RESUMEN
Single particle analysis (SPA) in cryo-electron microscopy (cryo-EM) is highly used to obtain the near-atomic structure of biological macromolecules. The current methods allow users to produce high-resolution maps from many samples. However, there are still challenging cases that require extra processing to obtain high resolution. This is the case when the macromolecule of the sample is composed of different components and we want to focus just on one of them. For example, if the macromolecule is composed of several flexible subunits and we are interested in a specific one, if it is embedded in a viral capsid environment, or if it has additional components to stabilize it, such as nanodiscs. The signal from these components, which in principle we are not interested in, can be removed from the particles using a projection subtraction method. Currently, there are two projection subtraction methods used in practice and both have some limitations. In fact, after evaluating their results, we consider that the problem is still open to new solutions, as they do not fully remove the signal of the components that are not of interest. Our aim is to develop a new and more precise projection subtraction method, improving the performance of state-of-the-art methods. We tested our algorithm with data from public databases and an in-house data set. In this work, we show that the performance of our algorithm improves the results obtained by others, including the localization of small ligands, such as drugs, whose binding location is unknown a priori.
Asunto(s)
Algoritmos , Imagen Individual de Molécula , Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/químicaRESUMEN
Computational approaches for predicting protein-protein interfaces are extremely useful for understanding and modelling the quaternary structure of protein assemblies. In particular, partner-specific binding site prediction methods allow delineating the specific residues that compose the interface of protein complexes. In recent years, new machine learning and other algorithmic approaches have been proposed to solve this problem. However, little effort has been made in finding better training datasets to improve the performance of these methods. With the aim of vindicating the importance of the training set compilation procedure, in this work we present BIPSPI+, a new version of our original server trained on carefully curated datasets that outperforms our original predictor. We show how prediction performance can be improved by selecting specific datasets that better describe particular types of protein interactions and interfaces (e.g. homo/hetero). In addition, our upgraded web server offers a new set of functionalities such as the sequence-structure prediction mode, hetero- or homo-complex specialization and the guided docking tool that allows to compute 3D quaternary structure poses using the predicted interfaces. BIPSPI+ is freely available at https://bipspi.cnb.csic.es.
Asunto(s)
Uso de Internet , Aprendizaje Automático , Mapeo de Interacción de Proteínas , Proteínas , Programas Informáticos , Sitios de Unión , Conjuntos de Datos como Asunto , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas/químicaRESUMEN
Cryo-electron microscopy (cryoEM) has become a well established technique to elucidate the 3D structures of biological macromolecules. Projection images from thousands of macromolecules that are assumed to be structurally identical are combined into a single 3D map representing the Coulomb potential of the macromolecule under study. This article discusses possible caveats along the image-processing path and how to avoid them to obtain a reliable 3D structure. Some of these problems are very well known in the community. These may be referred to as sample-related (such as specimen denaturation at interfaces or non-uniform projection geometry leading to underrepresented projection directions). The rest are related to the algorithms used. While some have been discussed in depth in the literature, such as the use of an incorrect initial volume, others have received much less attention. However, they are fundamental in any data-analysis approach. Chiefly among them, instabilities in estimating many of the key parameters that are required for a correct 3D reconstruction that occur all along the processing workflow are referred to, which may significantly affect the reliability of the whole process. In the field, the term overfitting has been coined to refer to some particular kinds of artifacts. It is argued that overfitting is a statistical bias in key parameter-estimation steps in the 3D reconstruction process, including intrinsic algorithmic bias. It is also shown that common tools (Fourier shell correlation) and strategies (gold standard) that are normally used to detect or prevent overfitting do not fully protect against it. Alternatively, it is proposed that detecting the bias that leads to overfitting is much easier when addressed at the level of parameter estimation, rather than detecting it once the particle images have been combined into a 3D map. Comparing the results from multiple algorithms (or at least, independent executions of the same algorithm) can detect parameter bias. These multiple executions could then be averaged to give a lower variance estimate of the underlying parameters.
