Tinkerbell—A Tool for Interactive Segmentation of 3D Data

A software system for interactive manipulation of three-dimensional data has been developed, based on the Open Inventor tool kit. The primary use of this software system is in the segmentation of tomographic reconstructions of subcellular structures. To this end, the reconstruction is represented by volume rendering and displayed in stereo. A three-dimensional cursor with adjustable shape and size is used to define and isolate regions of interest inside the volume, based on the user's expert knowledge. Once isolated, the region of interest can be conveniently analyzed and displayed.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

Fine Structure of the Cone of Heterodera schachtii with Emphasis on Musculature and Fenestration

Fine structure of the posterior cone of monoxenically cultured Heterodera schachtii is examined. The cone is not evident at the end of the fourth molt, but as the female matures the cone elongates, vulval lips enlarge, and cuticular patterns on the lips are modified. Body wall cuticle (BW) of the cone includes layers A and B, but C is modified or replaced by a network of fibers which correspond to the semifenestrae. Vaginal lining is continuous with the BW and terminates at the cuticular underbridge near the uterus. Vaginal musculature includes 48 dilatores vaginae (DV) as well as a sphincter vaginae (SV). The DV include a contractile and noncontractile region with abundant actin and glycogen. A distinct anal depressor muscle is present. In the cyst, only bullae, the underbridge, vagina lining, and traces of the SV muscle persist. Detailed morphology of the cone of H. schachtii provides insight into characters which, when compared with other heteroderines, will be useful in phylogenetic analysis of Heteroderinae.     

Evaluation of the Quantitative Accuracy of 3D Reconstruction of Edentulous Jaw Models with Jaw Relation Based on Reference Point System Alignment

ObjectivesTo apply contact measurement and reference point system (RPS) alignment techniques to establish a method for 3D reconstruction of the edentulous jaw models with centric relation and to quantitatively evaluate its accuracy.MethodsUpper and lower edentulous jaw models were clinically prepared, 10 pairs of resin cylinders with same size were adhered to axial surfaces of upper and lower models. The occlusal bases and the upper and lower jaw models were installed in the centric relation position. Faro Edge 1.8m was used to directly obtain center points of the base surface of the cylinders (contact method). Activity 880 dental scanner was used to obtain 3D data of the cylinders and the center points were fitted (fitting method). 3 pairs of center points were used to align the virtual model to centric relation. An observation coordinate system was interactively established. The straight-line distances in the X (horizontal left/right), Y (horizontal anterior/posterior), and Z (vertical) between the remaining 7 pairs of center points derived from contact method and fitting method were measured respectively and analyzed using a paired t-test.ResultsThe differences of the straight-line distances of the remaining 7 pairs of center points between the two methods were X: 0.074 ± 0.107 mm, Y: 0.168 ± 0.176 mm, and Z: −0.003± 0.155 mm. The results of paired t-test were X and Z: p >0.05, Y: p <0.05.ConclusionBy using contact measurement and the reference point system alignment technique, highly accurate reconstruction of the vertical distance and centric relation of a digital edentulous jaw model can be achieved, which meets the design and manufacturing requirements of the complete dentures. The error of horizontal anterior/posterior jaw relation was relatively large.     

A 3D Architectural and Process-based Model of Maize Development

A 3D architectural and process-based model of maize developmentwas implemented on the basis of the L-system software Graphtal,interfaced with physical models computing microclimate distributedon the 3D canopy structure. In a first step, we incorporatedin the software Graphtal additional functions that enable bi-directionalcommunication with external modules. A simple model for distributedphotosynthetically active radiation and the model for apex temperatureby Cellieret al. (Agricultural and Forest Meteorology63: 35–54,1993) were interfaced with Graphtal. In a second step we developeda L-system model for maize, where production rules for growthand development of organs are based on the current state ofknowledge of maize development as a function of temperature.Visual representation of the plant is based on the geometricalmodel of leaf shape by Prévot, Aries and Monestiez (Agronomie11:491–503, 1991). Finally, various data sets were used toevaluate the physiological aspects and the geometrical representation.It is concluded that environmental L-systems are a convenienttool to integrate biophysical processes from organ to canopylevel, and provide a framework to model growth of individualplants in relation to local conditions and ability to foragefor resources. However, progress is needed to improve both theknowledge of physiological processes at the organ level andthe calculation of physical environmental parameters; some directionsfor future research are proposed.Copyright 1998 Annals of BotanyCompany Growth model; 3D plant architecture;Zea maysL.; corn; temperature; L-system modelling; developmental physiology; virtual plant.     

