RESUMEN
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.
Asunto(s)
Imagenología Tridimensional , Microscopía , Algoritmos , Animales , Núcleo Celular , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Ratones , Programas InformáticosRESUMEN
We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects.
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Miembro Posterior/anomalías , Esbozos de los Miembros/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Condrogénesis , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Esbozos de los Miembros/citología , Esbozos de los Miembros/crecimiento & desarrollo , Células Madre Mesenquimatosas/metabolismo , Ratones , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.
Asunto(s)
Linaje de la Célula/genética , Corazón/embriología , Línea Primitiva/embriología , Animales , Linaje de la Célula/fisiología , Femenino , Gástrula , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/fisiología , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Masculino , Mesodermo , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Línea Primitiva/fisiología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
Perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIß as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIß in the brain remain poorly explored. Here, we reveal a novel function for this CaMKII isoform in vivo during neuronal development. By using in utero electroporation, we show that CaMKIIß is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIß causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIß inhibits migration, demonstrating that precise regulation of CaMKIIß expression is required for correct neuronal migration. More precisely, CaMKIIß controls the multipolar-bipolar transition in the intermediate zone and locomotion in the cortical plate through its actin-binding and -bundling activities. In addition, our data indicate that a fine-tuned balance between CaMKIIß and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIß, demonstrating that CaMKIIß has a major biological function in the developing brain.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/fisiología , Neurogénesis/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Corteza Cerebral/metabolismo , Embrión de Mamíferos/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Proteínas de Microfilamentos/genética , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Neuronas/metabolismo , Cultivo Primario de Células , Isoformas de Proteínas/metabolismo , Células Piramidales/metabolismoRESUMEN
Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.
Asunto(s)
Sistema Nervioso Entérico/metabolismo , Enfermedad de Hirschsprung/genética , Proteína Quinasa 10 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas c-ret/genética , Pez Cebra/genética , Animales , Colon/inervación , Colon/patología , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/patología , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Humanos , Mutación , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-ret/metabolismoRESUMEN
The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.
Asunto(s)
Linaje de la Célula , Mesodermo/citología , Células-Madre Neurales/citología , Tubo Neural/citología , Factores de Transcripción SOXB1/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Coristoma/embriología , Coristoma/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Placa Neural/citología , Placa Neural/embriología , Placa Neural/metabolismo , Tubo Neural/embriología , Tubo Neural/metabolismo , Factores de Transcripción SOXB1/genética , Proteínas de Dominio T Box , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMEN
To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.
Asunto(s)
Drosophila melanogaster/citología , Proteínas Luminiscentes/análisis , Red Nerviosa/citología , Neuronas/citología , Coloración y Etiquetado/métodos , Animales , Secuencia de Bases , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Unión al ADN/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas Luminiscentes/genética , Ratones , Datos de Secuencia Molecular , Red Nerviosa/embriología , Neuroglía/química , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/química , Neuronas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.
Asunto(s)
Antígenos CD8/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Núcleo Celular/genética , Regulación del Desarrollo de la Expresión Génica/inmunología , Timo/inmunología , Timo/metabolismo , Animales , Antígenos CD4/genética , Antígenos CD8/biosíntesis , Posicionamiento de Cromosoma/genética , Sondas de ADN/genética , Femenino , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Estructura Terciaria de Proteína/genética , Timo/citologíaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers mainly due to spatial obstacles to complete resection, early metastasis and therapy resistance. The molecular events accompanying PDAC progression remain poorly understood. SOX9 is required for maintaining the pancreatic ductal identity and it is involved in the initiation of pancreatic cancer. In addition, SOX9 is a transcription factor linked to stem cell activity and is commonly overexpressed in solid cancers. It cooperates with Snail/Slug to induce epithelial-mesenchymal transition (EMT) during neural development and in diseases such as organ fibrosis or different types of cancer. METHODS: We investigated the roles of SOX9 in pancreatic tumor cell plasticity, metastatic dissemination and chemoresistance using pancreatic cancer cell lines as well as mouse embryo fibroblasts. In addition, we characterized the clinical relevance of SOX9 in pancreatic cancer using human biopsies. RESULTS: Gain- and loss-of-function of SOX9 in PDAC cells revealed that high levels of SOX9 increased migration and invasion, and promoted EMT and metastatic dissemination, whilst SOX9 silencing resulted in metastasis inhibition, along with a phenotypic reversion to epithelial features and loss of stemness potential. In both contexts, EMT factors were not altered. Moreover, high levels of SOX9 promoted resistance to gemcitabine. In contrast, overexpression of SOX9 was sufficient to promote metastatic potential in K-Ras transformed MEFs, triggering EMT associated with Snail/Slug activity. In clinical samples, SOX9 expression was analyzed in 198 PDAC cases by immunohistochemistry and in 53 patient derived xenografts (PDXs). SOX9 was overexpressed in primary adenocarcinomas and particularly in metastases. Notably, SOX9 expression correlated with high vimentin and low E-cadherin expression. CONCLUSIONS: Our results indicate that SOX9 facilitates PDAC progression and metastasis by triggering stemness and EMT.
