Adsorption Characteristics and Mechanical Responses of Lubricants Containing Polymer Additives under Fluid Lubrication with a Narrow Gap.

The adsorption behavior of poly(methyl acrylate) (PMA)-based polymer additives and their mechanical response under fluid lubrication in narrow gaps were investigated by using neutron reflectometry, microchannel devices, and the narrow gap viscometer. The surface adsorption layer formed by the polymer additive in a stationary field that was investigated by neutron reflectometry was only about 3 nm thick. On the other hand, when the sample oil containing the polymer additive was flowed into the microchannel device with channels about 500 nm deep, the adsorption layer grew over a long period of time and eventually formed a layer that appeared to be more than 100 nm thick. The mechanical response was measured during one-directional rotation with a constant gap length by using the narrow gap viscometer. The results showed that the effective viscosity increased in the low shear rate range. The same behavior was also observed in the reciprocating rotational tests, where the mechanical response showed a distinctive distortion only when the shear rate was low near 0 rpm. The results of the neutron reflectometer, incorporating the narrow gap viscometer, showed no effect of the rotational speed with regard to the structure of the homogeneous layer over a large area. However, the discrepancy between the reflectivity profile and the fitting curve became progressively more pronounced with time, confirming the formation of inhomogeneous structures with time. It is finally suggested that the inhomogeneous structure is due to the formation of local aggregates by PMA molecules, and it acts as flow resistance only in the low shear rate, resulting in an increase in effective viscosity.

Achieving Ultrawide Tunability in Monolithically Fabricated Si Nanoresonator Devices.

Nanoresonators are powerful and versatile tools promising to revolutionize a wide range of technological areas by delivering unparalleled performance in physical, chemical, biological sensing, signal and information processing, quantum computation, etc., via their high-frequency resonant vibration and rich dynamic behavior. Having the ability to tune the resonance frequency and dynamic behavior at the application stage promises further improvement in their effectiveness and enables novel applications. However, achieving significant room-temperature tunability in conventional (monolithically fabricated) nanoresonators is considered challenging. Here we demonstrate ultrawide electrostatic tuning (∼70%) of (initial) resonance-frequency (∼7% V-1) at room temperature in a monolithically fabricated ultrathin Si nanoresonator (width ∼ 40 nm, length ∼ 200 µm) device. Extreme electrostatic tuning of nonlinear behavior is also demonstrated by canceling the cubic-nonlinear coefficient and subsequently flipping its sign. Thus, these results are expected to provide remarkable operational flexibility and new capabilities to microfabricated resonators, which will benefit many technological areas.

Integrated-gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) afflicts a significant percentage of the population; however, no effective treatments have yet been established because of the unsuitability of in vitro assays and animal experimental models. Here, we present an integrated-gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) by co-culturing human gut and liver cell lines interconnected via microfluidics in a closed circulation loop, for the initiation and progression of NAFLD by treatment with free fatty acids (FFAs) for 1 and 7 days, respectively. Co-cultured Caco-2 gut-mimicking cells and HepG2 hepatocyte-like cells demonstrate the protective effects from apoptosis against FFAs treatment, whereas mono-cultured cells exhibit induced apoptosis. Phenotype and gene expression analyses reveal that the FFAs-treated gut and liver cells accumulated intracellular lipid droplets and show an increase in gene expression associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating the mechanisms of NAFLD.

Gut-liver-axis microphysiological system for studying cellular fluidic shear stress and inter-tissue interaction.

To clarify the physiological and pathological roles of gut-liver-axis (GLA) in the human body, a GLA microphysiological system (GLA-MPS) holds great potential. However, in current GLA-MPSs, the importance of a physiologically relevant flow for gut and liver cells' cultivation is not fully addressed. In addition, the integration of individual organ perfusion, circulation flow, and organ tissue functions in a single device has not been achieved. Here, we introduce a GLA-MPS by integrating two cell-culture chambers with individually applied perfusion flows and a circulation channel with an on-chip pneumatic micropump under cell-culture chambers via a porous membrane for interconnecting them. We analyzed the fluid shear stress (FSS) with computational fluid dynamics simulations and confirmed that the physiologically relevant FSS could be applied to the gut (Caco-2) (8 × 10-3 dyn cm-2) and liver (HepG2) cells (1.2 × 10-7 dyn cm-2). Under the physiologically relevant flow, the Caco-2 and HepG2 cells in the GLA-MPS maintained a cell survival rate of 95% and 92%, respectively. Furthermore, the expression of functional proteins such as zonula occludens 1 (in Caco-2) and albumin (in HepG2) was enhanced. To demonstrate the GLA interaction, the inflammatory bowel disease was recapitulated by applying lipopolysaccharide for only Caco-2 cells. The inflammatory proteins, such as inducible nitric oxide synthase, were induced in Caco-2 and HepG2 cells. The presented GLA-MPS can be adapted as an advanced in vitro model in various applications for disease modeling associated with inter-tissue interactions, such as inflammatory disease.

