Sustainable mechanochemical growth of double-network hydrogels supported by vascular-like perfusion.

Double-network (DN) gels are unique mechanochemical materials owing to their structures that can be dynamically remodelled during use. The mechanical energy applied to DN gels is efficiently transferred to the chemical bonds of the brittle network, generating mechanoradicals that initiate the polymerisation of pre-loaded monomers, thereby remodelling the materials. To attain continuous remodelling or growth in response to repetitive mechanical stimuli, a sustainable supply of chemical reagents to such dynamic materials is essential. In this study, inspired by the vascular perfusion transporting nutrients to cells, we constructed a circulatory system for a continuous supply of chemicals to channel-containing DN hydrogels (c-DN gels). The perfusion of monomer solutions through the channel and permeability of the c-DN gels not only replenishes the monomers consumed by the polymerisation but also replenishes the water loss caused by the surface evaporation of hydrogel, thereby freeing the mechanochemical process of DN gels from the constraints of the underwater environment. The facile chemical supply enabled us to modulate the mechanical enhancement of the c-DN gel and attain muscle-like strengthening under repeated mechanical training in deoxygenated air. We also studied the kinetics of polymer growth and strengthening and deciphered unique features of mechanochemical reaction in DN gels including the extremely long-living radicals and delayed mechanical strengthening.

Force-triggered rapid microstructure growth on hydrogel surface for on-demand functions.

Living organisms share the ability to grow various microstructures on their surface to achieve functions. Here we present a force stamp method to grow microstructures on the surface of hydrogels based on a force-triggered polymerisation mechanism of double-network hydrogels. This method allows fast spatial modulation of the morphology and chemistry of the hydrogel surface within seconds for on-demand functions. We demonstrate the oriented growth of cells and directional transportation of water droplets on the engineered hydrogel surfaces. This force-triggered method to chemically engineer the hydrogel surfaces provides a new tool in addition to the conventional methods using light or heat, and will promote the wide application of hydrogels in various fields.

How chain dynamics affects crack initiation in double-network gels.

Double-network gels are a class of tough soft materials comprising two elastic networks with contrasting structures. The formation of a large internal damage zone ahead of the crack tip by the rupturing of the brittle network accounts for the large crack resistance of the materials. Understanding what determines the damage zone is the central question of the fracture mechanics of double-network gels. In this work, we found that at the onset of crack propagation, the size of necking zone, in which the brittle network breaks into fragments and the stretchable network is highly stretched, distinctly decreases with the increase of the solvent viscosity, resulting in a reduction in the fracture toughness of the material. This is in sharp contrast to the tensile behavior of the material that does not change with the solvent viscosity. This result suggests that the dynamics of stretchable network strands, triggered by the rupture of the brittle network, plays a role. To account for this solvent viscosity effect on the crack initiation, a delayed blunting mechanism regarding the polymer dynamics effect is proposed. The discovery on the role of the polymer dynamic adds an important missing piece to the fracture mechanism of this unique material.

Toughening hydrogels through force-triggered chemical reactions that lengthen polymer strands.

The utility and lifetime of materials made from polymer networks, including hydrogels, depend on their capacity to stretch and resist tearing. In gels and elastomers, those mechanical properties are often limited by the covalent chemical structure of the polymer strands between cross-links, which is typically fixed during the material synthesis. We report polymer networks in which the constituent strands lengthen through force-coupled reactions that are triggered as the strands reach their nominal breaking point. In comparison with networks made from analogous control strands, reactive strand extensions of up to 40% lead to hydrogels that stretch 40 to 50% further and exhibit tear energies that are twice as large. The enhancements are synergistic with those provided by double-network architectures and complement other existing toughening strategies.

Quantitative evaluation of macromolecular crowding environment based on translational and rotational diffusion using polarization dependent fluorescence correlation spectroscopy.

Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid-liquid phase separation.

Effects of Host-Adaptive Mutations on Hop Stunt Viroid Pathogenicity and Small RNA Biogenesis.

