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Recently, the electric mobility has emerged with great prospects as there has been a growing interest in and demand for eco-friendly energy. The electric vehicle uses a large number of lithium batteries as sources of power, and the lithium battery poses a risk of fire and explosion when the external impact is loaded. Therefore, in this study, an underbody shield (UBS) was designed and manufactured using carbon fiber reinforced thermoplastic composites for battery protection. The underbody shield is composed of two parts, which are the main shield plate and collision protection bar (CPB). Mechanical behavior against impact was analyzed through finite element analysis considering the position of the CPB and the shape of the underbody shield plate. For the UBS, the hybrid composites (Carbon fiber (CF)/polyethylene terephthalate (PET) and self-reinforced polypropylene (SRPP) composites) were used and the ratio between the CF/PET and SRPP was optimized. Through a finite element analysis, the optimal underbody shield was designed with a thickness of 2.4 mm and a hybrid ratio of CF/PET:SRPP as 1:2. Finally, the designed underbody shield was manufactured using a thermo-forming process and mounted to an electric vehicle, and then a vehicle crash test was performed with a concrete obstacle. The developed underbody shield could protect the battery against the impact damage successfully, showing that the deformation of a rear part was within 5 mm and no battery leakage occurred. [ABSTRACT FROM AUTHOR]

The electrical-resistance-change method (ERCM) is a potential smart-sensing technique for carbon-fiber-reinforced plastic (CFRP) structures. However, a practical way to fabricate electrodes on CFRP structures, such as ink-jet printing with metal nano-inks, is necessary to reduce the time required for the process. As metal nano-inks can be sintered in a few milliseconds under ambient conditions using white-flash-light irradiation from a xenon lamp, the parameters of flash-light sintering such as light energy, duration, and number of pulses were investigated. The light intensity, which is the light energy divided by duration, was found to be an indicator of whether low electrical resistance was attained along with strong adhesion to the CFRP plate. The contact resistance between the electrode and CFRP plate was also examined in a tensile test to confirm the durability. The electrode sintered by flash light with the properly selected parameters exhibited high quality and strain monitoring capability. [ABSTRACT FROM AUTHOR]

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes. [ABSTRACT FROM AUTHOR]

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes. [ABSTRACT FROM AUTHOR]

We report an extremely efficient method for fabricating high-performance supercapacitive CoO/NiCo 2 O 4 nanoflakes on Ni-foam substrate by using intense pulsed light (IPL) technology. Structural and morphological characterization is carried out using X-ray diffraction (XRD) and scanning and transmission electron microscopies (SEM and TEM). These reveal that hierarchically structured CoO/NiCo 2 O 4 nanoflakes of 150–200 nm in size and a thickness around 10 nm are formed on Ni-foam substrate by IPL irradiation with energy of 20 J cm −2 for 15 ms. The electrochemical behavior of the composites is analyzed by cyclic voltammetry and galvanostatic charge–discharge experiments. The IPL-induced CoO/NiCo 2 O 4 /Ni-foam electrode exhibits a very high specific capacitance of 2163 Fg −1 at a discharge current density of 1 Ag −1 and a good rate performance of 908 Fg −1 even at 50 Ag −1 . [ABSTRACT FROM AUTHOR]

The effect of poly (N-vinylpyrrolidone) (PVP) molecular weight (MW) on the flash light sintering of copper nanopaste was investigated. The copper nanopaste was coated on polyimide substrates using doctor blade method. PVP was used as reducing agent to functionalize the copper nanoparticles during flash light sintering process. To find the optimum sintering conditions of copper nanoparticles, the various MWs of PVP (10 000, 40 000 and 55 000) were used and the flash light irradiation energy was varied from 7.5 J/cm 2 to 17.5 J/cm 2 . Meanwhile, other flash light irradiation conditions (pulse numbers, on-time and off-time durations) and amounts of PVP were fixed to clarify the effect of the PVP MW. As the results, it was found that flash light sintered copper nanofilms have the resistivity of 54 μΩcm without any damages to the polymer substrate. To characterize the microstructures and transformation crystal phase of the sintered copper nanofilms, scanning electron microscopy and X-ray diffraction were conducted. In order to understand the interaction between oxidized copper and PVP, X-ray photoelectron spectroscopy was analyzed. To observe the electrical conductivity of flash light sintered copper nanofilms, the sheet resistance of the sintered copper nanofilms was measured using a four-point probe method and the sheet resistance changes during the flash light irradiation process were monitored using in-situ monitoring system. [ABSTRACT FROM AUTHOR]

