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A superconducting flux flow transistor (SFFT) with three channels and nanobridges was successfully fabricated by electron beam (e-beam) lithography and an Ar ion milling technique. The SFFT is composed of three weak links with a nearby gate current line. We explain the process to obtain the equation for the current-voltage characteristics and describe the method to induce external and internal magnetic fields by Biot–Savart's law. The equation can be used to predict the current-voltage curves for the 3-channel SFFT fabricated using e-beam lithography. I–V characteristics were simulated to analyze the SFFT with three channels and nanobridges by a Matlab program. From the I–V characteristics equation of the 3-channel SFFT, the drain currents and the output voltages as the gate current is applied are graphically compared with the measured value and the simulation value. The simulated I–V curves were in good agreement with the measured curves of the 3-channel SFFT with nanobridges. [ABSTRACT FROM AUTHOR]

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315I kinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 ?M. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I, in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity. [ABSTRACT FROM AUTHOR]

Cloud computing platforms have become very attractive for parallel applications, thanks to the system virtualization technology that allows versatile and pliable computing environments. However, owing to the virtualization overhead, parallel applications can suffer from poor performance when executing synchronization operations. In this paper, we propose sc scheduling, which is a synchronization-conscious scheduling algorithm that can mitigate the existing virtualization overhead. For this purpose, the proposed scheduler understands the synchronization phases of each parallel application. Based on this comprehension, it then eliminates unnecessary CPU spinning of parallel threads and its incurred waste of valuable CPU time. In addition, it prevents their long blocking, which otherwise causes unfairness between concurrent virtual machines (VMs) and other VMs. We implemented these simple concepts and thoroughly evaluated them in a recent Xen hypervisor release. Our results demonstrate that our approach can significantly improve the speed of concurrent virtual machines compared to the original credit scheduler in Xen. [ABSTRACT FROM AUTHOR]

In this paper, the cost of a light emitting diode (LED) package is lowered by using a silicon substrate as the base attached to the chip, in contrast to the conventional chip-on-board (COB) package. In addition we proposed an LED package with a new structure to promote reliability and lifespan by maximizing heat dissipation from the chip. We designed an LED package combining the advantages of COB based on conventional metal printed circuit board (PCB) and the merits of a silicon sub-mount as a substrate. When an input current 500–1000 mA was applied, the fabricated LED exhibited the light output of approximately 112 lm/W at 29 W. We also measured and compared the thermal resistance of the sub-mount package and conventional COB package. The measured thermal resistance of the sub-mount package with a reflective film of Ag and the COB package were 0.625 K/W and 1.352 K/W, respectively. [ABSTRACT FROM AUTHOR]

In this paper, we analyze the performance impact of JobTracker failure in Hadoop. A JobTracker failure is a serious problem that affects the overall job processing performance. We describe the cause of failure and the system behaviors because of failed job processing in the Hadoop. On the basis of the analysis, we build a job completion time model that reflects failure effects. Our model is based on a stochastic process with a node crash probability. With our model, we run simulation of performance impact with very credible failure data available from USENIX called computer failure data repository that have been collected for past 9 years. The results show that the performance impact is very severe in that the job completion time increases about four times typically, and in a worst case, it increases up to 68 times. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Bio-conjugated nanoparticles have emerged as novel molecular probes in nano-biotechnology and nanomedicine and chemical analyses of their surfaces have become challenges. The time-of-flight (TOF) secondary ion mass spectrometry (SIMS) has been one of the most powerful surface characterization techniques for both nanoparticles and biomolecules. When combined with various nanoparticle-based signal enhancing strategies, TOF-SIMS can probe the functionalization of nanoparticles as well as their locations and interactions in biological systems. Especially, nanoparticle-based SIMS is an attractive approach for label-free drug screening because signal-enhancing nanoparticles can be designed to directly measure the enzyme activity. The chemical-specific imaging analysis using SIMS is also well suited to screen nanoparticles and nanoparticle-biomolecule conjugates in complex environments. This review presents some recent applications of nanoparticle-based TOF-SIMS to the chemical analysis of complex biological systems. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 34: 237-247, 2015. [ABSTRACT FROM AUTHOR]

Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR), has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG) antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody) that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH) and light (VL) region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was the development of a bifunctional protein crosslinker-based surface plasmon resonance (SPR) biosensor for rapid detection of aflatoxin B1 (AFB1), a potent carcinogen. A fusion protein was obtained by genetically fusing gold binding protein (GBP) that binds strongly to gold surfaces to protein G (ProG) that interacts with the Fc portion of antibodies. It was used as a bifunctional crosslinker for rapid self-oriented immobilization of antibodies on gold substrates without any chemical treatment. SPR analyses demonstrated the binding of the GBP-ProG crosslinker to the gold surface was superior to that of an only ProG via currently used self-assembled monolayers of alkanethiol due to the GBP property. As a result, anti-AFB1 antibodies were 36% more immobilized on the GBP-ProG layer than the ProG layer. When the GBP-ProG crosslinker-based SPR chips were fabricated with the best density (100 μg/mL) of anti-AFB1 antibodies, they could detect AFB1 as low as 1 μg/mL in both buffer and corn extracts and selectively detect it with negligible SPR responses in control toxins (zearalenone and ochratoxin A). These results mean the GBP-ProG is more useful than the thiolated chemical linkers for development of gold substrate-based immunosensors, and this GBP-ProG crosslinker-based immunosensor could detect small molecules effectively. [Copyright &y& Elsevier]

