Self-Assembled Nanostructures of Homo-Oligopeptide as a Potent Ice Growth Inhibitor.

This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.

In Vitro Membrane Platform for the Visualization of Water Impermeability across the Liquid-Ordered Phase under Hypertonic Conditions.

Passive water penetration across the cell membrane by osmotic diffusion is essential for the homeostasis of cell volume, in addition to the protein-assisted active transportation of water. Since membrane components can regulate water permeability, controlling compositional variation during the volume regulatory process is a prerequisite for investigating the underlying mechanisms of water permeation and related membrane dynamics. However, the lack of a viable in vitro membrane platform in hypertonic solutions impedes advanced knowledge of cell volume regulation processes, especially cholesterol-enriched lipid domains called lipid rafts. By reconstituting the liquid-ordered (Lo) domain as a likeness of lipid rafts, we verified suppressed water permeation across the Lo domains, which had yet to be confirmed with experimental demonstrations despite a simulation approach. With the help of direct transfer of the Lo domains from vesicles to supported lipid membranes, the biological roles of lipid composition in suppressed water translocation were experimentally confirmed. Additionally, the improvement in membrane stability under hypertonic conditions was demonstrated based on molecular dynamics simulations.

[A Case of Type II Achalasia Occurring in a Nonagenarian Diagnosed with Acute Food Impaction].

Achalasia is an esophageal motility disorder characterized by loss of esophageal peristalsis and impaired relaxation of the lower esophageal sphincter. Patients with achalasia often complain of persistent symptoms for several years before diagnosis. On the other hand, achalasia diagnosed as a sudden esophageal food impaction is uncommon, and no report has been issued on the diagnosis and successful treatment of achalasia in a 95-year-old patient. We report a case of achalasia diagnosed by high-resolution esophageal manometry and timed barium esophagography after food material removal by endoscopy in a 95-year-old woman who visited the hospital due to sudden esophageal food impaction and was successfully treated by endoscopic balloon dilatation.

Tailoring the Interplay between Two Monomers in the Properties of Degradable Polyesters Synthesized via Ring-Opening Alternating Copolymerization.

Ring-opening alternating copolymerization (ROAC) of cyclic anhydrides and epoxides has emerged as a powerful strategy to produce degradable polyesters with a diverse array of structures from the combination of two distinct building blocks. In this work, we exploited the organocatalytic ROAC of cyclic anhydrides and a functional epoxide, t-butyl glycidoxy acetate, followed by acidic deprotection to access degradable polyesters with carboxylic acid pendants. To study the interplay between monomers, diglycolic anhydride and glutaric anhydride were used as cyclic anhydrides to prepare two polyesters. In particular, the effects of the oxygen heteroatom in the cyclic anhydrides on the properties of the carboxylic acid-containing polyesters were investigated. The introduction of the oxygen heteroatom into the cyclic anhydrides significantly influenced their thermal properties and pH-dependent self-association behavior in an aqueous solution. Furthermore, molecular dynamics simulations elucidate that the number and type of hydrogen bonds play a crucial role in the self-association behavior between the polymers both in the solution and bulk states. The findings of this study highlight the importance of the interplay between monomers in the design of functional polyesters with tunable properties.

Triple-Peak Photoluminescence of DNA-Hybrid Alq3 Crystals Emitting a Depressed Single Peak upon Bio-Recognition.

The green organic semiconductor, tris-(8-hydroxyquinoline)aluminum (Alq3), was hybridized with DNA growing in the shape of hexagonal prismatic crystals. In this study, we applied hydrodynamic flow to the fabrication of Alq3 crystals doped with DNA molecules. The hydrodynamic flow in the Taylor-Couette reactor induced nanoscale pores in the Alq3 crystals, especially at the side part of the particles. The particles exhibited distinctly different photoluminescence emissions divided into three parts compared to common Alq3-DNA hybrid crystals. We named this particle a "three-photonic-unit". After treatment with complementary target DNA, the three-photonic-unit Alq3 particles doped with DNAs were found to emit depressed luminescence from side parts of the particles. This novel phenomenon would expand the technological value of these hybrid crystals with divided photoluminescence emissions toward a wider range of bio-photonic applications.