Accidental transmission of hop stunt viroid (HSVd) from grapevine to hop has led to several epidemics of hop stunt disease with convergent evolution of HSVd-g(rape) into HSVd-h(op) containing five mutations. However, the biological function of these five mutations remains unknown. In this study, we compare the biological property of HSVd-g and HSVd-h by bioassay and analyze HSVd-specific small RNA (HSVd-sRNA) using high-throughput sequencing. The bioassay indicated an association of these five mutations with differences in infectivity, replication capacity, and pathogenicity between HSVd-g and HSVd-h, e.g., HSVd-g induced more severe symptoms than HSVd-h in cucumber. Site-directed mutagenesis of HSVd-g showed that the mutation at position 54 increased pathogenicity. HSVd-sRNA analysis of cucumber and hop plants infected with different HSVd variants showed that several sRNA species containing adaptive nucleotides were specifically down-regulated in plants infected with HSVd-h. Several HSVd-sRNAs containing adaptive mutations were predicted to target cucumber genes, but changes in the levels of these genes were not directly correlated with changes in symptom expression. Furthermore, expression levels of two other cucumber genes targeted by HSVd-RNAs, encoding ethylene-responsive transcription factor ERF011, and trihelix transcription factor GTL2, were altered by HSVd infection. The possible relationship between these two genes to HSVd pathogenicity is discussed.

Surface charge dominated protein absorption on hydrogels.

Soft tissue engineering requires antifouling materials that are biocompatible and mechanically flexible. Conventional hydrogels containing more than 70 wt% water are thus promising antifouling material candidates. However, some hydrogels are difficult to apply in internal body organs because of undesirable protein absorption on their surfaces. Due to the lack of an effective method for observing the true charge densities of hydrogels, the reason why electrostatic interactions dominate protein absorption behavior remains unclear. In this work, we adopt the microelectrode technique (MET) to study the electrical potentials of hydrogels with negative, positive, and neutral potentials and demonstrate the protein absorption behaviors on those hydrogels. The results show that MET is an effective method to obtain the surface charge densities of various hydrogels. Furthermore, the amounts of absorbed proteins on the gels were quantified with respect to the charge densities of the hydrogels. The results indicate that electrostatic absorption is quantitatively dominated by a combination of hydrogel charge density and overall protein charge. Based on the knowledge obtained in this work, the effects of hydrogel surface charges on protein absorption can be better understood. Thus, the results are expected to promote the application of hydrogels in tissue engineering.

Damage cross-effect and anisotropy in tough double network hydrogels revealed by biaxial stretching.

Anisotropy of strain-induced internal damage in tough double network (DN) hydrogels is characterized by a sequence of two tensile experiments. Firstly, the virgin DN gels are subjected to a single biaxial loading-unloading cycle using various combinations of the two maximum strains λx,m and λy,m in the x- and y-directions (λx,m ≥ λy,m). Secondly, the rectangular subsamples, which are cut out from the unloaded specimens so that the long axis can have an angle (θ) relative to the larger pre-strain (x-)axis, are stretched uniaxially along the long axis. Directional internal damage caused by various types of pre-stretching is evaluated by comparing the loading curves of the virgin gels and the subsamples with various θ. The modulus reduction (ΔEθ) and strain-energy reduction (Dθ) are characterized as functions of λx,m, λy,m and θ. The anisotropy of damage increases with the anisotropy of imposed pre-strain field as well as λx,m, which is also observed in the anisotropic re-swelling behavior of the subsamples. The damage and the extensibility of the subsamples with θ = 0° increase with λy,m, and the damage of the subsamples with θ = 90° significantly increases with λx,m. These results reveal the presence of a pronounced damage cross-effect: a finite portion of the chain fractures in the first brittle network in one direction is caused by loading in the other orthogonal direction. This feature is in contrast to the very modest damage cross-effect in the silica reinforced elastomers, which show apparently similar stress-softening behavior but with a different origin. The strong damage cross-effect is a key feature of the internal fracture mechanism of the tough DN gels.

Mechanoresponsive self-growing hydrogels inspired by muscle training.

Living tissues, such as muscle, autonomously grow and remodel themselves to adapt to their surrounding mechanical environment through metabolic processes. By contrast, typical synthetic materials cannot grow and reconstruct their structures once formed. We propose a strategy for developing "self-growing" polymeric materials that respond to repetitive mechanical stress through an effective mechanochemical transduction. Robust double-network hydrogels provided with a sustained monomer supply undergo self-growth, and the materials are substantially strengthened under repetitive loading through a structural destruction-reconstruction process. This strategy also endows the hydrogels with tailored functions at desired positions by mechanical stamping. This work may pave the way for the development of self-growing gel materials for applications such as soft robots and intelligent devices.

Tough Double-Network Gels and Elastomers from the Nonprestretched First Network.