Efficient binding of enzymes onto the electrode surface has been prerequisite for the construction of sensitive biosensors and biochips. Here, a simple and robust construction of electrochemical glucose biosensor based on pyrroloquinoline quinone-glucose dehydrogenase was demonstrated. The glucose biosensor was fabricated by binding the enzyme onto the anionic self-assembled monolayers on gold electrode via electrostatic interactions. The resulting glucose biosensor gave rise to twofold higher detection sensitivity than that by covalent conjugation under the same condition. Surface plasmon resonance and atomic force microscopy analyses revealed that electrostatic binding of the enzyme leads to much higher surface density of the enzyme. This approach will find wide applications to the development of robust enzyme-based biosensors and biochips. [ABSTRACT FROM AUTHOR]

Abstract: Lipid oxidation was effectively prevented by natural antioxidants (NA) for surface free and encapsulated oils in microencapsulated seed oil (MESO) coated with dextrin and milk protein. Three kinds of NA were extracted from rosemary, broccoli sprout, and citrus, respectively. The antioxidant effect was significantly enhanced by the use of a mixture composed of 0.05% (w/w, oil based) rosemary, 1% broccoli sprout, and 1% citrus extract. The peroxide value (POV) of the total oil in the MESO was shown to lower to about 60 meq/kg even under the accelerated storage condition at 60 ± 1 °C for 30 days. The POV of total oil from MESO was 28.1 meq/kg in the presence of antioxidants, whereas the POV of the control sample without antioxidants reached 78.8 meq/kg. The present approach can find widespread use for preventing various microencapsulated seed oils from lipid oxidation. [Copyright &y& Elsevier]

Abstract: Chlamydia is a bacterial genus that is comprised of obligate intracellular pathogens. The Chlamydia inclusion membrane proteins (Inc proteins) play a key role in host/pathogen interactions. To determine the integral membrane protein structure via NMR, milligram quantities of the protein must be produced and optimised for protein stability in detergent micelles. In this study, we successfully expressed and purified the full length Chlamydia pneumoniae IncC protein (IncC-Full, 2–203), as well as the IncC transmembrane domain (IncC-TM, 124–197) and analysed their biophysical characteristics. Specifically, we used NMR spectroscopy, CD spectroscopy, chemical cross-linking, size exclusion chromatography, and analytical ultracentrifugation (AUC) to evaluate the homogeneity, oligomeric state, and molecular mass of the recombinant proteins. The cross-linking and AUC data indicate that IncC-Full and IncC-TM form homo-dimers in DPC micelles. Based on NMR T 1 and T 2 relaxation, we estimate the molecular mass of IncC-Full and IncC-TM in DPC micelles to be approximately 60.2 and 42.7kDa, respectively, while the calculated AUC molecular masses in the buoyant density of the detergent were 37.6kDa and 14.7kDa, respectively. The 3-D 1H-15N-1H NOESY-HSQC data indicate that both IncC-Full and IncC-TM are primarily alpha helical structures. [Copyright &y& Elsevier]

Abstract: We investigated the leakage mechanism in the recently developed DRAM cell transistors having deeply recessed channels for sub-50nm technology using a gate-controlled diode method. The identification and modeling of the various leakage components in DRAM cell transistors with three-dimensional structures is of great importance for the estimation of their data retention characteristics. Our study reveals that there is a significant difference in the leakage mechanisms of planar and recessed channel MOSFETs, due to their different geometrical aspects. The leakage current at the extended gate–drain overlapping region in recessed channel MOSFETs is of particular importance from the viewpoint of their refresh modeling. The information on the leakage characteristics of three-dimensional DRAM cell transistors obtained herein will be very useful for refresh modeling and future DRAM device designs. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial histidine kinases (HKs) play a critical role in signal transduction for cellular adaptation to environmental conditions and stresses. YbdK from Bacillus subtilis is a 320-residue intra-membrane sensing HK characterized by a short input domain consisting of two transmembrane helices without an extracytoplasmic domain. While the cytoplasmic domains of HKs have been studied in detail, the intra-membrane sensing domain systems are still uncharacterized due to difficulties in handling the transmembrane domain. Here, we successfully obtained pure recombinant transmembrane domain of YbdK (YbdK-TM) from E. coli and analyzed the characteristics of YbdK-TM using nuclear magnetic resonance (NMR) and other biophysical methods. YbdK-TM was found to form homo-dimers in DPC micelles based on cross-linking assays and analytical ultracentrifugation analyses. We estimated the size of the YbdK-TM DPC complex to be 46kDa using solution state NMR T 1/T 2 relaxation analyses in DPC micelles. These results provide information that will allow functional and structural studies of intra-membrane sensing HKs to begin. [Copyright &y& Elsevier]

Abstract: We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions. [Copyright &y& Elsevier]

Abstract: We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner. [Copyright &y& Elsevier]

