Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of particulate samples, typically proteins or other large biological entities such as viruses. Individual images of stained or unstained particles are very noisy, making interpretation difficult. Combining several digitized images of similar particles together gives an image with stronger and more easily interpretable features. An extension of this technique uses single particle methods to build up a three-dimensional reconstruction of the particle. Using cryo-electron microscopy it has become possible to generate reconstructions with sub-nanometer, near-atomic resolution resolution first in the case of highly symmetric viruses, and now in smaller, asymmetric proteins as well. == Techniques == Single particle analysis can be done on both negatively stained and vitreous ice-embedded transmission electron cryomicroscopy (CryoTEM) samples. Single particle analysis methods are, in general, reliant on the sample being homogeneous, although techniques for dealing with conformational heterogeneity are being developed. Images (micrographs) are taken with an electron microscope using charged-coupled device (CCD) detectors coupled to a phosphorescent layer (in the past, they were instead collected on film and digitized using high-quality scanners). The image processing is carried out using specialized software programs, often run on multi-processor computer clusters. Depending on the sample or the desired results, various steps of two- or three-dimensional processing can be done. === Alignment and classification === Biological samples, and especially samples embedded in thin vitreous ice, are highly radiation sensitive, thus only low electron doses can be used to image the sample. This low dose, as well as variations in the metal stain used (if used) means images have high noise relative to the signal given by the particle being observed. By aligning several similar images to each other so they are in register and then averaging them, an image with higher signal-to-noise ratio can be obtained. As the noise is mostly randomly distributed and the underlying image features constant, by averaging the intensity of each pixel over several images only the constant features are reinforced. Typically, the optimal alignment (a translation and an in-plane rotation) to map one image onto another is calculated by cross-correlation. However, a micrograph often contains particles in multiple different orientations and/or conformations, and so to get more representative image averages, a method is required to group similar particle images together into multiple sets. This is normally carried out using one of several data analysis and image classification algorithms, such as multi-variate statistical analysis and hierarchical ascendant classification, or k-means clustering. Often data sets of tens of thousands of particle images are used, and to reach an optimal solution an iterative procedure of alignment and classification is used, whereby strong image averages produced by classification are used as reference images for a subsequent alignment of the whole data set. === Image filtering === Image filtering (band-pass filtering) is often used to reduce the influence of high and/or low spatial frequency information in the images, which can affect the results of the alignment and classification procedures. This is particularly useful in negative stain images. The algorithms make use of fast Fourier transforms (FFT), often employing Gaussian shaped soft-edged masks in reciprocal space to suppress certain frequency ranges. High-pass filters remove low spatial frequencies (such as ramp or gradient effects), leaving the higher frequencies intact. Low-pass filters remove high spatial frequency features and have a blurring effect on fine details. === Contrast transfer function === Due to the nature of image formation in the electron microscope, bright-field TEM images are obtained using significant underfocus. This, along with features inherent in the microscope's lens system, creates blurring of the collected images visible as a point spread function. The combined effects of the imaging conditions are known as the contrast transfer function (CTF), and can be approximated mathematically as a function in reciprocal space. Specialized image processing techniques such as phase flipping and amplitude correction / Wiener filtering can (at least partially) correct for the CTF, and allow high resolution reconstructions. === Three-dimensional reconstruction === Transmission electron microscopy images are projections of the object showing the distribution of density through the object, similar to medical X-rays. By making use of the projection-slice theorem a three-dimensional reconstruction of the object can be generated by combining many images (2D projections) of the object taken from a range of viewing angles. Proteins in vitreous ice ideally adopt a random distribution of orientations (or viewing angles), allowing a fairly isotropic reconstruction if a large number of particle images are used. This contrasts with electron tomography, where the viewing angles are limited due to the geometry of the sample/imaging set up, giving an anisotropic reconstruction. Filtered back projection is a commonly used method of generating 3D reconstructions in single particle analysis, although many alternative algorithms exist. Before a reconstruction can be made, the orientation of the object in each image needs to be estimated. Several methods have been developed to work out the relative Euler angles of each image. Some are based on common lines (common 1D projections and sinograms), others use iterative projection matching algorithms. The latter works by beginning with a simple, low resolution 3D starting model and compares the experimental images to projections of the model and creates a new 3D to bootstrap towards a solution. Methods are also available for making 3D reconstructions of helical samples (such as tobacco mosaic virus), taking advantage of the inherent helical symmetry. Both real space methods (treating sections of the helix as single particles) and reciprocal space methods (using diffraction patterns) can be used for these samples. === Tilt methods === The specimen stage of the microscope can be tilted (typically along a single axis), allowing the single particle technique known as random conical tilt. An area of the specimen is imaged at both zero and at high angle (~60-70 degrees) tilts, or in the case of the related method of orthogonal tilt reconstruction, +45 and −45 degrees. Pairs of particles corresponding to the same object at two different tilts (tilt pairs) are selected, and by following the parameters used in subsequent alignment and classification steps a three-dimensional reconstruction can be generated relatively easily. This is because the viewing angle (defined as three Euler angles) of each particle is known from the tilt geometry. 