correlation between focal epithelial thickness and anterior curvature changes, with wide variability between patients. Establishing the pattern of epithelial remodeling after CXL might help optimize future custom treatment protocols.
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Sustancia Propia/efectos de los fármacos , Reactivos de Enlaces Cruzados , Epitelio Corneal/fisiología , Queratocono/tratamiento farmacológico , Fármacos Fotosensibilizantes/uso terapéutico , Riboflavina/uso terapéutico , Adolescente , Adulto , Colágeno/metabolismo , Paquimetría Corneal , Sustancia Propia/metabolismo , Femenino , Humanos , Queratocono/metabolismo , Queratocono/fisiopatología , Masculino , Persona de Mediana Edad , Fotoquimioterapia/métodos , Estudios Prospectivos , Factores de Tiempo , Tomografía de Coherencia Óptica/métodos , Rayos Ultravioleta , Agudeza Visual/fisiología , Adulto JovenRESUMEN
Recent technological advances in high-content screening instrumentation have increased its ease of use and throughput, expanding the application of high-content screening to the early stages of drug discovery. However, high-content screens produce complex data sets, presenting a challenge for both extraction and interpretation of meaningful information. This shifts the high-content screening process bottleneck from the experimental to the analytical stage. In this article, the authors discuss different approaches of data analysis, using a phenotypic neurite outgrowth screen as an example. Distance measurements and hierarchical clustering methods lead to a profound understanding of different high-content screening readouts. In addition, the authors introduce a hit selection procedure based on machine learning methods and demonstrate that this method increases the hit verification rate significantly (up to a factor of 5), compared to conventional hit selection based on single readouts only.
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Neuritas/metabolismo , Análisis de Matrices Tisulares/normas , Análisis por Conglomerados , Análisis Multivariante , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.
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Crassostrea/genética , Perfilación de la Expresión Génica/veterinaria , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Animales , Crassostrea/fisiología , Expresión Génica/fisiología , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that exists as two isozymes, GSK3alpha and GSK3beta. It plays important roles in regulating cell structure, function, and survival, and dysregulation of its function is linked to disorders such as Alzheimer's disease and type II diabetes. In resting cells, GSK3 is active and regulates the function of many downstream targets, including beta-catenin. We describe the development of a cell-based assay designed to measure the activity of GSK3 by directly measuring the accumulation of beta-catenin in Chinese hamster ovary clone K1 (CHOK1) cells. Beta-catenin levels were assessed using an antibody-based staining protocol with a luminometric readout. The assay is set up in a 96-well format. The use of GSK3 inhibitors demonstrated that this assay could be used to compare the effects of various small molecules on GSK3 inhibition in CHOK1 cells.
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Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , beta Catenina/metabolismo , Aminofenoles/farmacología , Animales , Benzazepinas/farmacología , Bencimidazoles/farmacología , Western Blotting , Células CHO , Células Clonales , Cricetinae , Cricetulus , Dimetilsulfóxido/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Factor 2B Eucariótico de Iniciación/metabolismo , Glucógeno Sintasa Quinasa 3/química , Humanos , Imidazoles/farmacología , Immunoblotting , Indoles/farmacología , Cloruro de Litio/farmacología , Mediciones Luminiscentes/métodos , Maleimidas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Reproducibilidad de los Resultados , beta Catenina/químicaRESUMEN
Type II diabetes and its associated complications are a major health concern of the developed world. One of the hallmarks of diabetes is insulin resistance, where secreted insulin no longer has any effect on its target tissues, namely, liver, muscle, and fat. An important therapeutic strategy is to modulate blood glucose levels using pharmacological agents. Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that plays important roles in regulating glucose metabolism. It is a key negative regulator of insulin action and is an important contributing factor to insulin resistance in liver, muscle, and adipose tissue. We describe the development of a cell-based assay designed to measure glucose production in rat hepatoma cell line H4IIE liver cells in response to treatment with small molecule inhibitors, including GSK3 inhibitors. The assay is set up in a 96-well format, and glucose production is assessed using a convenient fluorescence-based readout. This disease-relevant cellular assay is a valuable tool for the progression of small molecules that modulate glucose production.