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Background and Purpose: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt‐3a in live CRISPR‐Cas9‐edited SW480 cells typifying colorectal cancer. Experimental Approach: SW480 cells were CRISPR‐Cas9‐edited to insert a HiBiT tag on the N‐terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP‐Wnt‐3a association with endogenous and overexpressed HiBiT‐FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results: With this new assay the binding of eGFP‐Wnt‐3a to endogenous HiBiT‐FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on‐rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion. [ABSTRACT FROM AUTHOR]

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events. [ABSTRACT FROM AUTHOR]

Most of the biological processes, including cell signaling, cancer invasion, embryogenesis, or neural development, are dependent on and guided by the complex architecture and composition of cellular microenvironments. Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations. The ability to combine different cell types grown as both two‐dimensional (2D) monolayers and three‐dimensional (3D) spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes. In this study, we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization. We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies. Furthermore, we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies. This method provides a general approach for the miniaturized, high‐throughput, and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro. [ABSTRACT FROM AUTHOR]

Author(s): Gary Davidson (corresponding author) [1, 2]; Wei Wu [1, 2]; Jinlong Shen [1]; Josipa Bilic [1]; Ursula Fenger [1]; Peter Stannek [1]; Andrei Glinka [1]; Christof Niehrs (corresponding author) [...]

Author(s): Bingyu Mao [1]; Wei Wu [1]; Gary Davidson [1]; Joachim Marhold [2]; Mingfa Li [2]; Bernard M. Mechler [2]; Hajo Delius [3]; Dana Hoppe [1]; Peter Stannek [1]; Carmen [...]

Abstract Background A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh. Results Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos. Conclusion We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis.

Development of non-viral gene vectors which can efficiently and safely transfect plasmid DNA and siRNA into cells is of great importance for gene therapy. Despite lots of efforts spent, it is still imperative to develop suitable gene vectors with better transfection efficiency and low cytotoxicity. To this end, we successfully designed, synthesized and screened a library of 120 polymers (via nucleophilic substitution reaction between dihalides and amines). With cell-based transfection screening assays, 120 polymers were tested to evaluate their transfection efficiency of transporting DNA and siRNA into cells. Our results indicated that hydrophobic modification could greatly enhance cationic polymers' transfection efficiency, and polymers with long linkers usually showed better transfection performance, especially for polymers with the linker of 1, 12-dibromododecane (L3 linker). Besides, polyalkylamines exhibited better transfection efficiency with the polymer particle size around 200 nm and the zeta potential in the range of + 40 mV to +50 mV. Interestingly, polymer particles made from N15HL3 not only exhibited better DNA transfection efficiency in HEK 293T cells but also showed higher siRNA transfection efficiency in U87 Luc-GFP cells together with low cell toxicity than Lipofectamine 2000 (one of commercial transfection reagents). Therefore, it is hoped that our study here not only provides promising gene vector candidates for further evaluation in gene therapy, but also provides valuable insights for better understanding of the relationship between the chemical structures and gene transfection efficiency to rationally design better non-viral gene vectors for gene therapy in the future. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Research has been conducted on growth factor antagonists which are important for mediating the inductive effects of Spemann organized in amphibian embryos. The authors have tested the hypothesis that the dual inhibition of Wnt and BMP signals could confer head organizer activity, and they report the results.

Advocates the use of online communication as a resource for meaningful research and shared observation by Montessorians. Makes suggestions regarding Web resources, purchasing a computer and hardware, obtaining Internet access, and using the Internet wisely. (KB)

The development of large medium-speed catamarans aims increasing economic viability and reducing the possible negative influence on the environment of fast sea transportation. These vessels are likely to operate at hump speed where wave-making can be the dominating component of the total resistance. Shallow water may considerably amplify the wave-making and hence the overall drag force. Computational fluid dynamics is used to predict the drag force of medium-speed catamarans at model and full scale in infinite and restricted water to study the impact on the resistance. Steady and unsteady shallow-water effects that occur in model testing or full-scale operation are taken into account using computational fluid dynamics as they are inherently included in the mathematical formulations. Unsteady effects in the ship-model response were recorded in model test experiments, computational fluid dynamics simulations and full-scale measurements and found to agree with each other. For a medium-speed catamaran in water that is restricted in width and depth, it was found that computational fluid dynamics is capable of accurately predicting the drag with a maximum deviation of no more than 6% when compared to experimental results in model scale. The influences of restricted depth and width were studied using computational fluid dynamics where steady finite width effects in shallow water and finite depth effects at finite width were quantified. Full-scale drag from computational fluid dynamics predictions in shallow water (h/L = 0.12 – 0.17) was found to be between full-scale measurements and extrapolated model test results. Finally, it is shown that current extrapolation procedures for shallow-water model tests over-estimate residuary resistance by up to 12% and underestimate frictional forces by up to 35% when compared to validated computational fluid dynamics results. This study concludes that computational fluid dynamics is a versatile tool to predict the full-scale ship resistance to a more accurate extent than extrapolation model test data and can also be utilised to estimate model sizes that keep finite-water effects to an agreed minimum. [ABSTRACT FROM AUTHOR]

