Edited by Jeffrey E. Pessin Alterations in endoplasmic reticulum (ER) calcium (Ca 2؉) levels diminish insulin secretion and reduce -cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca 2؉ loss in  cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP 3 R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca 2؉ imaging techniques in INS-1  cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP 3 R function. Our results revealed that the RyR is sensitive mainly to ER stress-induced dysfunction, whereas cytokine stress specifically alters IP 3 R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP 3 R with xestospongin C prevented ER Ca 2؉ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented -cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca 2؉ oscillations in tunicamycin-treated INS-1  cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca 2؉ transients that depend on both ER Ca 2؉ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca 2؉ dynamics in  cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca 2؉ may be an early pathogenic event in diabetes. This work was supported by National Institutes of Health Grants R01 DK093954 and UC4 DK 104166 (to C. E-M.); Department of Veterans Affairs Merit Award I01BX001733 (to C. E-M.); and Sigma Beta Sorority, Ball Brothers Foundation, and George and Frances Ball Foundation gifts (to C. E-M.).
Water or aqueous electrolytes are the dominant components in electrowetting on dielectric (EWOD)-based microfluidic devices. Low thermal stability, evaporation, and a propensity to facilitate corrosion of the metal parts of integrated circuits or electronics are drawbacks of aqueous solutions. The alternative use of ionic liquids (ILs) as electrowetting agents in EWOD-based applications or devices could overcome these limitations. Efficient EWOD devices could be developed using task-specific ILs. In this regard, a fundamental study on the electrowetting properties of ILs is essential. Therefore electrowetting properties of 19 different ionic liquids, including mono-, di-, and tricationic, plus mono- and dianionic ILs were examined. All tested ILs showed electrowetting of various magnitudes on an amorphous flouropolymer layer. The effects of IL structure, functionality, and charge density on the electrowetting properties were studied. The enhanced stability of ILs in electrowetting on dielectric at higher voltages was studied in comparison with water. Deviations from classical electrowetting theory were confirmed. The physical properties of ILs and their electrowetting properties were tabulated. These data can be used as references to engineer task-specific electrowetting agents (ILs) for future electrowetting-based applications.
Dynamic solvation of the dye coumarin 153 is studied in a phosphonium ionic liquid: hexadecyltributylphosphonium bromide, [(C4)3C16P+][Br-]. It forms micelles in water, and the bulk also exists as a liquid under our experimental conditions. This system permits a comparison with an imidazolium ionic liquid studied earlier, which also formed micelles in water (J. Phys. Chem. A 2006, 110, 10725−10730). We conclude that our analysis of the comparable situation in a phosphonium liquid is not as definitive as we had proposed earlier, i.e., that the majority of the early-time solvation arises from the organic cation. Part of the difficulty in performing this analysis is most likely due to the amount of water that is associated with the micelle. In the course of this work, we have focused on the calculation of the solvation correlation function, C(t), and investigated how it depends upon the methods with which the "zero-time" spectrum is constructed. Arlington, Box 19065, Arlington, Texas 76019 ReceiVed: NoVember 8, 2007; In Final Form: December 7, 2007 Dynamic solvation of the dye coumarin 153 is studied in a phosphonium ionic liquid: hexadecyltributylphosphonium bromide, [(C 4 ) 3 C 16 P + ][Br -]. It forms micelles in water, and the bulk also exists as a liquid under our experimental conditions. This system permits a comparison with an imidazolium ionic liquid studied earlier, which also formed micelles in water (J. Phys. Chem. A 2006, 110, 10725-10730). We conclude that our analysis of the comparable situation in a phosphonium liquid is not as definitive as we had proposed earlier, i.e., that the majority of the early-time solvation arises from the organic cation. Part of the difficulty in performing this analysis is most likely due to the amount of water that is associated with the micelle. In the course of this work, we have focused on the calculation of the solvation correlation function, C(t), and investigated how it depends upon the methods with which the "zero-time" spectrum is constructed. Keywords
Temperature sensitive (TS) missense mutants have been foundational for characterization of essentialgene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TSmissense mutants within the context of their functional proteomes is lacking. We applied massspectrometry (MS) based thermal proteome profiling (TPP) to investigate the proteome-wide effects ofmissense mutations in an application that we refer to as mutant Thermal Proteome Profiling (mTPP).This study characterized global impacts of temperature sensitivity-inducing missense mutations in twodifferent subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and globalproteomics were similar between the mutants, which could suggest that a similar functional disruption isoccurring in both missense variants. Results from mTPP, however, provide unique insights into themechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were notobtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is aprecise approach to measure changes in missense mutant containing proteomes without the requirementfor large amounts of starting material, specific antibodies against proteins of interest, and/or geneticmanipulation of the biological system. Although experiments were performed under permissiveconditions, mTPP provided insights into the underlying protein stability changes that cause dramaticcellular phenotypes observed at non-permissive temperatures. Overall, mTPP provides uniquemechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in arapid, non-biased fashion.
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