Highlights: [•] We prepared magnetic nanoparticles for capturing EGFR-expressing cancer cells. [•] Conjugation of antibodies using a PEG-linker leads to a high salt stability. [•] The use of the PEG-linker reduces the non-specific binding onto cells. [•] The EGFR-expressing cancer cells could be captured by simple magnetic isolation. [•] The captured cancer cells showed a high viability. [Copyright &y& Elsevier]

Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes. [ABSTRACT FROM AUTHOR]

In this study, the size effect of copper particles on the flash light sintering of copper (Cu) ink was investigated using Cu nanoparticles (20–50 nm diameter) and microparticles (2 μm diameter). Also, the mixed Cu nano-/micro-inks were fabricated, and the synergetic effects between the Cu nano-ink and micro-ink on flash light sintering were assessed. The ratio of nanoparticles to microparticles in Cu ink and the several flash light irradiation conditions (irradiation energy density, pulse number, on-time, and off-time) were optimized to obtain high conductivity of Cu films. In order to precisely monitor the milliseconds-long flash light sintering process, in situ monitoring of electrical resistance and temperature changes of Cu films was conducted during the flash light irradiation using a real-time Wheatstone bridge electrical circuit, thermocouple-based circuit, and a high-rate data acquisition system. Also, several microscopic and spectroscopic characterization techniques such as scanning electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy were used to characterize the flash light sintered Cu nano-/micro-films. In addition, the sheet resistance of Cu film was measured using a four-point probe method. This work revealed that the optimal ratio of nanoparticles to microparticles is 50:50 wt%, and the optimally fabricated and flash light sintered Cu nano-/micro-ink films have the lowest resistivity (80 μΩ cm) among nano-ink, micro-ink, or nano-micro mixed films. [ABSTRACT FROM AUTHOR]

Highlights: [•] We conduct docking simulation of VX onto the model structure of OPH. [•] Key residues are predicted to enhance the substrate access to the active site. [•] We assess the OPH variants for a substrate analogue. [•] The catalytic efficiency of L271/Y309A is increased 150-fold compared to wild-type. [•] The rational design approach improves the catalytic efficiency of OPH for VX. [Copyright &y& Elsevier]

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non-small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non-small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the repebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6Rα complex. Our results suggest that high-affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non-small cell lung cancer. [ABSTRACT FROM AUTHOR]

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g. [ABSTRACT FROM AUTHOR]

Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs) which consists of leucine-rich repeat (LRR) modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge. [ABSTRACT FROM AUTHOR]

Abstract: Exon 2 deletion in aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) has been suggested to be associated with the progression of various cancers such as lung and ovarian cancers. However, few studies have been conducted regarding detection and relevance of exon 2-deleted AIMP2 (AIMP2-DX2) mutation to a specific cancer. Here, we demonstrate the rapid and simple detection of the AIMP2-DX2 mutation by molecular beacons and its relation to lung cancer. Real-time PCR with molecular beacons allowed a sensitive detection of the AIMP2-DX2 mutation as low as 0.3pg initial template. Dual-conjugated liposomes with folate and molecular beacon enabled fluorescence imaging of cancer cells harboring the AIMP2-DX2 mutation with high resolution. Association of the AIMP2-DX2 mutation with lung cancer was shown by analyzing tissue samples from lung cancer patients using real-time PCR. Approximately, 60% of lung cancer patients harbored the AIMP2-DX2 mutation, which implies a potential of the AIMP2-DX2 mutation as a prognostic biomarker for lung cancer. Molecular beacon-based approaches will find applications in the simple and rapid detection of mutations on nucleotides for diagnosing and monitoring the progression of relevant cancer. [Copyright &y& Elsevier]

