A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR

Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an “indirect/reflected” detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a “chimeric membrane protein”-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained ¹⁵N-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.     

Heat transfer mechanisms in liquid pool fires

The heat transfer mechanisms in liquid pool fires were studied. The heat balance in the combustion system which includes both the liquid fuel and the burner vessel was discussed. The convective heat from the vessel wall to the fuel is dominant in small scale pool fires, and was calculated using the finite difference program. The heat flux between the wall and the fuel during flame spreading over the fuel below its flash point in the vessel of small width was calculated. From the results, it was found that the poorer the thermal conductivity of the vessel the larger the heat flux from the wall to the fuel immediately below the fuel surface. The liquid level at which a flame self-quenches was measured for various materials and wall thicknesses of the vessel. The concentration gradient of fuel at the self-quenching liquid level was found to be nearly constant for a given fuel.     

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.     

Development of an in-air RBS technique using a metal capillary

We have developed a simple Rutherford backscattering spectroscopic (RBS) method to analyze sample depth profiles in air. To avoid excessive energy loss of projectile ions in air, we have used a 3 MeV proton beam extracted into air with a metal capillary. Using this capillary, we were able to extract a sufficiently large proton beam current to perform in-air RBS and in-air PIXE without requiring any window, such as a thin film, between a vacuum chamber and air. We have validated our technique by analyzing the depth profiles for Au foils of various thicknesses, 0.25, 0.75, and 2.5 μm. A comparison of the experimental results with a simple theoretical calculation indicates that this technique is useful for analyzing the depth profile of any specimen in air.     

TEM,XPS and SIMS Analyzes on Grain Boundary of Lanthanum Chromites

The morphological characteristics, chromium valence state, and cation transport in the vicinity of grain boundary in La_1-xCa_xCrO₃ were investigated by using TEM/EDS, XPS and SIMS techniques. The width of grain boundary was around 1 nm where anomalous enrichment of calcium was observed. Higher valence state of chromium such as Cr⁶⁺(d⁰) was detected in the grain boundaries whereas Cr³⁺(d³) and Cr⁴⁺(d²) were dominant in the bulk. Very fast interdiffusion of alkaline earths was observed in the Sr²⁺-La_0.75Ca_0.25CrO₃ system. All observed phenomena were correlated by assuming the A-site vacancy which may be induced by the formation of Cr⁶⁺ at grain boundaries.     

Phase diagram calculations of ZrO2-based ceramics with an emphasis on the reduction/oxidation equilibria of cerium ions in the ZrO2-YO1.5-CeO2-CeO1.5 system

Phase diagram calculations that were made previously for the ZrO₂-MO _m/2 (m = 2, 3, 4) systems and for the ZrO₂-YO_1.5-MO _m/2 (M = transition metals) systems have been extended to the ZrO₂-YO_1.5-CeO₂(-CeO_1.5) system to make an attempt to explain (1) thermogravimetric (TG) results as a function of oxygen potential, (2) electronic conductivity as a function of oxygen potential, and (3) a miscibility gap observed in air. The interaction parameters for the CeO₂-CeO_1.5-YO_1.5 system were obtained from the reported oxygen nonstoichiometry in CeO_2−x and rate earth doped ceria, (Ce,RE)O_2−δ . The interaction parameters for the ZrO₂-CeO₂ subsystem were obtained so as to reproduce the observed miscibility gap at 1273 K. Those thermodynamic properties can reproduce consistently the experimental behaviors of the electronic conductivity and the TG results in the (Zr_1−x Ce _x )_0.8Y_0.2O_1.9 solid solutions; these indicate the enhancement of reduction of CeO₂.     

NUCLEAR MAGNETIC RESONANCE STUDIES OF ASPHALTENES AND COALS. A NEW APPROACH TO OBTAINING ASPHALTENE STRUCTURAL PARAMETERS WITH DIPOLAR DEPHASING

Apparent carbon aromaticities, fa, of some asphaltenes have been measured by n.m.r. in solution and compared with those obtained by the CP/MAS technique on solid samples. The aromaticities were also measured for several Alberta plains coals. From the 1H solution spectra and 13C solid nmr spectra of asphaltenes, some of their skeletal features, not obtainable from solution spectra only, have been suggested.     

Chemical Potential Diagrams for Rare Earth-Transition Metal-Oxygen Systems: I, Ln-V-O and Ln-Mn-O System

The thermodynamic properties of double oxides in rare earth-transition metal (V and Mn)-oxygen ternary systems have been collected and evaluated to examine their stability using chemical potential diagrams plotted as log (α_A/α_B) vs log ( P (O₂)). The thermodynamic regularities of perovskites, pyrochlores, and other compounds have been discussed in terms of the effective ionic radii across a series of rare earths. The obtained regularities have been used to estimate tentatively the thermodynamic properties of some rare-earth vanadium and manganese oxides.     

Adaptive control design for a class of nonsmooth nonlinear systems with matched and linearly parameterized uncertainty

This paper presents an adaptive control design for a class of nonsmooth nonlinear systems with matched uncertainty, which is linearly parameterized with a known discontinuous function. The design framework is based on the concept of the Filippov solution as the classical Lyapunov theory for smooth systems cannot be applied to establish the stability of the adaptive control system due to the presence of the discontinuity. It is proved that as an adaptive control system, the global Lyapunov stability with the convergence of the state of the controlled system to the origin can be achieved by evaluating the monotonicity of the Lyapunov function in the state space, particularly on the discontinuous surface, while the uniqueness of the solution of the closed‐loop system is not necessarily guaranteed. Some interesting numerical examples are demonstrated with simulation results. Copyright © 2008 John Wiley & Sons, Ltd.     

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.