Behaviors of Micro-Arcs, Bubbles, and Coating Growth during Plasma Electrolytic Oxidation of ß-Titanium Alloy.

The plasma electrolytic oxidation (PEO) of a titanium alloy, Ti-15V-3Cr-3Sn-3Al, was performed to develop mechanical applications by improving the tribological characteristics. The behaviors of micro-arcs, bubbles, and coating growth during the PEO process were investigated under three different operating conditions, constant voltage (CV) operation, constant current operation (CC), and short treatment time (ST) operation, to control the surface structure and function by the PEO process. A low friction coefficient was achieved by CV operation at 500 V and by CC operation at 3.0 kA/m2. The maximum coating thickness of 6.88 µm was achieved by CV operation at 500 V and 60 s. From the observation of micro-arcs, bubbles, and discharge craters by ST operation, the minimum discharge diameter of the micro-arc was 8 µm, and the discharge craters had a discharge pore size of 0.3 µm in diameter in the center with a petal-shaped burr around the discharge pore. During the PEO process, no bubble bursts around the micro-arcs and no backfilling of the discharge pores by the ejected materials were observed. Thus, the discharge pores remain a porous structure in the PEO coating for Ti. The utilization efficiency of the total charge density by CV operation above 300 V was lower than that by the conventional anodization process. The utilization efficiency of total charge density by CC operation was higher than that by the conventional anodization process.

An Efficient Framework for Estimating the Direction of Multiple Sound Sources Using Higher-Order Generalized Singular Value Decomposition.

This paper presents an efficient framework for estimating the direction-of-arrival (DOA) of wideband sound sources. The proposed framework provides an efficient way to construct a wideband cross-correlation matrix from multiple narrowband cross-correlation matrices for all frequency bins. In addition, the proposed framework is inspired by the coherent signal subspace technique with further improvement of linear transformation procedure, and the new procedure no longer requires any process of DOA preliminary estimation by exploiting unique cross-correlation matrices between the received signal and itself on distinct frequencies, along with the higher-order generalized singular value decomposition of the array of this unique matrix. Wideband DOAs are estimated by employing any subspace-based technique for estimating narrowband DOAs, but using the proposed wideband correlation instead of the narrowband correlation matrix. It implies that the proposed framework enables cutting-edge studies in the recent narrowband subspace methods to estimate DOAs of the wideband sources directly, which result in reducing computational complexity and facilitating the estimation algorithm. Practical examples are presented to showcase its applicability and effectiveness, and the results show that the performance of fusion methods perform better than others over a range of signal-to-noise ratios with just a few sensors, which make it suitable for practical use.

An Efficient Route to Highly Substituted Indoles via Tetrahydroindol-4(5H)-one Intermediates Produced by Ring-Opening Cyclization of Spirocyclopropanes with Amines.

An efficient route to highly substituted indoles was developed. It included regioselective functionalization of tetrahydroindol-4(5H)-ones, prepared by ring-opening cyclization of cyclohexane-1,3-dione-2-spirocyclopropanes with primary amines, and subsequent oxidation. The 6-substituted indoles were synthesized from a readily available 5-substituted cyclohexane-1,3-dione-2-spirocyclopropane. The synthesis of 5- and 7-substituted indoles was achieved by regioselective electrophilic alkylation of tetrahydroindol-4(5H)-one, followed by oxidation. The 4-substituted indoles were synthesized by nucleophilic alkylation of the corresponding pyrrole derivative, which was prepared by partial oxidation of tetrahydroindol-4(5H)-one, and sequential oxidation. The synthesis of 4-substituted indoles was also accomplished by palladium-catalyzed coupling of 4-hydroxyindole-derived triflates. Furthermore, the synthesis of 4,5,6,7-tetrasubstituted indoles was achieved by using these regioselective alkylations.

An Efficient Method for the Synthesis of 2',3'-Nonsubstituted Cycloalkane-1,3-dione-2-spirocyclopropanes Using (2-Bromoethyl)diphenylsulfonium Trifluoromethanesulfonate.