In this work, a flash-light sintering process for Cu nanoinks was studied. In order to precisely monitor the milliseconds flash-light sintering process, a real-time Wheatstone bridge electrical circuit and a high-rate data acquisition system were used. The effects of several flash-light irradiation conditions (irradiation energy, pulse number, on-time, and off-time) and the effects of the amount of poly(N-vinylpyrrolidone) in the Cu nanoink on the flash-light sintering process were investigated. The microstructures of the sintered Cu films were analyzed by scanning electron microscopy. To investigate the oxidation or reduction of the oxide-covered copper nanoparticles, a crystal phase analysis using x-ray diffraction was performed. In addition, the sheet resistance of Cu film was measured using a four-point probe method. From this study, it was found that the flash-light sintered Cu nanoink films have a conductivity of 72 Ωm/sq without any damage to the polyimide substrate. Similar nanoinks are expected to be widely used in printed and flexible electronics products in the near future. [ABSTRACT FROM AUTHOR]

Protein dynamics have been suggested to have a crucial role in biomolecular recognition, but the precise molecular mechanisms remain unclear. Herein, we performed single-molecule fluorescence resonance energy transfer measurements for wild-type maltose-binding protein (MBP) and its variants to demonstrate the interplay of conformational dynamics and molecular recognition. Kinetic analysis provided direct evidence that MBP recognizes a ligand through an 'induced-fit' mechanism, not through the generally proposed selection mechanism for proteins with conformational dynamics such as MBP. Our results indicated that the mere presence of intrinsic dynamics is insufficient for a 'selection' mechanism. An energetic analysis of ligand binding implicated the critical role of conformational dynamics in facilitating a structural change that occurs upon ligand binding. [ABSTRACT FROM AUTHOR]

Abstract: Malaria is the most common of the parasitic diseases in tropical and subtropical regions. Adverse side effects of anti-malarial drugs have precluded them as a potential clinical drug. In this study, novel derivatives of N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) based on a variety of dipeptidyl α,β-unsaturated amides containing lysine as a part were synthesized and evaluated. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. The synthesized compounds were evaluated for anti-malarial efficacy against Plasmodium falciparum and cytotoxicity in human epitheloid carcinoma cervix (HeLa cells) by estimating the therapeutic index (TI). N-Methyl amide with N′-Boc protection among them exhibited strong anti-malarial activity and N-methyl amide with N′-m-methylbenzyl amide showed excellent anti-malarial activity with much lower toxicity than the ALLN. Therefore, the two chemicals, as well as the underlying design rationale, could be useful in the discovery and development of new anti-malarial drugs. [Copyright &y& Elsevier]

In this paper, a two-step flash light sintering process for inkjet-printed Ag films is investigated with the aim of improving the quality of sintered Ag films. The flash light sintering process is divided into two steps: a preheating step and a main sintering step. The preheating step is used to remove the organic binder without abrupt vaporization. The main sintering step is used to complete the necking connections among the silver nanoparticles and achieve high electrical conductivity. The process minimizes the damage on the polymer substrate and the interface between the sintered Ag film and polymer substrate. The electrical conductivity is calculated by measuring the resistance and cross-sectional area with an LCR meter and 3D optical profiler, respectively. It is found that the resistivity of the optimal flash light-sintered Ag films (36.32 nΩm), which is 228.86% of that of bulk silver, is lower than that of thermally sintered ones (40.84 nΩm). Additionally, the polyimide film used as the substrate is preserved with the inkjet-printed pattern shape during the flash light sintering process without delamination or defects. [ABSTRACT FROM AUTHOR]

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Hence, effective methods for detoxification and decontamination of OPs are of great significance. Here we constructed and used a high-throughput screening (HTS) system that was based on phenolics-responsive transcription activator for directed evolution of OP-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting. Methyl parathion hydrolase from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after three rounds of directed evolution. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. The DmpR-based screening system is expected to be widely used for developing OP-degrading enzymes with greater potential. [ABSTRACT FROM PUBLISHER]

