RESUMO
Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.
Assuntos
Lista de Checagem , Editoração , Reprodutibilidade dos Testes , Processamento de Imagem Assistida por Computador , MicroscopiaRESUMO
The low reproducibility of scientific data published in articles has recently become a cause of concern in many scientific fields. Data involving light microscopy is no exception. The low awareness of researchers of the technologies they use in their research has been identified as one of the main causes of the problem. Potential solutions have hinted at the need to improve technological and methodological education within research. Despite the pivotal role of microscopy core facilities in the education of researchers being well documented, facility staff (FS) often learn their trade on the job, without receiving themselves any structured education about the technology they teach others to use. Additionally, despite endorsing an important role at the highest level of education, most FS never receive any training in pedagogy, the field of research on teaching and learning methods. In this article, we argue that the low level of awareness that researchers have of microscopy stems from a knowledge gap formed between them and microscopy FS during training routines. On the one hand, FS consider that their teaching task is to explain what is needed to produce reliable data. On the other, despite understanding what is being taught, researchers fail to learn the most challenging aspects of microscopy, those involving their judgement and reasoning. We suggest that the misunderstanding between FS and researchers is due to FS not being educated in pedagogy and thus often confusing understanding and learning. To bridge this knowledge gap and improve the quality of the microscopy education available to researchers, we propose a paradigm shift where training staff at technological core facilities be acknowledged as full-fledged teachers and offered structured education not only in the technology they teach but also in pedagogy. We then suggest that training routines at facilities be upgraded to follow the principles of the Constructive Alignment pedagogical method. We give an example of how this can be applied to existing microscopy training routines. We also describe a model to define where the responsibility of FS in training researchers begins and ends. This involves a major structural change where university staff involved in teaching research technologies themselves receive appropriate education. For this to be achieved, we advocate that funding agencies, universities, microscopy and core facility organisations mobilise resources of time and funding. Such changes may involve funding the creation and development of 'Train-the-trainer' type of courses and giving incentives for FS to upgrade their technological and pedagogical knowledge, for example by including them in career paths. We believe that this paradigm shift is necessary to improve the level of microscopy education and ultimately the reproducibility of published data.
RESUMO
Deep Convolutional Neural Networks (DCNN) have recently emerged as superior for many image segmentation tasks. The DCNN performance is however heavily dependent on the availability of large amounts of problem-specific training samples. Here we show that DCNNs trained on ground truth created automatically using fluorescently labeled cells, perform similar to manual annotations.
Assuntos
Técnicas Citológicas , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital , Aprendizado de Máquina , Redes Neurais de Computação , Automação Laboratorial , Linhagem Celular Tumoral , HumanosRESUMO
BACKGROUND: The dyslexia susceptibility 1 candidate 1 (DYX1C1) gene has recently been associated with dyslexia and reading scores in several population samples. The DYX1C1 has also been shown to affect neuronal migration and modulate estrogen receptor signaling. METHODS: We have analyzed the molecular networks of DYX1C1 by gene expression and protein interaction profiling in a human neuroblastoma cell line. RESULTS: We find that DYX1C1 can modulate the expression of nervous system development and neuronal migration genes such as RELN and associate with a number of cytoskeletal proteins. We also show by live cell imaging that DYX1C1 regulates cell migration of the human neuroblastoma cell line dependent on its tetratricopeptide repeat and DYX1 protein domains. The DYX1 domain is a novel highly conserved domain identified in this study by multiple sequence alignment of DYX1C1 proteins recovered from a wide range of eukaryotic species. CONCLUSIONS: Our results contribute to the hypothesis that dyslexia has a developmental neurobiological basis by linking DYX1C1 with many genes involved in neuronal migration disorders.
Assuntos
Movimento Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/genética , Proteína Reelina , Transcriptoma/genéticaRESUMO
BACKGROUND: Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS: A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 µg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS: We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION: Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING: European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.
