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As part of the child-rearing process, situations that invite difficult conversations will inevitably arise. Oftentimes, there are no guidelines or structure for how to discuss topics such as sex education, systemic racism, bullying, grieving, and gun violence. Accordingly, adults may feel at a loss for how to address difficult topics and may even avoid difficult conversations completely. When adults choose to have these conversations, they may imitate the conversations their caregivers had with them, and therefore further the cycle of systemic racism, often unknowingly and unintentionally. Racial injustice has been a core part of the American experience since the founding of the republic; hence, conversations about systemic racism are long overdue. The need has significantly increased, given the current socio-political climate. Social justice may be a sensitive topic for some, but it is a needed conversation for all, including children with Autism Spectrum Disorder (ASD). Currently available curricula and teaching manuals in the Applied Behavior Analysis (ABA) literature include little or no resources for caregivers on how to address systemic racism with their children on the spectrum. Children with ASD should be educated about how they, and their families, can combat systemic racism in their everyday lives. The present paper addresses this gap in available treatment resources by offering practical suggestions and guidelines for how adults can address the topic of systemic racism with children on the autism spectrum to educate them and prepare them to contribute to a more equitable and just future.
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Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.
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Proteínas Adaptadoras Transductoras de Señales/genética , Empalme Alternativo , Conductos Biliares Intrahepáticos/embriología , Conductos Biliares Intrahepáticos/metabolismo , Morfogénesis/genética , Alelos , Animales , Animales Modificados Genéticamente , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Orden Génico , Pruebas Genéticas , Variación Genética , Hígado/metabolismo , Modelos Biológicos , Mutación , Fenotipo , Isoformas de ARN , Pez Cebra , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Alemania , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Sensibilidad y Especificidad , Sierra Leona , Esputo , Tuberculosis Resistente a Múltiples Medicamentos/diagnósticoRESUMEN
The degree of organotin compounds (OTCs), including dibutyltin, tributyltin, triphenyltin and dioctyltin, contamination in seafood purchased in 2017 and 2018 from Hong Kong market was studied. Edible portions of 341 seafood samples, including fish, crustaceans and molluscs, were used for analysis by gas chromatograph coupled to an inductively coupled plasma mass spectrometry (GC-ICP/MS). The method detection limits and quantification limits of OTCs were below or equal to 0.25 and 1.0 µg Sn kg-1 respectively. Triphenyltin accounted for the majority amongst other OTCs and was detected in 53% of samples. In general, mean total OTCs levels of fish (24 µg Sn kg-1) were higher than crustaceans (14 µg Sn kg-1) and molluscs (15 µg Sn kg-1). The highest detected levels of triphenyltin, tributyltin, dibutyltin and dioctyltin were found to be 480, 24, 4.5 and 0.89 µg Sn kg-1 in a mangrove snapper, noodle fish, coral clam and giant grouper respectively.
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Compuestos Orgánicos de Estaño , Animales , Crustáceos , Peces , Hong Kong , Alimentos Marinos/análisisRESUMEN
This work demonstrates the potential for using a deformable mapping method to register lesions between dedicated breast computed tomography (bCT) and both automated breast ultrasound (ABUS) and digital breast tomosynthesis (DBT) images (craniocaudal [CC] and mediolateral oblique [MLO] views). Two multi-modality breast phantoms with external fiducial markers attached were imaged by the three modalities. The DBT MLO view was excluded for the second phantom. The automated deformable mapping algorithm uses biomechanical modeling to determine corresponding lesions based on distances between their centers of mass (dCOM) in the deformed bCT model and the reference model (DBT or ABUS). For bCT to ABUS, the mean dCOM was 5.2 ± 2.6 mm. For bCT to DBT (CC), the mean dCOM was 5.1 ± 2.4 mm. For bCT to DBT (MLO), the mean dCOM was 4.7 ± 2.5 mm. This application could help improve a radiologist's efficiency and accuracy in breast lesion characterization, using multiple imaging modalities.
