Atlas Toolkit: Fast registration of 3D morphological datasets in the absence of landmarks

Image registration is a gateway technology for Developmental Systems Biology, enabling computational analysis of related datasets within a shared coordinate system. Many registration tools rely on landmarks to ensure that datasets are correctly aligned; yet suitable landmarks are not present in many datasets. Atlas Toolkit is a Fiji/ImageJ plugin collection offering elastic group-wise registration of 3D morphological datasets, guided by segmentation of the interesting morphology. We demonstrate the method by combinatorial mapping of cell signalling events in the developing eyes of chick embryos, and use the integrated datasets to predictively enumerate Gene Regulatory Network states

Comparative study of four immortalised human brain capillary endothelial cell lines, hCMEC/D3, hBMED, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies

BACKGROUND: Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. METHODS: Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (C(CL)) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. RESULTS: The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. CONCLUSIONS: Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products

Inversion of Randomly Corrugated Surfaces Structure from Atom Scattering Data

The Sudden Approximation is applied to invert structural data on randomly corrugated surfaces from inert atom scattering intensities. Several expressions relating experimental observables to surface statistical features are derived. The results suggest that atom (and in particular He) scattering can be used profitably to study hitherto unexplored forms of complex surface disorder.Comment: 10 pages, no figures. Related papers available at http://neon.cchem.berkeley.edu/~dan

Detection of embryo mortality and hatch using thermal differences among incubated chicken eggs

Accurate diagnosis of both the stage of embryonic mortality and the hatch process in incubated eggs is a fundamental component in troubleshooting and hatchery management. However, traditional methods disturb incubation, destroy egg samples, risk contamination, are time and labour-intensive and require specialist knowledge and training. Therefore, a new method to accurately detect embryonic mortality and hatching time would be of significant interest for the poultry industry if it could be done quickly, cheaply and be fully integrated into the process. In this study we have continuously measured individual eggshell temperatures and the corresponding micro-environmental air temperatures throughout the 21 days of incubation using standard low-cost temperature sensors. Moreover, we have quantified the thermal interaction between eggs and air by calculating thermal profile changes (temperature drop time, drop length and drop magnitude) that allowed us to detect four categories of egg status (infertile/early death, middle death, late death and hatch) during incubation. A decision tree induction classification model accurately (93.3%) predicted the status of 105 sampled eggs in comparison to the classical hatch residue breakout analyses. With this study we have provided a major contribution to the optimisation of incubation processes by introducing an alternative method for the currently practiced hatch residue breakout analyses.status: publishe

The scattering from generalized Cantor fractals

We consider a fractal with a variable fractal dimension, which is a generalization of the well known triadic Cantor set. In contrast with the usual Cantor set, the fractal dimension is controlled using a scaling factor, and can vary from zero to one in one dimension and from zero to three in three dimensions. The intensity profile of small-angle scattering from the generalized Cantor fractal in three dimensions is calculated. The system is generated by a set of iterative rules, each iteration corresponding to a certain fractal generation. Small-angle scattering is considered from monodispersive sets, which are randomly oriented and placed. The scattering intensities represent minima and maxima superimposed on a power law decay, with the exponent equal to the fractal dimension of the scatterer, but the minima and maxima are damped with increasing polydispersity of the fractal sets. It is shown that for a finite generation of the fractal, the exponent changes at sufficiently large wave vectors from the fractal dimension to four, the value given by the usual Porod law. It is shown that the number of particles of which the fractal is composed can be estimated from the value of the boundary between the fractal and Porod regions. The radius of gyration of the fractal is calculated analytically.Comment: 8 pages, 4 figures, accepted for publication in J. Appl. Crys

A random cell motility gradient downstream of FGF controls elongation of amniote embryos

Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient1, which has been implicated in the control of cell motility in this tissue2. Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data indicate that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements

Apparent Fractality Emerging from Models of Random Distributions

The fractal properties of models of randomly placed $n$-dimensional spheres ($n$=1,2,3) are studied using standard techniques for calculating fractal dimensions in empirical data (the box counting and Minkowski-sausage techniques). Using analytical and numerical calculations it is shown that in the regime of low volume fraction occupied by the spheres, apparent fractal behavior is observed for a range of scales between physically relevant cut-offs. The width of this range, typically spanning between one and two orders of magnitude, is in very good agreement with the typical range observed in experimental measurements of fractals. The dimensions are not universal and depend on density. These observations are applicable to spatial, temporal and spectral random structures. Polydispersivity in sphere radii and impenetrability of the spheres (resulting in short range correlations) are also introduced and are found to have little effect on the scaling properties. We thus propose that apparent fractal behavior observed experimentally over a limited range may often have its origin in underlying randomness.Comment: 19 pages, 12 figures. More info available at http://www.fh.huji.ac.il/~dani

Hopping Conductivity of a Nearly-1d Fractal: a Model for Conducting Polymers

We suggest treating a conducting network of oriented polymer chains as an anisotropic fractal whose dimensionality D=1+\epsilon is close to one. Percolation on such a fractal is studied within the real space renormalization group of Migdal and Kadanoff. We find that the threshold value and all the critical exponents are strongly nonanalytic functions of \epsilon as \epsilon tends to zero, e.g., the critical exponent of conductivity is \epsilon^{-2}\exp (-1-1/\epsilon). The distribution function for conductivity of finite samples at the percolation threshold is established. It is shown that the central body of the distribution is given by a universal scaling function and only the low-conductivity tail of distribution remains $\epsilon$-dependent. Variable range hopping conductivity in the polymer network is studied: both DC conductivity and AC conductivity in the multiple hopping regime are found to obey a quasi-1d Mott law. The present results are consistent with electrical properties of poorly conducting polymers.Comment: 27 pages, RevTeX, epsf, 5 .eps figures, to be published in Phys. Rev.

Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-seq and ESTs

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct annotation is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3-prime untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3-prime polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3-prime UTR re-annotation (including extension of one 3-prime UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental dataComment: 44 pages, 9 figure