HLA-B*57:01-dependent intracellular stress in keratinocytes triggers dermal hypersensitivity reactions to abacavir.

Specific human leukocyte antigen (HLA) polymorphisms combined with certain drug administration strongly correlate with skin eruption. Abacavir hypersensitivity (AHS), which is strongly associated with HLA-B*57:01, is one of the most representative examples. Conventionally, HLA transmits immunological signals via interactions with T cell receptors on the cell surface. This study focused on HLA-mediated intracellular reactions in keratinocytes that might determine the onset of skin immunotoxicity by drug treatments. Abacavir exposure resulted in keratinocytes expressing HLA-B*57:01 exhibiting endoplasmic reticulum (ER) stress responses, such as immediate calcium release into the cytosol and enhanced HSP70 expression. In contrast, keratinocytes expressing HLA-B*57:03 (closely related to HLA-B*57:01) did not show these changes. This indicated that HLA-B*57:01 has a specific intracellular response to abacavir in keratinocytes in the absence of lymphocytes. Furthermore, abacavir exposure in HLA-B*57:01-expressing keratinocytes elevated the expression of cytokines/chemokines such as interferon-γ, interleukin-1ß, and CCL27, and induced T lymphoblast migration. These effects were suppressed by ER stress relief using 4-phenylbutyrate (4-PB). HLA-B*57:01-transgenic mice also exhibited ER stress in epidermal areas following abacavir administration, and abacavir-induced skin toxicity was attenuated by the administration of 4-PB. Moreover, abacavir bound to HLA-B*57:01 within cells and its exposure led to HLA-B*57:01 protein aggregation and interaction with molecular chaperones in the ER of keratinocytes. Our results underscore the importance of HLA-mediated intracellular stress responses in understanding the onset of HLA-B*57:01-mediated AHS. We provide the possibility that the intracellular behavior of HLA is crucial for determining the onset of drug eruptions.

Determining the Sequence Dependency of Self-Assembly of Elastin-Like Peptides Using Short Peptide Analogues with Shuffled Repetitive Sequences.

Synthetic elastin-like peptides (ELPs) that possess characteristic tropoelastin-derived hydrophobic repetitive sequences, such as (VPGVG)n, exhibit thermoresponsive reversible self-assembly. Although their thermoresponsive properties have been well-studied, the sequence-dependent and structural requirements for self-assembly remain ambiguous. In particular, it is still unclear whether the amino acid sequences derived from tropoelastin are necessary for self-assembly. In this study, 11 sequence-shuffled ELP analogues based on (FPGVG)5, which is a previously developed short ELP (sELP), were designed to elucidate the sequence-dependent and structural requirements for their self-assembly. Among them, eight shuffled peptides exhibited self-assembling properties, whereas the other three peptides were difficult to dissolve in water. Structural analyses revealed that the structural characteristics of the three insoluble peptides were different from those of their thermoresponsive analogues. Furthermore, the secondary structures of the peptide analogues possessing the self-assembly abilities were different from each other. These results suggest that the potential for self-assembly and water solubility of sELPs depend on the primary structure in each repeated unit. Moreover, several shuffled analogues exhibited more potent self-assembling properties than the original (FPGVG)5, indicating that shorter ELPs can be obtained using their novel motifs as repetitive units. We also observed that the presence of Pro-Gly sequence in the repeating units was advantageous in terms of peptide solubility. Although further analysis will be necessary to elucidate the molecular mechanism underlying the self-assembly of these sELPs, this study provides insights into the relationship between the amino acid sequence and the self-assembling ability of the peptides for developing new sELPs for various applications.

Weak complex formation of adverse drug reaction-associated HLAB57, B58, and B15 molecules.