Abstract: We synthesized Pt, Pt–Ru alloy and Pt–Ru–Mo alloy nanoparticles on the multi-walled carbon nanotubes (MWCNTs) using flash light irradiation and characterized these catalysts. Pt100, Pt50-Ru50 alloy and Pt43-Ru43-Mo14 alloy are coated onto MWCNTs followed by flash light irradiation to facilitate the formation of nanoparticle-alloys. The fabricated pure Pt and Pt-based alloy nanoparticle/MWCNTs were characterized by X-ray diffraction (XRD), raman spectroscopy and scanning electron microscopy (SEM). Cyclic voltammetry (CV) studies and electrochemical impedance spectroscopy (EIS) results revealed that the Pt43-Ru43-Mo14/MWCNT catalyst has higher activity and stability with regard to methanol electro-oxidation than the Pt100/MWCNT and Pt50-Ru50/MWCNT catalysts. [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: Selection of single cells capable of producing target proteins at high rates is crucial for the development of protein manufacturing processes. Here, we present the rapid selection of single cells producing immunoglobulin antibodies at high specific rates by microwell array and microengraving. Chinese hamster ovary (CHO) cells secreting chimeric antibodies were deposited in a microwell array in a manner such that each microwell contained a single cell. Secreted antibodies in the microwells were transferred onto a glass slide by microengraving, followed by interrogation using fluorescence-based immunoassay. Single cells displaying high signal intensities were selected, retrieved, and clonally expanded to assess their specific antibody production rates. Three successive rounds of the process resulted in the selection of single cells showing significantly increased antibody production rates. The present approach can be applied to the selection of single cells for producing other therapeutic proteins in a high-throughput manner. [Copyright &y& Elsevier]

Abstract: We demonstrate to decorate carbon nanotubes (CNTs) with metal alloy nanoparticles by flash light from a xenon lamp highlighting dramatically simplified process and ultrafast speed of millisecond. By managing the pulse energy of the irradiation, we customize the nucleation and growth rates of homogeneous binary and ternary nanoparticles with Pt–Ru and Pt–Ru–Mo thin layers that are e-beam deposited onto CNTs. No damage of CNTs during the light-induced nanogranulation is guaranteed by the rapid and surface-localized heating mechanism. The fabricated nanoparticle/CNT nanostructures are characterized using variety of microscopic and spectroscopic techniques such as SEM, TEM and Raman spectroscopy. [Copyright &y& Elsevier]

Abstract: We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6nAng−1 mL−1 and a detection limit of 0.19ngmL−1 (1.2pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months. [Copyright &y& Elsevier]

Abstract: Multiplexed immunoassay system was developed using the enzyme-immobilized mesoporous silica in a form of nanoscale enzyme reactors (NERs), which improve the enzyme loading, activity, and stability. Glucose oxidase (GO) and trypsin (TR) were adsorbed into mesoporous silica and further crosslinked for the construction of NERs, and antibody-conjugated NERs were employed for the analysis of target antigens in a sandwich-type magnetic bead-based immunoassay. This approach, called as NER-LISA (NER-linked immunosorbent assay), generated signals out of enzyme reactions that correlated well with the concentration of target antigens. The detection limit of NER-LISA using NER-GO and anti-human IgG was 67pM human IgG, and the sensitivity was 20 times higher than that of the conventional ELISA using anti-human IgG conjugated GO. Antibody-conjugated NER-GO and NER-TR were successfully employed for the simultaneous detection of two target antigens (human IgG and chicken IgG) in a solution by taking advantage of signals at different wavelengths (absorbances at 570nm and 410nm, respectively) from the assays of GO and TR activities, demonstrating the potential of NER-LISA in multiplexed immunoassay. The NER-LISA approach also enabled the successful use of a protease (trypsin), because the NER approach can effectively retain the protease molecules within the mesoporous silica and prevent the digestion of antibodies and enzymes during the whole process of NER-LISA. [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: This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude. [Copyright &y& Elsevier]