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Algoritmos , Neoplasias de la Mama/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador , Mamografía/métodos , Tomografía Computarizada por Rayos X/métodos , Ultrasonografía Mamaria/métodos , Fantasmas de ImagenRESUMEN
This work investigates the application of a deformable localization/mapping method to register lesions between the digital breast tomosynthesis (DBT) craniocaudal (CC) and mediolateral oblique (MLO) views and automated breast ultrasound (ABUS) images. This method was initially validated using compressible breast phantoms. This methodology was applied to 7 patient data sets containing 9 lesions. The automated deformable mapping algorithm uses finite element modeling and analysis to determine corresponding lesions based on the distance between their centers of mass (dCOM) in the deformed DBT model and the reference ABUS model. This technique shows that location information based on external fiducial markers is helpful in the improvement of registration results. However, use of external markers are not required for deformable registration results described by this methodology. For DBT (CC view) mapped to ABUS, the mean dCOM was 14.9⯱â¯6.8â¯mm based on 9 lesions using 6 markers in deformable analysis. For DBT (MLO view) mapped to ABUS, the mean dCOM was 13.7⯱â¯6.8â¯mm based on 8 lesions using 6 markers in analysis. Both DBT views registered to ABUS lesions showed statistically significant improvements (pâ¯≤â¯0.05) in registration using the deformable technique in comparison to a rigid registration. Application of this methodology could help improve a radiologist's characterization and accuracy in relating corresponding lesions between DBT and ABUS image datasets, especially for cases of high breast densities and multiple masses.
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Neoplasias de la Mama/diagnóstico por imagen , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Mamografía/métodos , Ultrasonografía Mamaria/métodos , Algoritmos , Fenómenos Biomecánicos , Conjuntos de Datos como Asunto , Femenino , Análisis de Elementos Finitos , Humanos , Aumento de la Imagen/métodos , Fantasmas de ImagenRESUMEN
PURPOSE: To develop a deformable mapping technique to match corresponding lesions between digital breast tomosynthesis (DBT) and automated breast ultrasound (ABUS) images. METHODS: External fiducial markers were attached to the surface of two CIRS multi-modality compressible breast phantoms (A and B) containing multiple simulated lesions. Both phantoms were imaged with DBT (upright positioning with cranial-caudal compression) and ABUS (supine positioning with anterior-to-chest wall compression). The lesions and markers were manually segmented by three different readers. Reader segmentation similarity and reader reproducibility were assessed using Dice similarity coefficients (DSC) and distances between centers of mass (dCOM ). For deformable mapping between the modalities each reader's segmented dataset was processed with an automated deformable mapping algorithm as follows: First, Morfeus, a finite element (FE) based multi-organ deformable image registration platform, converted segmentations into triangular surface meshes. Second, Altair HyperMesh, a FE pre-processor, created base FE models for the ABUS and DBT data sets. All deformation is performed on the DBT image data; the ABUS image sets remain fixed throughout the process. Deformation was performed on the external skin contour (DBT image set) to match the external skin contour on the ABUS set, and the locations of the external markers were used to morph the skin contours to be within a user-defined distance. Third, the base DBT-FE model was deformed with the FE analysis solver, Optistruct. Deformed DBT lesions were correlated with matching lesions in the base ABUS FE model. Performance (lesion correlation) was assessed with dCOM for all corresponding lesions and lesion overlap. Analysis was performed to determine the minimum number of external fiducial markers needed to create the desired correlation and the improvement of correlation with the use of external markers. RESULTS: Average DSC for reader similarity ranged from 0.88 to 0.91 (ABUS) and 0.57 to 0.83 (DBT). Corresponding dCOM ranged from 0.20 to 0.36 mm (ABUS) and 0.11 to 1.16 mm (DBT). Lesion correlation is maximized when all corresponding markers are within a maximum distance of 5 mm. For deformable mapping of phantom A, without the use of external markers, only two of six correlated lesions showed overlap with an average lesion dCOM of 6.8 ± 2.8 mm. With use of three external fiducial markers, five of six lesions overlapped and average dCOM improved to 4.9 ± 2.4 mm. For deformable mapping of Phantom B without external markers analysis, four lesions were correlated of seven with overlap between only one of seven lesions, and an average lesion dCOM of 9.7 ± 3.5 mm. With three external markers, all seven possible lesions were correlated with overlap between four of seven lesions. The average dCOM was 8.5 ± 4.0 mm. CONCLUSION: This work demonstrates the potential for a deformable mapping technique to relate corresponding lesions in DBT and ABUS images by showing improved lesion correspondence and reduced lesion registration errors with the use of external fiducial markers. The technique should improve radiologists' characterization of breast lesions which can reduce patient callbacks, misdiagnoses and unnecessary biopsies.
