Automatic particle selection from electron micrographs using machine learning techniques

The 3D reconstruction of biological specimens using Electron Microscopy is currently capable of achieving subnanometer resolution. Unfortunately, this goal requires gathering tens of thousands of projection images that are frequently selected manually from micrographs. In this paper we introduce a new automatic particle selection that learns from the user which particles are of interest. The training phase is semi-supervised so that the user can correct the algorithm during picking and specifically identify incorrectly picked particles. By treating such errors specially, the algorithm attempts to minimize the number of false positives. We show that our algorithm is able to produce datasets with fewer wrongly selected particles than previously reported methods. Another advantage is that we avoid the need for an initial reference volume from which to generate picking projections by instead learning which particles to pick from the user. This package has been made publicly available in the open-source package Xmipp.     

Automatic cryo-EM particle selection for membrane proteins in spherical liposomes

Random spherically constrained (RSC) single particle reconstruction is a method to obtain structures of membrane proteins embedded in lipid vesicles (liposomes). As in all single-particle cryo-EM methods, structure determination is greatly aided by reliable detection of protein “particles” in micrographs. After fitting and subtraction of the membrane density from a micrograph, normalized cross-correlation (NCC) and estimates of the particle signal amplitude are used to detect particles, using as references the projections of a 3D model. At each pixel position, the NCC is computed with only those references that are allowed by the geometric constraint of the particle’s embedding in the spherical vesicle membrane. We describe an efficient algorithm for computing this position-dependent correlation, and demonstrate its application to selection of membrane-protein particles, GluA2 glutamate receptors, which present very different views from different projection directions.     

Recent advances in gene expression data clustering: a case study with comparative results

Several advanced techniques have been proposed for data clustering and many of them have been applied to gene expression data, with partial success. The high dimensionality and the multitude of admissible perspectives for data analysis of gene expression require additional computational resources, such as hierarchical structures and dynamic allocation of resources. We present an immune-inspired hierarchical clustering device, called hierarchical artificial immune network (HaiNet), especially devoted to the analysis of gene expression data. This technique was applied to a newly generated data set, involving maize plants exposed to different aluminum concentrations. The performance of the algorithm was compared with that of a self-organizing map, which is commonly adopted to deal with gene expression data sets. More consistent and informative results were obtained with HaiNet.     

Rapid screening: a comparative study

Although rapid screening of negative and inadequate cervical smears is a quality assurance requirement for all UK laboratories, there has been little attempt to standardize the method and laboratories make use of a number of different techniques and times. The aim of this study was to assess the sensitivity of these various techniques by measuring their ability to pick out known false-negative smears. Completed questionnaires from 123 laboratories across England revealed that 52% of laboratories use a "step" technique, 19% use "turret", 15% use random paths and 34% attempt to rescreen the whole slide quickly. Twenty-two percent of laboratories use a mixture of techniques. Timings are also variable, with the majority of laboratories allowing screeners to review slides at a pace decided by themselves but usually between 1 and 2 min. The study involved 120 participants who performed a total of 24 000 rapid screens. The results showed that, of the 90 abnormal slides used in the study, 62 cases (69%) were identified as abnormal or needing review by more than 50% of participants. Overall rapid screening picked out 58% of high-grade squamous abnormalities, 59% of low-grade abnormalities and 72% of glandular lesions. Step screening performed best, followed by whole slide/random and then turret. One minute was the optimum time and there was a significant fall in performance once individuals attempted to rescreen large numbers (>50). The most significant finding was the marked variation in the performance of individuals using the same slide sets.     

Vertebrate liver cytokeratins: a comparative study

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40,000 to 60,000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.     

The sarcoplasmic reticulum: a comparative study

The diversity of cellular membrane structures associated with regulation of intracellular calcium level is described in several different groups of organisms and cells. All the instances reported refer to cellular processes related to movement, in which calcium ion acts as trigger and/or modulator. In addition, a simplified five-stage picture of the underlying view of evolution of these structures is presented. In short: the choice made by nature in using calcium as intracellular messenger was very early in the history of life; all cellular structures devoted to intracellular calcium regulation, from the simplest form of amoeba to the highly sophisticated apparatus of mammalian skeletal muscle, can be linked together in the chain of evolution. Because the evidence is still sparse, any conclusion more positive would be speculative and of little value. Hopefully, in the coming years, with a better understanding of membrane architecture as a whole and its protein components (i.e. calcium channels, calcium-binding proteins), we will be able to test the first segments of this evolutionary hypothesis.     

Orfordness and Dungeness: a comparative study

FULLER, R. M., 1989. Orfordness and Dungeness: a comparative study . Dungeness originally developed as a shingle spit from Winchelsea, across the bay which now forms the Romney Marshes and, 700–800 years ago, probably had its distal end between New Romney and Hythe. The structure seems to have been very similar to present-day Orfordness. Then a breach at the spit's origin produced an abrupt change in accretion and erosion patterns with the eventual development of the present-day cuspate foreland. It would seem that Orfordness is on the verge of such a breach, which is prevented by coastal defence works. Much can be learned by comparing physiographic and ecological processes at Orfordness with those at Dungeness, using Orfordness as an historical model for early Dungeness. This paper considers some of the evidence for these conclusions, examines the structures which survive to the present-day and discusses the ecological significance of the features, especially in terms of plant successional developments.     