A 3D Motile Rod-Shaped Monotrichous Bacterial Model

We introduce a 3D model for a motile rod-shaped bacterial cell with a single polar flagellum which is based on the configuration of a monotrichous type of bacteria such as Pseudomonas aeruginosa. The structure of the model bacterial cell consists of a cylindrical body together with the flagellar forces produced by the rotation of a helical flagellum. The rod-shaped cell body is composed of a set of immersed boundary points and elastic links. The helical flagellum is assumed to be rigid and modeled as a set of discrete points along the helical flagellum and flagellar hook. A set of flagellar forces are applied along this helical curve as the flagellum rotates. An additional set of torque balance forces are applied on the cell body to induce counter-rotation of the body and provide torque balance. The three-dimensional Navier–Stokes equations for incompressible fluid are used to describe the fluid dynamics of the coupled fluid–microorganism system using Peskin’s immersed boundary method. A study of numerical convergence is presented along with simulations of a single swimming cell, the hydrodynamic interaction of two cells, and the interaction of a small cluster of cells.     

A Theoretical Model of the Winter Survival Dynamics of Meloidogyne spp. Eggs and Juveniles

A theoretical model of the winter survival dynamics of Meloidagyne spp. was developed by considering the roles of egg hatching and juvenile mortality and the initial populations of eggs and juveniles at the onset of winter. Two distinct patterns of juvenile dynamics appear which depend upon the numerical values of the model parameters. The model predicts whether eggs or juveniles are the major component of overwintering nematode populations at any time prior to planting. The model could be elaborated to include egg viability and differential mortality of eggs but at some cost in ease of mathematical analysis. A general procedure for fitting the proposed model to experimental or observational data is outlined.     

PKD3 Is the Predominant Protein Kinase D Isoform in Mouse Exocrine Pancreas and Promotes Hormone-induced Amylase Secretion

