Plasma-on-chip device for stable irradiation of cells cultured in media with a low-temperature atmospheric pressure plasma.

We have developed a micro electromechanical systems (MEMS) device which enables plasma treatment for cells cultured in media. The device, referred to as the plasma-on-chip, comprises microwells and microplasma sources fabricated together in a single chip. The microwells have through-holes between the microwells and microplasma sources. Each microplasma source is located on the backside of each microwells. The reactive components generated by the microplasma sources pass through the through-holes and reach cells cultured in the microwells. In this study, a plasma-on-chip device was modified for a stable plasma treatment. The use of a dielectric barrier discharge (DBD) technique allowed a stable plasma treatment up to 3 min. The plasma-on-chip with the original electrode configuration typically had the maximum stable operation time of around 1 min. Spectral analysis of the plasma identified reactive species such as O and OH radicals that can affect the activity of cells. Plasma treatment was successfully performed on yeast (Saccharomyces cerevisiae) and green algae (Chlorella) cells. While no apparent change was observed with yeast, the treatment degraded the activity of the Chlorella cells and decreased their fluorescence. The device has the potential to help understand interactions between plasma and cells.

Non-thermal atmospheric-pressure plasma exposure as a practical method for improvement of Brassica juncea seed germination.

Here we report that non-thermal atmospheric-pressure plasma exposure can improve Brassica juncea (leaf mustard) seed germination rate from 50 % to 98 %. The commercially relevant germination rate was achieved by plasma exposure for only 10 minutes and the effect sustains at least for one month under an appropriate storage condition. Improved germination by plasma exposure was also observed for Brassica rapa subsp. pekinensis (Chinese cabbage) seeds. The plasma device used is simple. No pure gas flow system is necessary and it is easy to handle. A large number of seeds can be treated by simply scaling up the device. Plasma exposure can be a practical method for improving seed germination of crop plants important for agriculture.

Chiral meta-molecules consisting of gold nanoparticles and genetically engineered tobacco mosaic virus.

We demonstrate a chiral meta-molecule in the ultraviolet (UV) and visible (VIS) regions using a complex of Au nanoparticles (NPs) and rod-shaped tobacco mosaic virus (TMV). Au NPs five nm in diameter are uniformly formed on peptide-modified TMV. The peptide-modified TMV with uniform-sized Au NPs has improved dispersion in solution. A negative circular dichroism (CD) peak is produced around 540 nm, at plasmonic resonance wavelength of Au NPs. Additionally, modification of a CD peak in the UV region is observed. Attaching NPs to a virus causes the enhancement and modification of CD peaks in both the UV and VIS regions. Our results open a new avenue for the preparation of three dimensional chiral metamaterials at optical frequencies.

Non-thermal atmospheric-pressure plasma potentiates mesodermal differentiation of human induced pluripotent stem cells.

Non-thermal atmospheric-pressure plasma has been used for biological applications, including sterilization and stimulation of cell growth and differentiation. Here, we demonstrate that plasma exposure influences the differentiation pattern of human induced pluripotent stem cells (hiPSCs). We treated hiPSCs with dielectric barrier-discharge air plasma and found an exposure dose that does not kill hiPSCs. Immunohistochemical staining for E-CADHERIN showed that the exposure affected cell-cell attachment and doubled the average size of the hiPSCs. Analysis of mRNAs in embryoid bodies (EBs) from plasma-treated hiPSCs revealed repression of ectoderm genes, including WNT1, and increased expression of mesoderm genes. Importantly, hiPSCs deficient in DNA repair only displayed minimal damage after plasma exposure. Collectively, our results suggest that plasma treatment can be another tool for directing the fate of pluripotent stem cells without disrupting their genomic integrity.

Fabrication of aligned magnetic nanoparticles using tobamoviruses.

We used genetically modified tube-shaped tobamoviruses to produce 3 nm aligned magnetic nanoparticles. Amino acid residues facing the central channel of the virus were modified to increase the number of nucleation sites. Energy dispersive X-ray spectroscopy and superconducting quantum interference device analysis suggest that the particles consisted of Co-Pt alloy. The use of tobamovirus mutants is a promising approach to making a variety of components that can be applied to fabricate nanometer-scaled electronic devices.

Janus-like protein cages. Spatially controlled dual-functional surface modifications of protein cages.

Protein cages have been used both as size-constrained reaction vessels for nanomaterials synthesis and as nanoscale building blocks for higher order nanostructures. We generated Janus-like protein cages, which are dual functionalized with a fluorescent and an affinity label, and demonstrated control over both the stoichiometry and spatial distribution of the functional groups. The capability to toposelectively functionalize protein cages has allowed us to manipulate hierarchical assembly using the layer-by-layer assembly process. Janus-like protein cages expand the toolkit of nanoplatforms that can be used for directed assembly of nanostructured materials.

