Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its catalytic function. The predicted position on Chk1 of the phosphate group from Claspin corresponds to the location of activation-loop phosphorylation in various kinases. In addition, we have obtained evidence that the C-terminal regulatory domain of Chk1, which does not form a stable complex with Claspin under our assay conditions, nonetheless has some role in Claspin-dependent activation. Overall, these results indicate that Claspin docks with a phosphate-binding site in the catalytic domain of Chk1 during activation by ATR. Phosphorylated Claspin may mimic an activating phosphorylation of Chk1 during this process

THE ROLE OF TRAF6 PHOSPHORYLATION IN Src/TRAF6-MEDIATED IKK, JNK, Akt ACTIVATION AND TUMORIGENESIS

TRAF6 E3 ligase regulates numerous essential biological processes such as innate immune response, cell survival and osteoclast differentiation. Upon activation, it mediates activation of IKK/NF-κB and JNK signaling in response to engagement of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), and receptor activator of NF-κB (RANK) to their cognate ligands, including lipopolysaccharide (LPS), IL-1, and RANK ligand (RANKL). Recently, TRAF6 has also been shown to be involved in Akt signaling activation upon activation of insulin-like growth factor-1 receptor (IGF-1R), in turn orchestrating cell survival and tumorigenesis. Therefore, TRAF6 is a key player for the activation of IKK, JNK and Akt by diverse receptor signaling. Although TRAF6 activity is induced by these receptors, the mechanism by which TRAF6 is activated under these stimulating conditions remains largely unclear. Here, we show that TRAF6 Y473 phosphorylation is commonly induced and serves as an essential mode for TRAF6 activation by multiple signaling stimuli including IGF-1, IL-1, LPS, and RANKL. We identify Src tyrosine kinase as a direct kinase for TRAF6 and a mediator of TRAF6 Y473 phosphorylation. Notably, we find that Src or Traf6 deficiency impairs IKK, Akt, and JNK activation, which can be rescued by phosphomimetic TRAF6, but not by phosphorylation-dead TRAF6 mutant. Mechanistically, we show that TRAF6 phosphorylation is imperative for TRAF6 dimerization/oligomerization and its binding to E2 enzyme, UBC13. Significantly, we find that TRAF6 phosphorylation is critical for TRAF6-mediated cancer progression and represents a biomarker for poor survival outcome. Our study therefore resolves the long-standing puzzle of achievement of TRAF6 activation and elucidates TRAF6 phosphorylation as a central mode for Src/TRAF6-mediated downstream kinase activation and tumorigenesi

Hepatic Parasitic Abscess Caused by Clonorchiasis: Unusual CT Findings of Clonorchiasis

Clonorchiasis is caused by a chronic infestation of liver flukes, Clonorchis sinensis, and these reside mainly in the medium- and small-sized intrahepatic bile ducts. Therefore, diffuse, uniform, minimal or mild dilatation of these bile ducts, particularly in the periphery, without dilatation of the extrahepatic bile duct is the typical finding on several imaging modalities. We report here on the CT findings of an unusual case of hepatic parasitic abscess that was caused by clonorchiasis; this malady mimicked cholangiocarcinoma, and there was no dilatation of the intrahepatic bile ducts

Adaptive Fuzzy Dynamic Surface Sliding Mode Position Control for a Robot Manipulator with Friction and Deadzone

Precise tracking positioning performance in the presence of both the deadzone and friction of a robot manipulator actuator is difficult to achieve by traditional control methodology without proper nonlinear compensation schemes. In this paper, we present a dynamic surface sliding mode control scheme combined with an adaptive fuzzy system, state observer, and parameter estimator to estimate the uncertainty, friction, and deadzone nonlinearities of a robot manipulator system. We design a dynamic surface sliding mode basic controller by systematic recursive design steps that yields several adaptive laws for the compensation of nonlinear friction, deadzone, and other unknown nonlinear dynamics. The boundedness and convergence of this closed-loop system are guaranteed by the Lyapunov stability theorem. Experiments on the Scorbot robot manipulator demonstrate the validity and effectiveness of the proposed control scheme