Molecular doping of nucleic acids into light emitting crystals driven by multisite-intermolecular interaction.

We reveal the fundamental understanding of molecular doping of DNAs into organic semiconducting tris (8-hydroxyquinoline) aluminum (Alq3) crystals by varying types and numbers of purines and pyrimidines constituting DNA. Electrostatic, hydrogen bonding, and π-π stacking interactions between Alq3 and DNAs are the major factors affecting the molecular doping. Longer DNAs induce a higher degree of doping due to electrostatic interactions between phosphate backbone and Alq3. Among four bases, single thymine bases induce the multisite interactions of π-π stacking and hydrogen bonding with single Alq3, occurring within a probability of 4.37%. In contrast, single adenine bases form multisite interactions, within lower probability (1.93%), with two-neighboring Alq3. These multisite interactions facilitate the molecular doping into Alq3 particles compared to cytosines or guanines only forming π-π stacking. Thus, photoluminescence and optical waveguide phenomena of crystals were successfully tailored. This discovery should deepen our fundamental understanding of incorporating DNAs into organic semiconducting crystals.

Peptide-DNA origami as a cryoprotectant for cell preservation.

Cryopreservation of cells is essential for the conservation and cold chain of bioproducts and cell-based medicines. Here, we demonstrate that self-assembled DNA origami nanostructures have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotectant agents, because their nanoscale morphology and ice-philicity are tailored. In particular, a single-layered DNA origami nanopatch functionalized with antifreezing threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1 month of cryopreservation, surpassing dimethyl sulfoxide, which produced 38% viability. It also exhibited minimal dependence on the cryopreservation period and freezing conditions. We attribute this outcome to the fact that the peptide-functionalized DNA nanopatches exert multisite actions for the retardation of ice growth in both intra- and extracellular regions and the protection of cell membranes during cryopreservation. This discovery is expected to deepen our fundamental understanding of cell survival under freezing environment and affect current cryopreservation technologies.

Antimicrobial PEGtides: A Modular Poly(ethylene glycol)-Based Peptidomimetic Approach to Combat Bacteria.

Despite their high potency, the widespread implementation of natural antimicrobial peptides is still challenging due to their low scalability and high hemolytic activities. Herein, we address these issues by employing a modular approach to mimic the key amino acid residues present in antimicrobial peptides, such as lysine, leucine, and serine, but on the highly biocompatible poly(ethylene glycol) (PEG) backbone. A series of these PEG-based peptides (PEGtides) were developed using functional epoxide monomers, corresponding to each key amino acid, with several possessing highly potent bactericidal activities and controlled selectivities, with respect to their hemolytic behavior. The critical role of the composition and the structure of the PEGtides in their selectivities was further supported by coarse-grained molecular dynamic simulations. This modular approach is anticipated to provide the design principles necessary for the future development of antimicrobial polymers.

Ionic contrast across a lipid membrane for Debye length extension: towards an ultimate bioelectronic transducer.

Despite technological advances in biomolecule detections, evaluation of molecular interactions via potentiometric devices under ion-enriched solutions has remained a long-standing problem. To avoid severe performance degradation of bioelectronics by ionic screening effects, we cover probe surfaces of field effect transistors with a single film of the supported lipid bilayer, and realize respectable potentiometric signals from receptor-ligand bindings irrespective of ionic strength of bulky solutions by placing an ion-free water layer underneath the supported lipid bilayer. High-energy X-ray reflectometry together with the circuit analysis and molecular dynamics simulation discovered biochemical findings that effective electrical signals dominantly originated from the sub-nanoscale conformational change of lipids in the course of receptor-ligand bindings. Beyond thorough analysis on the underlying mechanism at the molecular level, the proposed supported lipid bilayer-field effect transistor platform ensures the world-record level of sensitivity in molecular detection with excellent reproducibility regardless of molecular charges and environmental ionic conditions.

