Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain.

Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.

Moiré-free fingerprint sensors based on multilayer oxide-metal-oxide electrodes.

The display quality of touchscreen devices with on-screen fingerprint sensors is reduced by moiré patterns, interference phenomena caused by an overlap between the pixel pattern of the display, and the electrode pattern of the fingerprint sensor. A promising strategy for resolving this issue is to reduce the visibility of the moiré pattern, by including a filling layer with a transmittance similar to that of the electrodes, between the different patterns. We propose a moiré-free fingerprint sensor that uses an oxide-metal-oxide (IZO/Ag/IZO) multilayer as a highly transparent electrode. To verify the moiré reduction effect, we conducted a two-dimensional spectral analysis to calculate the spatial frequencies of the superimposed image of the display and the sensor patterns, and demonstrated experimentally that the proposed electrode greatly reduces the undesirable moiré phenomenon.

Transdermal Properties of Cell-Penetrating Peptides: Applications and Skin Penetration Mechanisms.

Cell-penetrating peptides (CPPs) consist of 5-30 amino acids with intracellular transduction abilities and diverse physicochemical properties, origins, and sequences. Although recent developments in bioinformatics have facilitated the prediction of CPP candidates with the potential for transduction into cells, the mechanisms by which CPPs penetrate cells and various tissues have not yet been elucidated at the molecular interaction level. Recently, the skin-penetrating ability of CPPs has gained wide attention and emerged as a simple and effective strategy for the delivery of macromolecules into the skin. Studies on the skin structure have suggested that the penetration potential of CPPs is based on the molecular interactions and characteristics of the lipid lamellar structure between corneocytes in the stratum corneum. This review provides a brief overview of the general properties, transduction mechanisms, applications, and safety issues of CPPs, focusing on CPPs with transdermal properties, that are currently being used to develop therapeutics and cosmetics.

The Influence of Capping Layers on Tunneling Magnetoresistance and Microstructure in CoFeB/MgO/CoFeB Magnetic Tunnel Junctions upon Annealing.

This study investigates the effects of annealing on the tunnel magnetoresistance (TMR) ratio in CoFeB/MgO/CoFeB-based magnetic tunnel junctions (MTJs) with different capping layers and correlates them with microstructural changes. It is found that the capping layer plays an important role in determining the maximum TMR ratio and the corresponding annealing temperature (Tann). For a Pt capping layer, the TMR reaches ~95% at a Tann of 350 °C, then decreases upon a further increase in Tann. A microstructural analysis reveals that the low TMR is due to severe intermixing in the Pt/CoFeB layers. On the other hand, when introducing a Ta capping layer with suppressed diffusion into the CoFeB layer, the TMR continues to increase with Tann up to 400 °C, reaching ~250%. Our findings indicate that the proper selection of a capping layer can increase the annealing temperature of MTJs so that it becomes compatible with the complementary metal-oxide-semiconductor backend process.

Efficient transdermal delivery of functional protein cargoes by a hydrophobic peptide MTD 1067.

The skin has a protective barrier against the external environment, making the transdermal delivery of active macromolecules very difficult. Cell-penetrating peptides (CPPs) have been accepted as useful delivery tools owing to their high transduction efficiency and low cytotoxicity. In this study, we evaluated the hydrophobic peptide, macromolecule transduction domain 1067 (MTD 1067) as a CPP for the transdermal delivery of protein cargoes of various sizes, including growth hormone-releasing hexapeptide-6 (GHRP-6), a truncated form of insulin-like growth factor-I (des(1-3)IGF-I), and platelet-derived growth factor BB (PDGF-BB). The MTD 1067-conjugated GHRP-6 (MTD-GHRP-6) was chemically synthesized, whereas the MTD 1067-conjugated des(1-3)IGF-I and PDGF-BB proteins (MTD-des(1-3)IGF-I and MTD-PDGF-BB) were generated as recombinant proteins. All the MTD 1067-conjugated cargoes exhibited biological activities identical or improved when compared to those of the original cargoes. The analysis of confocal microscopy images showed that MTD-GHRP-6, MTD-des(1-3)IGF-I, and MTD-PDGF-BB were detected at 4.4-, 18.8-, and 32.9-times higher levels in the dermis, respectively, compared to the control group without MTD. Furthermore, the MTD 1067-conjugated cargoes did not show cytotoxicity. Altogether, our data demonstrate the potential of MTD 1067 conjugation in developing functional macromolecules for cosmetics and drugs with enhanced transdermal permeability.

Fabrication and MFM study of 60 nm track-pitch discrete track media.

