RESUMO
Heart development begins with the formation of a tube as cardiac progenitors migrate from opposite sides of the embryo. Abnormal cardiac progenitor movements cause congenital heart defects. However, the mechanisms of cell migration during early heart development remain poorly understood. Using quantitative microscopy, we found that in Drosophila embryos, cardiac progenitors (cardioblasts) migrated through a sequence of forward and backward steps. Cardioblast steps were associated with oscillatory non-muscle myosin II waves that induced periodic shape changes and were necessary for timely heart tube formation. Mathematical modeling predicted that forward cardioblast migration required a stiff boundary at the trailing edge. Consistent with this, we found a supracellular actin cable at the trailing edge of the cardioblasts that limited the amplitude of the backward steps, thus biasing the direction of cell movement. Our results indicate that periodic shape changes coupled with a polarized actin cable produce asymmetrical forces that promote cardioblast migration.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas de Drosophila/fisiologia , Actinas , Coração , Miosinas , Morfogênese , Drosophila melanogasterRESUMO
NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.
Assuntos
Células Secretoras de Insulina , NADP/metabolismo , Células Secretoras de Insulina/metabolismo , Oxirredução , Glucose/metabolismo , Mitocôndrias/metabolismoRESUMO
SUMMARY: Our increasing ability to resolve fine details using light microscopy is matched by an increasing need to quantify images in order to detect and measure phenotypes. Despite their central role in cell biology, many image analysis tools require a financial investment, are released as proprietary software, or are implemented in languages not friendly for beginners, and thus are used as black boxes. To overcome these limitations, we have developed PyJAMAS, an open-source tool for image processing and analysis written in Python. PyJAMAS provides a variety of segmentation tools, including watershed and machine learning-based methods; takes advantage of Jupyter notebooks for the display and reproducibility of data analyses; and can be used through a cross-platform graphical user interface or as part of Python scripts via a comprehensive application programming interface. AVAILABILITY AND IMPLEMENTATION: PyJAMAS is open-source and available at https://bitbucket.org/rfg_lab/pyjamas. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Microscopia , Software , Reprodutibilidade dos Testes , Processamento de Imagem Assistida por Computador , IdiomaRESUMO
PURPOSE: A clinical audit is a key component of the clinical governance framework. The rate of audit completion in general surgery has not been investigated. The purpose of this paper is to assess the rates of audit activity and completion and explore the barriers to successful audit completion. DESIGN/METHODOLOGY/APPROACH: This was a multi-centre study evaluating current surgical audit practice. A standardised audit proforma was designed. All clinical audits in general surgery during a two-year period were identified and retrospectively reviewed. Data held by the audit departments were collated, and individual audit teams were contacted to verify the data accuracy. Audit teams failing to complete the full audit cycle with a re-audit were asked to explain the underlying reasons behind this. FINDINGS: Of the six trusts approached, two refused to participate, and one failed to initiate the project. A total of 39 audits were registered across three surgical directorates. Only 15 out of 39 audits completed at least one audit cycle, with 4 deemed of no value to re-audit. Only seven audits were completed to re-audit. Achieving a publication or a presentation was the most cited reason for not completing the audit loop. ORIGINALITY/VALUE: This study demonstrates that the poor rates of audit completion rate found in other areas of clinical medicine pervade general surgery. Improved completion of an audit is essential and strategies to achieve this are urgently needed.
Assuntos
Cirurgia Geral/normas , Auditoria Médica/estatística & dados numéricos , Humanos , Estudos Retrospectivos , Medicina Estatal , Reino UnidoRESUMO
Augmented reality (AR) superimposes computer-generated virtual objects onto the user's view of the real world. Among medical disciplines, neurosurgery has long been at the forefront of image-guided surgery, and it continues to push the frontiers of AR technology in the operating room. In this systematic review, we explore the history of AR in neurosurgery and examine the literature on current neurosurgical applications of AR. Significant challenges to surgical AR exist, including compounded sources of registration error, impaired depth perception, visual and tactile temporal asynchrony, and operator inattentional blindness. Nevertheless, the ability to accurately display multiple three-dimensional datasets congruently over the area where they are most useful, coupled with future advances in imaging, registration, display technology, and robotic actuation, portend a promising role for AR in the neurosurgical operating room.