Abstract: Enhancement of signals in time-of-flight secondary ion mass spectrometry (ToF-SIMS) studies is necessary to many biological applications. We have developed an efficient method of enhancing the signals of secondary ions from peptides using gold nanoparticles (AuNPs) attached to a well-controlled surface such as self-assembled monolayers (SAMs). AuNPs function as both signal enhancers and effective binding sites for peptides, which allow the high signal intensity from the peptides to produce well-contrasted ToF-SIMS images of peptides that are micropatterned on the surface of the AuNPs. For application, this AuNP-enhanced SIMS (NE-SIMS) provided the basis for the spectrum and images to assay protein kinases and their inhibitors. Phosphorylation efficiency and inhibitor effect were quantified by detecting mass change of the peptide substrates by kinase reaction. Maximum efficiency of phosphorylation was achieved from cysteine-tethered peptides with a spacer linker, indicating that accessibility of kinase was dependent on the surface orientation and length of the peptide substrate on the three-dimensional AuNPs. The activities of other enzymes such as phosphatase and protease could also be assayed using NE-SIMS. Our study shows that NE-SIMS can be applied as a useful tool for enzyme assay in biochip surfaces. [Copyright &y& Elsevier]

Abstract: The response surface methodology (RSM) was employed to optimize the microencapsulation condition of sunflower oil (SO) as a typical seed oil. The microencapsulation efficiency (MEE) of microencapsulated sunflower oil (MESO) was investigated with respect to four variables including SO concentration (X 1), proportion of milk protein isolates (MPI) to coating wall (X 2), soy lecithin concentration (X 3), and homogenizing pressure (X 4). As a result, a polynomial regression model equation was fitted as follows: . Effect of homogenizing pressure was negligible. The optimal conditions for microencapsulation of SO were 23.6% SO, 19.0% MPI, 2.5% soy lecithin, and 54.8% dextrin, respectively. MESO under the optimized conditions gave rise to the highest MEE, approaching 96.6% of MEE. Compared to MESO showing a low MEE (70.2%), the peroxide value (POV) of the total oil from the MESO under the optimized conditions was significantly lowed even under the accelerated storage conditions at 60±1°C after 30 days, which indicates a promising feature of RSM-mediated microencapsulation process of seed oil. [Copyright &y& Elsevier]

Abstract: We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (α-pY) and Cy3-labeled secondary antibody (Cy3-α-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner. [Copyright &y& Elsevier]

Abstract: This study demonstrates that natural plants extract (NPE) such as rosemary, broccoli sprout and citrus can effectively inhibit the lipid oxidation of microencapsulated high oleic sunflower oil (MEHS). By employing a dextrin-coating method with supplements such as milk protein isolates (MPI), soy lecithin and sodium triphosphate emulsifier in the presence of NPE, MEHS with high microencapsulation efficiency was obtained. Similar to that of high oleic sunflower oil (HS) in liquid state, lipid oxidation of MEHS was remarkably reduced under the accelerated storage condition in the presence of a mixture of NPEs rather than a single component of NPE. Specifically, induction period of MEHS was significantly elongated in the presence of NPE when tested by using the Rancimat method, and the peroxide value (POV) and p-anisidine value (ASV) were also significantly lowered by addition of NPE even after storage for 30 days at 60±1°C. Based on the results, it is anticipated that NPEs find wide applications as an antioxidant for the elevated quality of microencapsulated oil products in food industries. [Copyright &y& Elsevier]

Abstract: ToF-SIMS analysis with principal component analysis (PCA) has been used for quantitatively studying the interaction between streptavidin and biotin on a dendrimer surface. A poly(amidoamine) dendrimer surface was used as a model amine surface for biotinylation. The surface streptavidin density was systematically varied and independently quantified using the surface plasmon resonance (SPR) technique. A good linear correlation of streptavidin density was observed between the ToF-SIMS and SPR results. [Copyright &y& Elsevier]

Abstract: Although the high mobility channel was formed with the strained Si layer on the fully relaxed Si0.8Ge0.2 buffer layer, the mobility was severely attenuated with increasing the gate bias due to the degraded interface. The quality of oxide grown on strained Si was found to be worse than that for the unstrained Si in terms of fixed oxide charge, interface state density, oxide traps. Also the degraded oxide quality induces high leakage current, it can be a severe source which gives rise to potential reliability issues and performance degradation in strained Si devices. [Copyright &y& Elsevier]

In this study, we examined the impact of environmental perturbation on the movement of the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor] between vegetative and resting cell phases of the life history. Resting state induction, in batch culture, was most effective when vegetative cells were subjected to low temperature (10° C) and darkness for extended time periods. Heterosigma cells in stasis had neither a cell wall nor scales but were surrounded by a calyx, most probably of polysaccharide composition. The resting cell was completely immobile, although both flagella remained attached. Heterosigma resting cells did not require a maturation period before successful activation to the vegetative state could occur. Cell division and motility were impacted sequentially during both the induction and activation phases of resting cell development. Our data show that Heterosigma had an obligate light requirement for resting cell activation. In replete medium, very low light fluences of 5 μmol photons·m- 2 ·s- 1 were as effective as 60 μmol photons·m- 2 ·s- 1 in the initiation of activation. Such sensitivity to extremely low light might give Heterosigma a competitive advantage for bloom formation in nature. Reduced nitrate levels significantly shortened the temporal transition of vegetative cells into the resting cell phase of the life history. Additionally, when resting cells induced in nitrate-limited medium were activated under nitrate-replete condition, the efficiency of the activation response was directly correlated to light availability. Both vegetative and resting cells maintained a haploid DNA complement. Rapid amplified polymorphic DNA (RAPD) analysis demonstrated variation in genetic identity among axenic Heterosigma strains. Strain identity influenced success in... [ABSTRACT FROM AUTHOR]

