Automated Correlation and Averaging of Three-Dimensional Reconstructions Obtained by Electron Tomography

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from anin situspecimen containingEscherichia coli70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.     

Artificial intelligence channelizing protein-peptide interactions pipeline for host-parasite paradigm in IL-10 and IL-12 reciprocity by SHP-1

Identification of molecular targets in any cellular phenomena is a challenge and a path that one endeavors upon independently. We have identified a phosphatase SHP-1 as a point of intervention of IL-10 and IL-12 reciprocity in leishmaniasis. The therapeutic model that we have developed uniquely targets this protein but the pipeline in general can be used by the researchers for their unique targets. Naturally occurring peptides are well known for their biochemical participation in cellular functions hence we were motivated to use this uniqueness of physico-chemical properties of peptides conferred by amino acids through machine learning to channelize a mode of therapeutic exploration in infectious disease. Using computational approaches, we identified high order sequence conservation and similarity in SHP-1 sequence which was also evolutionarily conserved, complete structure of Mouse SHP-1 was predicted and validated, a unique motif of the same was identified against which library of synthetic peptides were designed and validated followed by screening the library by docking them with MuSHP-1 protein structure. Our findings showed 3 peptides had high binding affinity and in future can be validated using cell based and in vivo assays.     

Depth perception in disparity-defined objects: finding the balance between averaging and segregation

Deciding what constitutes an object, and what background, is an essential task for the visual system. This presents a conundrum: averaging over the visual scene is required to obtain a precise signal for object segregation, but segregation is required to define the region over which averaging should take place. Depth, obtained via binocular disparity (the differences between two eyes’ views), could help with segregation by enabling identification of object and background via differences in depth. Here, we explore depth perception in disparity-defined objects. We show that a simple object segregation rule, followed by averaging over that segregated area, can account for depth estimation errors. To do this, we compared objects with smoothly varying depth edges to those with sharp depth edges, and found that perceived peak depth was reduced for the former. A computational model used a rule based on object shape to segregate and average over a central portion of the object, and was able to emulate the reduction in perceived depth. We also demonstrated that the segregated area is not predefined but is dependent on the object shape. We discuss how this segregation strategy could be employed by animals seeking to deter binocular predators.This article is part of the themed issue ‘Vision in our three-dimensional world’.     

Symmetry cues for matching mirrored objects

The study explores cues for matching pairs of objects. These objects were arranged into mirror-symmetrical displays and the task was to judge whether a pre-specified 180 degree rotation around the X, Y or Z axis carries one object into the mirror object. For some rotations, the object's mirror symmetry (M) and, for other rotations, the object's point-symmetry (P) could serve as a cue. The matching results suggest that M is a better cue than P, say M > P. Various attempts are made to explain this effect. The most promising one focuses on simplest structural object representations as these capture M and not P. It is furthermore plausible that M captured by reference frames at high hierarchical representation levels, say M1, serves as a better cue than M captured at low levels, say M2. The prediction M1 > M2 > P merely applies to the open surface objects in the experiment. For the closed solid objects in the experiment the expectation is M1 = M2 = P. Both predictions roughly agree with the accuracy and reaction-time data. The results suggest the perceptual relevance of representation cues and, aditionally, that cues stemming from reference frames at higher hierarchical representation levels are more effective than those from lower levels.     

Kinematics of Visually-Guided Eye Movements

One of the hallmarks of an eye movement that follows Listing’s law is the half-angle rule that says that the angular velocity of the eye tilts by half the angle of eccentricity of the line of sight relative to primary eye position. Since all visually-guided eye movements in the regime of far viewing follow Listing’s law (with the head still and upright), the question about its origin is of considerable importance. Here, we provide theoretical and experimental evidence that Listing’s law results from a unique motor strategy that allows minimizing ocular torsion while smoothly tracking objects of interest along any path in visual space. The strategy consists in compounding conventional ocular rotations in meridian planes, that is in horizontal, vertical and oblique directions (which are all torsion-free) with small linear displacements of the eye in the frontal plane. Such compound rotation-displacements of the eye can explain the kinematic paradox that the fixation point may rotate in one plane while the eye rotates in other planes. Its unique signature is the half-angle law in the position domain, which means that the rotation plane of the eye tilts by half-the angle of gaze eccentricity. We show that this law does not readily generalize to the velocity domain of visually-guided eye movements because the angular eye velocity is the sum of two terms, one associated with rotations in meridian planes and one associated with displacements of the eye in the frontal plane. While the first term does not depend on eye position the second term does depend on eye position. We show that compounded rotation - displacements perfectly predict the average smooth kinematics of the eye during steady- state pursuit in both the position and velocity domain.     