Assuntos
Processamento de Imagem Assistida por Computador/tendências , Procedimentos Neurocirúrgicos/tendências , Cirurgia Assistida por Computador/tendências , Interface Usuário-Computador , Realidade Virtual , Encefalopatias/diagnóstico por imagem , Encefalopatias/cirurgia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Procedimentos Neurocirúrgicos/métodos , Doenças da Coluna Vertebral/diagnóstico por imagem , Doenças da Coluna Vertebral/cirurgia , Cirurgia Assistida por Computador/métodosRESUMO
The fruit fly Drosophila melanogaster has recently emerged as an excellent system to investigate the genetics of cardiovascular development and disease. Drosophila provides an inexpensive and genetically-tractable in vivo system with a large number of conserved features. In addition, the Drosophila embryo is transparent, and thus amenable to time-lapse fluorescence microscopy, as well as biophysical and pharmacological manipulations. One of the conserved aspects of heart development from Drosophila to humans is the initial assembly of a tube. Here, we review the cellular behaviours and molecular dynamics important for the initial steps of heart morphogenesis in Drosophila, with particular emphasis on the cell-cell adhesion and cytoskeletal networks that cardiac precursors use to move, coordinate their migration, interact with other tissues and eventually sculpt a beating heart.
Assuntos
Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Coração/crescimento & desenvolvimento , Morfogênese/fisiologia , Organogênese/fisiologia , Animais , Proteínas de Drosophila/metabolismo , HumanosRESUMO
Embryos extend their anterior-posterior (AP) axis in a conserved process known as axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern-recognition methods to track dividing cells from confocal microscopy movies in a generally applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from the influence of other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.
Assuntos
Padronização Corporal , Divisão Celular , Rastreamento de Células/métodos , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Animais , Automação , Fenômenos Biomecânicos , Contagem de Células , Forma Celular , Ectoderma/citologia , Mesoderma/citologiaRESUMO
We present the design and construction of a versatile, open frame inverted microscope system for wide-field fluorescence and single molecule imaging. The microscope chassis and modular design allow for customization, expansion, and experimental flexibility. We present two components which are included with the microscope which extend its basic capabilities and together create a powerful microscopy system: A Convex Lens-induced Confinement device provides the system with single-molecule imaging capabilities, and a two-color imaging system provides the option of imaging multiple molecular species simultaneously. The flexibility of the open-framed chassis combined with accessible single-molecule, multi-species imaging technology supports a wide range of new measurements in the health, nanotechnology, and materials science research sectors.
Assuntos
Microscopia/instrumentação , Imagem Molecular/instrumentação , Imagem Óptica/instrumentação , Bacteriófago lambda/genética , DNA Viral/química , Difusão , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/instrumentação , Corantes Fluorescentes , Lasers , Oligonucleotídeos/química , Fotodegradação , Polietilenoglicóis , Soluções , Estreptavidina/químicaRESUMO
We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub-30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.
Assuntos
Lentes , Nanoestruturas/química , Nanotecnologia/métodos , DNA/química , Imageamento Tridimensional , Desnaturação de Ácido NucleicoRESUMO
We present the conception, fabrication, and demonstration of a versatile, computer-controlled microscopy device which transforms a standard inverted fluorescence microscope into a precision single-molecule imaging station. The device uses the principle of convex lens-induced confinement [S. R. Leslie, A. P. Fields, and A. E. Cohen, Anal. Chem. 82, 6224 (2010)], which employs a tunable imaging chamber to enhance background rejection and extend diffusion-limited observation periods. Using nanopositioning stages, this device achieves repeatable and dynamic control over the geometry of the sample chamber on scales as small as the size of individual molecules, enabling regulation of their configurations and dynamics. Using microfluidics, this device enables serial insertion as well as sample recovery, facilitating temporally controlled, high-throughput measurements of multiple reagents. We report on the simulation and experimental characterization of this tunable chamber geometry, and its influence upon the diffusion and conformations of DNA molecules over extended observation periods. This new microscopy platform has the potential to capture, probe, and influence the configurations of single molecules, with dramatically improved imaging conditions in comparison to existing technologies. These capabilities are of immediate interest to a wide range of research and industry sectors in biotechnology, biophysics, materials, and chemistry.
Assuntos
Lentes , Microscopia/instrumentação , Bacteriófago lambda , DNA Viral/metabolismo , Difusão , Desenho de Equipamento , Modelos TeóricosRESUMO
Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.