The protein kinase D (PKD) family of serine/threonine kinases, which can be activated by gastrointestinal hormones, consists of three distinct isoforms that modulate a variety of cellular processes including intracellular protein transport as well as constitutive and regulated secretion. Although isoform-specific functions have been identified in a variety of cell lines, the expression and function of PKD isoforms in normal, differentiated secretory tissues is unknown. Here, we demonstrate that PKD isoforms are differentially expressed in the exocrine and endocrine cells of the pancreas. Specifically, PKD3 is the predominant isoform expressed in exocrine cells of the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly expressed in the pancreatic islets. Within isolated mouse pancreatic acinar cells, PKD3 undergoes rapid membrane translocation, trans-activating phosphorylation, and kinase activation after gastrointestinal hormone or cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs viaaCa²⁺-independent, diacylglycerol- and protein kinase C-dependent mechanism. PKD phosphorylation can also be induced by physiologic concentrations of secretagogues and by in vivo stimulation of the pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK, ribosomal S6 kinase) signaling and significantly enhances cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a novel distinction between the exocrine and endocrine cells of the pancreas and further identify PKD3 as a signaling molecule that promotes hormone-stimulated amylase secretion.Protein kinase D (PKD),² a serine/threonine kinase family with a catalytic domain homologous to the Ca²⁺/calmodulin-dependent kinase domain and two cysteine-rich phorbol ester binding domains similar to those of protein kinase C (PKC), is a physiologically important downstream mediator of diacylglycerol (DAG) signal transduction (1, 2). The mammalian PKDs include three members, PKD1, PKD2, and PKD3, which demonstrate different expression patterns and functions depending on the cell type and external signal stimuli. PKDs are ubiquitously expressed, but levels of individual isoforms vary with developmental stage and cell type (3). PKD proteins are reported to localize in the cytosol, Golgi, nucleus, and vesicle structures (4-9). Activation of PKDs results in a dynamic translocation among subcellular compartments (10, 11). Expression of multiple isoforms in different cell types and in different subcellular localizations suggests that individual PKD isoforms may serve specific functions. The majority of findings demonstrating the diverse expression patterns and functions of PKD have been described using established cell lines (4-9, 12). However, little is known about PKD isoform expression and function in normal differentiated cells and tissues.Recent functional studies have shown that PKD isoforms differentially regulate exocytic protein trafficking and cargo specificity (9, 12-14). Furthermore, PKD isoforms are differentially activated by oxidative stress signaling via PKCδ-mediated tyrosine phosphorylation (15). In each of these studies, PKD3 was found to have a regulatory mechanism or cellular function distinct from that of PKD1 and PKD2. Unlike the other two isoforms, PKD3 lacks the N terminus hydrophobic domain or the C terminus PDZ binding motif and contains divergent PH (pleckstrin homology) and C1 domains, which are important for regulating its catalytic activity (12, 16, 17). Current knowledge of the physiologic function of PKD3 is limited. It has been demonstrated using kinase-inactive mutants that PKD3 activity is required for basolateral exocytosis in Madin-Darby canine kidney cells (13). PKD3 has also been implicated in the epigenetic control of chromatin by regulating class II histone deacetylases in B lymphocytes (18). Furthermore, PKD3 was found to be a specific regulator of glucose transport in skeletal muscle cells (19).The exocrine pancreas is highly specialized for the synthesis, storage, and exocrine secretion of digestive enzymes and bicarbonate-rich fluid (20). More than 90% of the newly synthesized proteins in the pancreas is targeted to the secretory pathway (21). In addition, the pancreas contains a variety of endocrine cells localized to the islets which secrete peptide hormones. Numerous steps in the secretory pathway are modulated by DAG signaling, which promotes secretion by maintaining Golgi function and/or activating DAG receptor kinases such as PKCs, which are regulators of exocytic proteins (1, 22-25). PKD is also critical for DAG-mediated secretion, as it is recruited by DAG to the trans-Golgi network, where it phosphorylates the lipid kinase phosphatidylinositol 4-kinase to initiate the process of vesicle fission (9, 26). Gastrointestinal (GI) hormones such as cholecystokinin (CCK), gastrin, neurotensin (NT), and bombesin (BBS)/gastrin-releasing peptide are potent regulatory peptides that modulate pancreatic function (27, 28). They are known to activate PKDs to promote cell proliferation and survival in gut epithelial cells (29-32); however, the role of PKDs in modulating the secretory actions of GI hormones is unknown.Although the PKD isoforms have been reported to be expressed in secretory tissues such as salivary glands, adrenal glands, intestinal mucosa, and the pituitary (3, 5, 33), the role of PKD in the process of regulated secretion remains poorly understood. Previously, we demonstrated that PKD1 mediates NT peptide secretion from a pancreas-derived neuroendocrine cell line, BON, and that PKD1 activation is regulated by PKC and Rho/Rho kinase pathways (4); PKD1 and PKD2 isoforms are highly expressed in this endocrine cell line with little to no PKD3 expression, thus suggesting that PKD1/2 may be the predominant isoforms for endocrine secretion. The distribution and role of PKD isoforms in the pancreas, an organ with both exocrine and endocrine functions, is not known. Interestingly, we demonstrate that in both human and mouse pancreas, PKD3 is the predominant PKD isoform expressed in the exocrine acini, whereas PKD1 and PKD2 are more highly expressed in endocrine islets. PKD3 is catalytically activated by GI hormone stimulation of the pancreas, and its activation is dependent on CCK1/2 receptor binding and on DAG/PKC activity. PKD3 overexpression in mouse pancreatic acinar cells significantly increased CCK-mediated pancreatic amylase secretion, suggesting that PKD3, in concert with other signaling molecules, contributes to stimulated amylase secretion. Our findings reveal a distinct expression pattern in the exocrine and endocrine cells of the mouse and human pancreas and identify PKD3 as a novel DAG-activated mediator of the exocrine secretory process in response to GI hormone signaling.     