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Neoplasias de la Mama/diagnóstico por imagen , Mama/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Mamografía , Ultrasonografía Mamaria , Algoritmos , Automatización , Marcadores Fiduciales , Humanos , Procesamiento de Imagen Asistido por Computador/normas , Fantasmas de ImagenRESUMEN
Experimental evolution studies have characterized the genetic strategies microbes utilize to adapt to their environments, mainly focusing on how microbes adapt to constant and/or defined environments. Using a system that incubates Escherichia coli in different complex media in long-term batch culture, we have focused on how heterogeneity and environment affects adaptive landscapes. In this system, there is no passaging of cells, and therefore genetic diversity is lost only through negative selection, without the experimentally-imposed bottlenecking common in other platforms. In contrast with other experimental evolution systems, because of cycling of nutrients and waste products, this is a heterogeneous environment, where selective pressures change over time, similar to natural environments. We determined that incubation in each environment leads to different adaptations by observing the growth advantage in stationary phase (GASP) phenotype. Re-sequencing whole genomes of populations identified both mutant alleles in a conserved set of genes and differences in evolutionary trajectories between environments. Reconstructing identified mutations in the parental strain background confirmed the adaptive advantage of some alleles, but also identified a surprising number of neutral or even deleterious mutations. This result indicates that complex epistatic interactions may be under positive selection within these heterogeneous environments.
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Adaptación Biológica , Técnicas de Cultivo Celular por Lotes , Medios de Cultivo , Escherichia coli/fisiología , Fenómenos Fisiológicos de la Nutrición , Alelos , Epistasis Genética , Frecuencia de los Genes , Interacción Gen-Ambiente , Aptitud Genética , Variación Genética , MutaciónRESUMEN
AIMS: Distal intestinal obstruction syndrome (DIOS) and constipation in cystic fibrosis (CF) are conditions associated with impaction and/or obstruction by abnormally viscid mucofaecal material within the intestinal lumen. Dehydration has been proposed as a risk factor for DIOS and constipation in CF. The study primarily aimed to determine whether warmer ambient temperature and lower rainfall are risk factors for DIOS and constipation in CF. METHODS: Hospitalisations for DIOS (incomplete or complete) and/or constipation were retrospectively identified (2000-2012). Genotype, phenotype, temperatures and rainfall data (for the week preceding and season of hospitalisation) were collected. RESULTS: Twenty-seven DIOS (59.3% incomplete; 40.7% complete) and 44 constipation admissions were identified. All admitted patients were pancreatic insufficient. Meconium ileus was significantly more likely in DIOS than constipation (64.7% vs. 33.3%; P = 0.038) and in complete than incomplete DIOS (100% vs. 57.1%; P = 0.04). The maximum temperature of the week before DIOS admission (mean (standard deviation) = 28.0 (5.8) °C) was significantly higher than the maximum temperature of the season of admission (25.2 (3.4) °C; P = 0.002). Similarly, the maximum temperature of the week before hospitalisation for constipation (mean (standard deviation) = 27.9 (6.3) °C) was significantly warmer compared with the season of admission (24.0 (4.1) °C; P < 0.0001). There were no significant differences between levels of rainfall during the week before hospitalisation and the season of admission for both DIOS and constipation. CONCLUSIONS: Relatively high ambient temperature may play a role in the pathogenesis of DIOS and constipation in CF.
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Fibrosis Quística/complicaciones , Deshidratación/complicaciones , Hospitalización/estadística & datos numéricos , Calor/efectos adversos , Obstrucción Intestinal/epidemiología , Adolescente , Niño , Preescolar , Estudios de Cohortes , Fibrosis Quística/diagnóstico , Deshidratación/diagnóstico , Femenino , Estudios de Seguimiento , Hospitales Pediátricos , Humanos , Incidencia , Obstrucción Intestinal/etiología , Masculino , Queensland , Estudios Retrospectivos , Factores de Riesgo , Estaciones del Año , SíndromeRESUMEN
Intravital imaging by multiphoton microscopy is a powerful tool to gain invaluable insight into tissue biology and function. Here, we provide a step-by-step tissue preparation protocol for imaging the mouse tibialis anterior skeletal muscle. Additionally, we include steps for jugular vein catheterization that allow for well-controlled intravenous reagent delivery. Preparation of the tibialis anterior muscle is minimally invasive, reducing the chances of inducing damage and inflammation prior to imaging. The tibialis anterior muscle is useful for imaging leukocyte interaction with vascular endothelium, and to understand muscle contraction biology. Importantly, this model can be easily adapted to study neuromuscular diseases and myopathies.