Assuntos
Neoplasias Brônquicas/cirurgia , Leucócitos Mononucleares/transplante , Engenharia Tecidual/métodos , Alicerces Teciduais , Neoplasias da Traqueia/cirurgia , Adulto , Reatores Biológicos , Prótese Vascular , Transplante de Medula Óssea , Broncoscopia , Carcinoma Mucoepidermoide/cirurgia , Proliferação de Células , Epoetina alfa , Eritropoetina/uso terapêutico , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , MicroRNAs/metabolismo , Nanocompostos/química , Recidiva Local de Neoplasia/cirurgia , Neovascularização Fisiológica , Polietilenotereftalatos , Proteínas Recombinantes/uso terapêutico , Regeneração , Transplante AutólogoRESUMO
Cell anchorage is required for cell proliferation of untransformed cells, whereas anchorage-independent growth can be induced by oncogenes and is a hallmark of transformation. Whereas anchorage-dependent control of the progression of the G(1) phase of the cell cycle has been extensively studied, it is less clear whether and how anchorage may control other cell cycle phases and whether oncogenes may affect such controls. Here, we found that lack of cell anchorage did not influence progression through the cell cycle S phase, G(2) phase, or most of mitosis of primary human fibroblasts. However, unanchored fibroblasts could not complete cytokinesis. The cleavage furrow and central spindle were still formed in the absence of anchorage, but cells were unable to complete ingression, causing binucleation. Importantly, V12 H-Ras-transformed fibroblasts and two cancer cell lines progressed through the entire cell cycle without anchorage, including through cytokinesis. This indicates that oncogenic signaling may contribute to anchorage-independent growth and tumorigenesis by promoting the final cleavage furrow ingression during cytokinesis.
Assuntos
Adesão Celular , Transformação Celular Neoplásica/genética , Citocinese , Genes ras/fisiologia , Oncogenes/fisiologia , Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinese/genética , Fibroblastos/citologia , HumanosRESUMO
Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.
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Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Axônios/fisiologia , Olho/embriologia , Cones de Crescimento/metabolismo , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/fisiologia , Retina/embriologia , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética , Animais , Mapeamento Cromossômico , Clonagem Molecular , Drosophila , Genótipo , Hibridização In Situ , Ligantes , Microscopia de Fluorescência , Modelos Biológicos , Modelos Genéticos , Fenótipo , Fosforilação , RNA/metabolismo , Proteínas Repressoras/biossíntese , Retina/metabolismo , Transdução de Sinais , Transgenes , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. Here, we investigate the pigment phenotype and, from this, provide evidence for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biopterinas/análogos & derivados , Regulação Enzimológica da Expressão Gênica , Oxigenases de Função Mista/metabolismo , Pigmentação/fisiologia , Pterinas/metabolismo , Retina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Biopterinas/metabolismo , Cromatografia em Camada Fina , Embrião não Mamífero/embriologia , Embrião não Mamífero/enzimologia , Embrião não Mamífero/ultraestrutura , Microscopia Eletrônica , Oxigenases de Função Mista/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Members of the Eph-B family of receptors tyrosine kinase and their transmembrane ligands have been implicated in dorsoventral patterning of the vertebrate retinotectal projection. In the zebrafish retinotectal system, however, ephrinB2a is expressed strongly in the posterior tectum, in tectal neurons that form physical contacts with retinal ganglion cell (RGC) axons. In the gnarled mutant, where tectal neurons form ectopically in the pretectum, RGC axons stall before entering the tectum, or else are misrouted or branch aberrantly in the tectal neuropil. Ectopic expression of ephrinB2a in the anterior midbrain of wild-type embryos, with the aid of baculovirus, also inhibits RGC axon entry into the tectum. In vitro, zebrafish RGC axons are repelled by stripes of purified ephrinB2a. It is proposed that ephrinB2a may signal a subpopulation of RGC axons that they have reached their target neurons in the tectum.
Assuntos
Efrina-A2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Retina/metabolismo , Colículos Superiores/metabolismo , Vias Visuais/metabolismo , Animais , Axônios/metabolismo , Axônios/virologia , Baculoviridae/fisiologia , Carbocianinas/metabolismo , Embrião não Mamífero , Efrina-A2/genética , Olho/metabolismo , Olho/transplante , Hibridização In Situ/métodos , Técnicas In Vitro , Mutação , Transplante de Órgãos/métodos , Retina/citologia , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Colículos Superiores/citologia , Colículos Superiores/embriologia , Vias Visuais/embriologia , Peixe-ZebraRESUMO
We have identified two simple methods to analyse xanthophore and pterinosome biogenesis in zebrafish. The first uses methylene blue (methylthionium chloride), a redox dye which specifically labels xanthophores and pterinosomes, while the second uses autofluorescence to detect pteridine levels; these methods may be used to detect the number, location and shape of xanthophores and pterinosomes. These assays were applied to two zebrafish mutants--brie and yobo--and revealed that both mutants have pterinosome biogenesis and pteridine synthesis defects. Additionally, using capillary electrophoresis, we provide evidence that sepiapterin is responsible for the yellow colour and blue-light induced fluorescence in zebrafish embryos.