The combination of certain human leukocyte antigen (HLA) polymorphisms with administration of certain drugs shows a strong correlation with developing drug hypersensitivity. Examples of typical combinations are HLA-B*57:01 with abacavir and HLA-B*15:02 with carbamazepine. However, despite belonging to the same serotype, HLA-B*57:03 and HLA-B*15:01 are not associated with drug hypersensitivity. Recent studies have shown that several HLA polymorphisms are associated with multiple drugs rather than a single drug, all resulting in drug hypersensitivity. In this study, we compared the molecular structures and intracellular localization of HLA-B*57:01, HLA-B*58:01, and HLA-B*15:02, which pose risks for developing drug hypersensitivity, as well as HLA-B*57:03 and HLA-B*15:01 that do not present such risks. We found that HLA molecules posing risks have a low affinity for the subunit ß2-microglobulin; notably, the weak hydrogen bond formed via Gln96 of the HLA molecule contributes to this behavior. We also clarified that these HLA molecules are easily accumulated in the endoplasmic reticulum, exhibiting a low expression on the cell surface. Considering that these hypersensitivity risk-associated HLA molecules form complexes with ß2-microglobulin and peptides in the endoplasmic reticulum, we assumed that their low complex formation ability in the endoplasmic reticulum facilitates the interaction with multiple drugs.

Adaptive Ranking-Based Constraint Handling for Explicitly Constrained Black-Box Optimization.

We propose a novel constraint-handling technique for the covariance matrix adaptation evolution strategy (CMA-ES). The proposed technique is aimed at solving explicitly constrained black-box continuous optimization problems, in which the explicit constraint is a constraint whereby the computational time for the constraint violation and its (numerical) gradient are negligible compared to that for the objective function. This method is designed to realize two invariance properties: invariance to the affine transformation of the search space, and invariance to the increasing transformation of the objective and constraint functions. The CMA-ES is designed to possess these properties for handling difficulties that appear in black-box optimization problems, such as non-separability, ill-conditioning, ruggedness, and the different orders of magnitude in the objective. The proposed constraint-handling technique (CHT), known as ARCH, modifies the underlying CMA-ES only in terms of the ranking of the candidate solutions. It employs a repair operator and an adaptive ranking aggregation strategy to compute the ranking. We developed test problems to evaluate the effects of the invariance properties, and performed experiments to empirically verify the invariance of the algorithm. We compared the proposed method with other CHTs on the CEC 2006 constrained optimization benchmark suite to demonstrate its efficacy. Empirical studies reveal that ARCH is able to exploit the explicitness of the constraint functions effectively, sometimes even more efficiently than an existing box-constraint handling technique on box-constrained problems, while exhibiting the invariance properties. Moreover, ARCH overwhelmingly outperforms CHTs by not exploiting the explicit constraints in terms of the number of objective function calls.

Cloning and functional characterization of medaka TRPV4.

Medaka, Oryzias latipes is distributed in fresh water of South Asia. To study roles of TRPV4 in osmosensing and adaption mechanism of medaka during changes in salinity environment, we isolated the cDNA for TRPV4 from medaka (olTRPV4) and characterized it. The electrophysiological analysis using Xenopus oocytes revealed that olTRPV4 can be activated by a TRPV4-specific agonist, GSK1016790A and acid at pH 5. Further, olTRPV4 was sensitive to 2-APB. Although warm temperatures activate mammalian TRPV4, olTRPV4 was activated by cold and hot stimulation. The threshold for cold activation was determined as 13.13 ± 0.60 °C, and the heat-activation threshold was 40.26 ± 0.25 °C. Further, when olTRPV4-expressing oocytes were stimulated by hypotonic solution, an apparent activation was observed. We further found that the expression of this hypotonic sensor, olTRPV4 was significantly down-regulated in gills but up-regulated in brains of sea water-acclimated medaka. Results demonstrated that olTRPV4 must function as an osmosensor and play important roles in adaption mechanism of medaka fish in various salinity environments.

Monitoring Fluorescence Response of Amphiphilic Flapping Molecules in Compressed Monolayers at the Air-Water Interface.

