Topological analysis and interactive visualization of biological networks and protein structures

Computational analysis and interactive visualization of biological networks and protein structures are common tasks for gaining insight into biological processes. This protocol describes three workflows based on the NetworkAnalyzer and RINalyzer plug-ins for Cytoscape, a popular software platform for networks. NetworkAnalyzer has become a standard Cytoscape tool for comprehensive network topology analysis. In addition, RINalyzer provides methods for exploring residue interaction networks derived from protein structures. The first workflow uses NetworkAnalyzer to perform a topological analysis of biological networks. The second workflow applies RINalyzer to study protein structure and function and to compute network centrality measures. The third workflow combines NetworkAnalyzer and RINalyzer to compare residue networks. The full protocol can be completed in ～2 h.     

Quantitative analysis of nucleic acid three-dimensional structures

A new computer program to annotate DNA and RNA three-dimensional structures, MC-Annotate, is introduced. The goals of annotation are to efficiently extract and manipulate structural information, to simplify further structural analyses and searches, and to objectively represent structural knowledge. The input of MC-Annotate is a PDB formatted DNA or RNA three-dimensional structure. The output of MC-Annotate is composed of a structural graph that contains the annotations, and a series of HTML documents, one for each nucleotide conformation and base-base interaction present in the input structure. The atomic coordinates of all nucleotides and the homogeneous transformation matrices of all base-base interactions are stored in the structural graph. Symbolic classifications of nucleotide conformations, using sugar puckering modes and nitrogen base orientations around the glycosyl bond, and base-base interactions, using stacking and hydrogen bonding information, are introduced. Peculiarity factors of nucleotide conformations and base-base interactions are defined to indicate their marginalities with all other examples. The peculiarity factors allow us to identify irregular regions and possible stereochemical errors in 3-D structures without interactive visualization. The annotations attached to each nucleotide conformation include its class, its torsion angles, a distribution of the root-mean-square deviations with examples of the same class, the list of examples of the same class, and its peculiarity value. The annotations attached to each base-base interaction include its class, a distribution of distances with examples of the same class, the list of examples of the same class, and its peculiarity value. The distance between two homogeneous transformation matrices is evaluated using a new metric that distinguishes between the rotation and the translation of a transformation matrix in the context of nitrogen bases. MC-Annotate was used to build databases of nucleotide conformations and base-base interactions. It was applied to the ribosomal RNA fragment that binds to protein L11, which annotations revealed peculiar nucleotide conformations and base-base interactions in the regions where the RNA contacts the protein. The question of whether the current database of RNA three-dimensional structures is complete is addressed.     

Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.     

Quantitative analysis of cellular senescence phenotypes using an imaging cytometer

Until now, various stimuli as well as serial passaging have been known to induce cellular senescence in normal human diploid fibroblasts. However, in many cases, we have encountered difficulty in quantitatively analyzing the cellular senescence phenotypes of senescent cells in a physiological condition. High-content screening (HCS)-based image analysis is becoming an important and powerful research tool. In the present study, an automated and quantitative cellular image-analysis system was employed to quantify the cellular senescence phenotypes induced in normal human diploid fibroblasts, TIG-1 cells, and found to be a powerful tool in the cellular senescence study.     

Quantitative analysis of cellular differentiation during early embryogenesis ofPlatynereis dumerilii

Summary As in many spiralian embryos with unequal cleavage, cleavage inPlatynereis follows an invariant pattern. Preceding each cleavage the cytoplasm is reorganized, allowing the spiral cleavage mode to produce cells with different cytoplasmic composition. The fertilized egg undergoes a dramatic ooplasmic segregation after the completion of the cortical reaction. As a consequence, a plug of clear cytoplasm becomes located at the animal pole. Once the four quadrants of the embryo have been established, the cleavage sequence of the D quadrant differs clearly from that of the other three quadrants. The results presented here suggest that differential distribution of the clear cytoplasm governs this sequence. The first quartet of micromeres, which will form the ectoderm and the cerebral ganglia of the head, is clearly bilaterally symmetrical from the onset of the third cleavage. Dorsoventral polarity and bilateral symmetry in the ectoderm of the trunk is expressed most markedly by the dorsal location of the large 2d cell, whose rapid proliferation is bilaterally symmetrical with respect to the median plane. As a result of this proliferation it comes to fill most of the posttrochal region (ectoderm, three pairs of anlagen for the setal sacs, and the ventral plate which forms the nerve cord). The other micromeres contribute only a minor portion of the ventral ectoderm and are involved in the formation of the stomodaeum. The mesentoblast, 4d, i.e. the stem cell of the primary mesoderm, forms at the sixth cleavage, also in a position on the dorsal mid-line. The daughter cells, which arise from 4d by strictly bilaterally symmetrical cleavage, form the mesodermal germ bands, which lie beneath the ectoderm. The trochoblasts are formed by asynchronously cleaving founder cells, but further cleavages in these cells are synchronous. This suggests that cell-cell interaction is involved in the development of this alleged mosaic embryo.     

