Cytonemes with complex geometries and composition extend into invaginations of target cells

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.     

R/mpMap: a computational platform for the genetic analysis of multiparent recombinant inbred lines

Multiparent crosses of recombinant inbred lines provide opportunity to map markers and quantitative trait loci (QTL) with much greater resolution than is possible in biparental crosses. Realizing the full potential of these crosses requires computational tools capable of handling the increased statistical complexity of the analyses. R/mpMap provides a flexible and extensible environment, which interfaces easily with other packages to satisfy this demand. Functions in the package encompass simulation, marker map construction, haplotype reconstruction and QTL mapping. We demonstrate the easy-to-use features of mpMap through a simulated data example. AVAILABILITY: www.cmis.csiro.au/mpMap.     

Numerical evaluation of changes in the cytoplasmic microtubule complex of C3H mouse cells by optical diffractometry and of changes in cell shape by Fourier analysis

MO mouse cells in culture on glass were treated with taxol, or nocodazole, or incubated at 4 degrees C to alter their cytoplasmic microtubule complex (CMTC). From each treated group and from an untreated group, 30 cells stained with an antiserum against tubulin, were photographed under the photomicroscope, and negatives were analysed by optical diffractometry. Differences between groups of cells were tested by variance analysis. Phase-contrast micrographs of the same cells were used for Fourier analysis of cell shape. Both types of analyses provided numerical objective data about changes in the CMTC and in cell shape that were typical for the kind of treatment. We conclude that optical diffractometry of immunostained cells and Fourier analysis of cell shape are complementary to photomicroscopy for the study of the CMTC in cell populations cultured on an artificial substrate.     

Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex

Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure \(C_{2}^{n}(S)\) for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.     

A stopped-flow laser turbidimeter for studying changes in the shape of cells stimulated by external agents

A laser turbidimeter suitable for following rapid morphological changes which may accompany the stimulation of cells suspended in an adequate medium is described. Stopped-flow mixing is used to initiate the reaction and the changes in light transmission are followed with a fast recording technique. The apparatus has been used to examine the ADP-induced shape change reaction of human blood platelets.     

A computational model of the analysis of some first-order and second-order motion patterns by simple and complex cells.

Although spatio-temporal gradient schemes are widely used in the computation of image motion, algorithms are ill conditioned for particular classes of input. This paper addresses this problem. Motion is computed as the space-time direction in which the difference in image illuminance from the local mean is conserved. This method can reliably detect motion in first-order and some second-order motion stimuli. Components of the model can be identified with directionally asymmetric and directionally selective simple cells. A stage in which we compute spatial and temporal derivatives of the difference between image illuminance and the local mean illuminance using a truncated Taylor series gives rise to a phase-invariant output reminiscent of the response of complex cells.     

A microscope-based screening platform for large-scale functional protein analysis in intact cells

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.     

3D virtual human atria: A computational platform for studying clinical atrial fibrillation

Despite a vast amount of experimental and clinical data on the underlying ionic, cellular and tissue substrates, the mechanisms of common atrial arrhythmias (such as atrial fibrillation, AF) arising from the functional interactions at the whole atria level remain unclear. Computational modelling provides a quantitative framework for integrating such multi-scale data and understanding the arrhythmogenic behaviour that emerges from the collective spatio-temporal dynamics in all parts of the heart. In this study, we have developed a multi-scale hierarchy of biophysically detailed computational models for the human atria - the 3D virtual human atria. Primarily, diffusion tensor MRI reconstruction of the tissue geometry and fibre orientation in the human sinoatrial node (SAN) and surrounding atrial muscle was integrated into the 3D model of the whole atria dissected from the Visible Human dataset. The anatomical models were combined with the heterogeneous atrial action potential (AP) models, and used to simulate the AP conduction in the human atria under various conditions: SAN pacemaking and atrial activation in the normal rhythm, break-down of regular AP wave-fronts during rapid atrial pacing, and the genesis of multiple re-entrant wavelets characteristic of AF. Contributions of different properties of the tissue to mechanisms of the normal rhythm and arrhythmogenesis were investigated. Primarily, the simulations showed that tissue heterogeneity caused the break-down of the normal AP wave-fronts at rapid pacing rates, which initiated a pair of re-entrant spiral waves; and tissue anisotropy resulted in a further break-down of the spiral waves into multiple meandering wavelets characteristic of AF. The 3D virtual atria model itself was incorporated into the torso model to simulate the body surface ECG patterns in the normal and arrhythmic conditions. Therefore, a state-of-the-art computational platform has been developed, which can be used for studying multi-scale electrical phenomena during atrial conduction and AF arrhythmogenesis. Results of such simulations can be directly compared with electrophysiological and endocardial mapping data, as well as clinical ECG recordings. The virtual human atria can provide in-depth insights into 3D excitation propagation processes within atrial walls of a whole heart in vivo, which is beyond the current technical capabilities of experimental or clinical set-ups.     