In this paper, the fault current limiting (FCL) characteristics of a flux-coupled type superconducting fault current limiter (SFCL) with parallel connection between two windings in a DC system were analyzed. The flux-coupled type SFCL was composed of two coils connected in parallel and a superconducting element (SE), which was connected in series with the secondary coil. The flux-coupled type SFCL works in DC systems similar to those in AC systems. Before a fault occurs, the respective magnetic fluxes generated by the two coils connected in parallel offset each other, maintaining the voltage induced in the two coils at zero. In case of a fault, however, resistance is generated in the SE, preventing the magnetic fluxes generated by the two coils from offsetting each other. Thus, some voltage is induced in the two coils, and this starts to limit the fault current. DC short circuit tests were conducted, and the test results confirmed that the flux-coupled type SFCL with the two parallel connected coils was effective in limiting the fault current in a DC system. Additionally, the effect of the wiring direction of the two coils on the SFCL's FCL performance and operating current, limiting impedance, and instantaneous power load was further analyzed, and as a result, the performance conditions of the SFCL in a DC system were determined. [ABSTRACT FROM AUTHOR]

Abstract: Interaction between streptavidin and biotin on poly(amidoamine) (PAMAM) dendrimer-activated surfaces and on self-assembled monolayers (SAMs) was quantitatively studied by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface protein density was systematically varied as a function of protein concentration and independently quantified using the ellipsometry technique. Principal component analysis (PCA) and principal component regression (PCR) were used to identify a correlation between the intensities of the secondary ion peaks and the surface protein densities. From the ToF-SIMS and ellipsometry results, a good linear correlation of protein density was found. Our study shows that surface protein densities are higher on dendrimer-activated surfaces than on SAMs surfaces due to the spherical property of the dendrimer, and that these surface protein densities can be easily quantified with high sensitivity in a label-free manner by ToF-SIMS. [Copyright &y& Elsevier]

Glucagon, a peptide hormone clinically used to treat acute hypoglycemia in diabetes patients, is readily degenerated by undergoing fibrillation under pharmaceutical conditions. Since glucagon is employed as a typical model system for structural investigation of amyloid fibril formation of proteins, production of recombinant glucagon is in demand to facilitate mutagenesis and isotope labeling for nuclear magnetic resonance (NMR) studies. In this study, we established a method to produce recombinant glucagon in Escherichia coli. Recombinant plasmids were constructed to express a maltose-binding protein-fused glucagon, which was cleaved by factor-Xa protease to yield glucagon peptide without any N-terminal extra residues. The final product was clearly identified and characterized by immunoblotting, mass spectrometry, circular dichroism spectroscopy, and backbone NMR assignments of the [13C/15N] isotope-enriched sample. Cellular activity of the recombinant glucagon in hepatocytes was confirmed by monitoring characteristic gene expressions. Site-directed mutagenesis was successfully performed using our recombinant production system. Our bacterial production system and the recombinant glucagon not only provide an economical route of glucagon manufacturing, but also enable NMR-based structural investigation of glucagon fibrillation. [Display omitted] • Bacterial production of a recombinant glucagon was established using Escherichia coli. • MBP used as a fusion tag was cleaved by factor-Xa to yield isolated glucagon. • Final product was validated by mass spectrometry and activity assay in hepatocytes. • Mutagenesis and isotope labeling were successful using the production system. • Backbone NMR assignments were completed by heteronuclear multidimensional NMR. [ABSTRACT FROM AUTHOR]

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To resolve the problem of target specificity and light transmission to deep-seated tissues in photodynamic therapy (PDT), we report a cancer cell-targeted photosensitizer using photoprotein-conjugated upconversion nanoparticles (UCNPs) with high target specificity and efficient light transmission to deep tissues. Core-shell UCNPs with low internal energy back transfer were conjugated with recombinant proteins that consists of a photosensitizer (KillerRed; KR) and a cancer cell-targeted lead peptide (LP). Under near infrared (NIR)-irradiating condition, the UCNP-KR-LP generated superoxide anion radicals as reactive oxygen species via NIR-to-green light conversion and exhibited excellent specificity to target cancer cells through receptor-mediated cell adhesion. Consequently, this photosensitizing process facilitated rapid cell death in cancer cell lines (MCF-7, MDA-MB-231, and U-87MG) overexpressing integrin beta 1 (ITGB1) receptors but not in a cell line (SK-BR-3) with reduced ITGB1 expression and a non-invasive normal breast cell line (MCF-10A). In contrast to green light irradiation, NIR light irradiation exhibited significant PDT efficacy in cancer cells located beneath porcine skin tissues up to a depth of 10 mm, as well as in vivo tumor xenograft mouse models. This finding suggests that the designed nanocomposite is useful for sensing and targeting various deep-seated tumors. [ABSTRACT FROM AUTHOR]