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High grade gliomas (HGG) are classified into four subgroups based on transcriptional signatures and phenotypic characteristics. In particular, the proneural-to-mesenchymal transition (PMT) is associated with increased malignancy, poor prognosis, and disease recurrence, but the underlying causes of PMT are still unclear. In this study, we investigated whether radiotherapy promotes PMT using a genetically engineered mouse model of proneural HGG. We found that cranial ionizing radiation induced robust and durable PMT in tumors. Additionally, we isolated primary proneural HGG cells from mouse and human tumors and demonstrate that radiation induced a sustained cell-intrinsic mesenchymal transition associated with increased invasiveness and resistance to the alkylating agent temozolomide. Expectedly, irradiation-induced PMT was also associated with activation of the STAT3 transcription factor, and the combination of STAT3 blockade using JAK2 inhibitors with radiation abrogated the mesenchymal transition and extended survival of mice. Taken together, our data suggest that clinical JAK2 inhibitors should be tested in conjunction with radiation in patients with proneural HGG as a new strategy for blocking the emergence of therapy-resistant mesenchymal tumors at relapse.
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Glioma/metabolismo , Glioma/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Aloinjertos , Animales , Biomarcadores , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Células Madre Mesenquimatosas/efectos de la radiación , Ratones , Ratones Noqueados , Clasificación del Tumor , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Radiación , Factor de Transcripción STAT3/metabolismoRESUMEN
Glioma is the most common primary malignant brain tumor and arises throughout the central nervous system. Recent focus on stem-like glioma cells has implicated neural stem cells (NSCs), a minor precursor population restricted to germinal zones, as a potential source of gliomas. In this review, we focus on the relationship between oligodendrocyte progenitor cells (OPCs), the largest population of cycling glial progenitors in the postnatal brain, and gliomagenesis. OPCs can give rise to gliomas, with signaling pathways associated with NSCs also playing key roles during OPC lineage development. Gliomas can also undergo a switch from progenitor- to stem-like phenotype after therapy, consistent with an OPC-origin even for stem-like gliomas. Future in-depth studies of OPC biology may shed light on the etiology of OPC-derived gliomas and reveal new therapeutic avenues.
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Transformación Celular Neoplásica , Glioma/patología , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Animales , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Transformación Celular Neoplásica/patología , Glioma/genética , Glioma/terapia , Humanos , Terapia Molecular Dirigida , Células-Madre Neurales/patología , Neuroglía/patologíaRESUMEN
EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document coexpression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.
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Neoplasias Encefálicas/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Alelos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Trasplante de Neoplasias , Fosforilación , Transducción de SeñalRESUMEN
The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally stabilized murine N-myc(T58A) into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem, and forebrain. Transplantation of N-myc(WT) NSCs was insufficient for tumor formation. N-myc(T58A) cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating Sonic Hedgehog (SHH) dependence and SHH independence, respectively. These differences were regulated in part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal.
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Neoplasias Encefálicas/metabolismo , Linaje de la Célula , Transformación Celular Neoplásica/metabolismo , Células-Madre Neurales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Biomarcadores/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Tronco Encefálico/embriología , Tronco Encefálico/metabolismo , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Cerebelo/embriología , Cerebelo/metabolismo , Femenino , Edad Gestacional , Glioma/metabolismo , Glioma/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Mutación , Proteína Proto-Oncogénica N-Myc , Células-Madre Neurales/patología , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Esferoides Celulares , Factores de Tiempo , Transducción Genética , Proteína Gli2 con Dedos de ZincRESUMEN
INTRODUCTION: Medulloblastoma, the largest group of embryonal brain tumors, has historically been classified into five variants based on histopathology. More recently, epigenetic and transcriptional analyses of primary tumors have subclassified medulloblastoma into four to six subgroups, most of which are incongruous with histopathological classification. DISCUSSION: Improved stratification is required for prognosis and development of targeted treatment strategies, to maximize cure and minimize adverse effects. Several mouse models of medulloblastoma have contributed both to an improved understanding of progression and to developmental therapeutics. In this review, we summarize the classification of human medulloblastoma subtypes based on histopathology and molecular features. We describe existing genetically engineered mouse models, compare these to human disease, and discuss the utility of mouse models for developmental therapeutics. Just as accurate knowledge of the correct molecular subtype of medulloblastoma is critical to the development of targeted therapy in patients, we propose that accurate modeling of each subtype of medulloblastoma in mice will be necessary for preclinical evaluation and optimization of those targeted therapies.