Double-network (DN) gels and elastomers, which consist of two (or more) rubbery polymer networks with contrasting physical properties, have received significant attention as they are extremely tough soft materials. The first network of tough DN materials should be more brittle and weaker than the second network. In this paper, we re-examined the structural requirements of the covalently cross-linked first network of tough DN materials and established a nonprestretching strategy. While prestretching of network strands has been considered necessary for the preparation of the brittle and weak first network, we found that a nonprestretched network having a short strand length and low strand density can be used as the brittle and weak first network for preparation of both tough DN gels and elastomers. This work can further expand the chemical and mechanical diversity of DN materials.

Network elasticity of a model hydrogel as a function of swelling ratio: from shrinking to extreme swelling states.

In this work, we intended to investigate the relationship between the swelling ratio Q and Young's modulus E of hydrogels from their contracted state to extreme swelling state and elucidate the underlining molecular mechanism. For this purpose, we used tetra-poly(ethylene glycol) (tetra-PEG) gel, whose network parameters are well known, as the polymer backbone, and we succeeded in tuning the swelling of the gel by a factor of 1500 times while maintaining the topological structure of the network unchanged, using an approach combining a molecular stent method and a PEG dehydration method. A master curve of Q-E, independent of the method of obtaining Q, was obtained. Using the worm-like chain model, the experimentally determined master curve can be well reproduced. We also observed that the uniaxial stress-strain curve of the hydrogel can be well predicted by the worm-like chain model using the structure parameters determined from the fitting of the Q-E experimental curve.

Validation of Simultaneous Analytical Method of Veterinary Drugs in Foods Using New Automatic Pretreatment Equipment.

New automatic pretreatment equipment (FASVED; Food Automatic Analytical Systems for Veterinary Drugs) was developed. FASVED consists of ten main units: reagent dispenser, homogenizer, transfer hand, lid opening/closing device, centrifugal separator, pipette, shaker, column purification device, centrifugal evaporator and cooling device, and it is capable of freely combining operations by these units. A validation study was performed on two methods for determination of 178 veterinary drugs in livestock products, swine muscle, egg and shrimp, according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. The numbers of analytes that satisfied the criteria of the guideline were 148 in swine muscle, 160 in egg and 151 in shrimp.

Taxonomic revision of Pseudolachnea and Pseudolachnella and establishment of Neopseudolachnella and Pseudodinemasporium gen. nov.

The taxonomy of Pseudolachnea and Pseudolachnella is controversial. Some authors have regarded them as congeneric, whereas others have considered them to be distinct genera differentiated merely on the number of conidial septa. A total of 26 isolates of Pseudolachnea-like fungi were subjected to morphological examination and phylogenetic analyses of nuc rDNA internal transcribed spacers 1 and 2 and partial 28S sequences and partial sequence of the translation elongation factor 1α gene. The results indicated that our materials should be classified in four genera: Pseudolachnea, Pseudolachnella, and two new genera, Neopseudolachnella and Pseudodinemasporium. Although the monophyly of both Pseudolachnea and Pseudolachnella was confirmed, it was concluded that differences observed in the conidiomatal structure, such as thickness of basal stroma and the excipulum, were more reliable for their circumscription, instead of conidial septation. Neopseudolachnella was similar to Pseudolachnea and Pseudolachnella in conidial morphology but was characterized by the conidiomata lacking an excipulum, unlike members of the latter two genera. Pseudodinemasporium bore conidia morphologically similar to those of Dinemasporium but was differentiated from the latter by the conidiomata, which was composed of a well developed peridial structure. A total of 12 new species, namely three in Neopseudolachnella (N. acutispora, N. magnispora, N. uniseptata), one in Pseudodinemasporium (P. fabiforme) and eight in Pseudolachnella (P. asymmetrica, P. botulispora, P. brevicoronata, P. campylospora, P. complanata, P. falcatispora, P. fusiformis and P. pachyderma) are described and illustrated.

[Validation study on a multi-residue method for determination of pesticide residues in agricultural products by new automatic pretreatment equipment (FASRAC) and GC-MS/MS].

A validation study was performed on a multiresidue method for determination of pesticide residues in agricultural products according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. FASRAC (Food Automatic Analytical Systems for Residual Agricultural Chemicals) automatically performs extraction of pesticide residues from agricultural products with acetonitrile, filtration, constant volume, mixing with the use of air, mixing acetonitrile with buffer solvent, separation, and dehydration with sodium sulfate. The extract was purified with a GC/NH2 column. For wheat flour and soybeans, a purification step with a C18 column was added before a GC/NH2 column. After removal of the solvent, the extract was resolved in n-hexane/acetone solvent for GC-MS/MS analysis. In the case of manual analysis, pesticide residues were analyzed according to official multiresidue methods and purification steps were the same as in FASRAC. Recovery tests were performed with wheat flour, soybeans, spinach and apples, by addition of 302 pesticides at the concentrations 0.01 mg/kg. The results indicate that automatic extraction using FASRAC is superior to manual analysis in trueness, repeatability and within-run reproducibility. Specially, automatic extraction using FASRAC is superior to manual analysis in trueness because it is optimized in various respects, for example reextraction at salting-out.