A computational study of co-inhibitory immune complex assembly at the interface between T cells and antigen presenting cells

The activation and differentiation of T-cells are mainly directly by their co-regulatory receptors. T lymphocyte-associated protein-4 (CTLA-4) and programed cell death-1 (PD-1) are two of the most important co-regulatory receptors. Binding of PD-1 and CTLA-4 with their corresponding ligands programed cell death-ligand 1 (PD-L1) and B7 on the antigen presenting cells (APC) activates two central co-inhibitory signaling pathways to suppress T cell functions. Interestingly, recent experiments have identified a new cis-interaction between PD-L1 and B7, suggesting that a crosstalk exists between two co-inhibitory receptors and the two pairs of ligand-receptor complexes can undergo dynamic oligomerization. Inspired by these experimental evidences, we developed a coarse-grained model to characterize the assembling of an immune complex consisting of CLTA-4, B7, PD-L1 and PD-1. These four proteins and their interactions form a small network motif. The temporal dynamics and spatial pattern formation of this network was simulated by a diffusion-reaction algorithm. Our simulation method incorporates the membrane confinement of cell surface proteins and geometric arrangement of different binding interfaces between these proteins. A wide range of binding constants was tested for the interactions involved in the network. Interestingly, we show that the CTLA-4/B7 ligand-receptor complexes can first form linear oligomers, while these oligomers further align together into two-dimensional clusters. Similar phenomenon has also been observed in other systems of cell surface proteins. Our test results further indicate that both co-inhibitory signaling pathways activated by B7 and PD-L1 can be down-regulated by the new cis-interaction between these two ligands, consistent with previous experimental evidences. Finally, the simulations also suggest that the dynamic and the spatial properties of the immune complex assembly are highly determined by the energetics of molecular interactions in the network. Our study, therefore, brings new insights to the co-regulatory mechanisms of T cell activation.     

A computational study of a host-guest complex

The geometry and energetics of a complex involving pyrazine and an acridine diacid cleft-like host designed by Rebek were investigated at several levels of theory. Molecular mechanics (using the Tripos and CHARMm force fields), semiempirical quantum chemical approaches (with the AM1 and PM3 methods), and an ab initio quantum chemical method (RHF/STO-3G) were used in the complete relaxation of the complex. The geometry of the complex optimized by the RHF/STO-3G method is in excellent agreement with a published X-ray structure; upon superposition, the rms deviation between the corresponding cleft heavy atoms is only 0.17 Å and the pyrazine molecules are superimposable. In addition, ab initio quantum chemical techniques were used to study the complex when the cleft is modeled by a pair of acetic acid molecules. All the calculations presented herein support a two-point interaction mechanism. The similarities found in the results for the full complex and the truncated model are consistent with a purely structural role for the acridine linker of the host. © 1997 John Wiley & Sons, Ltd.     

A computational platform for MALDI-TOF mass spectrometry data: Application to serum and plasma samples

BackgroundMass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data.ResultsMASCAP integrates denoising and feature extraction algorithms, which have already shown to provide consistent peaks across mass spectra; furthermore, it relies on statistical analysis and graphical tools to compare the results between groups. The effectiveness in mass spectrum processing is demonstrated using MALDI-TOF data, as well as SELDI-TOF data. The usefulness in detecting potential protein biomarkers is shown comparing MALDI-TOF mass spectra collected from serum and plasma samples belonging to the same clinical population.ConclusionsThe analysis approach implemented in MASCAP may simplify biomarker detection, by assisting the recognition of proteomic expression signatures of the disease. A MATLAB implementation of the software and the data used for its validation are available at http://www.unich.it/proteomica/bioinf.     