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Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/terapia , Modelos Animales de Enfermedad , Meduloblastoma/genética , Meduloblastoma/terapia , Animales , Antineoplásicos/administración & dosificación , Neoplasias Cerebelosas/patología , Sistemas de Liberación de Medicamentos/tendencias , Humanos , Meduloblastoma/patología , Ratones , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias de Células Germinales y Embrionarias/terapia , Especificidad de la EspecieRESUMEN
PURPOSE: We evaluated the effect of roscovitine (Sigma-Aldrich®), a pharmacological inhibitor of cyclin dependent kinase, on renal cell carcinoma cell lines in vitro. MATERIALS AND METHODS: We exposed several renal cell carcinoma cell lines to roscovitine and examined apoptotic signaling pathways using immunoblotting and immunohistochemistry. RESULTS: As expected, roscovitine caused dose and time dependent inhibition of cyclin dependent kinase 2 autophosphorylation, and of cyclin dependent kinase mediated Pol II phosphorylation in the ACHN (p53-wt) and 786-O (p53 inactive) renal cell carcinoma cell lines (ATCC®). Roscovitine also induced apoptosis in each cell line within a narrow concentration range (about 10 µg/ml). Apoptosis induction was more efficient in ACHN than in 786-O cells and at least partly due to p53 activity. In ACHN cells roscovitine induced apoptosis was associated with p21 induction, and decreased Akt1, XIAP and phospho-Rb expression. These changes also depended on p53 and were not present (p21) or showed a different dose pattern (Akt1, XIAP and phospho-Rb) in 786-O cells. Partial restoration of roscovitine induced apoptosis in 786-O cells by the Mdm-2 inhibitor nutlin-3 (Sigma-Aldrich) suggests that the inactivating mutation of VHL in these cells and its destabilizing effect on p53 are responsible for the decreased sensitivity to apoptosis. CONCLUSIONS: Our data extend previous studies documenting the pro-apoptotic effect of roscovitine and to our knowledge show for the first time that this activity is restricted to a narrow dose range in renal cell carcinoma cells and partly depends on p53. Thus, roscovitine is a novel potential chemotherapy in a subset of patients with renal cell carcinoma if a narrow therapeutic window is used. These data also provide insight into the role of VHL mutation and p53 in the renal cell carcinoma response to therapeutic cyclin dependent kinase manipulation.
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Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Neoplasias Renales/enzimología , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiología , Roscovitina , Células Tumorales CultivadasRESUMEN
Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.