Assuntos
Biofísica/métodos , Lentes , Microscopia de Fluorescência/métodos , Microscopia/instrumentação , Miosinas/química , Imagem Óptica/métodos , Actinas/química , Trifosfato de Adenosina/química , Animais , Simulação por Computador , Citoesqueleto/metabolismo , Dineínas/química , Desenho de Equipamento , Insetos , Cinesinas/química , Microscopia/métodos , Microtúbulos/química , Física/métodosRESUMO
It is estimated that pancreatic cancer has a familial component in approximately 5-10% of cases. Some of these cases are part of a defined cancer syndrome with a known gene mutation but in the remaining the causative gene remains unknown. In recent years, a better understanding of the molecular events that occur in the progression model of pancreatic cancer has lead to the development of secondary screening programmes with the aim of identifying early precursor lesions or pre-invasive cancer at a stage amenable to curative resection. High-risk groups who have an inherited predisposition for pancreatic cancer form the ideal group to study in developing a robust screening programme. Multimodality screening using computed tomography and endoluminal ultrasound in combination with molecular analysis of pancreatic juice are proving promising as diagnostics tools or at least serving as predictors of risk over a defined period.
Assuntos
Predisposição Genética para Doença , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/etiologia , Biomarcadores Tumorais/análise , Fibrose Cística/complicações , Complicações do Diabetes , Humanos , Programas de Rastreamento/métodos , Pancreatite Crônica/complicações , Polimorfismo Genético , Grupos Raciais , Fatores de Risco , Fatores SexuaisRESUMO
Cystic fibrosis (CF) is a recessive disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. The risk of idiopathic chronic pancreatitis (ICP) is increased in individuals who have CFTR genotypes containing a CF-causing mutation plus a second pathogenic allele. It is unknown whether the risk of ICP is increased in CF carriers who have one CF-causing mutation plus one normal allele. In this study, 52 sporadic cases of ICP were ascertained through the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer. Individuals with pathogenic cationic trypsinogen mutations were excluded. DNA was comprehensively tested for CFTR mutations using a robotically enhanced, multiplexed, and highly redundant form of single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing. Fifteen subjects had a total of 18 pathogenic CFTR alleles. Eight subjects had common CF-causing mutations. This group included seven CF carriers in whom the second CFTR allele was normal (4.3 times the expected frequency, P=0.0002). Three subjects had compound heterozygotes genotypes containing two pathogenic alleles (31 times the expected frequency, P<0.0001). A variant allele of uncertain significance (p.R75Q) was detected in eight of the 52 ICP subjects and at a similar frequency (13/96) in random donors. ICP differs from other established CFTR-related conditions in that ICP risk is increased in CF carriers who have one documented normal CFTR allele. Having two CFTR mutations imparts a higher relative risk, while having only one mutation imparts a higher attributable risk.
Assuntos
Fibrose Cística/genética , Heterozigoto , Mutação/fisiologia , Adulto , Fibrose Cística/metabolismo , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Mutação/genética , Pancreatite Crônica , Fatores de RiscoRESUMO
BACKGROUND & AIMS: Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. METHODS: Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16(INK4a) promoter methylation was estimated using comparative methylation-specific real-time PCR. RESULTS: p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16(INK4a) promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. CONCLUSIONS: Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.
Assuntos
Adenocarcinoma/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Marcadores Genéticos , Neoplasias Pancreáticas/diagnóstico , Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Estudos de Casos e Controles , Metilação de DNA , Análise Mutacional de DNA , Diagnóstico Diferencial , Genes p16 , Genes p53 , Genes ras , Humanos , Suco Pancreático , Neoplasias Pancreáticas/genética , Pancreatite/diagnóstico , Pancreatite/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Fatores de RiscoRESUMO
Familial pancreatic cancer (FPC) (approximately 3% of all cases) has not been linked to defects in any specific gene. Germline inactivation of the gene LKB1/STK11 have been shown to cause Peutz-Jeghers syndrome (PJS) associated with a approximately 100-fold higher risk for the development of pancreatic cancer. We have analysed 39 index patients from European FPC families for mutations of LKB1/STK11 by sequencing of their DNA. No germline mutation was found within the complete coding region. Therefore, our results indicate that LKB1/STK11 is not altered in the germline of patients with hereditary pancreatic cancer.