A reporter platform for the monitoring of in vivo conformational changes in AcrB

AcrB is an inner membrane protein in Escherichia coli that is a component of a triplex AcrA-AcrB-TolC (AcrAB-TolC) multidrug efflux pump. The AcrAB-TolC complex and its homologues are highly conserved among Gram-negative bacteria and are major players in conferring multidrug resistance (MDR) in many pathogens. In this study we developed a disulfide trapping method that may reveal AcrB conformational changes under the native condition in the cell membrane. Specifically, we created seven disulfide bond pairs in the periplasmic domain of AcrB, which can be used as probes to determine local conformational changes. We have developed a rigorous protocol to measure the extent of disulfide bond formation in membrane proteins under the native condition. The rigorousness of the method was verified through examining the extent of disulfide bond formation in Cys pairs separated by different distances. The blocking-reducing-labeling scheme combined with fluorescence labeling made the current method convenient, reliable, and quantitative.     

Systems Biology Toolbox for MATLAB: a computational platform for research in systems biology

We present a Systems Biology Toolbox for the widely used general purpose mathematical software MATLAB. The toolbox offers systems biologists an open and extensible environment, in which to explore ideas, prototype and share new algorithms, and build applications for the analysis and simulation of biological and biochemical systems. Additionally it is well suited for educational purposes. The toolbox supports the Systems Biology Markup Language (SBML) by providing an interface for import and export of SBML models. In this way the toolbox connects nicely to other SBML-enabled modelling packages. Models are represented in an internal model format and can be described either by entering ordinary differential equations or, more intuitively, by entering biochemical reaction equations. The toolbox contains a large number of analysis methods, such as deterministic and stochastic simulation, parameter estimation, network identification, parameter sensitivity analysis and bifurcation analysis.     

A computational image analysis glossary for biologists

Recent advances in biological imaging have resulted in an explosion in the quality and quantity of images obtained in a digital format. Developmental biologists are increasingly acquiring beautiful and complex images, thus creating vast image datasets. In the past, patterns in image data have been detected by the human eye. Larger datasets, however, necessitate high-throughput objective analysis tools to computationally extract quantitative information from the images. These tools have been developed in collaborations between biologists, computer scientists, mathematicians and physicists. In this Primer we present a glossary of image analysis terms to aid biologists and briefly discuss the importance of robust image analysis in developmental studies.     

Healthy and diseased coronary bifurcation geometries influence near-wall and intravascular flow: A computational exploration of the hemodynamic risk

Local hemodynamics has been identified as one main determinant in the onset and progression of atherosclerotic lesions at coronary bifurcations. Starting from the observation that atherosensitive hemodynamic conditions in arterial bifurcation are majorly determined by the underlying anatomy, the aim of the present study is to investigate how peculiar coronary bifurcation anatomical features influence near-wall and intravascular flow patterns. Different bifurcation angles and cardiac curvatures were varied in population-based, idealized models of both stenosed and unstenosed bifurcations, representing the left anterior descending (LAD) coronary artery with its diagonal branch. Local hemodynamics was analyzed in terms of helical flow and exposure to low/oscillatory shear stress by performing computational fluid dynamics simulations.Results show that bifurcation angle impacts lowly hemodynamics in both stenosed and unstenosed cases. Instead, curvature radius influences the generation and transport of helical flow structures, with smaller cardiac curvature radius associated to higher helicity intensity. Stenosed bifurcation models exhibit helicity intensity values one order of magnitude higher than the corresponding unstenosed cases. Cardiac curvature radius moderately affects near-wall hemodynamics of the stenosed cases, with smaller curvature radius leading to higher exposure to low shear stress and lower exposure to oscillatory shear stress. In conclusion, the proposed controlled benchmark allows investigating the effect of various geometrical features on local hemodynamics at the LAD/diagonal bifurcation, highlighting that cardiac curvature influences near wall and intravascular hemodynamics, while bifurcation angle has a minor effect.