An efficient and practical synthesis of 2',3'-nonsubstituted cyclohexane-1,3-dione-2-spirocyclopropanes using a sulfonium salt was achieved. The reaction of 1,3-cyclohexanediones and (2-bromoethyl)diphenylsulfonium trifluoromethanesulfonate with powdered K2CO3 in EtOAc at room temperature (r.t.) provided the corresponding spirocyclopropanes in high yields. The synthetic method was also applied to 1,3-cyclopentanedione, 1,3-cycloheptanedione, 1,3-indanedione, acyclic 1,3-diones, ethyl acetoacetate, and Meldrum's acid.

Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network.

Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/ß-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks.

Ring-opening cyclization of cyclohexane-1,3-dione-2-spirocyclopropanes with amines: rapid access to 2-substituted 4-hydroxyindole.

An efficient ring-opening cyclization of cyclohexane-1,3-dione-2-spirocyclopropanes with primary amines has been developed. The reaction proceeded at room temperature without any additives to provide 2-substituted tetrahydroindol-4-ones in good to excellent yields without the formation of the 3-substituted isomers. The obtained product was readily converted into a 2-substituted 4-hydroxyindole derivative via a synthetically useful indoline intermediate.

Cross-talk between distinct nuclear import pathways enables efficient nuclear import of E47 in conjunction with its partner transcription factors.

Nuclear import of karyophilic proteins is carried out by a variety of mechanisms. We previously showed that two basic helix-loop-helix proteins, NeuroD1 and E47, synergistically affect each other's nuclear import. In this study, we dissected the molecular pathways underlying nuclear import of the NeuroD1/E47 heterodimer. In vitro nuclear import assays indicated that importin α family members are the major nuclear import receptors for E47. However, inhibition of importin α resulted in cytoplasmic retention of E47 that could be rescued by its binding partner, NeuroD1, through heterodimerization. In addition, nuclear import of NeuroD1 was importin α independent but importin ß1 dependent. In primary neurons, localization of endogenous E47 was not affected by importin α inhibition, suggesting that neuronal E47 could be imported into the nucleus as a heterodimer with NeuroD1 by using importin ß1 alone. We also found that E47 had similar nuclear import characteristics in C2C12 cells, where E47 heterodimerized with MyoD, another helix-loop-helix protein, suggesting functional conservation within the same family of transcription factors. Collectively, our data reveal that E47 is imported into the nucleus via multiple pathways, depending on the molecular binding mode, establishing a previously uncharacterized cross-talk between two distinct nuclear import pathways.

Proteomic analysis of importin α-interacting proteins in adult mouse brain.

Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor ß1 (TRß1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TRß1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin α5 together with importin ß and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin α5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.

Altered protein expression in endometrial carcinogenesis.

We have discovered several protein biomarkers that are altered during carcinogenesis of the human uterine endometrium. Proteins prepared from 19 endometrial carcinomas (Group A), and 20 normal endometria obtained from benign gynecological conditions (Group B), were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). Two proteins, EC1 and EC2, were consistently expressed differentially. EC1 had an increased level of expression in carcinomas (P<0.001), while EC2 was expressed at a lower level (P=0.004). The isoelectric points of EC1 and EC2 were approximately pH 5.0 and 7.0, respectively. These proteins are thus potential biomarkers for detection of endometrial carcinoma.

14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1).

p27(Kip1) (p27), a CDK inhibitor, migrates into the nucleus, where it controls cyclin-CDK complex activity for proper cell cycle progression. We report here that the classical bipartite-type basic amino-acid cluster and the two downstream amino acids of the C-terminal region of p27 function as a nuclear localization signal (NLS) for its full nuclear import activity. Importin alpha3 and alpha5, but not alpha1, transported p27 into the nucleus in conjunction with importin beta, as evidenced by an in vitro transport assay. It is known that Akt phosphorylates Thr 157 of p27 and this reduces the nuclear import activity of p27. Using a pull-down experiment, 14-3-3 was identified as the Thr157-phosphorylated p27NLS-binding protein. Although importin alpha5 bound to Thr157-phosphorylated p27NLS, 14-3-3 competed with importin alpha5 for binding to it. Thus, 14-3-3 sequestered phosphorylated p27NLS from importin alpha binding, resulting in cytoplasmic localization of NLS-phosphorylated p27. These findings indicate that 14-3-3 suppresses importin alpha/beta-dependent nuclear localization of Thr157-phosphorylated p27, suggesting implications for cell cycle disorder in Akt-activated cancer cells.

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner.

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.