Spin transport in a lateral spin valve with a suspended Cu channel.

We study spin transport through a suspended Cu channel by an electrical non-local 4-terminal measurement for future spin mechanics applications. A magnetoresistance due to spin transport through the suspended Cu channel is observed, and its magnitude is comparable to that of a conventional fixed Cu lateral spin valve. The spin diffusion length in the suspended Cu channel is estimated to be 340 nm at room temperature from the spin signal dependence on the distance between the ferromagnetic injector and detector electrodes. This value is found to be slightly shorter than in a fixed Cu. The decrease in the spin diffusion length in the suspended Cu channel is attributed to an increase in spin scattering originating from naturally oxidized Cu at the bottom of the Cu channel.

Laser-driven optothermal microactuator operated in water.

This paper proposes and studies the characteristics of a laser-driven optothermal microactuator (OTMA) directly operated in water. A theoretical model of optothermal temperature rise and expansion is established, and simulations on a 1000 µm long OTMA are conducted, revealing that its arm is able to expand and contract in response to the laser pulses in a water environment. Microactuating experiments are further carried out using a microfabricated OTMA. The results demonstrate that the OTMA can be practically actuated in water by a 650 nm laser beam and that the OTMA's deflection amplitude increases linearly with laser power. When irradiated by laser pulses with 9.9 mW power and 0.9-25.6 Hz frequencies, the OTMA achieves deflection amplitude ranging from 3.9 to 3.2 µm, respectively. The experimental results match well with theoretical model when taking the damping effect of water into account. This research may be conducive to developing particular micro-electromechanical systems or micro-optoelectromechanical devices such as underwater optothermal micromotors, micro-pumps, micro-robots, and other underwater microactuators.

Rhombic-Shaped Nanostructures and Mechanical Properties of 2D DNA Origami Constructed with Different Crossover/Nick Designs.

DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency-modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic-shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one-third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3-turn crossover design rhombic-shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force-controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties.

Crystal orientation-dependent fatigue characteristics in micrometer-sized single-crystal silicon.

Repetitive bending fatigue tests were performed using five types of single-crystal silicon specimens with different crystal orientations fabricated from {100} and {110} wafers. Fatigue lifetimes in a wide range between 100 and 1010 were obtained using fan-shaped resonator test devices. Fracture surface observation via scanning electron microscope (SEM) revealed that the {111} plane was the primary fracture plane. The crack propagation exponent n was estimated to be 27, which was independent of the crystal orientation and dopant concentration; however, it was dependent on the surface conditions of the etched sidewall. The fatigue strengths relative to the deflection angle were orientation dependent, and the ratios of the factors obtained ranged from 0.86 to 1.25. The strength factors were compared with those obtained from finite element method stress analyses. The calculated stress distributions showed strong orientation dependence, which was well-explained by the elastic anisotropy. The comparison of the strength factors suggested that the first principal stress was a good criterion for fatigue fracture. We include comparisons with specimens tested in our previous report and address the tensile strength, initial crack length, volume effect, and effects of surface roughness such as scallops.

Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation.

Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17-20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2-4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.

Application of the improved BALB/c 3T3 cell transformation assay to the examination of the initiating and promoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic carcinogen study group of Japan.

The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1microg/ml 12-O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the twostage assay (pretreated with 0.2microg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-beta1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative - again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the determination of the maximum dose, are adequately considered. The application of this two-stage assay for screening the initiating and promoting potential of chemicals is recommended for consideration by other research groups and regulatory authorities.

Technical modification of the Balb/c 3T3 cell transformation assay: the use of serum-reduced medium to optimise the practicability of the protocol.