Abstract: Enzymes that exhibit superior catalytic activity, stability and substrate specificity are highly desirable for industrial applications. These goals prompted the designed substrate specificity of Bacillus stearothermophilus d-hydantoinase toward the target substrate hydroxyphenylhydantoin (HPH). Positions crucial to substrate specificity were selected using structural and mechanistic information on the structural loops at the active site. The size and hydrophobicity of the involved amino acids were rationally changed, and the substrate specificities of the designed d-Hyd mutants were investigated. As a result, M63I/F159S exhibited about 200-fold higher specificity for HPH than the wild-type enzyme. Systematic mutational analysis and computational modeling also supported the rationale used in the design. [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 anti-inflammatory effects of glycyrol, a benzofuran coumarin isolated from Glycyrrhizae Radix, were studied. Glycyrol of 5, 25 and 50 µM dose-dependently inhibited nitric oxide (NO) production by down-regulating inducible nitric oxide synthase (iNOS), and alleviated cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages, in both the mRNA and the protein. Furthermore, glycyrol dose-dependently decreased the mRNA of the pro-inflammatory cytokines IL-1β and IL-6. LPS-induced NF-κB activation was prevented in RAW264.7 macrophages by inhibition of I-κBα phosphorylation. In addition, administration of glycyrol (30 and 100 mg/kg, i.p) reduced the thickness of carrageenan-induced mouse-paw edema swelling. Taken together, our results indicate that glycyrol is an important anti-inflammatory constituent of Glycyrrhizae Radix, and that its anti-inflammatory effect is attributed to the inhibition I-κBα phosphorylation. [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: 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: Vibriocidal antibody assay has been a surrogate standard assay in the evaluation of cholera vaccine efficacy because it has a good correlation with protection. Although the optical density-based vibriocidal assay in a 96-well microtiter-plate format is widely used in clinical trials, it has limitations as vibriocidal titers are altered by incubation time and samples with the same end-point titers could have potentially different vibriocidal kinetics. In the present study, we developed an improved agar-plate assay coupled with an automated colony counting system. Through testing 30 pairs of human sera from vaccinees administered with a cholera vaccine or placebo, these two assays showed good correlations for the vibriocidal titers and fold increases in titers between pre- and post-vaccinated sera as determined by the Pearson correlation coefficient and the Regression coefficient. Notably, the newly-developed semi-automated assay demonstrated that serum samples with the same end-point titers turned out to have distinct vibriocidal kinetics that were not distinguishable by the microtiter-plate assay. The semi-automated assay responded specifically to Vibrio cholerae but not to irrelevant bacteria such as Salmonella typhi and Escherichia coli. These results demonstrate that the semi-automated assay provides better sensitivity, accuracy, and stability of the assay results with minimized efforts than conventional microtiter-plate assay and could provide a useful tool as an in vitro surrogate assay for the evaluation of cholera vaccine efficacy. [Copyright &y& Elsevier]

Conformationally locked uridine in the ‘southern’ conformation, which could be an important precursor for corresponding nucleotides, was stereoselectively synthesized. Southern uridine nucleotides are expected to be full agonists for the P2Y6 receptor. Poor diastereoselectivity in the osmium-mediated dihydroxylation on the allyl amine was overcome by introduction of an allyl azide on which osmium medium hydroxylation, and subsequent cyclization yielded 6-oxabicyclo[3.2.0]heptane in a 9:1 ratio. High regio�selectivity between the 2′- and 3′-hydroxyl groups in the intramolecular O-alkylation and construction of uracil moiety from the azido group provided the final target, a southern 2′-benzoylated uridine derivative. [ABSTRACT FROM AUTHOR]

Abstract: Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress. [Copyright &y& Elsevier]

Abstract: A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation of Ran GTPase-activating protein 1 (RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of sumoylation, fluorescent detection of sumoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner. [Copyright &y& Elsevier]

Abstract: The nozzle parts of solid rocket motors must endure both the internal pressure generated by high temperature exhaust gas and the mechanical load generated by steering operation. Therefore, the nozzle parts of solid rocket motors are fabricated with thick carbon fiber phenolic resin composites. When the thick-walled phenolic composite cylinder is cooled down from the curing temperature of about 155°C to the room temperature, thermal residual stresses are created due to the anisotropic thermal deformation of the composite structure. In this paper, a smart cure method with cooling and reheating was developed to reduce residual stresses in thick-wound composite cylinders made of carbon phenolic woven composite. The optimal cure cycle was obtained to reduce the residual stresses without increasing processing time and applied to fabrication of the thick-walled composite cylinder. From the residual stresses measured by the radial-cut-cylinder-bending method, it was found that the residual stresses were reduced 30% by using the smart cure method. [Copyright &y& Elsevier]