Phenotypic spectrum of Parachlorella kessleri (Chlorophyta) mutants produced by heavy-ion irradiation.

Heavy-ion mutagenesis is a technology used for effective production of genetic mutants. This study demonstrates that algal breeding using a unicellular alga, Parachlorella kessleri, by heavy-ion mutagenesis can improve lipid yield in laboratory experiments. The primary screening yielded 23 mutants among which a secondary screening yielded 7 strains, which were subjected to phenotypic assays. P. kessleri strains produced by heavy-ion radiation spanned a broad spectrum of phenotypes that differed in lipid content and fatty acid profiles. Starch grain morphology was distinctively altered in one of the mutants. The growth of strain PK4 was comparable to that of the wild type under stress-free culture conditions, and the mutant also produced large quantities of lipids, a combination of traits that may be of commercial interest. Thus, heavy-ion irradiation is an effective mutagenic agent for microalgae and may have potential in the production of strains with gain-of-function phenotypes.

[Analytical method of cereulide in processed cereal-based foods and milk powder by LC-MS/MS].

An LC-MS/MS method for analysis of cereulide, an emetic toxin produced by Bacillus cereus, was developed. Cereulide was extracted from samples, fried rice, pan-fried noodles, red bean paste and baby formula, with methanol and purified using Oasis HLB cartridges. LC separation was performed on a C18 column with a mixture of formic acid solution and methanol containing ammonium formate as a mobile phase, and the mass spectrometer was operated in the positive electrospray ionization mode. Performance evaluation showed that trueness was higher than 70% and repeatability and reproducibility were within 10%. The limits of quantification were lower than 1 µg/kg.

[Determination of vedaprofen in livestock products and seafoods by liquid chromatography-tandem mass spectrometry].

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the trace residue determination of vedaprofen (VPF) in livestock products and seafoods. VPF was extracted from each sample with acidified acetone, and the crude extract was re-extracted with ethyl acetate and NaCl solution. Clean-up was performed using a weak anion exchange cartridge (Bond Elut DEA). The LC separation was performed on a C18 column using acetonitrile-0.0025 mol/L formic acid (3 : 2) as the mobile phase and MS was run in the negative ion electrospray ionization mode. The calibration curve was linear in the range of 0.001-0.1 µg/mL VPF. The mean recoveries from equine muscle, cattle muscle, cattle liver, cattle fat, salmon, eel, corbicula, milk, egg and buckwheat honey were 72-94%, and the relative standard deviations (RSDs) were 1.1-2.0%. Limits of quantitation (LOQs) ranged from 0.001 to 0.007 µg/g.

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic glands of Bombyx and Drosophila.

Ecdysteroids mediate a wide variety of developmental and physiological events in insects. In the postembryonic development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although many studies have revealed the biochemical and physiological properties of the enzymes for ecdysteroid biosynthesis, most of the molecular identities of these enzymes have not been elucidated. Here we describe an uncharacterized cytochrome P450 gene, designated Cyp306a1, that is essential for ecdysteroid biosynthesis in the PGs of the silkworm Bombyx mori and fruit fly Drosophila melanogaster. Using the microarray technique for analyzing gene expression profiles in PG cells during Bombyx development, we identified two PG-specific P450 genes whose temporal expression patterns are correlated with changes in ecdysteroid titer during development. Amino acid sequence analysis showed that one of the Bombyx P450 genes belongs to the CYP306A1 subfamily. The temporal and spatial expression pattern of the Drosophila Cyp306a1 homolog is essentially the same as that of Bombyx Cyp306a1. We also found that Drosophila Cyp306a1 is disrupted in the phantom (phm) mutant, known also as the Halloween mutant. The morphological defects and decreased expression of ecdysone-inducible genes in phm suggest that this mutant cannot produce a high titer of ecdysone. Finally we demonstrate that S2 cells transfected with Cyp306a1 convert ketodiol to ketotriol via carbon 25 hydroxylation. These results strongly suggest that CYP306A1 functions as a carbon 25 hydroxylase and has an essential role in ecdysteroid biosynthesis during insect development.