3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms.

Assembling models of embryo development: Image analysis and the construction of digital atlases

Digital atlases of animal development provide a quantitative description of morphogenesis, opening the path toward processes modeling. Prototypic atlases offer a data integration framework where to gather information from cohorts of individuals with phenotypic variability. Relevant information for further theoretical reconstruction includes measurements in time and space for cell behaviors and gene expression. The latter as well as data integration in a prototypic model, rely on image processing strategies. Developing the tools to integrate and analyze biological multidimensional data are highly relevant for assessing chemical toxicity or performing drugs preclinical testing. This article surveys some of the most prominent efforts to assemble these prototypes, categorizes them according to salient criteria and discusses the key questions in the field and the future challenges toward the reconstruction of multiscale dynamics in model organisms.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule

Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the Ig domains of a prototypical Ig superfamily CAM that contains intradomain disulfide bonds. The Ig domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin Ig-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five Ig domains of Mel-CAM (melanoma CAM)—a protein that is overexpressed in metastatic melanomas—under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's Ig-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell–cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's Ig domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions.

Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments

Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, β-catenin, and α-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.

Cell Adhesion Molecule L1 in Folded (Horseshoe) and Extended Conformations

We have investigated the structure of the cell adhesion molecule L1 by electron microscopy. We were particularly interested in the conformation of the four N-terminal immunoglobulin domains, because x-ray diffraction showed that these domains are bent into a horseshoe shape in the related molecules hemolin and axonin-1. Surprisingly, rotary-shadowed specimens showed the molecules to be elongated, with no indication of the horseshoe shape. However, sedimentation data suggested that these domains of L1 were folded into a compact shape in solution; therefore, this prompted us to look at the molecules by an alternative technique, negative stain. The negative stain images showed a compact shape consistent with the expected horseshoe conformation. We speculate that in rotary shadowing the contact with the mica caused a distortion of the protein, weakening the bonds forming the horseshoe and permitting the molecule to extend. We have thus confirmed that the L1 molecule is primarily in the horseshoe conformation in solution, and we have visualized for the first time its opening into an extended conformation. Our study resolves conflicting interpretations from previous electron microscopy studies of L1.

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Efeito do tratamento térmico do titânio sobre a proliferação de células pré-osteoblásticas

Titanium is a biomaterial widely employed in biomedical applications (implants, prostheses, valves, stents). Several heat treatments are usually used in order to obtain physical properties required to different applications. This work studied the influence of the heat treatment on microstructure of commercial pure titanium, and their consequences in growth and proliferation of MC3T3-E1 cells. Discs of titanium were treated in different temperatures, and characterized by optical microscopy, image analysis, wettabillity, roughness, hardness and X-ray diffraction. After the heat treatment, significant modifications in these properties were observed. Pattern images of titanium, before and after the cell culture, were compared by overlapping to analyze the influence of microstructure in microstructure and preferences guidance cells. However, in general, titanium discs that showed a higher residual strength also presented an increase of cells numbers on surface

Disarray Of Glomerular And Tubular Cell Adhesion Molecules In The Course Of Experimental Bothrops Moojeni Envenomation.

In this study, we show that administration of Bothrops moojeni venom in rats induces a general disturbance in the distribution and content of the tight junctional protein ZO-1, the cell-matrix receptor beta 1 integrin, the cytoskeletal proteins, vinculin and F-actin, and of the extracellular matrix component laminin in renal corpuscles and cortical nephron tubules. These findings suggest that cell-cell and cell-matrix adhesion proteins may be molecular targets in the B. moojeni-induced kidney injury.

Characterization of micro-bubble size distribution and flow configuration in DAF contact zone by a non-intrusive image analysis system and tracer tests

This paper aims to investigate the influence of some dissolved air flotation (DAF) process variables (specifically: the hydraulic detention time in the contact zone and the supplied dissolved air concentration) and the pH values, as pretreatment chemical variables, on the micro-bubble size distribution (BSD) in a DAF contact zone. This work was carried out in a pilot plant where bubbles were measured by an appropriate non-intrusive image acquisition system. The results show that the obtained diameter ranges were in agreement with values reported in the literature (10-100mm), quite independently of the investigated conditions. The linear average diameter varied from 20 to 30mm, or equivalently, the Sauter (d(3,2)) diameter varied from 40 to 50mm. In all investigated conditions, D(50) was between 75% and 95%. The BSD might present different profile (with a bimodal curve trend), however, when analyzing the volumetric frequency distribution (in some cases with the appearance of peaks in diameters ranging from 90-100mm). Regarding volumetric frequency analysis, all the investigated parameters can modify the BSD in DAF contact zone after the release point, thus potentially causing changes in DAF kinetics. This finding prompts further research in order to verify the effect of these BSD changes on solid particle removal efficiency by DAF.

Quantification of Clay Dispersion in Nanocomposites of Styrenic Polymers

Polymer-clay nanocomposites are materials with many interesting structures, properties, and potential applications. Microstructural evaluation of a nanocomposite is not an easy task, as clay may form hierarchical structures which may look different when observed at various magnifications under a microscope, and also as the concepts of ""intercalation"" and ""exfoliation"" are not self-sufficient to describe its morphology. In this work polymer-clay nanocomposites of polystyrene and two styrene-containing block copolymers (styrene-butadiene-styrene and styrene-ethylene/butylene-styrene) were prepared using three different techniques. Clay dispersion was evaluated by a recently developed microscopy image analysis procedure, combining the analysis of optical and transmission electron micrographs, and the characterization was complemented by X-ray diffraction and rheological measurements. The results showed better clay dispersion for both block copolymers nanocomposites, mainly due to their molecular architectures. Moreover, the techniques which showed the best results involved mixing the materials in a solvent medium. POLYM. ENG. SCI., 50:257-267, 2010. (C) 2009 Society of Plastics Engineers

CALCINED SLUDGE SINTERING EVALUATION BY HEATING MICROSCOPY THERMAL ANALYSIS

Aiming the use of the sewage sludge produced in one of the largest Brazilian wastewater treatment stations as a raw material for the ceramic industry, the sintering process of the ashes produced from its calcination was evaluated by heating microscopy thermal analysis (HMTA). From the microprocessed images, a method was developed to obtain HMTA dimensional change curves as a function of temperature, equivalent to those usually obtained from dilatometers or by thermomechanical analysis (TMA). The final product after sintering at 1050 degrees C, characterized by X-ray fluorescence spectrometry, scanning electron microscopy and X-ray dispersive energy, indicates the presence of a vitreous phase containing phosphorus, which explains the good sintering properties of the studied calcined sludge, as shown from its HMTA dimensional change curve.