Protein nanoarrays using the annexin A5 two-dimensional crystal on supported lipid bilayers.

Protein nanoarrays are regularly ordered patterns of proteins fixed on a solid surface with a periodicity on the order of nanometers. They have significant potential applications as highly sensitive bioassays and biosensors. While several researchers have demonstrated the fabrication of protein nanoarrays with lithographic techniques and programmed DNA nanostructures, it has been difficult to fabricate a protein nanoarray containing a massive number of proteins on the surface. We now report the fabrication of nanoarrays of streptavidin molecules using a two-dimensional (2D) crystal of annexin A5 as a template on supported lipid bilayers that are widely used as cell membranes. The 2D crystal of annexin A5 has a six-fold symmetry with a period of about 18 nm. There is a hollow of a diameter of about 10 nm in the unit cell, surrounded by six trimers of annexin A5. We found that a hollow accommodates up to three streptavidin molecules with their orientation controlled, and confirmed that the molecules in the hollow maintain their specific binding capability to biotinylated molecules, which demonstrates that the fabricated nanoarray serves as an effective biosensing platform. This methodology can be directly applied to the fabrication of nanoarrays containing a massive number of any other protein molecules.

Structured Water Molecules on Membrane Proteins Resolved by Atomic Force Microscopy.

Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.

ß-hydroxybutyrate suppresses NLRP3 inflammasome-mediated placental inflammation and lipopolysaccharide-induced fetal absorption.

The immune system contributes to the regulation of pregnancy, and the disruption of well-controlled immune functions leads to pregnancy complications. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome mechanisms [(a protein complex of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1)] have been reported to play roles in controlling placental inflammation involved in pregnancy pathologies. The ketone body ß-hydroxybutyrate (BHB) can suppress NLRP3 inflammasome activation and improve various inflammatory diseases. Therefore, we hypothesized that BHB could suppress activation of the NLRP3 inflammasome in the placenta, resulting in the improvement of pregnancy complications. In human placental tissue culture, treatment with BHB suppressed the secretion levels of inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and IL-8, but did not affect the mRNA expression levels of NLRP3 inflammasome-associated factors. Treatment with BHB reduced IL-1ß secretion and the amount of mature IL-1ß protein induced by lipopolysaccharide (LPS) stimulation in the placenta. In human trophoblast cells, BHB reduced ASC and activated-caspase-1 expression, resulting in the inhibition of IL-1ß secretion. To investigate the effect of BHB during pregnancy, we used an animal model of LPS (100 µg/kg intraperitoneally [i.p.] on gestational day 14)-induced pregnancy complications. Administration of BHB (100 mg/kg i.p.) clearly suppressed the absorption rate and IL-1ß production in the placenta induced by LPS in pregnant mice. Moreover, LPS-induced pregnancy abnormalities were improved in NLRP3-deficient mice. These findings suggest that BHB play a role in reducing placental inflammation and pregnancy complications via inhibition of NLRP3 inflammasome activation.

Advanced maternal age induces fetal growth restriction through decreased placental inflammatory cytokine expression and immune cell accumulation in mice.

Advanced maternal age is a risk factor for female infertility, and placental dysfunction is considered one of the causes of pregnancy complications. We investigated the effects of advanced maternal aging on pregnancy outcomes and placental senescence. Female pregnant mice were separated into three groups: young (3 months old), middle (8-9 months old), and aged (11-13 months old). Although the body weights of young and middle dams gradually increased during pregnancy, the body weight of aged dams only increased slightly. The placental weight and resorption rate were significantly higher, and live fetal weights were reduced in a maternal age-dependent manner. Although mRNA expression of senescence regulatory factors (p16 and p21) increased in the spleen of aged dams, mRNA expression of p16 did not change and that of p21 was reduced in the placenta of aged dams. Using a cytokine array of proteins extracted from placental tissues, the expression of various types of senescence-associated secretory phenotype (SASP) factors was decreased in aged dams compared with young and middle dams. The aged maternal placenta showed reduced immune cell accumulation compared with the young placenta. Our present results suggest that models using pregnant mice older than 8 months are more suitable for verifying older human pregnancies. These findings suggest that general cellular senescence programs may not be included in the placenta and that placental functions, including SASP production and immune cell accumulation, gradually decrease in a maternal age-dependent manner, resulting in a higher rate of pregnancy complications.

Deficiency of the RIß subunit of protein kinase A causes body tremor and impaired fear conditioning memory in rats.