Asunto(s)
Imagenología Tridimensional , Sesgo , Consenso , Microscopía por Crioelectrón/métodos , Imagenología Tridimensional/métodos , Reproducibilidad de los ResultadosRESUMEN
Electron cryomicroscopy (cryo-EM) has emerged as a powerful structural biology instrument to solve near-atomic three-dimensional structures. Despite the fast growth in the number of density maps generated from cryo-EM data, comparison tools among these reconstructions are still lacking. Current proposals to compare cryo-EM data derived volumes perform map subtraction based on adjustment of each volume grey level to the same scale. We present here a more sophisticated way of adjusting the volumes before comparing, which implies adjustment of grey level scale and spectrum energy, but keeping phases intact inside a mask and imposing the results to be strictly positive. The adjustment that we propose leaves the volumes in the same numeric frame, allowing to perform operations among the adjusted volumes in a more reliable way. This adjustment can be a preliminary step for several applications such as comparison through subtraction, map sharpening, or combination of volumes through a consensus that selects the best resolved parts of each input map. Our development might also be used as a sharpening method using an atomic model as a reference. We illustrate the applicability of this algorithm with the reconstructions derived of several experimental examples. This algorithm is implemented in Xmipp software package and its applications are user-friendly accessible through the cryo-EM image processing framework Scipion.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Sustancias Macromoleculares/ultraestructura , Cápside/química , Cápside/ultraestructura , Virus de la Hepatitis B/ultraestructura , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Reproducibilidad de los Resultados , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/ultraestructuraRESUMEN
Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Imagen Individual de Molécula , Microscopía por Crioelectrón , Sustancias Macromoleculares , Flujo de TrabajoRESUMEN
The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achieved.
Asunto(s)
Microscopía por Crioelectrón/métodos , Imagen Individual de Molécula/métodos , Algoritmos , Artefactos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodosRESUMEN
Advances in cryo-electron microscopy (cryo-EM) have made it possible to obtain structures of large biological macromolecules at near-atomic resolution. This "resolution revolution" has encouraged the use and development of modeling tools able to produce high-quality atomic models from cryo-EM density maps. Unfortunately, many practical problems appear when combining different packages in the same processing workflow, which make difficult the use of these tools by non-experts and, therefore, reduce their utility. We present here a major extension of the image processing framework Scipion that provides inter-package integration in the model building area and full tracking of the complete workflow, from image processing to structure validation.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Programas Informáticos , Microscopía por Crioelectrón , Flujo de TrabajoRESUMEN
Electron microscopy of macromolecular structures is an approach that is in increasing demand in the field of structural biology. The automation of image acquisition has greatly increased the potential throughput of electron microscopy. Here, the focus is on the possibilities in Scipion to implement flexible and robust image-processing workflows that allow the electron-microscope operator and the user to monitor the quality of image acquisition, assessing very simple acquisition measures or obtaining a first estimate of the initial volume, or the data resolution and heterogeneity, without any need for programming skills. These workflows can implement intelligent automatic decisions and they can warn the user of possible acquisition failures. These concepts are illustrated by analysis of the well known 2.2â Å resolution ß-galactosidase data set.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/métodos , Imagen Individual de Molécula/métodos , Programas Informáticos , Automatización , beta-Galactosidasa/químicaRESUMEN
Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.
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Electrones , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/estadística & datos numéricos , Sustancias Macromoleculares/ultraestructura , Microscopía Electrónica/métodos , Proteínas/ultraestructura , Algoritmos , Humanos , Sustancias Macromoleculares/química , Microscopía Electrónica/instrumentación , Conformación Molecular , Simulación de Dinámica Molecular , Análisis de Componente Principal , Proteínas/química , TermodinámicaRESUMEN
With the advent of high throughput techniques like Next Generation Sequencing, the amount of biological information for genes and proteins is growing faster than ever. Structural information is also rapidly growing, especially in the cryo Electron Microscopy area. However, in many cases, the proteomic and genomic data are spread in multiple databases and with no simple connection to structural information. In this work we present a new web platform that integrates EMDB/PDB structures and UniProt sequences with different sources of protein annotations. The application provides an interactive interface linking sequence and structure, including EM maps, presenting the different sources of information at sequence and structural level. The web application is available at http://3dbionotes.cnb.csic.es.