Approximation and visualization of large-scale motion of protein surfaces

We present a method for the approximation and real-time visualization of large-scale motion of protein surfaces. A molecular surface is represented by an expansion of spherical harmonic functions, and the motion of protein atoms around their equilibrium positions is computed by normal mode analysis. The motion of the surface is approximated by projecting the normal mode vectors of the solvent-accessible atoms to the spherical harmonic representation of the molecular surface. These surface motion vectors are represented by a separated spherical harmonic expansion. Representing the surface geometry and the surface motion vectors by spherical harmonic expansions allows variable-resolution analysis and real-time display of the large-scale surface motion. This technique has been applied to interactive visualization, interactive surface manipulation, and animation.     

Quantitative aspects of cellular turnover

Living organisms do not just grow by synthesizing cellular components. As part of the necessary steps for existence, some components are degraded after synthesis. Even for bacteria in balanced, exponential growth some substances, under some conditions, are turned over. In other phases of growth turnover can be much more extensive, but it is still selective. This review covers studies with animals as a way to put the studies on microorganisms in perspective. The history, the mathematics, and experimental design of turnover experiments are reviewed. The important conclusion is that most of the proteins during balanced growth are very stable in bacteria, although ribosomal proteins are degraded under starvation conditions. Another generalization is that the process of wall enlargement in general is associated with obligatory turnover of the peptidoglycan.     

Quantification and visualization of coordination during non-cyclic upper extremity motion

There are many design challenges in creating at-home tele-monitoring systems that enable quantification and visualization of complex biomechanical behavior. One such challenge is robustly quantifying joint coordination in a way that is intuitive and supports clinical decision-making. This work defines a new measure of coordination called the relative coordination metric (RCM) and its accompanying normalization schemes. RCM enables quantification of coordination during non-constrained discrete motions. Here RCM is applied to a grasping task. Fifteen healthy participants performed a reach, grasp, transport, and release task with a cup and a pen. The measured joint angles were then time-normalized and the RCM time-series were calculated between the shoulder-elbow, shoulder-wrist, and elbow-wrist. RCM was normalized using four differing criteria: the selected joint degree of freedom, angular velocity, angular magnitude, and range of motion. Percent time spent in specified RCM ranges was used as a composite metric and was evaluated for each trial. RCM was found to vary based on: (1) chosen normalization scheme, (2) the stage within the task, (3) the object grasped, and (4) the trajectory of the motion. The RCM addresses some of the limitations of current measures of coordination because it is applicable to discrete motions, does not rely on cyclic repetition, and uses velocity-based measures. Future work will explore clinically relevant differences in the RCM as it is expanded to evaluate different tasks and patient populations.     

Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy

The motion of subchromosomal foci and of whole chromosome territories in live human cell nuclei was investigated in four-dimensional space-time images. Visualization of subchromosomal foci was achieved by incorporating Cy3-dUTP into the nuclear DNA of two different cell types after microinjection. A subsequent segregation of the labeled cell nuclei led to the presence of only a few labeled chromosome territories on a background of nonlabeled chromatin (. Hum. Genet. 102:241-251). This procedure yielded many distinct signals in a given cell nucleus. Motion analysis in four-dimensional space-time images was performed using single-particle tracking and a statistical approach to the detection of a possible directional motion of foci relative to the center of mass of a chromosome territory. The accuracy of the analysis was tested using simulated data sets that closely mirrored the experimental setup and using microparticles of known size. Application of the analysis tools to experimental data showed that mutual diffusion-like movements between foci located on different chromosomes were more pronounced than inside the territories. In the time range observed, movements of individual foci could best be described by a random diffusion process. The statistical test for joint directed motion of several foci inside chromosome territories revealed that foci occasionally switched from random to directional motion inside the territories.     

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include ¹⁵N T ₁ relaxation times measured at two different magnetic fields as well as ¹H–¹⁵N dipole, ¹⁵N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T ₂ in the solid-state. In addition, global order parameters are included from a ¹H,¹⁵N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore–Lipari–Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10° for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken α-spectrin SH3 domain.     