The two-stage Balb/c 3T3 model of cell transformation can mimic the two-stage carcinogenicity bioassay, and has been recognised as a screening method for detecting potential tumour initiators and promoters. A technical modification to the original protocol (which involved the use of M10F medium, consisting of MEM plus 10% fetal bovine serum [FBS]) has been previously proposed, in order to increase its efficacy, namely: the introduction of enriched, serum-reduced medium (DF2F medium, comprising DMEM/F12 plus 2% FBS and other supplements). The aim of this study was to further modify the protocol, so as to attain higher practicability for the assay. The protocol was further optimised by: a) reducing the number of plates required, through the use of larger plates; b) reducing the cost of the assay by retaining the reduced serum concentration and by using 2microg/ml insulin, rather than the more-complex insulin-transferrin-ethanolamine-sodium selenite (ITES) supplement (i.e. DF2F2I medium); and c) extending the culture period from 24-25 days to 31-32 days, resulting in clearer foci (the number of medium changes did not increase, as less-frequent medium changes were performed during the extended culture period). Growth curve construction revealed that variations in the saturation densities of the parental Balb/c 3T3 cell line and its three transformed clones were highest when M10F medium was replaced with DF2F2I medium just before cells reached confluence. We applied this newly-optimised protocol to the assessment of: a) the tumour initiating activity of 3-methylcholanthrene (MCA), N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methylmethane sulphonate, CdCl(2) and phenacetin, combining a post-treatment of 100ng/ml 12-O-tetradecanoylphorbol-13-acetate at the promotion stage; and b) the tumour promoting activity of insulin, lithocholic acid, CdCl(2) and phenobarbital, with pre-treatment of 0.2microg/ml MCA at the initiation stage. In the present study, only phenobarbital was negative when tested by using the modified protocol.

Use of whole sheep red blood cells in ELISA to assess immunosuppression in vivo.

An enzyme-linked immunosorbent assay (ELISA) using whole sheep red blood cells (SRBC) has been reported as one of the methods for detecting a T-lymphocyte-dependent antibody response. However, it has not been widely used because of SRBC problems such as the weak attachment to ELISA plates, specificity and short-term stability. The objectives of this study were to address these issues and to validate the SRBC-specific antibody response assay. Male Sprague-Dawley rats were bled after 6 days of SRBC immunization. In our new procedure, glutaraldehyde was added before discarding the supernatant of inoculated SRBC suspension to attach SRBC firmly to the plate, while in the original method it was added after discarding. As a result, the attached SRBC was maintained throughout the ELISA procedures. No interference was observed in the titration curve of IgM and IgG antibodies in rats and IgM-antibody in mice when control sera were analyzed to evaluate specificity of this method. The short-term stability of SRBC was overcome by using the different lots of SRBC. They provided antibody titers, which were consistent with those measured using the same lot for immunization. In addition, cyclophosphamide, cyclosporine, prednisolone and methotrexate, well-known immunosuppressive agents, were tested to confirm the applicability of the improved ELISA method to detect the T-lymphocyte-dependent antibody response. All four compounds inhibited the IgM antibody responses dose-dependently. These results demonstrate that the improved whole SRBC-ELISA method provides reproducible and reliable results in the T-lymphocyte-dependent antibody response assay.

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells.

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.

Inorganic arsenic compounds and methylated metabolites induce morphological transformation in two-stage BALB/c 3T3 cell assay and inhibit metabolic cooperation in V79 cell assay.

We have performed two-stage transformation assay using BALB/c 3T3 cells to determine initiating and promoting activities of disodium arsenate, sodium arsenite, monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA). Treatment with these arsenic compounds at the initiating stage induced significant numbers of transformed foci when cells were post-treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Disodium arsenate was active at the concentrations of 15-30 microM, sodium arsenite 5-20 microM, and DMAA 1-2 mM. MMAA required 10 mM to induce cell transformation. The concentrations of these compounds (except DMAA) that induced transformation were highly growth-inhibitory (more than 50%). DMAA induced transformation foci at growth inhibition levels of 66 to 84%. In experiments on promoting activity, cells pretreated with a sub-threshold dose of 20-methylcholanthrene (MCA, 0.2 microg/ml) or sodium arsenite (10 microM) were used. Transformation was enhanced by post-treatment with disodium arsenate (1-10 microM), sodium arsenite (0.5-2 microM), and MMAA (200-1000 microM), but not with DMAA. Studies of gap junctional intercellular communication using the V79 cell metabolic cooperation assay showed that the arsenic compounds (except DMAA) exhibited inhibitory activity. Thus, most arsenicals were shown to have not only initiating activity, but also promoting activity. In addition, inorganic arsenicals, especially trivalent sodium arsenite, were more active than organic ones and exhibited promoting activity at one-order of magnitude lower than initiating activity. These results suggest that from the viewpoint of human hazard, more attention should be paid to the tumor promoting activity of inorganic arsenic compounds.