Asunto(s)
Proteómica/estadística & datos numéricos , Programas Informáticos , Proteína de la Poliposis Adenomatosa del Colon/química , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Secuencia de Aminoácidos , Antígenos CD , Secuencia de Bases , Cadherinas/química , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Bases de Datos de Proteínas , Proteínas F-Box/química , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Expresión Génica , Humanos , Internet , Modelos Moleculares , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Relación Estructura-ActividadRESUMEN
PURPOSE: Suffering is a complex multifaceted phenomenon, which has received limited attention in relation to children with terminal illness. As part of a wider study we interviewed parents of children with terminal illness to elicit their perspectives on suffering, in order to provide initial understanding from which to develop observational indicators and further research. METHODS: Qualitative descriptive study with semi-structured interviews made "ad hoc". Selection through deliberate sampling of mothers and fathers of hospitalised children (0-16 years old) with a terminal illness in Granada (Spain). KEY RESULTS: 13 parents were interviewed. They described children's suffering as manifested through sadness, apathy, and anger towards their parents and the professionals. The isolation from their natural environment, the uncertainty towards the future, and the anticipation of pain caused suffering in children. The pain is experienced as an assault that their parents allow to occur. CONCLUSIONS: The analysis of the interview with the parents about their perception of their ill children's suffering at the end of their lives is a valuable source of information to consider supportive interventions for children and parents in health care settings. An outline summary of the assessed aspects of suffering, the indicators and aspects for health professional consideration is proposed.
Asunto(s)
Neoplasias/psicología , Padres/psicología , Estrés Psicológico/psicología , Enfermo Terminal/psicología , Adaptación Psicológica , Adulto , Actitud Frente a la Muerte , Niño , Femenino , Humanos , Entrevistas como Asunto , Masculino , Neoplasias/diagnóstico , Neoplasias/terapia , Relaciones Padres-Hijo , Investigación Cualitativa , Calidad de Vida , España , Estrés Psicológico/prevención & control , Cuidado Terminal/métodos , Cuidado Terminal/psicologíaRESUMEN
Multisensory perception and action in 3-ball cascade juggling was investigated in intermediate-skilled performers by manipulating vision (full or lower field restricted) or ball weight (equal or different). There were main effects for both independent variables but no interactions. Manipulation of ball weight had a more pervasive effect on performance outcome, as well as central tendency and dispersion of kinematic measures of the juggling action. A common finding to both manipulations was that balls were tossed to higher zeniths, thus increasing parabola height and flight time. For intermediate-skilled jugglers, proprioception-haptics available when the balls were in the hands and vision of the balls and hands around the moment of ball toss and catch both make a contribution to the juggling action.
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Destreza Motora/fisiología , Propiocepción/fisiología , Desempeño Psicomotor/fisiología , Percepción Visual/fisiología , Femenino , Humanos , Masculino , Soporte de Peso/fisiologíaAsunto(s)
Conductas Relacionadas con la Salud , Conocimientos, Actitudes y Práctica en Salud , Infarto del Miocardio/epidemiología , Anciano , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Femenino , Humanos , Masculino , Infarto del Miocardio/prevención & control , Prevención Primaria , Estudios Retrospectivos , Factores de RiesgoRESUMEN
OBJECTIVE: To identify the problems nurses encounter in a Neonatal Unit and adapt them to the corresponding NANDA (North American Nursing Diagnosis Association) diagnostic categories for their validation in our environment. DESIGN: A Nominal Group Technique (NGT) was developed to identify the problems that nurses detect and solve. Identified problems were catalogued with their corresponding diagnostic label using the NANDA diagnostic taxonomy. A concordance study was carried out between two observers, using the Kappa coefficient. RESULTS: During the study period, 36 different NANDA diagnostic categories were identified for use. From these, 5 have been validated with an excellent or good concordance (K > 0.70) and 2 with an average concordance (0.70 > K > 0.40). The remaining categories did not occur with enough frequency during the study period to apply statistical treatment. CONCLUSIONS: The diagnostic categories validated in our Unit occur frequently, are easily identified by the nurses, and can be used in patient care plans. Non-validated categories may become valid in subsequent studies.