Despite the simplicity and usability of gold nanoparticle (AuNP)-based colorimetry in a large panel of bioassays, this technique still suffers from a poor detection limit. To overcome this issue, we report an improved AuNP-based colorimetric assay in combination with simple centrifugation and silver enhancement. As a proof-of-concept, two types of enzymes (i.e., protein phosphatase and protease) were constructed based on the self-assembly of thiol-stabilized AuNPs in the presence of peptides and metal ions. When the AuNP solutions were subjected to a short cycle of centrifugation and silver enhancement, the signal-to-background ratio (SBR) was distinctly improved by a factor of 5–10 in both enzyme activity assays, as compared to that of AuNP-based colorimetry. The detection limit achieved by using the silver enhancement was determined to be improved by a factor of 1.0–3.4. Furthermore, grayscale images of the silver enhancement allowed for a rapid and simple enzyme assay that did not require measuring the absorbance. Due to its ability to improve the detection senstivity in a facile way, we anticipate that this approach will be suitable for use in many AuNP colorimetric assays. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the spatial and energetic distributions of electrons trapped within a SiO2/HfO2 dual layer gate stack (EOT = 1 nm) of fully processed high-k/metal gate nFETs were investigated by discharging-sweep mode amplitude charge pumping (DSACP). DSACP enables the separate energy profiling of the traps in the SiO2 and HfO2 layers of a SiO2/HfO2 gate stack. The electrical measurement of the spatial/energetic trap profiles with DSACP is based on the electron de-trapping mechanism, which allows scanning to be performed below the conduction band of Si in terms of both the depth and energy. The results show that shallower traps appear in the HfO2 layer with increasing discharging time and a significant correlation exists between the density of the shallow traps and positive bias temperature instability (PBTI) characteristics. [Copyright &y& Elsevier]

Despite the therapeutic potential of recombinant proteins, their cell permeabilities and stabilities remain significant challenges. Here we demonstrate that cyclized recombinant proteins can be used as universal cargos for permeable and stable delivery into cells and polydiacetylene liposomes. Utilizing a split intein-mediated process, cyclized model fluorescent proteins containing short tetraarginine (R 4) and hexahistidine (H 6) tags were generated without compromising their native protein functions. Strikingly, as compared to linear R 4 /H 6 -tagged proteins, the cyclized counterparts have substantially increased permeabilities in both cancer cells and synthetic liposomes, as well as higher resistances to enzymatic degradation in cancer cells. These properties are likely a consequence of structural constraints imposed on the proteins in the presence of short functional peptides. Additionally, photodynamic therapy by cyclized photoprotein-loaded liposomes in cancer cells was significantly improved in comparison to that by their non-cyclized counterparts. These findings suggest that our strategy will be universally applicable to intercellular delivery of proteins and therapeutics. • Cyclization of proteins with tags enhances membrane permeability and stability. • Functions of cyclized proteins are effective in both cells and PCDA liposomes. • This strategy allows liposome-mediated cytosolic delivery of proteins. • Cyclized photoproteins enable photodynamic therapeutic effect on cancer cells. • This strategy serves as a universal cargo platform for protein delivery. [ABSTRACT FROM AUTHOR]

Despite the increasing concern regarding bisphenol A (BPA) as an endocrine disrupting chemical (EDC) upon environmental or human exposure, development of simple method for BPA detection has been hampered, due to the lack of a stable bioreceptor and signal generator. Here, we report a nucleic acid-based rapid and sensitive method for BPA detection, which constitutes a ssDNA aptamer and ssDNAzyme. When the peroxidase-like DNAzyme sequence was split into two parts (one incorporated into the anti-BPA aptamer as a target recognition element and the other into the complementary sequence as a bait), the presence of BPA hindered the association of the split DNA sequence, leading to a reduced signal in the DNAzyme-triggered chemiluminescence (CL). Thus, this NA-based CL measurement permitted the detection of BPA at as low as 5 nM with a broad dynamic range of five orders and with high selectivity towards BPA over other EDCs with structural similarity. With the development of aptamers, our detection method is expected to facilitate studies to monitor EDCs with high simplicity and sensitivity in the field of environmental science. • Rapid and sensitive detection of BPA was developed using coupled nucleic acids. • Split peroxidase-like ssDNAzyme was used with an anti-BPA ssDNA aptamer. • BPA-induced chemiluminescence showed high detection sensitivity and selectivity. • This method will promote applications for monitoring EDCs. [ABSTRACT FROM AUTHOR]

Highlights • Plasma copolymerized (PcP) method with PEG/EDA created antifoulding surfaces. • Protease activity in cell culture media was detected on a PcP/SPR sensor chip. • This approach will be useful for bioassays with minimal nonspecific binding. Abstract We report polyethylene glycol (PEG)-grafting antifouling surfaces using a plasma copolymerized (PcP) technique to monitor protease activity in complex media. By varying the mixing ratio of the PEG and ethylenediamine (EDA) precursors, the PcP-PEG-EDA (PcP-PE) film was able to easily control surface amine density with good preservation of the internal PEG structure. We found that nonspecific protein adsorption was dramatically reduced in serum-containing media on the PcP-PE films, as opposed to that on plasma polymerized-EDA (PP-E) films without PEG. When SPR sensor chips coated with PcP-PE film were employed to detect protease activity, biotinylated luciferase probes (luciferase-peptide-biotin) on streptavidin-conjugated SPR chips enabled real-time and label-free measurement of matrix metalloproteinase activity in cell culture media. Owing to its excellent antifouling ability, this newly developed method boasts minimal nonspecific binding and can serve as a biochip platform to promote a wide range of applications in the biological field. [ABSTRACT FROM AUTHOR]