RESUMEN
Sensory hair cells and their associated non-sensory supporting cells in the inner ear are fundamental for hearing and balance. They arise from a common progenitor, but little is known about the molecular events specifying this cell lineage. We recently identified two allelic mouse mutants, light coat and circling (Lcc) and yellow submarine (Ysb), that show hearing and balance impairment. Lcc/Lcc mice are completely deaf, whereas Ysb/Ysb mice are severely hearing impaired. We report here that inner ears of Lcc/Lcc mice fail to establish a prosensory domain and neither hair cells nor supporting cells differentiate, resulting in a severe inner ear malformation, whereas the sensory epithelium of Ysb/Ysb mice shows abnormal development with disorganized and fewer hair cells. These phenotypes are due to the absence (in Lcc mutants) or reduced expression (in Ysb mutants) of the transcription factor SOX2, specifically within the developing inner ear. SOX2 continues to be expressed in the inner ears of mice lacking Math1 (also known as Atoh1 and HATH1), a gene essential for hair cell differentiation, whereas Math1 expression is absent in Lcc mutants, suggesting that Sox2 acts upstream of Math1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Oído Interno/embriología , Oído Interno/metabolismo , Transactivadores/metabolismo , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Oído Interno/anomalías , Oído Interno/patología , Células Ciliadas Auditivas Internas/anomalías , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patología , Ratones , Ratones Mutantes , Mutación/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1 , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.
Asunto(s)
Células del Tejido Conectivo/citología , Extremidades/fisiología , Desarrollo de Músculos , Músculo Esquelético/fisiología , Animales , Tipificación del Cuerpo , Agregación Celular , Eliminación de Gen , Integrasas/metabolismo , Ratones Transgénicos , Morfogénesis , Células Musculares/citología , Fibras Musculares Esqueléticas/citología , Proteínas de Dominio T Box/metabolismo , Tendones/citología , Factores de Transcripción/metabolismoRESUMEN
The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.
Asunto(s)
Cóclea/embriología , Neuronas Aferentes/citología , Vestíbulo del Laberinto/embriología , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Proteínas Aviares/biosíntesis , Proteínas Aviares/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Linaje de la Célula , Movimiento Celular/fisiología , Embrión de Pollo , Cóclea/citología , Cóclea/inervación , Epitelio/embriología , Epitelio/inervación , Colorantes Fluorescentes , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas Aferentes/fisiología , Neuropéptidos/biosíntesis , Neuropéptidos/genética , Células Madre/citología , Células Madre/metabolismo , Vestíbulo del Laberinto/citología , Vestíbulo del Laberinto/inervaciónRESUMEN
The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Asunto(s)
Microscopía/métodos , Morfogénesis , Imagen de Lapso de Tiempo/métodos , Microscopía/instrumentación , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
The enteric nervous system (ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units founded by postmigratory neural crest-derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment. Clonally related enteric neurons exhibit synchronous activity in response to network stimulation. Thus, lineage relationships underpin the organization of the peripheral nervous system.
Asunto(s)
Linaje de la Célula , Sistema Nervioso Entérico/citología , Animales , Linaje de la Célula/genética , Proliferación Celular , Células Clonales/citología , Sistema Nervioso Entérico/metabolismo , Mucosa Intestinal/citología , Ratones , Mosaicismo , Mutagénesis , Cresta Neural/citología , Neurogénesis , Neuroglía/fisiología , Neuronas/citología , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Células Madre/citología , TranscriptomaRESUMEN
Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro.
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Técnica de Placa Hemolítica/métodos , Malaria Falciparum/parasitología , Parásitos/aislamiento & purificación , Plasmodium falciparum/aislamiento & purificación , Animales , Eritrocitos/parasitología , Humanos , Estadios del Ciclo de Vida , Proteína 1 de Superficie de Merozoito/metabolismo , Mutación/genética , Fenotipo , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Despite great strides during the preceding 3 decades, the ability to consistently eliminate postoperative nausea and vomiting (PONV) continues to elude anesthesia practitioners. The occurrence of PONV related to anesthesia and surgery prolongs hospital stays and increases healthcare costs. Protracted recovery times place constraints on patients, healthcare systems, and healthcare financiers. Many pharmacological antiemetics have been developed and are in use in the attempt to alleviate PONV. Side effects and cost profiles of many of these interventions, however, reinforce the broadly held belief that there remains opportunity for improvement. Because the Western culture almost exclusively favors evidence-based scientific practice and interventions, the search continues for an ideal, cost-effective, safe, and efficacious pharmacological agent to prevent PONV. Eastern culture, on the other hand, relies heavily on naturopathic remedies whose successful use has spanned thousands of years. Increasing attention has been given to the potential benefits of nonpharmacological intervention for the prevention of PONV in association with anesthesia care. Therefore, the purpose of this AANA Journal course will be to focus attention on what is known and what is unknown in the literature regarding use of the nonallopathic remedy of acupressure as a nonpharmacological alternative to commonly utilized antiemetic prophylaxis.
