Nedd4-2-dependent ubiquitination potentiates the inhibition of human NHE3 by cholera toxin and enteropathogenic Escherichia coli

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Naþ/Hþ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Naþ/ Hþ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2–hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans

Quasinormal modes in the background of charged Kaluza-Klein black hole with squashed horizons

We study the scalar perturbation in the background of the charged Kaluza-Klein black holes with squashed horizons. We find that the position of infinite discontinuities of the heat capacities can be reflected in quasinormal spectrum. This shows the possible non-trivial relation between the thermodynamical and dynamical properties of black holes.Comment: revised version, accepted for publication in Phys.Lett.

Study in Wire Feedability-Related Properties of Al-5Mg Solid Wire Electrodes Bearing Zr for High-Speed Train

This work offers an analysis of the wire feedability-related properties of Al-5Mg solid wire electrodes bearing Zr. Effects of Zr content on microstructures and mechanical properties of the Al-5Mg alloys were studied. Experimental results have demonstrated that α-Al dendrites of the as-cast Al-5Mg alloy are refined, and the tensile strength, microhardness and roughness of the 1.2 mm wire electrode are improved with an appropriate addition of Zr. In addition, the tensile strength and elongation of the welded joints welded using Al-5Mg wire electrodes bearing Zr reach the maximum value when 0.12% Zr is added into the wire alloy. However, when excess Zr is added, α-Al phases of the wire alloy and welded joint are coarsened, and the mechanical properties are deteriorated. Moreover, the structure and principle of a novel apparatus, which can enhance the feedability of the wire electrode, are introduced and the apparatus can achieve the rough and fine adjustments of cast and helix of the wire electrode

Influence of Hydrogen Content on the Microstructure and Mechanical Properties of ER5183 Wires

This work focused on the influence of hydrogen content on the microstructure and mechanical properties of ER5183 Al-Mg-Mn alloy wires for aluminum alloy welding. The hydrogen content of the ER5183 wires was measured, the macroscopic and microscopic morphologies of fractures were observed as well as the microstructure of the wires, and the tensile strength of the wires was also tested and investigated. The experimental results demonstrated three typical irregular macroscopic fractures of the wires appeared during the drawing process when the hydrogen content exceeded 0.23 μg/g. In the meantime, the aggregated pores were observed in the microstructure of the ϕ5.2 mm wire with the hydrogen content of 0.38 μg/g. Such defects may become the origin of cracks in subsequent processing and tensile tests. Moreover, higher hydrogen content in the ϕ5.2 mm welding wire will bring obvious changes in the fracture surface, which are internal cracks and micropores replacing the original uniform and compact dimples. With the higher hydrogen content, the tensile strength and plastic strain rate of ϕ1.2 mm wires would decrease. At the same time, unstable crack propagation would occur during the process of plastic deformation, leading to fracture. Considering the mechanical properties and microstructure, the hydrogen content of the ER5183 wires should be controlled below 0.23 μg/g

Effects of Porosity, Heat Input and Post-Weld Heat Treatment on the Microstructure and Mechanical Properties of TIG Welded Joints of AA6082-T6

Various heat input conditions and post-weld heat treatments were adopted to investigate the microstructure evolution and mechanical properties of tungsten inert gas (TIG) welded joints of AA6082-T6 with porosity defects. The results show that the fracture location is uncertain when an as-welded joint has porosities in the weld zone (WZ), and overaging in the heat-affected zone (HAZ) at the same time. When the fracture of the as-welded joint occurs in the HAZ, the total heat input has a linear relation with the tensile strength of the joint. An excess heat input induces the overgrowth of Mg2Si precipitates in HAZ and the coarsening of α-Al grains in WZ, resulting in a decrease in the microhardness of the corresponding areas. After artificial aging treatment, the tensile strength of the welded joint is increased by approximately 9–13% as compared to that of as-welded joint, and fracture also occurs in HAZ. In contrast, for solution treated and artificial aging treated joint, fracture occurs suddenly at the rising phase of the tensile curve due to porosity defects throughout the weld metal. Furthermore, the eutectic Si particles of WZ coarsen and spheroidize after solution treatment and artificial aging treatment, due to the diffusion of Si to the surface of the original Si phases when soaking at high temperature

Evaluation of Reliability and Validity of the Hendrich II Fall Risk Model in a Chinese Hospital Population.

