A new algorithm for particle weighted subtraction to decrease signals from unwanted components in single particle analysis.

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.

BIPSPI+: Mining Type-Specific Datasets of Protein Complexes to Improve Protein Binding Site Prediction.

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.

On bias, variance, overfitting, gold standard and consensus in single-particle analysis by cryo-electron microscopy.

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.

Cryo-EM density maps adjustment for subtraction, consensus and sharpening.

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.

Cryo-EM and Single-Particle Analysis with Scipion.

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.

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM.

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.

Integration of Cryo-EM Model Building Software in Scipion.

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.

Flexible workflows for on-the-fly electron-microscopy single-particle image processing using Scipion.

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 2.2 Šresolution ß-galactosidase data set.

Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy.

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.

3DBIONOTES: A unified, enriched and interactive view of macromolecular information.

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.

Suffering indicators in terminally ill children from the parental perspective.

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.

Multisensory perception and action in 3-ball cascade juggling.

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.

Anticoncepción de emergencia: perfil de las usuarias y características de la demanda / Emergency contraceptive: profile of the users and demand characteristics

OBJETIVOS. Describir el perfil de las usuarias de la anticoncepción de emergencia así como las características de la demanda de dicho medicamento. MATERIAL Y MÉTODOS. Estudio descriptivo, transversal, realizado en el ámbito de las urgencias de Atención Primaria en un medio urbano. Las participantes fueron las pacientes que solicitaron la píldora del día después en dichas urgencias. RESULTADOS. Características generales: 132 mujeres, cuya media de edad fue 22,9 años (rango 14-46 años); referido a sus características sociales, un 85,6% estaban solteras, un 12,1% casadas y el 2,3% separadas. En cuanto a su nivel de estudios, un 45,8% eran estudiantes, tenían estudios primarios un 18,2%, secundarios un 66,7% y superiores un 15,2%. Las razones de la demanda en un 75,8% se debió a un fallo en el método de barrera o preservativo, el 17,7% no usó ningún método anticonceptivo y un 6,5% lo atribuyó a otras razones. Los meses de mayor demanda fueron agosto, septiembre y diciembre; los sábados y domingos los días en que más se solicitó. La media de horas transcurridas desde el coito sin protección hasta la solicitud de este medicamento fueron 14,5. Hasta un 24,4% de las mujeres ya habían utilizado previamente la anticoncepción de emergencia. CONCLUSIÓN. El perfil de las usuarias de la anticoncepción de emergencia se ajusta al descrito en otros estudios. Existe un uso abusivo o mal uso de la píldora del día después y un bajo uso de los métodos de barrera

OBJECTIVE. Describe user profile of emergency contraceptions and the characteristics of demand for this drug. MATERIAL AND METHODS. Descriptive, cross-sectional study conducted in primary care emergency setting in urban area. Participants were patients who requested the day after pill in such emergencies. RESULTS. General characteristics: 132 women, whose mean age was 22.9 years (range 14-46 years). Social characteristics: 85.6% were single, 12.1% married and 2.3% separated. Regarding study level, 45.8% were students, 18.2% had primary studies, 66.7% secondary and 15.2% upper education. Reasons for the demand were due to failure of barrier method or condom in 75.8%, 17.7% did not use any contraceptive method and 6.5% attributed it to other reasons. The months of greatest demand were August, September and December. Saturday and Sunday were the days on which it was requested most. Mean hours since the intercourse without protection until request of the medication was 14.5. Up to 24.4% of the women had already previously used the emergency contraception. CONCLUSION. The user profile of emergency contraceptives adjusts to its description in other studies. There is an abusive or bad use of the day after pill and low use of barrier methods

Conocimiento y grado de control de los factores de riesgo cardiovascular antes y después de un infarto / Knowledge and degree of control of cardiovascular risk factors before and after a heart attack

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NANDA nursing diagnoses identification and validation in a neonatal unit.

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.

[Perioperative risk in heart surgery. An original material and review of difficulties in correct comparisons with other materials]. / Perioperativ risiko ved hjerteoperationer. Et originalmateriale og en gennemgang af vanskelighederne ved korrekt sammenligning med andre materialer.

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.

[Non-traumatic dissection of the thoracic aorta. A review with special emphasis on postoperative paraplegia]. / Ikketraumatisk torakal aortadissektion. En oversigt med soerlig voegt på postoperativ paraplegi.

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.

[The battle against leprosy in Spain in the first half of the XX century]. / La lucha contra la lepra en la España de la primera mitad del siglo XX. Evolución de las estrategias preventivas basada en los avances científicos, sanitarios y sociales.

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.