Injectable Single-Component Peptide Depot: Autonomously Rechargeable Tumor Photosensitization for Repeated Photodynamic Therapy.

The general practice of photodynamic therapy (PDT) comprises repeated multiple sessions, where photosensitizers are repeatedly administered prior to each operation of light irradiation. To address potential problems arising from the total overdose of photosensitizer by such repeated injections, we here introduce an internalizing RGD peptide (iRGD) derivative (Ppa-iRGDC-BK01) that self-aggregates into an injectable single-component supramolecular depot. Ppa-iRGDC-BK01 is designed as an in situ self-implantable photosensitizer so that it forms a depot by itself upon injection, and its molecular functions (cancer cell internalization and photosensitization) are activated by sustained release, tumor targeting, and tumor-selective proteolytic/reductive cleavage of the iRGD segment. The experimental and theoretical studies revealed that when exposed to body temperature, Ppa-iRGDC-BK01 undergoes thermally accelerated self-assembly to form a supramolecular depot through the hydrophobic interaction of the Ppa pendants and the reorganization of the interpeptide hydrogen bonding. It turned out that the self-aggregation of Ppa-iRGDC-BK01 into a depot exerts a multiple-quenching effect on the photosensitivity to effectively prevent nonspecific phototoxicity and protect it from photobleaching outside the tumor, while enabling autonomous tumor rephotosensitization by long sustained release, tumor accumulation, and intratumoral activation over time. We demonstrate that depot formation through a single peritumoral injection and subsequent quintuple laser irradiations at intervals resulted in complete eradication of the tumor. During the repeated PDT, depot-implanted normal tissues around the tumor exhibited no phototoxic damage under laser exposure. Our approach of single-component photosensitizing supramolecular depot, combined with a strategy of tumor-targeted therapeutic activation, would be a safer and more precise operation of PDT through a nonconventional protocol composed of one-time photosensitizer injection and multiple laser irradiations.

Identification of a fibrinolytic enzyme by Bacillus vallismortis and its potential as a bacteriolytic enzyme against Streptococcus mutans.

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg(-1). Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.

An R-peak detection method that uses an SVD filter and a search back system.

In this paper, we present a method for detecting the R-peak of an ECG signal by using an singular value decomposition (SVD) filter and a search back system. The ECG signal was detected in two phases: the pre-processing phase and the decision phase. The pre-processing phase consisted of the stages for the SVD filter, Butterworth High Pass Filter (HPF), moving average (MA), and squaring, whereas the decision phase consisted of a single stage that detected the R-peak. In the pre-processing phase, the SVD filter removed noise while the Butterworth HPF eliminated baseline wander. The MA removed the remaining noise of the signal that had gone through the SVD filter to make the signal smooth, and squaring played a role in strengthening the signal. In the decision phase, the threshold was used to set the interval before detecting the R-peak. When the latest R-R interval (RRI), suggested by Hamilton et al., was greater than 150% of the previous RRI, the method of detecting the R-peak in such an interval was modified to be 150% or greater than the smallest interval of the two most latest RRIs. When the modified search back system was used, the error rate of the peak detection decreased to 0.29%, compared to 1.34% when the modified search back system was not used. Consequently, the sensitivity was 99.47%, the positive predictivity was 99.47%, and the detection error was 1.05%. Furthermore, the quality of the signal in data with a substantial amount of noise was improved, and thus, the R-peak was detected effectively.

Reactive processing of recycled polycarbonate/acrylonitrile butadiene styrene.

Cellular phone housings were ground to make original particulates using a knife mill. Foams and adhesives with a lighter density than water were removed from ground mixtures using a sink-float process in water; ground metals, button rubbers, and wires were separated from desired materials by using a sink float process in salt All housing materials, consisting of seven thermoplastics included in cellular phone housings, showed better tensile properties than pure housing materials made of polycarbonate/acrylonitrile butadiene styrene, but they only had about half of the impact strength. In contrast, the low impact strength for all housing materials was improved by adding 25 wt % polyethylene elastomer and/or 2.4 wt % ground epoxy circuit boards for batch mixing. Impact strengths, tensile strengths, and the energy absorption ability of all housing materials were improved by adding 5.4wt% glycidyl methacrylate for twin screw extrusion.