The air-water interface, which is the boundary of two phases with a large difference in polarity, gives a distinct environment compared with bulk water or air. Since the interface provides a field for various biomolecules to work, it is important to understand the molecular behaviors at the interface. Here, polarity-independent flapping viscosity probes (FLAP) equipped with hydrophobic/hydrophilic substituents have been synthesized and studied at the air-water interface. In situ fluorescence (FL), which is related to the internal motion and orientation, of three different FLAPs were investigated at the interface, and the internal motion of the molecule was indicated to be suppressed at the interface. In addition, the molecular response was compared with that of conventional viscosity probes (molecular rotors), which indicates the different behaviors of FLAP probably due to the distinct molecular orientation as well as molecular motion.

Molecular rotors confined at an ordered 2D interface.

Intramolecular rotation of molecules contained in a two-dimensional monolayer or a three-dimensional collapsed film at an air-water interface was investigated by in situ fluorescence spectroscopy of twisted intramolecular charge transfer (TICT) type 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) derivatives. The TICT type molecules, CCVJ-C12 and CCVJ-Chol, that contain a linear alkyl dodecyl chain or a cholesteryl group, respectively, as their hydrophobic group, were designed and synthesized to manipulate them at the air-water interface. These lipophilized molecular rotors showed the general properties of TICT molecules in solutions that the fluorescence intensity increases with increasing viscosity of the solvent, which is induced by inhibition of internal molecular rotations. The molecular rotors CCVJ-C12 and CCVJ-Chol formed monolayers at the air-water interface and in situ fluorescence spectroscopy was performed during the in-plane compression of the monolayers. It was revealed that the monomer emissions were suppressed and only after the collapse of monolayers, excimer emission from both layers consisting of CCVJ-C12 or CCVJ-Chol was observed. Suppressed monomer emission from monolayers suggests that intramolecular rotation is not inhibited in dense ordered monolayers. Furthermore, fluorescence spectroscopy of Langmuir-Blodgett (LB) films indicated that molecular rotations are not inhibited in the monolayer transferred on the solid substrates.

Relationship between interfacial hydrophobicity and hydroxylation activity of fungal cells located on an organic-aqueous interface.

In a liquid-liquid interface bioreactor, fungal cells locate in a hydrophilic polyacrylonitrile microsphere layer on an aqueous-organic interface. In this article, effects of hydrophobicity of the interface on n-decane hydroxylation activity of Monilliera sp. NAP 00702 was examined. (-)-4-Decanol production was significantly enhanced to 132% by addition of polytetrafluoroethylene.

Enhancement of 6-pentyl-α-pyrone fermentation activity in an extractive liquid-surface immobilization (Ext-LSI) system by mixing anion-exchange resin microparticles.

The addition of anion-exchange resin microparticles into a polyacrylonitrile (PAN) ballooned microsphere layer drastically enhanced the fermentative activity of Trichoderma atroviride AG2755-5NM398 in an extractive liquid-surface immobilization (Ext-LSI) system. The production of 6-pentyl-α-pyrone (6PP), a fungicidal secondary metabolite, was 1.92-fold higher than the control (PAN alone).

Profiles of ion beams and plasma parameters on a multi-frequencies microwaves large bore electron cyclotron resonance ion source with permanent magnets.

In order to contribute to various applications of plasma and beams based on an electron cyclotron resonance, a new concept on magnetic field with all magnets on plasma production and confinement has been proposed with enhanced efficiency for broad and dense ion beam. The magnetic field configuration consists of a pair of comb-shaped magnet surrounding plasma chamber cylindrically. Resonance zones corresponding for 2.45 GHz and 11-13 GHz frequencies are positioned at spatially different positions. We launch simultaneously multiplex frequencies microwaves operated individually, try to control profiles of the plasma parameters and the extracted ion beams, and to measure them in detail.