Discrete track magnetic recording media with a 60 nm track pitch and prewritten servo patterns were fabricated and tested for read/write performance, and a feasibility analysis of the embedded servo was performed. The fabrication process consisted of ultraviolet nanoimprint lithography (UV-NIL) and sequential ion beam etching on a conventional perpendicular magnetic recording medium. Magnetic patterns were written to the fabricated tracks at 700 kilo flux changes per inch (kFCI) using a spin stand and were read using magnetic force microscopy (MFM), with a resulting signal-to-noise ratio (SNR) of 12.15 dB. The servo pattern was also visualized with MFM. These results demonstrated the feasibility of writing to a 30 nm wide discrete data track and the workability of the embedded servo pattern.

On-screen fingerprint sensor with optically and electrically tailored transparent electrode patterns for use on high-resolution mobile displays.

In this study, a mutual capacitive-type on-screen fingerprint sensor, which can recognize fingerprints on a display screen to provide smartphones with full-screen displays with a minimal bezel area, is fabricated. On-screen fingerprint sensors are fabricated using an indium tin oxide transparent conductor with a sheet resistance of ~10 Ω/sq. and a transmittance of ~94% (~86% with the substrate effect) in the visible wavelength range, and assembled onto a display panel. Even at this high transmittance, the electrodes can degrade the display quality when they are placed on the display. The interference between periodic display pixel arrays and sensor patterns can lead to the Moiré phenomenon. It is necessary to find an appropriate sensor pattern that minimizes the Moiré pattern, while maintaining the signal sensitivity. To search for appropriate patterns, a numerical calculation is carried out over wide ranges of pitches and rotation angles. The range is narrowed for an experimental evaluation, which is used to finally determine the sensor design. As the selected sensor pitches are too small to detect capacitance variations, three unit patterns are electrically connected to obtain a unit block generating a larger signal. By applying the selected sensor pattern and circuit driving by block, fingerprint sensing on a display is demonstrated with a prototype built on a commercial smartphone.

The fabrication of Co-Pt electro-deposited bit patterned media with nanoimprint lithography.

Bit patterned media with 25 nm hole diameter and 50 nm pitch size were fabricated with serial processes comprising master patterning with electron-beam lithography, a Si etching process, multi-layer soft stamp replication, and UV nanoimprinting, followed by Co-Pt magnetic material filling by electro-deposition. From these processes, the designed patterns were well defined, and perpendicular magnetic anisotropy of the fabricated bit patterned media was obtained.

Comparison of chemical compositions and antimicrobial activities of essential oils from three conifer trees; Pinus densiflora, Cryptomeria japonica, and Chamaecyparis obtusa.

The chemical compositions, and antibacterial and antifungal effects of essential oils extracted from three coniferous species, Pinus densiflora, Cryptomeria japonica, and Chamaecyparis obtusa, were investigated. Gas chromatography mass analysis of the essential oils revealed that the major components and the percentage of each essential oil were 16.66% beta-phellandrene and 14.85% alpha-pinene in P. densiflora; 31.45% kaur-16-ene and 11.06% sabinene in C. japonica; and 18.75% bicyclo [2, 2, 1] heptan-2-ol and 17.41% 2-carene in Ch. obtusa. The antimicrobial assay by agar disc diffusion method showed that 2.2 microg of Ch. obtusa oil inhibited most effectively the growth of Escherichia coli ATCC 33312 and Klebsiella oxytoca ATCC 10031, whereas the C. japonica oil gave weak antimicrobial activity. The minimal inhibitory concentration (MIC) values for bacterial strains were in the range of 5.45-21.8 mg/ml depending on essential oils, but most Gram-negative bacteria were resistant even at 21.8 mg oil/ml. P. densiflora oil showed the most effective antifungal activity and the MIC values for Cryptococcus neoformans B42419 and Candida glabrata YFCC 062CCM 11658 were as low as 0.545 and 2.18 mg/ml, respectively. Cryp. neoformans B42419 was the most sensitive to all essential oils in the range of 0.545-2.18 mg/ml. Our data clearly showed that the essential oils from the three conifers had effective antimicrobial activity, especially against fungi.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity.

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta. TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (K(D)) of approximately 5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.

Work environments and exposure to hazardous substances in korean tire manufacturing.