Abstract: The electrochemical behavior of needle-like and forest-like single-walled carbon nanotube (SWCNT) electrodes, wet-chemically assembled on a gold substrate, were investigated by cyclic voltammetry and single potential time-based techniques. The results indicate that a SWCNT electrode system exhibits quasi-reversible behavior, and SWCNT electrodes exhibit better electrochemical behavior than bare gold electrodes. A forest-like SWCNT electrode possesses the best electrochemical behavior. This is explained using Randle’s equivalent circuit unit during the electron-transfer process between the redox centre and the SWCNT electrode surface, and an alternative electron-transfer mechanism was suggested for the observed electrochemical properties. [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: Carbon–phenolic woven composite materials are employed in heavy duty bearings due to their inherent self-lubricating properties and thermal stability. In this work, a hybrid composite hemispherical bearing (CHB) composed of carbon–phenolic composite and aluminum back-up for the suspension of high mobility tracked vehicles, is developed. In order to reduce the damage generated during machining of the spherical surface of CHB, a near net-shape molding process was used. The thermal residual stresses in the composite and aluminum generated during molding process were calculated by finite element analysis with respect to the fiber types and material stacking sequences. From the calculated data and tested results for CHB, it was found that the developed CHB performed better than the conventional carbon-PEEK CHB. [Copyright &y& Elsevier]

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce β-lactamase activity into the αβ/βα metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo β-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (k[sub cat]/K[sub m])[sup app] of 1.8 x 10[sup 2] (mole/liter)[sup -1] second[sup -1], thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100. [ABSTRACT FROM AUTHOR]

Abstract: Novel cyclopentane analogues of fumagillol were synthesized and their endothelial cell proliferation inhibitory activities were evaluated. The cyclopentane-fumagillol derivatives were synthesized from (−)-2,3-O-isopropylidene-d-erythronolactone via stereoselective glycolate Claisen rearrangement and intramolecular ester enolate alkylation as key steps. [Copyright &y& Elsevier]

Abstract: The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of l-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates. [Copyright &y& Elsevier]

This paper describes the synthesis of a tri(ethylene oxide)-attached fourth-generation poly(amidoamine) dendrimer (EO3-dendrimer) and the characterization of its layers on gold. NMR analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that about 61 amine groups of a G4 PAMAM dendrimer were covalently conjugated with tri(ethylene oxide) units, accounting for a 95% modification level. Layers of the EO3-dendrimer were formed on gold, and the resulting surface was characterized by infrared reflection absorption spectroscopy, ellipsometry, and contact angle goniometry. The EO3-dendrimer resulted in more hydrophilic and less compact layers with no substantial deformation of the molecule during layer formation by virtue of the EO3 units, compared to a PAMAM dendrimer. Interestingly, the specific binding of avidin to the biotinylated layers of the EO3-dendrimer approached a surface density of 5.2±0.2 ng mm−2, showing about 92% of full surface coverage. The layers of the EO3-dendrimer were found to be more resistant to nonspecific adsorption of proteins than PAMAM dendrimer layers when bovine serum albumin and serum proteins were tested. [Copyright &y& Elsevier]