Cetuximab is a murine-human chimeric monoclonal antibody (mAb) that is clinically used to treat epidermal growth factor receptor (EGFR)-positive cancers. As antibody fragment engineering has emerged as an economic alternative to mAb drugs via bacterial production, we have previously generated FM318, a recombinant Fab adopted from cetuximab. Here, in an effort to facilitate industrial development, we searched for useful mutations that could increase its production yields. Amino acid substitutions were selected to resemble the humanized sequence to avoid unexpectedly raising immunogenic problems by the mutations. As a result, FM329, which accommodates L3Q/L4 M mutations in the light chain and S15G/Q16G in the heavy chain Fd, showed a high production yield in a fed-batch operation, reaching approximately 3.5 times FM318 productivity, with its structure and EGFR-binding affinity being maintained. Additionally, for a potential application to antibody-drug conjugates, a cytotoxic agent, DM1 was successfully linked to FM329 with an average drug-to-antibody ratio of 1.4. The conjugate showed dramatically increased anticancer activity with retention of EGFR-binding affinity. Collectively, we suggest that the partially humanized recombinant cetuximab Fab, FM329, and its DM1 conjugate would serve as promising platforms to develop an economic alternative to cetuximab and/or an improved drug candidate for a potent anti-cancer therapy. [ABSTRACT FROM AUTHOR]

An insufficient sensitivity of aptamer-integrated colorimetric gold nanoparticles (AuNPs) is a common challenge in the detection of environmental chemicals. We report the use of a modified aptamer/AuNP-based sensor in conjunction with a fluorescing single-stranded DNA aptamer for the sensitive detection of bisphenol A (BPA), which is a major endocrine disruptor (EDC). While an anti-BPA single-stranded DNA aptamer was bound with BPA, a weak fluorescence signal was observed upon the addition of SYBR Green-I (SG-I), which is specific to a duplex region of the free aptamer. This reaction was accompanied by a subsequent aggregation of unmodified AuNPs (citrate-stabilized AuNPs) at high salt concentrations, leading to a color change from reddish to purple. In contrast, the absence of BPA elicited a high fluorescence signal from SG-I and produced no color change in the AuNP solution even upon the addition of salt ions. Unlike aptamers that are labeled at their ends with fluorophores, our strategy gave rise to neither a loss of target-binding affinity nor a perturbation of the AuNP colorimetry. Thus, a quantitative analysis with a broad dynamic range was achieved by combining the fluorescent and colorimetric measurements at BPA concentrations ranging over four orders of magnitude. Our approach also yielded a greater detection sensitivity (as low as 9 pg·mL −1 ) than classical AuNP colorimetry or other aptamer-combined methods Moreover, this method enabled the detection of BPA in a small fraction extracted from thermal paper with a high specificity among EDCs. We anticipate that this approach will facilitate further advances in the design of traditional AuNP colorimetric sensors using different aptamers for chemical sensing in the environmental and clinical fields. [ABSTRACT FROM AUTHOR]

Despite the major role of cancer antigen 125 (CA125) in cancer progression, its structural diversity makes antibody-based immunoassay of this protein difficult. Here we report the selection of an anti-CA125 ssDNA aptamer and its application to sensitive detection of CA125 when used together with a CA125-specific antibody immobilized on a three-dimensional network of carbon nanotubes (3DN-CNTs). With exceptional stability and easy modifications over antibodies, the selected ssDNA aptamer (rCAA-8) showed high binding affinity (166 nM) for recombinant CA125, which enabled CA125-specific imaging in ovarian cancer cells (OVCAR-3). Furthermore, when compared to other fluorescent assays based on graphene oxide or flat surface and traditional enzyme-linked immunosorbent assay, a chip-based assay using a 3DN-CNT surface and anti-CA125 antibody-aptamer pair resulted in higher sensitivity and broader dynamic range as a function of CA125 concentration, due to high target specificity and high surface loading density. Our on-chip aptamer-based assay will facilitate sensitive and specific monitoring of CA125 in the biological and clinical fields. [ABSTRACT FROM AUTHOR]

We report bioluminescence analysis of matrix metalloproteinase (MMP) activity in biological substances using a surface-bound luciferase probe. Intein-fused luciferase protein enables site-specific biotinylation of luciferase in the presence of N-terminus cysteine-biotin via intein-mediated splicing process, resulting in a strong association with high bioluminescence signal onto a NeutrAvidin-coated surface. When the peptide substrate for MMP-7 was inserted into a region between luciferase and intein, the biotinylated probe detected MMP-7 activity by cleaving the peptide, and surface-induced bioluminescence signal was strongly reduced in the MMP-secreted media or mouse tissue extracts, compared with that in MMP-deficient control set. Our approach is anticipated to be useful for generating biotinylated proteins and for their applications in diagnosing MMP activity in human diseases. [ABSTRACT FROM AUTHOR]