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Regulación Neoplásica de la Expresión Génica , Meduloblastoma/fisiopatología , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Ciclo Celular/fisiología , Senescencia Celular/fisiología , Cerebelo/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Inestabilidad Genómica , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/patología , Ratones , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc , Metástasis de la Neoplasia/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genéticaRESUMEN
The linker for activation of T cells (LAT) is an adaptor protein that couples TCR engagement to downstream signaling cascades. LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the double-negative (DN) 3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by the Cre recombinase. Analysis of these mice showed that deletion of LAT after the DN3 stage allowed thymocytes to develop past the DN3 to DN4 checkpoint and to generate double-positive thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to differentiate into single-positive thymocytes efficiently. Consequently, few LAT-deficient mature T cells could be found in the periphery. These T cells had undergone extensive homeostatic proliferation and expressed low levels of the TCR on their surface. Collectively, our data indicate that in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during transition from the double-positive to single-positive stage.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/inmunología , Proteínas de la Membrana/fisiología , Fosfoproteínas/fisiología , Timo/citología , Timo/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD4/genética , Antígenos CD8/genética , Diferenciación Celular/genética , Eliminación de Gen , Técnicas de Sustitución del Gen , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Timo/metabolismoRESUMEN
The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76) is a cytosolic adaptor protein essential for thymocyte development and T-cell activation. It contains a sterile-alpha motif (SAM) domain, 3 phosphotyrosine motifs, a proline-rich region, and a Src homology 2 domain. Whereas the other domains have been extensively studied, the role of the SAM domain in SLP-76 function is not known. To understand the function of this domain, we generated SLP-76 knockin mice with the SAM domain deleted. Analysis of these mice showed that thymocyte development was partially blocked at the double-positive to single-positive transition. Positive and negative thymic selection was also impaired. In addition, we analyzed T-cell receptor (TCR)-mediated signaling in T cells from these mutant mice. TCR-mediated inositol 1,4,5-triphosphate production, calcium flux, and extracellular signal-regulated kinase activation were decreased, leading to defective interleukin-2 production and proliferation. Moreover, despite normal association between Gads and SLP-76, TCR-mediated formation of SLP-76 microclusters was impaired by the deletion of the SAM domain. Altogether, our data demonstrated that the SAM domain is indispensable for optimal SLP-76 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Fosfoproteínas/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Secuencia de Bases , Señalización del Calcio , Diferenciación Celular , Proliferación Celular , Cartilla de ADN/genética , Interleucina-2/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Fosfoproteínas/química , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Eliminación de Secuencia , Transducción de Señal , Linfocitos T/citología , Timo/citología , Timo/inmunologíaRESUMEN
The reaction of [Os3Rh(mu-H)3(CO)12] with an excess amount of 4-vinylphenol (as hydride acceptor) in refluxing m-xylene, chlorobenzene or benzene yielded the three new clusters [Os5Rh2(mu-CO){eta6-C6H4(CH3)2}(CO)16] 1, [Os5Rh2(mu-CO)(eta6-C6H5Cl)(CO)16] 2 and [Os5Rh2(mu-CO)(eta6-C6H6)(CO)16] 3. The treatment of [Os3Rh(mu-H)3(CO)12] 4 in refluxing toluene with an excess amount of 4-vinylphenol afforded a new complex, [Os4Rh(mu-H)(eta6-C6H5CH3)(CO)12], which was isolated as a brown complex in 20% yield together with two known compounds, [Os5Rh2(eta6-C6H5CH3)(mu-CO)(CO)16] in 10% yield and [Os3Rh4(mu3-eta1:eta1:eta1-C6H5CH3)(CO)13] in 5% yield. Complexes 1-4 were fully characterized by IR, 1H NMR spectroscopy, mass spectroscopy, elemental analysis and X-ray crystallography. The molecular structures of compounds 1-3 are isomorphous, and only differ in the arene-derivatives that attach to the same metal core. Their metal cores can be viewed as a monocapped octahedral, in which an osmium atom caps one of the Os-Os-Os triangular faces of the Os4Rh2 metal framework. Complex 4 has a trigonal-bipyramidal metal core with a C6H5Me ligand that is terminally bound to the Rh atom that lies in the trigonal plane of the metal core. The hydrogenation of [Os5Rh2(eta6-C6H5CH3)(mu-CO)(CO)16] with [Os3(mu-H)2(CO)10] in chloroform under reflux resulted in two hydrogen-rich compounds: [Os7Rh3(mu-H)11(CO)23] 5 and [Os5Rh3Cl(mu-H)8(CO)18] 6, both in moderate yields. The reaction of [Os5Rh2(eta6-C6H5CH3)(mu-CO)(CO)16] with hydrogen in refluxing chloroform yielded a new cluster compound, [Os5Rh(mu-H)5(CO)18] 7, in 20% yield, together with a known osmium-rhodium cluster, [Os6Rh(mu-H)7(mu-CO)(CO)18], as a major compound. Clusters 5, 6, and 7 have been fully characterized by both spectroscopic and crystallographic methods. Additionally, a deuterium-exchange experiment was performed on [Os7Rh3(mu-H)11(CO)23] 5 and [Os5Rh3Cl(mu-H)8(CO)18] 6. Both the compounds proved to be able to exchange the H atom with D in the presence of D2SO4, and the absence of the hydride signal in the 1H NMR spectrum is consistent with this. Therefore, clusters 5 and 6 may serve as appropriate new hydrogen storage models.