The RIß subunit of cAMP-dependent protein kinase (PKA), encoded by Prkar1b, is a neuronal isoform of the type I regulatory subunit of PKA. Mice lacking the RIß subunit exhibit normal long-term potentiation (LTP) in the Schaffer collateral pathway of the hippocampus and normal behavior in the open-field and fear conditioning tests. Here, we combined genetic, electrophysiological, and behavioral approaches to demonstrate that the RIß subunit was involved in body tremor, LTP in the Schaffer collateral pathway, and fear conditioning memory in rats. Genetic analysis of WTC-furue, a mutant strain with spontaneous tremors, revealed a deletion in the Prkar1b gene of the WTC-furue genome. Prkar1b-deficient rats created by the CRISPR/Cas9 system exhibited body tremor. Hippocampal slices from mutant rats showed deficient LTP in the Schaffer collateral-CA1 synapse. Mutant rats also exhibited decreased freezing time following contextual and cued fear conditioning, as well as increased exploratory behavior in the open field. These findings indicate the roles of the RIß subunit in tremor pathogenesis and contextual and cued fear memory, and suggest that the hippocampal and amygdala roles of this subunit differ between mice and rats and that rats are therefore beneficial for exploring RIß function.

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Properties of modified surface for biosensing interface.

Properties of modified surface, behavior against salting-out effect, suppressive effect for protein nonspecific adsorption, and wettability were examined using various mercapto compounds bearing methyloligoethylene glycol, oligoethylene glycol, alkyl oligoethylene glycol, alkyl phosphoryl choline, alkyl inverse phosphoryl choline, and alkyl sulfobetaine moieties. The behavior against salting-out effect was examined using gold nanoparticle with PBS and NaCl aqueous solution. The suppressive effect for protein nonspecific adsorption was evaluated by SPR, and the wettability was measured on the SPR chip. The gold nanoparticle modified with 8C3EG, 12C4EG, 12CPC, 6CCP, and 12CCP showed excellent behavior against salting-out effect. The suppression of protein nonspecific adsorption was effective with 6EG, 12C4EG, 12CPC, and 12CS. On the other hand, the modified surface possessed high wettability except for the surface modified with M6EG. The results indicate that incorporation of alkyl group into surface modification materials is effective for the enhancement of behavior against salting-out effect and suppressive effect for protein nonspecific adsorption regardless of wettability. Among the zwitter ionic derivatives, inverse phosphoryl choline derivatives showed intriguing properties, high behavior against salting-out effect with high wettability but low suppressive effect for protein nonspecific adsorption.

Enzyme and Mediator-coadsorbed Carbon Felt Electrode for Electrochemical Detection of Glucose Covered with Polymer Layers Based on Layer-by-Layer Technique.

Glucose dehydrogenase (GlDH) and ferrocene were coadsorbed on a carbon felt (CF) sheet (5 × 10 mm, 2 mm thickness), which was used to construct an electrode for the electrochemical detection of glucose. A potential of +0.3 V vs. Ag/AgCl was applied on the base CF, and the current was measured. After the addition of glucose, the current increased and reached a steady state within 50 s. The current response was proportional to the glucose concentration up to 20 µM, with a lower detection limit of 1 µM. The surface of the CF electrode was covered by layers of polystyrene sulfonate and poly-L-lysine using layer-by-layer technique. Again the current response was proportional to glucose concentration up to 20 µM, with a lower detection limit of 2 µM. The oxidation current owing to electrochemical interferents such as L-ascorbate and acetaminophen was 1/8 times of the current observed on the unprotected electrode. In addition, the protection imparted stability to the electrode. Our work demonstrates that a GlDH/ferrocene CF electrode, protected with polystyrene sulfonate and poly-L-lysine, could be used for the electrochemical detection of glucose.

Long-Term Stability of a Cellulose-Based Glucose Oxidase Membrane.

A cellulose-based glucose oxidase membrane was prepared on a glassy carbon (GC) electrode. The current response of the electrode to glucose was measured by applying a potential of 1.0 V vs. Ag/AgCl on the base GC and was proportional to the concentration of glucose up to 1 mM. The long-term stability of the electrode was examined by measuring the daily glucose response. Over four months, the response magnitude was maintained and then gradually decreased. After 11 months, though the response magnitude decreased to 50% of the initial value, the linear response range did not change. Therefore, the electrode could be used as a glucose biosensor even after 11 months of use. The entrapment of the enzyme in the cellulose matrix promoted the stability of the enzyme, as revealed by data on the enzyme activity after the enzyme electrode was immersed in urea. Therefore, the cellulose matrix may be used to improve the performance of biosensors, bioreactors and bio-fuel cells.

Design and fabrication of biosensing interface for waveguide-mode sensor.

In order to develop a biosensing system with waveguide-mode sensor, fabrication of a biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize the antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfactant (0.05% Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into antibody solution to show response of antigen-antibody reaction, where the chip was modified with triethoxysilane derivatives bearing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the response of antigen-antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succinimide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after 1-month storage in a refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens of nanograms per milliliter of biomarkers in human serum with an unlabeled detection method.