Solid–liquid interface temperature measurement of evaporating droplet using thermoresponsive polymer aqueous solution

The present study aims to measure the solid–liquid interface temperature of an evaporating droplet on a heated surface using a thermoresponsive polymer. Poly(N-isopropylacrylamide) (pNIPAM) was used owing to its sensitive optical and mechanical properties to the temperature. We also measured the refractive index variation of the pNIPAM solution by using the surface plasmon resonance imaging (SPRi). In particular, the present study proposed a new method to measure the solid–liquid interface temperature using the correlation among reflectance, refractive index, and temperature. It was found that the reflectance of a pNIPAM solution decreased after the droplet deposition. The solid–liquid interface temperature, estimated from the reflectance, showed a lower value at the center of the droplet, and it gradually increased along the radial direction. The lowest temperature at the contact line region is present because of the maximum evaporative cooling. Moreover, the solid–liquid interface temperature deviation increased with the surface temperature, which means solid–liquid interface temperature should be considered at high temperature to predict the evaporation flux of the droplet accurately

Overexpression of Arabidopsis thaliana brassinosteroid-related acyltransferase 1 gene induces brassinosteroid-deficient phenotypes in creeping bentgrass

Brassinosteroids (BRs) are naturally occurring steroidal hormones that play diverse roles in various processes during plant growth and development. Thus, genetic manipulation of endogenous BR levels might offer a way of improving the agronomic traits of crops, including plant architecture and stress tolerance. In this study, we produced transgenic creeping bentgrass (Agrostis stolonifera L.) overexpressing a BR-inactivating enzyme, Arabidopsis thaliana BR-related acyltransferase 1 (AtBAT1), which is known to catalyze the conversion of BR intermediates to inactive acylated conjugates. After putative transgenic plants were selected using herbicide resistance assay, genomic integration of the AtBAT1 gene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by northern blot analysis. The transgenic creeping bentgrass plants exhibited BR-deficient phenotypes, including reduced plant height with shortened internodes (i.e., semi-dwarf), reduced leaf growth rates with short, wide, and thick architecture, high chlorophyll contents, decreased numbers of vascular bundles, and large lamina joint bending angles (i.e., erect leaves). Subsequent analyses showed that the transgenic plants had significantly reduced amounts of endogenous BR intermediates, including typhasterol, 6-deoxocastasterone, and castasterone. Moreover, the AtBAT1 transgenic plants displayed drought tolerance as well as delayed senescence. Therefore, the results of the present study demonstrate that overexpression of an Arabidopsis BR-inactivating enzyme can reduce the endogenous levels of BRs in creeping bentgrass resulting in BR-deficient phenotypes, indicating that the AtBAT1 gene from a dicot plant is also functional in the monocot crop.111Ysciescopu

PD-1 deficiency protects experimental colitis via alteration of gut microbiota

Programmed cell death-1 (PD-1) is a coinhibitory molecule and plays a pivotal role in immune regulation. Here, we demonstrate a role for PD-1 in pathogenesis of inflammatory bowel disease (IBD). Wild-type (WT) mice had severe wasting disease during experimentally induced colitis, while mice deficient for PD-1 (PD-1(-/-)) did not develop colon inflammation. Interestingly, PD-1(-/-) mice cohoused with WT mice became susceptible to colitis, suggesting that resistance of PD-1(-/-) mice to colitis is dependent on their gut microbiota. 16S rRNA gene-pyrosequencing analysis showed that PD-1(-/-) mice had altered composition of gut microbiota with significant reduction in Rikenellaceae family. These altered colon bacteria of PD-1(-/-) mice induced less amount of inflammatory mediators from colon epithelial cells, including interleukin (IL)-6, and inflammatory chemokines. Taken together, our study indicates that PD-1 expression is involved in the resistance to experimental colitis through altered bacterial communities of colon.112Ysciescopuskc