Self-assembly process of peptide amphiphile worm-like micelles.

Peptide amphiphile molecules (PA) are remarkably versatile and useful as building blocks for construction of complex supramolecular structures in a bottom-up fashion. Worm-like micelles of PA have been demonstrated to have successful application to creation of synthetic extracellular matrix materials for tissue engineering and regenerative medicine. However, the pathway of the self-assembly process of the PA worm-like micelle has not been fully characterized or understood. This work analyzes the self-assembly process leading to worm-like micelle formation in our designed PA with small-angle neutron scattering and atomic force microscopy. The experimental results demonstrate the existence of transient spherical micelles in the early stage of the process and subsequent micelle chain elongation by attachment of spherical micelles to the end of growing cylindrical micelles to form worm-like micelles in a process mimicking chain-growth polymerization.

Production of electron cyclotron resonance plasma by using multifrequencies microwaves and active beam profile control on a large bore electron cyclotron resonance ion source with permanent magnets.

A new concept on magnetic field with all magnets on plasma production and confinement has been proposed to enhance efficiency of an electron cyclotron resonance (ECR) plasma for broad and dense ion beam source under the low pressure. The magnetic field configuration is constructed by a pair of magnets assembly, i.e., comb-shaped magnet which cylindrically surrounds the plasma chamber. The resonance zones corresponding to the fundamental ECR for 2.45 GHz and 11-13 GHz frequencies are constructed at different positions. The profiles of the plasma parameters in the ECR ion source are different from each frequency of microwave. Large bore extractor is set at the opposite side against the microwave feeds. It is found that differences of their profiles also appear at those of ion beam profiles. We conducted to launch simultaneously multiplex frequencies microwaves controlled individually, and tried to control the profiles of the plasma parameters and then those of extracted ion beam.

Characterization of water/AOT/benzene microemulsions during photoreduction to produce silver particles.

Colloidal dispersions of silver (Ag) particles were prepared by the photoreduction of silver perchlorate (AgClO(4)) in water/AOT/benzene water-in-oil (w/o) microemulsions consisting of sodium bis(2-ethylhexyl) sulfosuccinate (AOT), water and benzene. Formation mechanisms of Ag particles prepared in the presence of benzoin by photoreduction from Ag(+)-containing w/o microemulsions were investigated by UV-vis, transmission electron microscopy (TEM), extended X-ray absorption fine structure (EXAFS) and small-angle X-ray scattering (SAXS) measurements. By a combination of TEM and EXAFS results, Ag particles show a metallic nature after the photoreduction and the diameter of Ag particles ranges between 5 and 30 nm. In situ time-resolved SAXS measurements show that the integrated rate equation can be applied in the reduction process of Ag(+) ions to Ag(0) atoms, and the autocatalytic Ag particle growth proceeds in the association process of Ag(0) atoms during the photoreduction of Ag(+)-containing w/o microemulsions. The nanometer-sized water droplets microemulsified by AOT in benzene continuous-phase regulated the size of Ag particles, and the size and shape of water droplets were retained during the Ag particle formation.

Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase.

The formaldehyde-fixing enzymes, 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), are the key enzymes catalyzing sequential reactions in the ribulose monophosphate (RuMP) pathway. In this study, we generated two fused gene constructs of the hps and phi genes (i.e., hps-phi and phi-hps) from a methylotrophic bacterium Mycobacterium gastri MB19. The gene product of hps-phi exhibited both HPS and PHI activities at room temperature and catalyzed the sequential reactions more efficiently than a simple mixture of the individual enzymes. The gene product of phi-hps failed to display any enzyme activity. Escherichia coli strains harboring the hps-phi gene consumed formaldehyde more efficiently and exhibited better growth in a formaldehyde-containing medium than the host strain. Our results demonstrate that the engineered fusion gene has the possibility to be used to establish a formaldehyde-resistance detoxification system in various organisms.