Pollen capture media: a comparative study

Research laboratories, part of international aerobiological networks are often using different collection media on their exposed tape (or slides). However, their relative pollen capture efficiency has rarely been tested. A single Hirst-trap was used, in Bologna, in February and March 1993, for this experiment. Melinex tapes were divided longitudinally in two equal parts, so that two different media could be tested simultaneously. The tested media were: gelatine/glycerine, hexane/vaseline, silicone/carbon tetrachloride, and paraffin. Each comparisons were repeated on 4 different days. At the time of the sampling, the dominant airborne particles were Cupressaceae andCladosporium. The differences between the different media were non-significant (meanF of different days of 1.720, mean probability of 0.241 (min. of 0.077, max. of 0.356)). The highest probabilities of a significative difference all involved glycerine/gelatine either in comparison with paraffin (0.76), with silicone (0.75), or with vaseline (0.75), while the lowest was for the couple silicone-vaseline (0.59). In general, even if these differences were non-significant, gelatine underestimated spore counts in comparison to silicone, but overestimated them if compared to vaseline or paraffin. However, vaseline looked more efficient then silicone for spore capture. For pollen counts, the same ratios were found, except for the couple silicone-gelatine, which has given similar results. The lowest differences were found withAlternaria andDreschlera, while the highest were found withCladosporium andEpicoccum. The relationship between spore diameter and differences between media was highly significant (τ=?0.786,P=0.027). This was not the case for pollen, withPopulus andAlnus giving the highest differences, andBetula andUlmus the lowest (τ=0.048,P=0.873).     

Sex differences in heritability of BMI: a comparative study of results from twin studies in eight countries.

Body mass index (BMI), a simple anthropometric measure, is the most frequently used measure of adiposity and has been instrumental in documenting the worldwide increase in the prevalence of obesity witnessed during the last decades. Although this increase in overweight and obesity is thought to be mainly due to environmental changes, i.e., sedentary lifestyles and high caloric diets, consistent evidence from twin studies demonstrates high heritability and the importance of genetic differences for normal variation in BMI. We analysed self-reported data on BMI from approximately 37,000 complete twin pairs (including opposite sex pairs) aged 20-29 and 30-39 from eight different twin registries participating in the GenomEUtwin project. Quantitative genetic analyses were conducted and sex differences were explored. Variation in BMI was greater for women than for men, and in both sexes was primarily explained by additive genetic variance in all countries. Sex differences in the variance components were consistently significant. Results from analyses of opposite sex pairs also showed evidence of sex-specific genetic effects suggesting there may be some differences between men and women in the genetic factors that influence variation in BMI. These results encourage the continued search for genes of importance to the body composition and the development of obesity. Furthermore, they suggest that strategies to identify predisposing genes may benefit from taking into account potential sex specific effects.     

SwarmPS: rapid, semi-automated single particle selection software

Single particle analysis (SPA) coupled with high-resolution electron cryo-microscopy is emerging as a powerful technique for the structure determination of membrane protein complexes and soluble macromolecular assemblies. Current estimates suggest that approximately 10(4)-10(5) particle projections are required to attain a 3A resolution 3D reconstruction (symmetry dependent). Selecting this number of molecular projections differing in size, shape and symmetry is a rate-limiting step for the automation of 3D image reconstruction. Here, we present Swarm(PS), a feature rich GUI based software package to manage large scale, semi-automated particle picking projects. The software provides cross-correlation and edge-detection algorithms. Algorithm-specific parameters are transparently and automatically determined through user interaction with the image, rather than by trial and error. Other features include multiple image handling (approximately 10(2)), local and global particle selection options, interactive image freezing, automatic particle centering, and full manual override to correct false positives and negatives. Swarm(PS) is user friendly, flexible, extensible, fast, and capable of exporting boxed out projection images, or particle coordinates, compatible with downstream image processing suites.     

A comparative study of host selection in the European cuckoo Cuculus canorus

Certain kinds of hosts are commonly regarded as being more suitable than other for rearing European cuckoos (Cuculus canorus) – insectivores that lay small eggs and have open, shallow nests – although empirical tests of cuckoo host selection are lacking. We analysed host use by the European cuckoo in 72 British passerines that are potential hosts and for which there was information available on life-history variables and variables related to cuckoo-host coevolution, such as rate of parasitism, rejection rate of non-mimetic model eggs and degree of cuckoo-egg mimicry of host eggs. The relative population size of the host species affected parasitism rate most strongly, followed by relatively short duration of the nestling period, and the kind of nest, with cuckoos selecting open-nesting hosts. However, the effect of the nestling period could be related to host body size and the kind of nest used, because hole-nesting species normally have longer nestling periods than open-nesters. We re-analysed the data excluding hole nesters and corvid species (species with larger body mass), but the results remained identical. The European cuckoo may benefit from selecting hosts with short nestling periods because such hosts provide food for their nestlings at a very high rate. When only those species known as cuckoo hosts were analysed, the variable that best accounted for the parasitism rate was duration of the breeding season. Therefore, availability of potential hosts in both time and space is important for cuckoos in selecting hosts. Received: 16 July 1998 / Accepted: 27 October 1998     

The surgical marking pen: a comparative study

A study has been undertaken to objectively evaluate the available surgical marking pens. Representative samples were requested from manufacturers of marking pens. Standardized skin preparations were utilized in accordance with the recommendation of the manufacturer of the skin-preparation substance (Betadine, pHisoHex, Hibiclens). Evaluation of the marking pens was done by the ability to make an easily discernible mark and maintain this ability over a 1-year storage interval. Objective evaluation demonstrated a marked variation in effectiveness of the marking pens. Consideration of skin-preparation solutions and types of marking pens are discussed.     

Assays for mammalian tyrosinase: a comparative study

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.