We previously proposed the hydrophobic and bulky residues of the three loops, designated stereochemistry gate loops (SGLs), to constitute a hydrophobic substrate binding pocket of d-hydantoinase from Bacillus stearothermophilus SD1. Simulation of substrate binding in the active site of d-hydantoinase and sequence alignment of various d-hydantoinases revealed the critical hydrophobic residues closely located around the exocyclic substituent of substrate. To evaluate the roles of these residues in substrate binding pocket, site-directed mutagenesis was performed specifically for Leu 65, Tyr 155, and Phe 159. When Tyr 155 was mutated to Phe and Glu, both mutants Y155F and Y155E were totally inactive for nonsubstituted hydantoin and d,l-5-hydroxyphenyl hydantoin (HPH), which indicates that Tyr 155 is involved in substrate binding via a hydrogen bond with the hydantoinic ring. Furthermore, replacement of the hydrophobic residues Leu 65 and Phe 159 with Glu, a charged amino acid, resulted in a significant decrease in activity for nonsubstituted hydantoin, but not for HPH. The Kcat values of both mutants for nonsubstituted hydantoin also severely decreased, but a slight change in the Kcat values was observed towards HPH. These results suggest that the hydrophobic residues in SGLs play an essential role in substrate binding, and differentially interact according to the property of the exocyclic substituent. [Copyright &y& Elsevier]

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric d-hydantoinase by mutating five residues in the hydrophobic α-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of d-hydantoinase. The specific activity of the dimeric d-hydantoinase corresponds to 5.3% of the activity of tetrameric d-hydantoinase. This low specific activity of the dimeric d-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer. [Copyright &y& Elsevier]

In this paper, the inter-ply frictional behavior between woven and unidirectional (UD) carbon/epoxy prepregs was studied and its effect on the forming process was investigated. The coefficient of friction (COF) was obtained according to the fiber direction of the prepreg, the temperature, the sliding speed and the pressure. Also, the forming properties such as shear modulus and bending stiffness were measured. In addition, hybrid woven/UD prepregs were formed by U-shape mold and the forming process was implemented by commercial software PAM-FORM. Then, the influence of the frictional resistance along the fiber direction was observed. As a result, it was found that the difference in the interlayer displacement was significantly affected by the frictional behavior according to the fiber direction, and it changed the shear angle distribution and wrinkles after the forming process. [ABSTRACT FROM AUTHOR]

Polymers are highly affected by moisture and temperature in terms of reliability of packaging. Especially, interface delamination between dissimilar materials with polymer is one of the major issues to the structural integrity and reliability, which is affected significantly by temperature and moisture concentration. In this paper, moisture absorption characteristics and thermo-mechanical delamination of semiconductor packages were predicted by finite element analysis considering moisture and temperature changes during the reflow process simultaneously based on a home-made user's subroutine. To implement the delamination through finite element analysis, moisture absorption experiments were conducted to evaluate hygroscopic properties (diffusivity, concentration) of the package material with different relative humidities (RH) and temperatures. In addition, adhesion strength of bi-material was evaluated by a micro-scale shear test. The measured adhesion strength of bi-material such as Epoxy molding compound (EMC)/Chip, Chip/substrate, and substrate/EMC were implemented in the user's subroutine with respect to the moisture concentration and temperature. The user's subroutine based finite element analysis code was developed to analyze the combined effect of hygroscopic and thermal deformation. As a result, interface delamination was successfully predicted considering the in-situ moisture desorption and temperature increase during the reflow process and interfacial failure strength considering the temperature and moisture concentration. • Prediction of moisture absorption characteristics and thermo-mechanical delamination of semiconductor packages • Evaluation of hygroscopic properties of package material with different relative humidity and temperatures • Adhesion strength measurement of bi-material with respect to the moisture concentration and temperature [ABSTRACT FROM AUTHOR]

Thermoplastic differs in crystallinity depending on the manufacturing conditions such as temperature and cooling rate, which affect the mechanical properties of the thermoplastic-based composites. In this work, the crystallinity of polyethylene terephthalate (PET) was varied according to the cooling rate to investigate the mechanical properties of PET matrix-based carbon fiber (CF)/PET composites. The thermal characteristics and crystallinity were analyzed through the differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis. The mechanical behavior of CF/PET composites with different degrees of crystallinity was studied with varied temperature conditions. The failure modes of the fractured CF/PET composites were observed by using a digital microscope and scanning electron microscope (SEM). As a result, the crystallized CF/PET composites were improved 11.6 times higher in in-plane shear (±45° laminated) strength and 3.78 times higher in shear modulus than that of amorphous CF/PET composites at high temperature. [Display omitted] [ABSTRACT FROM AUTHOR]