Assessment of village health worker training program in Tuguegarao, Philippine.

OBJECTIVES: This study was performed to evaluate the effectiveness of 'village health worker training program' which aimed to build community participatory health promotion capacity of community leaders in villages of low developed country and to develop methods for further development of the program. METHODS: The intervention group were 134 community leaders from 25 barangays (village). Control group were 149 form 4 barangays. Intervention group participated 3-day training program. Questionnaire was developed based on 'Health Promotion Capacity Checklist' which assessed capacity in 4 feathers; 'knowledge', 'skill', 'commitment', and 'resource'. Each feather was assessed in 4 point rating scale. Capacity scores between intervention group and control group were examined to identify changes between the pre- and post-intervention periods. A qualitative evaluation of the program was conducted to assess the appropriateness of the program. The program was conducted in Tuguegarao city, Philippine in January, 2009. RESULTS: The result showed significant increases in the total health promotion capacity and each feather of health promotion capacities between pre and post assessment of intervention group. But there was no significant change in that of control group. Participants marked high level of satisfaction for preparedness, selection of main subjects and education method. Qualitative evaluation revealed that training program facilitated community participatory health promotion capacity of participants. CONCLUSIONS: This study suggested that the Village health worker training program is effective for building health promotion capacity of community leaders and it can be a main method for helping low developed countries with further development.

Identification of an alternative translation initiation site for the Pantoea ananatis lycopene cyclase (crtY) gene in E. coli and its evolutionary conservation.

Previous sequence analyses of the lycopene cyclase gene (crt Y) from Pantoea ananatis revealed that translation of its protein product in Escherichia coli began at the ATG start codon. We found, however, that this enzyme could also be produced in E. coli without the ATG start codon present. Results of experiments using crt Y mutants revealed that a GTG (Val) sequence, located in-frame and 24 bp downstream of the ATG, could act as a potential start codon. Additionally, a point-mutated GTA (Val), replaced from alternative GTG start codon, also displayed its potential as a start codon although the strength as a translation initiation codon was considerably weak. This finding suggests that non-ATG codons, especially one base pairing with the anticodon (3'-UAC-5') in fMet-tRNA, might be also able to function as start codon in translation process. Furthermore, amino acid sequence alignment of lycopene cyclases from different sources suggested that a Val residue located within the N-terminus of these enzymes might be used as an alternative translation initiation site. In particular, presence of a conserved Asp, located in-frame and 12 bp upstream of potential start codon, supports this assumption in view of the fact that Asp (GAT or GAC) can function as part of the Shine-Dalgano sequence (AGGAGG).

Structural insight into bioremediation of triphenylmethane dyes by Citrobacter sp. triphenylmethane reductase.

Triphenylmethane dyes are aromatic xenobiotic compounds that are widely considered to be one of the main culprits of environmental pollution. Triphenylmethane reductase (TMR) from Citrobacter sp. strain KCTC 18061P was initially isolated and biochemically characterized as an enzyme that catalyzes the reduction of triphenylmethane dyes. Information from the primary amino acid sequence suggests that TMR is a dinucleotide-binding motif-containing enzyme; however, no other functional clues can be derived from sequence analysis. We present the crystal structure of TMR in complex with NADP+ at 2.0-angstroms resolution. Despite limited sequence similarity, the enzyme shows remarkable structural similarity to short-chain dehydrogenase/reductase (SDR) family proteins. Functional assignments revealed that TMR has features of both classic and extended SDR family members and does not contain a conserved active site. Thus, it constitutes a novel class of SDR family proteins. On the basis of simulated molecular docking using the substrate malachite green and the TMR/NADP+ crystal structure, together with site-directed mutagenesis, we have elucidated a potential molecular mechanism for triphenylmethane dye reduction.

Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides.

Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3.IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3.IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3.IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3.IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3.IL-2/HE molecule. The G3.IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16).