Asunto(s)
Enfermería Neonatal/métodos , Diagnóstico de Enfermería/métodos , Diagnóstico de Enfermería/normas , Humanos , Recién Nacido , Investigación en Evaluación de Enfermería , Variaciones Dependientes del Observador , Solución de Problemas , Reproducibilidad de los Resultados , Terminología como AsuntoRESUMEN
The purpose of this study was to present data concerning morbidity and mortality after cardiac surgery and to establish a method to make the presentation comparable to other reports. The main difficulty in comparing results of surgery of one institution with those of another is the lack of a simple and widely acceptable quantification of risk. A preoperative risk classification of patients requires readily available and objective data. The shortage of standardized criteria for comparing outcome was obvious as only a few comprehensive reports regarding preoperative predictors were found in the literature. The method of Tuman et al is based on 12 preoperative risk factors that are reasonably free of observer bias and practically obtainable. This method was used to report the results of 628 consecutive patients undergoing coronary revascularization or valvular surgery. Total in-hospital morbidity was 3.5% and mortality 1.0%. The most important predictors for postoperative morbidity were valvular surgery, advanced age, renal dysfunction, recent myocardial infarction and pulmonary hypertension. The system is most useful in predicting good outcome in low-risk patients. The identification of high-risk patients is valuable in spite of the limited predictive ability, by allowing special attention to be directed to the patient at risk.
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Procedimientos Quirúrgicos Cardíacos/efectos adversos , Complicaciones Intraoperatorias/mortalidad , Complicaciones Posoperatorias/mortalidad , Adulto , Anciano , Procedimientos Quirúrgicos Cardíacos/mortalidad , Puente de Arteria Coronaria/efectos adversos , Puente de Arteria Coronaria/mortalidad , Circulación Extracorporea/efectos adversos , Circulación Extracorporea/mortalidad , Femenino , Prótesis Valvulares Cardíacas/efectos adversos , Prótesis Valvulares Cardíacas/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de RiesgoRESUMEN
Non-traumatic dissection of the thoracic aorta is a degenerative vascular disease with a high morbidity and mortality. The main symptom is stabbing thoracic pain, but the diagnosis is made difficult when the clinical picture is dominated by symptoms caused by the aorta's neighbouring structures. The definitive diagnosis is made by aortography. Treatment and prognosis depend on the anatomic placement of the dissection. In dissection of the ascending aorta (type A) the treatment is solely surgical, whereas a combined medical and surgical treatment is recommended for dissections of the descending aorta (type B). The most common postoperative complication in type B dissection is paraplegia, which occurs in up to 40% of cases. The cause of paraplegia is postoperative ischaemia of the spinal medulla. Many methods and treatments have been used to avoid medullary ischaemic damage. The most important ones are reducing the time length of aortic clamping, maintaining an adequate perfusion pressure, and the use of moderate hypothermia. Spinal drainage with or without pharmacological intervention possibly has a preventive effect, but convincing clinical documentation is lacking.
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Aneurisma de la Aorta Torácica/cirugía , Disección Aórtica/cirugía , Paraplejía/etiología , Complicaciones Posoperatorias , Disección Aórtica/diagnóstico , Disección Aórtica/etiología , Aneurisma de la Aorta Torácica/diagnóstico , Aneurisma de la Aorta Torácica/etiología , Humanos , Paraplejía/prevención & control , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/prevención & control , PronósticoRESUMEN
The present work deals with the measures taken in Spain during the first half of the XX century to fight leprosy. The main policy to curb this infectious and contagious disease consisted first in isolating the patients; nevertheless, as knowledge concerning the disease grew up considerably, its preventive measures started to evolve along the period. Another point of decisive influence regarding the fight against leprosy was the intervention of the health authorities in the different historical moments and the social response to this evil.
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Lepra/historia , Medicina Preventiva/historia , Historia del Siglo XX , Humanos , Salud Pública/historia , EspañaRESUMEN
During an 18-month period, 82 consecutive patients (37 women and 45 men), with a mean age of 50 yr (range 15 to 74), underwent hemodialysis with 91 polyurethane double-lumen subclavian superior vena cava catheters inserted via the right subclavian vein. Upon catheter removal, venograms were performed in 53 which revealed thrombosis in 13. Autopsy performed in 13 other patients revealed thrombosis in three. Mean catheter duration was 27 days (range 1 to 138). Catheter duration was significantly (p less than .01) longer in the 16 cases complicated by thrombosis. There was no significant difference in the frequency of thrombosis between men and women. Two patients developed symptoms of subclavian vein occlusion after the creation of an arteriovenous fistula on the same side as a previous hemodialysis catheter. Overall, thrombosis was found in 23% and increased with longer catheter duration. We recommend phlebography to exclude thrombosis before creating an arteriovenous fistula on the same side as the previous hemodialysis catheter. There was no case of clinical discernible pulmonary embolism.