Protein local conformations arise from a mixture of Gaussian distributions

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures, which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF’s corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end, we have analyzed the correlation between sequence features and the conformational classes.     

A Large-Insert (130 kbp) Bacterial Artificial Chromosome Library of the Rice Blast FungusMagnaporthe grisea:Genome Analysis, Contig Assembly, and Gene Cloning

Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request.     

Progressive background in moths, and a quantitative measure of crypsis

A method is presented for quantitative estimation of the degree of crypsis of species seen by visual predators against known backgrounds. It is based upon a comparison between transects taken across animal and background colour patterns. The method was applied to day-resting moths in deciduous forest in New Jersey. Each species is found for two to four weeks at characteristic dates, and there is a constant turnover of species. In both moths and backgrounds there is a regular change in the colour pattern parameters from winter through spring to early summer. Moths are on average more cryptic at their normal dates than they would be if present earlier or later in the year. Species with known resting sites are on average more cryptic on their resting sites than other background habitats. Species that rest on more than one background habitat are less cryptic on their preferred habitats than are specialists. Species that rest under leaves and are not visible from above are not very cryptic. Specific v. general resemblance, disruptive coloration, and factors affecting 'aspect diversity' are discussed. The new method of estimating crypsis is useful for studies of crypsis as well as in sexual selection. It is necessary to know much about the resting sites and behaviour of moths, as well as other functions of colour patterns, to understand colour pattern evolution.     

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

 The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae. Received: 28 July 1998 / Accepted: 8 October 1998     

Protein local conformations arise from a mixture of Gaussian distributions

The classical approaches for protein structure prediction rely either on homology of the protein sequence with a template structure or on ab initio calculations for energy minimization. These methods suffer from disadvantages such as the lack of availability of homologous template structures or intractably large conformational search space, respectively. The recently proposed fragment library based approaches first predict the local structures,which can be used in conjunction with the classical approaches of protein structure prediction. The accuracy of the predictions is dependent on the quality of the fragment library. In this work, we have constructed a library of local conformation classes purely based on geometric similarity. The local conformations are represented using Geometric Invariants, properties that remain unchanged under transformations such as translation and rotation, followed by dimension reduction via principal component analysis. The local conformations are then modeled as a mixture of Gaussian probability distribution functions (PDF). Each one of the Gaussian PDF's corresponds to a conformational class with the centroid representing the average structure of that class. We find 46 classes when we use an octapeptide as a unit of local conformation. The protein 3-D structure can now be described as a sequence of local conformational classes. Further, it was of interest to see whether the local conformations can be predicted from the amino acid sequences. To that end,we have analyzed the correlation between sequence features and the conformational classes.     

Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

A bacterial artificial chromosome (BAC) library was constructed for Gossyplum hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carded out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of pdmere for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were Identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clonee are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with Important agronomic traits.     

Predicting CNS permeability of drug molecules: comparison of neural network and support vector machine algorithms.

Two different machine-learning algorithms have been used to predict the blood-brain barrier permeability of different classes of molecules, to develop a method to predict the ability of drug compounds to penetrate the CNS. The first algorithm is based on a multilayer perceptron neural network and the second algorithm uses a support vector machine. Both algorithms are trained on an identical data set consisting of 179 CNS active molecules and 145 CNS inactive molecules. The training parameters include molecular weight, lipophilicity, hydrogen bonding, and other variables that govern the ability of a molecule to diffuse through a membrane. The results show that the support vector machine outperforms the neural network. Based on over 30 different validation sets, the SVM can predict up to 96% of the molecules correctly, averaging 81.5% over 30 test sets, which comprised of equal numbers of CNS positive and negative molecules. This is quite favorable when compared with the neural network's average performance of 75.7% with the same 30 test sets. The results of the SVM algorithm are very encouraging and suggest that a classification tool like this one will prove to be a valuable prediction approach.     

Exploratory analysis of kinetic solubility measurements of a small molecule library

Kinetic solubility measurements using prototypical assay buffer conditions are presented for a ～58,000 member library of small molecules. Analyses of the data based upon physical and calculated properties of each individual molecule were performed and resulting trends were considered in the context of commonly held opinions of how physicochemical properties influence aqueous solubility. We further analyze the data using a decision tree model for solubility prediction and via a multi-dimensional assessment of physicochemical relationships to solubility in the context of specific 'rule-breakers' relative to common dogma. The role of solubility as a determinant of assay outcome is also considered based upon each compound's cross-assay activity score for a collection of publicly available screening results. Further, the role of solubility as a governing factor for colloidal aggregation formation within a specified assay setting is examined and considered as a possible cause of a high cross-assay activity score. The results of this solubility profile should aid chemists during library design and optimization efforts and represent a useful training set for computational solubility prediction.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