Background: The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. Methods: Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. Results: More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. Conclusions: Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway. [ABSTRACT FROM AUTHOR]

Impact testing of a typical high-speed ship section has shown that the mode of vibration most likely to fall in the frequency range of excitation resulting from propeller or rotor blade passing is that where the ship frame rocks in a fore and aft direction about its base connection to the hull plate. This vibration has significant amplitude to either side of the keel and it is found that connection between the inboard and outboard sides is weak. As a consequence, vibration can occur in two modes in which the opposite sides of the hull either move together or in antiphase. This leads to the structural strain energy in the two modes being slightly different, the mode with rather less strain energy having slightly lower frequency. Because the frequencies of the two modes are close together, the transient response exhibits beating in which vibration energy is exchanged between the two sides of the hull at the low beat frequency. Vibration of the ship frames in this manner appears to have been the cause of minor weld cracking where the top of stiffeners pass through cutouts in the web of the ship frames within the fuel tank areas of the hull. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling controls many aspects of cell behavior throughout development and in adults. One of its best-known and cancer-relevant functions is to stimulate cell proliferation. Recent work has implicated Wnt components in regulating mitotic events, suggesting that the cell cycle and Wnt signaling are directly linked. This concept has now been substantially strengthened with the finding that the mitotic CDK14/cyclin Y complex promotes Wnt signaling through phosphorylation of the LRP6 co-receptor, a key regulatory nexus in the Wnt/β-catenin pathway. Thus, an unexpectedly tight collaboration between the mitotic cell cycle machinery and Wnt signaling is emerging, suggesting that this pathway might orchestrate mitotic processes. [ABSTRACT FROM AUTHOR]

Summary: Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, we show that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling. [Copyright &y& Elsevier]

To exert its activity, anthrax toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the anthrax toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in anthrax toxin endocytosis, but is not essential. We indeed found that toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR–LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling. [ABSTRACT FROM AUTHOR]

Focuses on the superimposition of Barrovian-type metamorphism on low-pressure aureoles in magmatic arcs on the northeast side of Mount Stuart batholith in Washington, that is subject to the kinetic constraints of temperature and influx of water. Spatial distribution of detectable overprint; Characteristics suggestive of magma loading.

This study shows that tile incorporation of radiolabelled UTP into RNA in Percoll-gadient-purified potato mitochondria is regulated by the redox state of the mitochondrial electron transport chain. An early indication that there might be a redox effect on RNA synthesis was a decrease in UTP incorporation in incubates containing an oxidisable substrate, such as succinate or malate. Subsequent use of a variety of electron transport inhibitors acting at different points in the electron transport chain established that the redox state of the Rieske iron-sulphur protein was the major determinant of UTP incorporation. Inhibitors acting on the substrate side of the Rieske iron-sulphur protein, and causing oxidation of components on the oxygen side of their site of action, increased UTP incorporation into RNA. These included antimycin A, myxothiazole, and undecylhydroxydioxobenzothiazole at 500 nM. Inhibitors acting on the oxygen side of the Rieske iron-sulphur protein, and causing a reduction of components on the substrate side of the block, decreased UTP incorporation. These inhibitors were undecylhydroxydioxobenzothiazole at 25 nM and KCN. When phenazine methosulphate was present as an auto-oxidisable electron sink the effect of KCN was diminished. The conclusion from the inhibitor experiments that the redox state of the Rieske iron-suphur protein was important was supported when RNA synthesis was measured at a range of redox potentials. This gave a measured redox potential for the control of UTP incorporation into RNA of +270 mV and the slope of the curve indicated an n = 1 carrier. This value is close to the reported value of the Rieske iron-suphur protein. UTP incorporation was decreased by some 50% in the presence of low concentrations of okadaic acid (5 nM), an inhibitor of protein phosphatases PP1 and PP2A, and α-naphthyl acid phosphate, a broad-spectrum phosphatase inhibitor, indicating that the redox effect on RNA synthesis may be mediated via protein phosphorylation. We did not, however, detect an expected increase in RNA synthesis when protein kinase inhibitors were used, so the involvement of protein phosphorylation in the redox regulation of RNA synthesis is as yet uncertain. [ABSTRACT FROM AUTHOR]