3D reconstructions from random conical tilt suffer from missing information resulting from a restricted range of orientations. Known as the missing cone (due to the shape in reciprocal space), this causes distortions in the 3D maps. However, the missing cone problem can often be overcome by combining several tilt reconstructions. Tilt methods are best suited to negatively stained samples, and can be used for particles that adsorb to the carbon support film in preferred orientations. The phenomenon known as charging or beam-induced movement makes collecting high-tilt images of samples in vitreous ice challenging. === Map visualization and fitting === Various software programs are available that allow viewing the 3D maps. These often enable the user to manually dock in protein coordinates (structures from X-ray crystallography, NMR, or a computational model such as one found in the AlphaFold Protein Structure Database) of subunits into the electron density. Several programs can also fit subunits computationally; as of the 2020s using these programs tend to produce better accuracy than manual docking because they can perform labor-intensive tasks such as: The scale of SPA-derived maps depends on knowing the pixel size (angstorms per pixel), which is not always accurate. Programs can automatically correct for this difference by using coordinate data or by using knowledge of chemical bonds. Many proteins are made up of several roughly rigid protein domains linked by flexible parts. Pre-existing coordinate data, whether experimental or computational, may not exactly match the inter-domain positioning of the cyro-EM map. Modern programs can automatically "chop" pre-existing coordinate data into individual domains and fit them in individually. For higher-resolution structures, it is pos
Tweak programming environment
Tweak is a graphical user interface (GUI) layer written by Andreas Raab for the Squeak development environment, which in turn is an integrated development environment based on the Smalltalk-80 computer programming language. Tweak is an alternative to an earlier graphic user interface layer called Morphic. Development began in 2001. Applications that use the Tweak software include Sophie (version 1), a multimedia and e-book authoring system, and a family of virtual world systems: Open Cobalt, Teleplace, OpenQwaq, 3d ICC's Immersive Terf and the Croquet Project. == Influences == An experimental version of Etoys, a programming environment for children, used Tweak instead of Morphic. Etoys was a major influence on a similar Squeak-based programming environment known as Scratch.
Optimal discriminant analysis and classification tree analysis
Optimal Discriminant Analysis (ODA) and the related classification tree analysis (CTA) are exact statistical methods that maximize predictive accuracy. For any specific sample and exploratory or confirmatory hypothesis, optimal discriminant analysis (ODA) identifies the statistical model that yields maximum predictive accuracy, assesses the exact Type I error rate, and evaluates potential cross-generalizability. Optimal discriminant analysis may be applied to > 0 dimensions, with the one-dimensional case being referred to as UniODA and the multidimensional case being referred to as MultiODA. Optimal discriminant analysis is an alternative to ANOVA (analysis of variance) and regression analysis.
GeWorkbench
geWorkbench (genomics Workbench) is an open-source software platform for integrated genomic data analysis. It is a desktop application written in the programming language Java. geWorkbench uses a component architecture. As of 2016, there are more than 70 plug-ins available, providing for the visualization and analysis of gene expression, sequence, and structure data. geWorkbench is the Bioinformatics platform of MAGNet, the National Center for the Multi-scale Analysis of Genomic and Cellular Networks, one of the 8 National Centers for Biomedical Computing funded through the NIH Roadmap (NIH Common Fund). Many systems and structure biology tools developed by MAGNet investigators are available as geWorkbench plugins. == Features == Computational analysis tools such as t-test, hierarchical clustering, self-organizing maps, regulatory network reconstruction, BLAST searches, pattern-motif discovery, protein structure prediction, structure-based protein annotation, etc. Visualization of gene expression (heatmaps, volcano plot), molecular interaction networks (through Cytoscape), protein sequence and protein structure data (e.g., MarkUs). Integration of gene and pathway annotation information from curated sources as well as through Gene Ontology enrichment analysis. Component integration through platform management of inputs and outputs. Among data that can be shared between components are expression datasets, interaction networks, sample and marker (gene) sets and sequences. Dataset history tracking - complete record of data sets used and input settings. Integration with 3rd party tools such as GenePattern, Cytoscape, and Genomespace. Demonstrations of each feature described can be found at GeWorkbench-web Tutorials. == Versions == geWorkbench is open-source software that can be downloaded and installed locally. A zip file of the released version Java source is also available. Prepackaged installer versions also exist for Windows, Macintosh, and Linux.