Preparation of a cellulose-based enzyme membrane using ionic liquid to lengthen the duration of enzyme stability.

A novel method for preparing enzyme membranes was developed. The enzyme was attached onto the electrode surface by dropping the enzyme solution and allowing it to dry. Glucose oxidase was used for entrapment. Then, the electrode surface was coated with an ionic liquid containing cellulose, and the ionic liquid was removed by immersing the electrode into water. Enzyme activity was retained in the membrane; the enzyme electrode can be used for detecting glucose in the range of 10 µM to 1 mM, and the response time was ~10 s. The stability duration of the electrode was examined: the enzyme electrode could be used for glucose detection for 6 months. The membrane was observed by atomic force microscopy in the force modulation mode; crystalline and amorphous parts were intermingled. In conclusion, the cellulose membrane can be a suitable immobilization matrix for enzymes.

Visualizing water molecule distribution by atomic force microscopy.

Hydration structures at biomolecular surfaces are essential for understanding the mechanisms of the various biofunctions and stability of biomolecules. Here, we demonstrate the measurement of local hydration structures using an atomic force microscopy system equipped with a low-noise deflection sensor. We applied this method to the analysis of the muscovite mica/water interface and succeeded in visualizing a hydration structure that is site-specific on a crystal. Furthermore, at the biomolecule/buffer solution interface, we found surface hydration layers that are more packed than those at the muscovite mica/water interface.

Development of atomic force microscope with wide-band magnetic excitation for study of soft matter dynamics.

In order to probe dynamical properties of mesoscopic soft matter systems such as polymers, structured liquid, etc., a new atomic force microscopy apparatus with a wide-band magnetic cantilever excitation system was developed. Constant-current driving of an electromagnet up to 1 MHz was implemented with a closed-loop driver circuit. Transfer function of a commercial cantilever attached with a magnetic particle was measured in a frequency range of 1-1000 kHz in distilled water. Effects of the laser spot position, distribution of the force exerted on the cantilever, and difference in the detection scheme on the obtained transfer function are discussed in comparison with theoretical predictions by other research groups. A preliminary result of viscoelasticity spectrum measurement of a single dextran chain is shown and is compared with a recent theoretical calculation.

Permeation regulation of charged species by the component change of polyion complex membranes.

Three kinds of polyion complex membranes were prepared on a glassy carbon electrode: polycation (poly-L-lysine)-rich membrane, polyanion (DNA)-rich membrane, and equivalent membrane. The permeation of electroactive species (e.g., hydrogen peroxide, L-ascorbate, urate, dopamine) through the membrane was measured by the oxidation current of species at base electrode. Permeation of the anionic species can be depressed through the anion-rich membrane, and permeation of the cation can also be regulated through the cation-rich membrane. It is obvious that the charge exclusion can be controlled by changing the component ratio of polycation and polyanion during preparation.

Heavy phosphate adsorption on amorphous ITO film electrodes: nano-barrier effect for highly selective exclusion of anionic species.

We prepared an amorphous indium tin oxide (ITO) film and studied it with respect to its surface characterization and the effect of phosphate adsorption on its electrochemical properties. The film was deposited using RF sputtering under ambient low-oxygen conditions at room temperature. The XPS results revealed that the amount of phosphate adsorbed on the amorphous ITO film was more than 4.6 times greater than that adsorbed on commercially available polycrystalline ITO film in spite of the smaller microscopic surface area of the former. Electrochemical responses for anionic species such as L-ascorbic acid (AA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the phosphate-adsorbed ITO film electrodes were more effectively suppressed at the amorphous ITO film electrode than at the polycrystalline ITO film electrode when a phosphate-containing electrolyte was used. Such suppression could be attributed to the electrostatic repulsion between the anionic species and more heavily adsorbed phosphate on our amorphous ITO film electrode surface. This effect is made more pronounced by increasing the phosphate concentration to 1 mM. With 1 mM phosphate, the amorphous ITO film electrode showed the highest selectivity for dopamine (DA) against the anionic species, namely, 880 for DA/AA and 330 for DA/DOPAC, respectively. In contrast, the selectivity was 120 for DA/AA and 20 for DA/DOPAC with the polycrystalline ITO film electrode.

Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces.

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.

Permselective monolayer membrane based on two-dimensional cross-linked polysiloxane LB films for hydrogen peroxide detecting glucose sensors.

Novel two-dimensional (2D) cross-linked polysiloxane LB films were prepared and applied for glucose sensing as H2O2-permselective films in order to block other electroactive interferences, such as L-ascorbic acid, L-cysteine, uric acid and acetaminophen; the 2D cross-linked polysiloxane monolayers were remarkably effective in eliminating interfering responses and had a rapid response for glucose, even though the films were only a monolayer thick.