We report the development of amplification-free surface-enhanced Raman scattering (SERS)-based DNA assays for the rapid and highly sensitive detection of prostate cancer antigen 3 (PCA3) mimic DNA. This technique does not require any DNA amplification process using thermo-cycles in conventional polymerase chain reaction (PCR) due to its highly sensitive detection capability. Herein, the PCA3 mimic DNA, composed of 45 nucleotide sequences, was sandwiched between two probe DNA-immobilized particles (ASO 735 -conjugated detection HGNs and ASO 683 -immobilized capture magnetic beads) by hybridization reactions. Its quantitative analysis was performed by monitoring the characteristic Raman peak intensity of sandwich DNA complexes. The limit of detection (LOD) is estimated to be 2.7 fM, which is about four orders of magnitude more sensitive than that of conventional PCR. This SERS-based DNA assay technique is expected to be a potentially useful tool for early disease diagnosis. [ABSTRACT FROM AUTHOR]

Although cyclic AMP receptor protein ( CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo- CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain ( CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP. [ABSTRACT FROM AUTHOR]

Despite therapeutic benefits of singlet oxygen (1O 2), the development of reliable 1O 2 detection methods is challenging due to its short lifespan and fast reactivity. Herein, we report a photodynamic color sensor for visualizing photoinduced 1O 2 using a 10,12-pentacosadiynoic acid (PCDA) vesicle and a mini singlet oxygen generator (miniSOG). The miniSOG produced 1O 2 by photoactivation, which subsequently reacted with the PCDA vesicle, resulting in a rapid color change from colorless monomeric PCDA to blue-colored polydiacetylene (PDA) vesicles (from yellow to green in the miniSOG-mixed solutions) through electron delocalization-mediated polymerization. Therefore, the color change from PCDA to PDA enabled the rapid and simple detection of photoinduced 1O 2 and its scavengers on a microplate. Notably, the PCDA vesicles exhibited better sensitivity and stability for photoinduced 1O 2 than classical chromogenic reagents. Along with the unique chromatic property and structural versatility of PCDA, we suggest that our approach is highly suitable for visualizing 1O 2 in a rapid and simple way. [Display omitted] • Rapid determination of 1O 2 is highly demanded for its therapeutic applications. • A photodynamic color sensor for 1O 2 was developed using diacetylene vesicles. • Colorless PCDA vesicles turned into blue PDA vesicles by photoinduced 1O 2. • This platform is useful for visualizing 1O 2 in a rapid and simple way. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the effect of the oligomerization between BSU1 family members on brassinosteroid signaling in Arabidopsis.

We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the μIEF channel based on a change in the isoelectric point (p I ) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by μIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways. [ABSTRACT FROM AUTHOR]

Enterprise servers require customized solid-state drives (SSDs) to satisfy their specialized I/O performance and reliability requirements. For effective use of SSDs for enterprise purposes, SSDs must be designed considering requirements such as those related to performance, lifetime, and cost constraints. However, SSDs have numerous hardware and software design options, such as flash memory types and block allocation methods, which have not been well analyzed yet, but on which the SSD performance depends. Furthermore, there is no methodology for determining the optimal design for a particular I/O workload. This paper proposes SSD-Tailor, a customization tool for SSDs. SSD-Tailor determines a near-optimal set of design options for a given workload. SSD designers can use SSD-Tailor to customize SSDs in the early design stage to meet the customer requirements. We evaluate SSD-Tailor with nine I/O workload traces collected from real-world enterprise servers. We observe that SSD-Tailor finds near-optimal SSD designs for these workloads by exploring only about 1% of the entire set of design candidates. We also show that the near-optimal designs increase the average I/O operations per second by up to 17% and decrease the average response time by up to 163% as compared to an SSD with a general design. [ABSTRACT FROM PUBLISHER]

We demonstrate that carbon nitride nanotube (CNNT) hybrids modified with CoPt nanoparticles (CoPt NPs) oxidize reduced nicotinamide adenine dinucleotide (NADH) to a greater extent than carbon nanotubes (CNTs) or CNNTs alone due to synergistic interactions between the CNNTs and the CoPt NPs through the improved internal network. Heterogeneous nitrogen atoms in the sp 2 carbon network provide strong binding sites for in-situ synthesis of CoPt NPs of homogeneous size. The increase in heterogeneous charge transfer on the CoPt-CNNT hybrid electrode enhanced the electrochemical oxidation of NADH at a low applied potential compared to those for pristine CNTs or CNNTs. Our results suggest that CoPt-CNNT hybrids are highly stable and ideal for use in electrochemical sensing. [ABSTRACT FROM AUTHOR]

Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells. [ABSTRACT FROM AUTHOR]

Robust composite nanofibers (NFs) are prerequisite for highly efficient electrochemical sensors. We report the electrochemical application of gold nanoparticle (Au NP)-composite Nafion NFs using a facile electrospinning technique. Owing to the uniform distribution and large surface area of the Au NPs in the NFs, the Au NP-composite electrodes gave rise to greatly improved electrochemical properties, compared to AuNP-free composite electrodes. When they were employed as reservoirs for immobilizing horseradish peroxidase (HRP), reliable and sensitive electrochemical detection by the enzyme reaction was achieved. The detection sensitivity for H2O2 was determined to be as low as 38 nM, which was one order higher than that of previous electrochemical sensors. In addition, there was no change in the enzyme stability over three weeks. In this regard, the developed NP/NF-based electrochemical sensors are anticipated to be very suitable for monitoring other enzyme reactions with high sensitivity and stability. [ABSTRACT FROM AUTHOR]