Quantitative underwater 3D motion analysis using submerged video cameras: accuracy analysis and trajectory reconstruction

In this study we aim at investigating the applicability of underwater 3D motion capture based on submerged video cameras in terms of 3D accuracy analysis and trajectory reconstruction. Static points with classical direct linear transform (DLT) solution, a moving wand with bundle adjustment and a moving 2D plate with Zhang's method were considered for camera calibration. As an example of the final application, we reconstructed the hand motion trajectories in different swimming styles and qualitatively compared this with Maglischo's model. Four highly trained male swimmers performed butterfly, breaststroke and freestyle tasks. The middle fingertip trajectories of both hands in the underwater phase were considered. The accuracy (mean absolute error) of the two calibration approaches (wand: 0.96 mm – 2D plate: 0.73 mm) was comparable to out of water results and highly superior to the classical DLT results (9.74 mm). Among all the swimmers, the hands' trajectories of the expert swimmer in the style were almost symmetric and in good agreement with Maglischo's model. The kinematic results highlight symmetry or asymmetry between the two hand sides, intra- and inter-subject variability in terms of the motion patterns and agreement or disagreement with the model. The two outcomes, calibration results and trajectory reconstruction, both move towards the quantitative 3D underwater motion analysis.     

Proteomics of organelles and large cellular structures

The mass-spectrometry-based identification of proteins has created opportunities for the study of organelles, transport intermediates and large subcellular structures. Traditional cell-biology techniques are used to enrich these structures for proteomics analyses, and such analyses provide insights into the biology and functions of these structures. Here, we review the state-of-the-art proteomics techniques for the analysis of subcellular structures and discuss the biological insights that have been derived from such studies.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

Quantitative characterization of cellular differentiation of Streptomyces ambofaciensin submerged culture by image analysis

Image analysis methods were developed for light and epifluorescence microscopy of Streptomyces ambofaciens undergoing differentiation in submerged culture. Grey level images were obtained with an integration controlled CCD camera and allowed three parameters to be measured: occurence of empty zones in mycelium, number of septations, mycelium thickness.     

Generation and visualization of root-like structures in a three-dimensional space

In his Notebooks Leonardo da Vinci describes his diameter squared rule for the branching of above-ground tree parts. We now apply this branching-rule to the study of root-branching. Repeated application of this rule in a recursive computer program (ArtRoot) produces the diameters of the branches. An algorithm in this recursive program calculates the orientation of the branches in a three-dimensional space. From these data three-dimensional root-like structures are visualized by a computer program (PLUTON) designed to draw three-dimensional molecular structures. The visualization shows that starting from a relatively simple, symmetrical structure of dichotomous branching, more complex, asymmetrical root-like structures arise even if only a few parameter values are changed. The introduction of randomness into parameter values produces structures that differ considerably in their architecture. An extra force has been added in order to influence the orientation of new branches in space and to increase the flexibility of the structure formation.     

Metabolic flux analysis and visualization

One of the ultimate goals of systems biology research is to obtain a comprehensive understanding of the control mechanisms of complex cellular metabolisms. Metabolic Flux Analysis (MFA) is a important method for the quantitative estimation of intracellular metabolic flows through metabolic pathways and the elucidation of cellular physiology. The primary challenge in the use of MFA is that many biological networks are underdetermined systems; it is therefore difficult to narrow down the solution space from the stoichiometric constraints alone. In this tutorial, we present an overview of Flux Balance Analysis (FBA) and (13)C-Metabolic Flux Analysis ((13)C-MFA), both of which are frequently used to solve such underdetermined systems, and we demonstrate FBA and (13)C-MFA using the genome-scale model and the central carbon metabolism model, respectively. Furthermore, because such comprehensive study of intracellular fluxes is inherently complex, we subsequently introduce various pathway mapping and visualization tools to facilitate understanding of these data in the context of the pathways. Specific visualization of MFA results using the BioCyc Omics Viewer and Pathway Projector are shown as illustrative examples.     

Ultrastructural visualization of low-density lipoproteins during receptor binding and cellular endocytosis

Human fibroblasts possess surface receptors which have a high affinity for low-density lipoproteins (LDL). However, previous studies have not provided direct ultrastructural visualization of LDL bound to the receptor. To permit direct observation of unlabeled LDL during receptor binding and cellular endocytosis, we examined several fixative regimens which employ lipophilic stains. Staining with tannic acid, an oxidized form of ruthenium red, or potassium ferrocyanide imparted sufficient contrast to individual molecules of LDL to permit high-resolution electron microscopy of receptor binding and endocytosis. The LDL molecule was observed in immediate contact with the receptor and the coated vesicle, indicating that receptor-ligand binding occurs by short-range interactions.