To translate, validate and examine the reliability and validity of a Chinese version of the Hendrich II Fall risk Model (HFRM) in predicting falls in elderly inpatient. A sample of 989 Chinese elderly inpatients was recruited upon admission at the Peking Union Medical College Hospital. The inpatients were assessed for fall risk using the Chinese version of the HFRM at admission. The reliability of the Chinese version of the HFRM was determined using the internal consistency and test-rested methods. Validity was determined using construct validity and convergent validity. Receiver operating characteristic (ROC) curves were created to determine the sensitivity and specificity. The Chinese version of the HFRM showed excellent repeatability with an intra-class correlation coefficient (ICC) of 0.9950 (95% confidence interval (CI): 0.9923-0.9984). The inter-rater reliability was high with an ICC of 0.9950 (95%CI: 0.9923-0.9984). Cronbach's alpha coefficient was 0.366. Content validity was excellent, with a content validity ratio of 0.9333. The Chinese version of the HFRM had a sensitivity of 72% and a specificity of 69% when using a cut-off of 5 points on the scale. The area under the curve (AUC) was 0.815 (P<0.001). The Chinese version of the HFRM showed good reliability and validity in assessing the risk of fall in Chinese elderly inpatients

Human and non-human intestinal NHE3 : human NHE3 demonstrates increased susceptibility to inhibition and unique regulation by ubiquitin

Introduction: The Na+/H+ Exchanger 3 (NHE3) is responsible for the majority of the electroneutral sodium absorption occurring in the intestine. As such, NHE3 has a major role in controlling electrolyte and fluid balance and is a frequent target of inhibition in many diarrheal diseases. While mice and rabbits have been used to investigate the mechanisms of diarrhea, they are less prone to develop diarrhea than humans. Recently, we have shown that human NHE3, but not non-human NHE3s, interacts with the ubiquitin E3 ligase Nedd4-2. We hypothesize that this property of human NHE3 contributes to the increased severity of diarrhea. Method and Materials: To investigate this hypothesis, we determined human and non-human NHE3 activities and ubiquitination levels in response to the NHE3 inhibitors forskolin (FSK), cholera toxin (CTX) or Enteropathogenic E.coli (EPEC). In vitro, we generated Caco-2/bbe cells transfected with human or rabbit NHE3, and in vivo we generated transgenic mice expressing human NHE3 in the intestine (hNHE3int). For in vitro experiments, inhibitor treatments of 30 and 90 min were directly applied to cells diluted with normal growth media. NHE3 activity was measured by Na+ dependent intracellular pH recovery. NHE3 ubiquitination was evaluated by immunoprecipitation of NHE3 followed by western blot of ubiquitin. Nedd4-2 was knocked down in cells via electroporation. For in vivo experiments, mice were anesthetised with a ketamine/xylazine cocktail and a 2 - 5 cm section of ileum was tied off and injected with inhibitor treatment or Hanks Buffered Saline Solution (HBSS) vehicle buffer. Mice were allowed to recover for 5h post-injection before cervical dislocation was performed. Closed loops were removed, measured and weighed, and villi were dissected and used for NHE3 activity analysis. Results: In vitro, we found that 10uM CTX significantly increased human NHE3 ubiquitination. Both CTX (1-10 uM) and EPEC treatments induced significantly more inhibition of human NHE3 activity in Caco-2/bbe cells than what was observed in rabbit NHE3 transfected cells. Nedd4-2 knockdown blunted the inhibitory effect on human NHE3, demonstrating the importance of Nedd4-2 in regulating human NHE3. In vivo, NHE3 knockout mice (NHE3-/-) have previously been shown to display symptoms of diarrhea. However, our model of hNHE3int mice did not show any signs of diarrhea, indicating that the transgenic hNHE3 is functional. In anesthetised hNHE3int mice, we found that both 5h closed-loop intestinal treatment with inhibitors EPEC (2 ×108 CFU) and CTX (10ug) significantly increased water accumulation in the small intestine and significantly reduced NHE3 activity compared to wild type mice. Conclusion: These findings demonstrate that human and non-human NHE3s are differentially regulated, suggesting that the characteristics of human NHE3 regulation may contribute to increased diarrhea severity in humans