Acteoside, an active phenylethanoid glycoside, has been used traditionally as an anti-inflammatory agent. The molecular mechanism by which acteoside reduces inflammation was investigated in lipopolysaccharide (LPS)-induced Raw264.7 cells and in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. In vitro, acteoside inhibits high mobility group box 1 (HMGB1) release and iNOS/NO production and induces heme oxygenase-1 (HO-1) expression in a concentration-dependent manner, while HO-1 siRNA antagonizes the inhibition of HMGB1 and NO. The effect of acteoside is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and Nfr2 siRNA, indicating that acteoside induces HO-1 via p38 MAPK and NF-E2-related factor 2 (Nrf2). In vivo, acteoside increases survival and decreases serum and lung HMGB1 levels in CLP-induced sepsis. Overall, these results that acteoside reduces HMGB1 release and may be beneficial for the treatment of sepsis. [ABSTRACT FROM AUTHOR]

Abstract: Among proteases, matrix metalloproteinases (MMPs) have been of significant interest because they are considered as one of the promising biomarkers in association with cancer metastasis, inflammation and other degenerative diseases. Many attempts based on the optical sensing have been made to analyze the activity of MMPs, but most of them require an expensive fluorescence readout and a labor-intensive process. To circumvent this issue, we demonstrated a simple colorimetric detection of protease activity by using carboxy gold nanoparticles (AuNPs) and histidine-containing peptides via metal-affinity coordination. Due to their higher surface-to-volume ratio, the nanometer size of AuNPs enables the surface ligands to function like a chelator, providing greater affinity with metal ions, even in the absence of chelators. With no additional modification by multidentate ligands, the carboxy AuNPs were easily aggregated and changed in color (from reddish-brown to violet) after adding peptide substrates with hexahistidine at both ends and metal ions, whereas the presence of proteases in solution prevented NP aggregation by cleaving the peptides, thereby retaining the original color of the AuNPs. When the extinction ratio (E 520/E 700) of the AuNP solution was measured as a function of matrix metalloproteinase concentration in a single reaction, there was good linearity from as low as 3nM to 52nM. This approach is anticipated to be useful in designing other diagnostic nanosensors. [Copyright &y& Elsevier]

Abstract: We report the novel activity-based detection of antifreeze protein (AFP), also known as ice-binding protein (IBP), using freeze-labile gold nanoparticles (AuNPs) in order to overcome labor-intensive and low throughput issues of the current method based on thermal hysteresis (TH). Upon the addition of either CnAFP from the Antarctic diatom Chaetoceros neogracile or LeIBP from the Arctic yeast Leucosporidium sp. to mercaptosuccinic acid-capped AuNP, the self-assembly of AuNPs was highly inhibited after a freezing/thawing cycle, leading to no color change in the AuNP solution. As a result, the aggregation parameter (E 520/E 650) of AuNP presented the rapid detection of both the concentration-dependent activity and stability of two AFPs with high sensitivity, where the detection range was 100-fold lower than that of the TH-based method. We suggest that our newly developed method is very suitable for simple and high-throughput measurement of AFP activity. [Copyright &y& Elsevier]

We report a gold nanoparticle (AuNP)-based fluorescence quenching system via metal coordination for the simple assay of protease activity. Carboxy AuNPs (5 nm in core diameter) functioned as both quenchers and metal chelators without requiring further modification with multidentate ligands; therefore, they were strongly associated with the hexahistidine regions of dye-tethered peptides in the presence of Ni(II) ions, leading to notable fluorescence quenching over the varying molar ratios of dye to AuNP. Upon the addition of matrix metalloproteinase-7 (MMP-7), the fluorescent intensity was efficiently recovered in one-pot mixture especially at 10:1-100:1 molar ratios of dye to AuNP. Consequently, the dequenching degree was dependent on the MMP-7 concentration in a hyperbolic manner, ranging from as low as 10 to 1,000 ng mL. In this regard, we anticipate that the developed system will give us a general way to construct nanoparticle-dye conjugates and will find applications in the analyses of many other proteases mediating significant biological processes with low background and high sensitivity. [ABSTRACT FROM AUTHOR]

To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual, proofreading, and self-monitoring platform and its application to small molecule-binding single-stranded DNA (ssDNA) aptasensors. Through the colorimetric changes between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor extra monitoring process. We identified ssDNA aptamers with high selectivity and binding affinity by targeting two small molecules (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is useful as a self-monitoring platform to discover ssDNA aptamers as well as to develop aptasensors for diverse targets in a rapid and simple way. • GNP-SELEX as a colorimetric SELEX platform was demonstrated. • GNP-SELEX enabled visual self-monitoring in and between SELEX rounds. • We discovered novel ssDNA aptamers to brassinolide and bisphenol A. • This platform facilitated the development of aptasensors against two small molecules. [ABSTRACT FROM AUTHOR]