Operational taxonomic unit
An operational taxonomic unit (OTU) is an operational definition used to classify groups of closely related individuals. The term was originally introduced in 1963 by Robert R. Sokal and Peter H. A. Sneath in the context of numerical taxonomy, where an "operational taxonomic unit" is simply the group of organisms currently being studied. In this sense, an OTU is a pragmatic definition to group individuals by similarity, equivalent to but not necessarily in line with classical Linnaean taxonomy or modern evolutionary taxonomy. Nowadays, however, the term is commonly used in a different context and refers to clusters of (uncultivated or unknown) organisms, grouped by DNA sequence similarity of a specific taxonomic marker gene (originally coined as mOTU; molecular OTU). In other words, OTUs are pragmatic proxies for "species" at different taxonomic levels, in the absence of traditional systems of biological classification as are available for macroscopic organisms. For several years, OTUs have been the most commonly used units of diversity, especially when analysing small subunit 16S (for prokaryotes) or 18S rRNA (for eukaryotes) marker gene sequence datasets. == Molecular OTU by clustering of marker gene sequences == In the approach represented by DNA barcoding, a particular locus is chosen to be used as the marker gene for classification. This locus should be universally present in the scope selected, variable enough to be different among close-related species, and be flanked by conservative sequences that allow for easy amplification and detection. There are databases containing sequences for such marker genes from many different species, allowing for comparison. (Sometimes only using one locus does not provide sufficient resolution, so multiple marker genes are used. This is the case for plants, where rbcL+matK is common.) Sequences obtained this way can be clustered according to their similarity to one another, and operational taxonomic units are defined based on the similarity threshold set by the researcher. The exact threshold depends on the taxa in question and the mutational rates of the selected locus in the taxon. 97–99% are commonly used, but "it is now recognized to be somewhat arbitrary as sequence variation within and among species varies across taxa". 100% similarity (fully identical) is also common, also known as single variants. It remains debatable how well this commonly used method recapitulates true microbial species phylogeny or ecology. Although OTUs can be calculated differently when using different algorithms or thresholds, research by Schmidt et al. (2014) demonstrated that 16S-derived microbial OTUs were generally ecologically consistent across habitats and several clustering approaches. The number of OTUs defined may be inflated due to errors in DNA sequencing. === OTU clustering approaches === There are three main approaches to clustering OTUs: De novo, for which the clustering is based on similarities between sequencing reads. Closed-reference, for which the clustering is performed against a reference database of sequences. Open-reference, where clustering is first performed against a reference database of sequences, then any remaining sequences that could not be mapped to the reference are clustered de novo. Using a reference provides taxonomic context for the OTUs found. Alternatively, taxonomic context can be found after the construction of clusters by comparing representative sequences from clusters against a reference database. There are also specialized classifiers for this purpose which are much faster than naive comparison using BLAST. === OTU clustering algorithms === Hierarchical clustering algorithms (HCA): uclust & cd-hit & ESPRIT Bayesian clustering: CROP == Molecular OTU by other methods == In addition to similarity-based grouping, marker gene sequences can be sorted into OTUs using molecular phylogeny, k-mer composition, or hybrid methods combining these methods with similarity. There are also Bayesian tree-less methods and machine learning approaches. Using phylogeny often involves manually assigning terminal clades or single nodes to an OTU, so this is usually only done for refinement. Genome skimming can be used to obtain high-copy DNA without the need to choose marker genes or to design PCR primers for the chosen genes. It can provide fairly good coverage of organelle DNA and repetitive elements such as ribosomal DNA, both of which can be used like marker genes in OTU analysis. Whole-genome sequencing is more expensive and involves the production and processing of more data. By considering the entire genome, many (sometimes over 100) marker genes can be used at the same time, producing highly resolved phylogenies that correctly identify problematic taxa. It is also possible to use entire genomes for OTU assignment. For example, genomes from different bacterial species almost always have an average nucleotide identity lower than 95%, a fact that can be used to define new OTUs (and likely new species).