Iris: Interactive all‐in‐one graphical validation of 3D protein model iterations

Iris validation is a Python package created to represent comprehensive per‐residue validation metrics for entire protein chains in a compact, readable and interactive view. These metrics can either be calculated by Iris, or by a third‐party program such as MolProbity. We show that those parts of a protein model requiring attention may generate ripples across the metrics on the diagram, immediately catching the modeler's attention. Iris can run as a standalone tool, or be plugged into existing structural biology software to display per‐chain model quality at a glance, with a particular emphasis on evaluating incremental changes resulting from the iterative nature of model building and refinement. Finally, the integration of Iris into the CCP4i2 graphical user interface is provided as a showcase of its pluggable design.     

Interactive Effects of Ocean Acidification and Nitrogen-Limitation on the Diatom Phaeodactylum tricornutum

Climate change is expected to bring about alterations in the marine physical and chemical environment that will induce changes in the concentration of dissolved CO₂ and in nutrient availability. These in turn are expected to affect the physiological performance of phytoplankton. In order to learn how phytoplankton respond to the predicted scenario of increased CO₂ and decreased nitrogen in the surface mixed layer, we investigated the diatom Phaeodactylum tricornutum as a model organism. The cells were cultured in both low CO₂ (390 μatm) and high CO₂ (1000 μatm) conditions at limiting (10 μmol L⁻¹) or enriched (110 μmol L⁻¹) nitrate concentrations. Our study shows that nitrogen limitation resulted in significant decreases in cell size, pigmentation, growth rate and effective quantum yield of Phaeodactylum tricornutum, but these parameters were not affected by enhanced dissolved CO₂ and lowered pH. However, increased CO₂ concentration induced higher rETR_max and higher dark respiration rates and decreased the CO₂ or dissolved inorganic carbon (DIC) affinity for electron transfer (shown by higher values for K_{1/2 DIC} or K_{1/2 CO2}). Furthermore, the elemental stoichiometry (carbon to nitrogen ratio) was raised under high CO₂ conditions in both nitrogen limited and nitrogen replete conditions, with the ratio in the high CO₂ and low nitrate grown cells being higher by 45% compared to that in the low CO₂ and nitrate replete grown ones. Our results suggest that while nitrogen limitation had a greater effect than ocean acidification, the combined effects of both factors could act synergistically to affect marine diatoms and related biogeochemical cycles in future oceans.     

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.     

A Predictive Computational Model of the Dynamic 3D Interphase Yeast Nucleus

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Highlights? We describe a predictive computational model of dynamic chromosomes in the yeast nucleus ? The model quantitatively recapitulates experimental data on nuclear organization ? The model predicts nuclear reorganization in response to treatment by rapamycin ? Large-scale nuclear organization is dominated by unspecific effects of crowded polymers     

Primary Human Osteoblasts in Response to 25-Hydroxyvitamin D3, 1,25-Dihydroxyvitamin D3 and 24R,25-Dihydroxyvitamin D3

The most biologically active metabolite 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D₃ (25(OH)D₃) can affect osteoblast function via conversion to 1,25(OH)₂D₃, however, it is largely unknown whether 25(OH)D₃ can affect primary osteoblast function on its own. Furthermore, 25(OH)D₃ is not only converted to 1,25(OH)₂D₃, but also to 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D₃ itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)₂D₃ can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D₃ and 1,25(OH)₂D₃ by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)₂D₃ synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D₃. We demonstrated that 24R,25(OH)₂D₃ increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)₂D₃ strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D₃, 1,25(OH)₂D₃ and 24R,25(OH)₂D₃ can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)₂D₃, but also to 24R,25(OH)₂D₃ by enhancing osteoblast differentiation. This suggests that 25(OH)D₃ can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)₂D₃, but also via conversion to 24R,25(OH)₂D₃. Whether 25(OH)D₃ has direct actions on osteoblast function needs further investigation.