OBJECTIVES: The purpose of this study is to evaluate the tire manufacturing work environments extensively and to identify workers' exposure to hazardous substances in various work processes. METHODS: Personal air sampling was conducted to measure polycyclic aromatic hydrocarbons, carbon disulfide, 1,3-butadiene, styrene, methyl isobutyl ketone, methylcyclohexane, formaldehyde, sulfur dioxide, and rubber fume in tire manufacturing plants using the National Institute for Occupational Safety Health Manual of Analytical Methods. Noise, carbon monoxide, and heat stress exposure were evaluated using direct reading instruments. Past concentrations of rubber fume were assessed using regression analysis of total particulate data from 2003 to 2007, after identifying the correlation between the concentration of total particulate and rubber fume. RESULTS: Workers were exposed to rubber fume that exceeded 0.6 mg/m(3), the maximum exposure limit of the UK, in curing and production management processes. Forty-seven percent of workers were exposed to noise levels exceeding 85 dBA. Workers in the production management process were exposed to 28.1℃ (wet bulb globe temperature value, WBGT value) even when the outdoor atmosphere was 2.7℃ (WBGT value). Exposures to other substances were below the limit of detection or under a tenth of the threshold limit values given by the American Conference of Governmental Industrial Hygienists. CONCLUSION: To better classify exposure groups and to improve work environments, examining closely at rubber fume components and temperature as risk indicators in tire manufacturing is recommended.

Characteristics of Occupational Exposure to Benzene during Turnaround in the Petrochemical Industries.

OBJECTIVES: The level of benzene exposure in the petrochemical industry during regular operation has been well established, but not in turnaround (TA), where high exposure may occur. In this study, the characteristics of occupational exposure to benzene during TA in the petrochemical companies were investigated in order to determine the best management strategies and improve the working environment. This was accomplished by evaluating the exposure level for the workers working in environments where benzene was being produced or used as an ingredient during the unit process. METHODS: From 2003 to 2008, a total of 705 workers in three petrochemical companies in Korea were studied. Long- and short-term (< 1 hr) samples were taken during TAs. TA was classified into three stages: shut-down, maintenance and start-up. All works were classified into 12 occupation categories. RESULTS: The long-term geometric mean (GM) benzene exposure level was 0.025 (5.82) ppm (0.005-42.120 ppm) and the short-term exposure concentration during TA was 0.020 (17.42) ppm (0.005-61.855 ppm). The proportions of TA samples exceeding the time-weighted average, occupational exposure level (TWA-OEL in Korea, 1 ppm) and the short-term exposure limit (STEL-OEL, 5 ppm) were 4.1% (20 samples of 488) and 6.0% (13 samples of 217), respectively. The results for the benzene exposure levels and the rates of exceeding the OEL were both statistically significant (p < 0.05). Among the 12 job categories of petrochemical workers, mechanical engineers, plumbers, welders, fieldman and scaffolding workers exhibited long-term samples that exceeded the OEL of benzene, and the rate of exceeding the OEL was statistically significant for the first two occupations (p < 0.05). CONCLUSION: These findings suggest that the periodic work environment must be assessed during non-routine works such as TA.

Involvement of Ca2+/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion.

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca(2+) channel opener Bay K8644. Genistein at a concentration of 50 µM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca(2+)/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca(2+)]i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.

Role of the RAM network in cell polarity and hyphal morphogenesis in Candida albicans.

RAM (regulation of Ace2p transcription factor and polarized morphogenesis) is a conserved signaling network that regulates polarized morphogenesis in yeast, worms, flies, and humans. To investigate the role of the RAM network in cell polarity and hyphal morphogenesis of Candida albicans, each of the C. albicans RAM genes (CaCBK1, CaMOB2, CaKIC1, CaPAG1, CaHYM1, and CaSOG2) was deleted. All C. albicans RAM mutants exhibited hypersensitivity to cell-wall- or membrane-perturbing agents, exhibiting cell-separation defects, a multinucleate phenotype and loss of cell polarity. Yeast two-hybrid and in vivo functional analyses of CaCbk1p and its activator, CaMob2p, the key factors in the RAM network, demonstrated that the direct interaction between the SMA domain of CaCbk1p and the Mob1/phocein domain of CaMob2p was necessary for hyphal growth of C. albicans. Genome-wide transcription profiling of a Camob2 mutant suggested that the RAM network played a role in serum- and antifungal azoles-induced activation of ergosterol biosynthesis genes, especially those involved in the late steps of ergosterol biosynthesis, and might be associated, at least indirectly, with the Tup1p-Nrg1p pathway. Collectively, these results demonstrate that the RAM network is critically required for hyphal growth as well as normal vegetative growth in C. albicans.

Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms.

Tumor necrosis factor-alpha (TNFalpha)-blocking therapy, using biologic TNFalpha antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFalpha and clinically relevant antagonists, particularly Infliximab (Remicade) and Etanercept (Enbrel). Here we characterized the binding stoichiometry and size of soluble TNFalpha-antagonist complexes and identified energetically important binding sites on TNFalpha for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFalpha neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFalpha-antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFalpha were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFalpha, which was further confirmed for solubly purified TNFalpha mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFalpha (Etanercept1-TNFalpha1), Infliximab6-TNFalpha3, and YHB1411-2(4)-TNFalpha2. The distinct features of the soluble antagonist-TNFalpha complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.