MegaHAL
MegaHAL is a computer conversation simulator, or "chatterbot", created by Jason Hutchens. == Background == In 1996, Jason Hutchens entered the Loebner Prize Contest with HeX, a chatterbot based on ELIZA. HeX won the competition that year and took the $2000 prize for having the highest overall score. In 1998, Hutchens again entered the Loebner Prize Contest with his new program, MegaHAL. MegaHAL made its debut in the 1998 Loebner Prize Contest. Like many chatterbots, the intent is for MegaHAL to appear as a human fluent in a natural language. As a user types sentences into MegaHAL, MegaHAL will respond with sentences that are sometimes coherent and at other times complete gibberish. MegaHAL learns as the conversation progresses, remembering new words and sentence structures. It will even learn new ways to substitute words or phrases for other words or phrases. Many would consider conversation simulators like MegaHAL to be a primitive form of artificial intelligence. However, MegaHAL doesn't understand the conversation or even the sentence structure. It generates its conversation based on sequential and mathematical relationships. In the world of conversation simulators, MegaHAL is based on relatively old technology and could be considered primitive. However, its popularity has grown due to its humorous nature; it has been known to respond with twisted or nonsensical statements that are often amusing. == Theory of Operation == MegaHal is based at least in part on a so-called "hidden Markov Model", so that the first thing that Megahal does when it "trains" on a script or text is to build a database of text fragments encompassing every possible subset of perhaps 4, 5, or even 6 consecutive words, so that for example - if MegaHal trains on the Declaration of Independence, then MegaHal will build a database containing text fragments such as "When in the course", "in the course of", "the course of human", "course of human events", "of human events, one", "human events, one people", and so on. Then if Megahal is fed another text, such has "Superman, Yes! It's Superman - he can change the course of mighty rivers, bend steel with his bare hands - and who disguised at Clark Kent …" IT MIGHT induce Megahal to apparently bemuse itself to proffer whether Superman can change the course of human events, or something else altogether - such as some rambling about "when in the course of mighty rivers", and so on. Thus likewise - if a phrase like "the White house said" comes up a lot in some text; then Megahal's ability to switch randomly between different contexts which otherwise share some similarity can result at times in some surprising lucidity, or else it might otherwise seem quite bizarre. == Examples == There are some sentences that MegaHAL generated: CHESS IS A FUN SPORT, WHEN PLAYED WITH SHOT GUNS. and COWS FLY LIKE CLOUDS BUT THEY ARE NEVER COMPLETELY SUCCESSFUL. == Distribution == MegaHAL is distributed under the Unlicense. Its source code can be downloaded from the Github repository.
Fitness approximation
Fitness approximation aims to approximate the objective or fitness functions in evolutionary optimization by building up machine learning models based on data collected from numerical simulations or physical experiments. The machine learning models for fitness approximation are also known as meta-models or surrogates, and evolutionary optimization based on approximated fitness evaluations are also known as surrogate-assisted evolutionary approximation. Fitness approximation in evolutionary optimization can be seen as a sub-area of data-driven evolutionary optimization. == Approximate models in function optimization == === Motivation === In many real-world optimization problems including engineering problems, the number of fitness function evaluations needed to obtain a good solution dominates the optimization cost. In order to obtain efficient optimization algorithms, it is crucial to use prior information gained during the optimization process. Conceptually, a natural approach to utilizing the known prior information is building a model of the fitness function to assist in the selection of candidate solutions for evaluation. A variety of techniques for constructing such a model, often also referred to as surrogates, metamodels or approximation models – for computationally expensive optimization problems have been considered. === Approaches === Common approaches to constructing approximate models based on learning and interpolation from known fitness values of a small population include: Low-degree polynomials and regression models Fourier surrogate modeling Artificial neural networks including Multilayer perceptrons Radial basis function network Support vector machines Due to the limited number of training samples and high dimensionality encountered in engineering design optimization, constructing a globally valid approximate model remains difficult. As a result, evolutionary algorithms using such approximate fitness functions may converge to local optima. Therefore, it can be beneficial to selectively use the original fitness function together with the approximate model.