IMPACT OF NIGHT-FLOAT ROTATION ON SLEEP,MOOD, AND ALERTNESS: THE RESIDENT'S PERCEPTION

Night-float rotations were designed to alleviate the workload of residents on night call and thereby improve patient safety. However, the impact of the night float on residents is yet to be surveyed. We assessed the impact of the night-float rotation on pediatric residents using an anonymous questionnaire that covered topics, based on recall, about sleep, mood, alertness, adjustment, and others. The study was conducted in a major tertiary pediatric teaching hospital in the United States. Participants were pediatric residents who had completed one or two night-float rotations and were in active training at our teaching hospital at the time of the study. Fifty-two of 60 eligible residents (87%) responded. Sleep duration during the night-float rotation was shorter than during day-shift work in 24 residents (46%), longer in 20 (38%), and unchanged in eight (15%). A higher proportion of residents took longer to fall asleep, had more difficulty falling asleep, had more sleep interruptions, and felt less rested upon awakening. Twenty-four residents (46%) felt that their bodies never adjusted to the night shift. Also, 22 residents (43%) felt moody or depressed in contrast to seven (14%) who felt depressed during the daytime rotation (p=0.0001). Twenty-one residents (41%) felt they were slower in their thinking during the night float than daytime rotations. The results suggest that disturbances of sleep and mood and decreased alertness, typical of night shift, are present in the night-float rotation. Residency programs should monitor closely the impact of the night-float rotation on resident well being and patient safety. The impact of night-shift work should be considered in the design of night-float schedules, and teaching should be provided for residents to learn coping strategies for night-shift work.     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.     

Integration Profiling of Gene Function With Dense Maps of Transposon Integration

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.     

Effect of polynomial,radial basis,and Pearson VII function kernels in support vector machine algorithm for classification of crayfish

Freshwater crayfish are one of the most important aquatic organisms that play a pivotal role in the aquatic food chain as well as serving as bioindicators for the aquatic ecosystem health assessment. Hemocytes, the blood cells of crustaceans, can be considered stress and health indicators in crayfish, and are used to evaluate the health response. Therefore, total hemocyte cell numbers (THCs) are useful parameters to show the health of crustaceans and serve as stress indicators to decide the quality of the habitat. Since, catching the fish and the other aquatic organisms, and collecting the data for further assessments are time-consuming and frustrating, today, scientists tend to use swift, more sophisticated, and more reliable methods for modeling the ecosystem stressors based on bioindicators. One tool which has attracted the attention of science communities in the last decades is machine learning algorithms that are reliable and accurate methods to solve classification and regression problems. In this study, a support vector machine is carried out as a machine learning algorithm to classify healthy and unhealthy crayfish based on physiological characteristics. To solve the non-linearity problem of the data by transporting data to high-dimensional space, different kernel functions including polynomial (PK), Pearson VII function-based universal (PUK), and radial basis function (RBF) kernels are used and their effect on the performance of the SVM model was evaluated. Both PK and PUK functions performed well in classifying the crayfish. RBF, however, had an adverse impact on the performance of the model. PUK kernel exhibited an outstanding performance (Accuracy = 100%) for the classification of the healthy and unhealthy crayfish.     

Developmental Models for Estimating Ecological Responses to Environmental Variability: Structural,Parametric, and Experimental Issues

Developmental models that account for the metabolic effect of temperature variability on poikilotherms, such as degree-day models, have been widely used to study organism emergence, range and development, particularly in agricultural and vector-borne disease contexts. Though simple and easy to use, structural and parametric issues can influence the outputs of such models, often substantially. Because the underlying assumptions and limitations of these models have rarely been considered, this paper reviews the structural, parametric, and experimental issues that arise when using degree-day models, including the implications of particular structural or parametric choices, as well as assumptions that underlie commonly used models. Linear and non-linear developmental functions are compared, as are common methods used to incorporate temperature thresholds and calculate daily degree-days. Substantial differences in predicted emergence time arose when using linear versus non-linear developmental functions to model the emergence time in a model organism. The optimal method for calculating degree-days depends upon where key temperature threshold parameters fall relative to the daily minimum and maximum temperatures, as well as the shape of the daily temperature curve. No method is shown to be universally superior, though one commonly used method, the daily average method, consistently provides accurate results. The sensitivity of model projections to these methodological issues highlights the need to make structural and parametric selections based on a careful consideration of the specific biological response of the organism under study, and the specific temperature conditions of the geographic regions of interest. When degree-day model limitations are considered and model assumptions met, the models can be a powerful tool for studying temperature-dependent development.