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Acupresión/instrumentación , Acupresión/métodos , Náusea y Vómito Posoperatorios/prevención & control , Antieméticos/efectos adversos , Humanos , Meridianos , Atención PerioperativaRESUMEN
The proneural factor Ascl1 controls multiple steps of neurogenesis in the embryonic brain, including progenitor division and neuronal migration. Here we show that Cenpj, also known as CPAP, a microcephaly gene, is a transcriptional target of Ascl1 in the embryonic cerebral cortex. We have characterized the role of Cenpj during cortical development by in utero electroporation knockdown and found that silencing Cenpj in the ventricular zone disrupts centrosome biogenesis and randomizes the cleavage plane orientation of radial glia progenitors. Moreover, we show that downregulation of Cenpj in post-mitotic neurons increases stable microtubules and leads to slower neuronal migration, abnormal centrosome position and aberrant neuronal morphology. Moreover, rescue experiments shows that Cenpj mediates the role of Ascl1 in centrosome biogenesis in progenitor cells and in microtubule dynamics in migrating neurons. These data provide insights into genetic pathways controlling cortical development and primary microcephaly observed in humans with mutations in Cenpj.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Corteza Cerebral/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , División Celular , Movimiento Celular , Centrosoma/metabolismo , Centrosoma/ultraestructura , Corteza Cerebral/citología , Electroporación , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Inyecciones Intraventriculares , Ratones , Ratones Transgénicos , Microtomía , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Células-Madre Neurales/ultraestructura , Neuronas/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Técnicas de Cultivo de TejidosRESUMEN
This cross-sectional, double-blind study reports the prevalence of Salmonella enterica serotype enteritidis (SE) on California egg layer premises using single vs. pooled manure drag swabs and presents a description of egg production and management systems in the state and an initial analysis of risk factors for SE. The study included 91% of all known eligible egg premises in California, representing the majority of eggs produced in the state. The overall prevalence of SE on California egg layer premises was 10.5%, while 1.1% of all rows sampled were positive for SE. The percentage of positive rows for SE on any premises never exceeded 25% of the 16 swabs collected per premises. A description of egg production and management on California egg layer premises is presented. Statistically significant associations for SE were not evident and were limited because of sample size and the low prevalence of SE on California egg layer premises. Several biological and management factors, such as flock health, stage of production, manure management, ventilation, and watering systems, show trend associations with premises positive for SE and require further investigation. Manure drag swabs serve as a useful tool to validate the core components of an egg quality assurance program for SE based on process control principles.
Asunto(s)
Crianza de Animales Domésticos/normas , Pollos , Huevos , Heces/microbiología , Vivienda para Animales , Salmonella enteritidis/aislamiento & purificación , Animales , California , Estudios Transversales , Método Doble Ciego , Microbiología Ambiental , Estudios Prospectivos , Factores de RiesgoRESUMEN
Between the summer of 1998 and the winter of 2000, Salmonella analysis was performed on 2128 single and 532 pooled manure drag swabs obtained from 133 California commercial egg laying farms. The isolation of Salmonella from all rows and from all flocks using single or pooled swabs was 80% and 92%, respectively. Hence, there was no statistical difference between single vs. pooled swabs in terms of identifying Salmonella on a row or flock basis. A total of 14 serogroups comprising 44 serotypes were isolated from 123 of 133 farms. When the top 10 serotypes were considered, there was no significant difference in the range of serotypes isolated by the two culturing methods. The overall S. enteritidis prevalence for California flocks was 10.5% (14/133). The overall row prevalence for S. enteritidis for all the farms was 1.1% (24/2128), and the overall pool prevalence was 2.4% (13/532). Sixty percent (12/20) of the S. enteritidis isolates from the positive farms were phage type 4, and 40% (8/20) represented five other phage types (1, 6B, 7, 8, and 28).
Asunto(s)
Pollos , Huevos , Heces/microbiología , Vivienda para Animales , Salmonella enteritidis/aislamiento & purificación , Manejo de Especímenes/métodos , Animales , California , Microbiología Ambiental , SerotipificaciónRESUMEN
This AANA Journal course discusses the American College of Cardiology (ACC) and American Heart Association (AHA) guideline on perioperative cardiovascular evaluation for noncardiac surgery. The intent of the ACC/AHA guideline is to assist clinicians in clinical decision making by describing a range of generally acceptable approaches for the diagnosis, management, and prevention of cardiac diseases. Optimizing the anesthetic management of the cardiac patient undergoing noncardiac surgery is becoming increasingly important: as the percentage of Americans older than 65 years continues to grow, so does the prevalence of cardiac disease in this population. Simply accepting a preoperative cardiology clearance for the cardiac patient undergoing noncardiac surgery provides little information that can be used for risk assessment and management of anesthesia. While national practice patterns vary significantly, there is an important need to standardize cost-effective preoperative cardiac evaluation. By using evidence-based studies, the ACC/AHA guideline delineates methods to objectively categorize cardiovascular risk and use data from the cardiology consultation to refine anesthetic management. Use of the guideline can lead to more efficient evaluation of the noncardiac patient with cardiac disease, which can decrease morbidity, mortality, and cost.