A self-assembled protein nanotube with high aspect ratio.

Production of a self-assembled protein nanotube achieved through engineering of the 11mer ring protein trp RNA-binding attenuation protein is described. The produced mutant protein is able to stack in solution to produce an extremely narrow, uniform nanotube apparently stabilized by a mixture of disulfide bonds and hydrophobic interactions. Assembly is reversible and the length of tube can potentially be controlled. Large quantities of hollow tubes 8.5 nm in overall diameter with lengths varying from 7 nm to over 1 microm are produced. The structure is analyzed using transmission electron microscopy, atomic force microscopy, mass spectrometry, and single-particle analysis and it is found that component rings stack in a head-to-head fashion. The internal diameter of the tube is 2.5 nm, and the amino acid residues lining the central cavity can be mutated, raising the possibility that the tube can be filled with a variety of conducting or semiconducting materials.

Optical control of cell differentiation on synthetic collagen-like scaffolds.

We have developed biocompatible scaffolds that enable cell fate control with visible light. The scaffolds are based on synthetic collagen-like polypeptide, poly(prolyl-hydroxyprolyl-glycyl) {poly(Pro-Hyp-Gly)} which has been used for cosmetics and other healthcare applications. Bioactive peptides were conjugated to the scaffolds via photoactivation reaction utilizing 488 nm visible light. In addition, the use of a photocleavable crosslinker enables dissociation of chemical moieties by 405 nm laser irradiation. The synthesis scheme enables optical control to attach and detach functional peptides in pre-patterned shapes. Using bone forming peptide (BFP), we demonstrate that calcium deposition by rat bone stromal cells can be directed on the scaffold. Using other signaling molecules and three-dimensional scaffolds, controlled differentiation of stem cells can be achieved by spatio-temporally specific irradiation of confocal microscope laser.

Calcium deposition in photocrosslinked poly(Pro-Hyp-Gly) hydrogels encapsulated rat bone marrow stromal cells.

Reproducing the features of the extracellular matrix is important for fabricating three-dimensional (3D) scaffolds for tissue regeneration. A collagen-like polypeptide, poly(Pro-Hyp-Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro-Hyp-Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible-light photocrosslinking at various concentrations of methacrylated poly(Pro-Hyp-Gly) (20-50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro-Hyp-Gly) with 5-min irradiation. These findings demonstrate that poly(Pro-Hyp-Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro-Hyp-Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.

Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment.

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.

Real-time imaging of DNA-streptavidin complex formation in solution using a high-speed atomic force microscope.

The direct observation of individual molecules in action is required for a better understanding of the mechanisms of biological reactions. We used a high-speed atomic force microscope (AFM) in solution to visualize short DNA fragments in motion. The technique represents a new approach in analyzing molecular interactions, and it allowed us to observe real-time images of biotinylated DNA binding to/dissociating from streptavidin protein. Our results show that high-speed AFMs have the potential to reveal the mechanisms of molecular interactions, which cannot be determined by analyzing the average value of mass reactions.

Cytocompatible polyion complex gel of poly(Pro-Hyp-Gly) for simultaneous rat bone marrow stromal cell encapsulation.

Polyion complex (PIC) gel of poly(Pro-Hyp-Gly) was successfully fabricated by simply mixing polyanion and polycation derivatives of poly(Pro-Hyp-Gly), a collagen-like polypeptide. The polyanion, succinylated poly(Pro-Hyp-Gly), and the polycation, arginylated poly(Pro-Hyp-Gly), contain carboxy (pKa = 5.2) and guanidinium (pKa = 12.4) groups, respectively. Mixing the polyanion and the polycation at physiological pH (pH = 7.4) resulted in PIC gel. The hydrogel formation was optimum at an equimolar ratio of carboxy to guanidinium groups, suggesting that ionic interaction is the main determinant for the hydrogel formation. The hydrogel was successfully used for simultaneous rat bone marrow stromal cell encapsulation. The encapsulated cells survived and proliferated within the hydrogel. In addition, the cells exhibited different morphology in the hydrogel compared with cells cultured on a tissue culture dish as a two-dimensional (2D) control. At day one, a round morphology and homogeneous single cell distribution were observed in the hydrogel. In contrast, the cells spread and formed a fibroblast-like morphology on the 2D control. After three days, the cells in the hydrogel maintained their morphology and some of them formed multicellular aggregates, which is similar to cell morphology in an in vivo microenvironment. These results suggest that the PIC gel of poly(Pro-Hyp-Gly) can serve as a cytocompatible three-dimensional scaffold for stem cell encapsulation, supporting their viability, proliferation, and in vivo-like behavior.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.

Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy.

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.

Protein classification using comparative molecular interaction profile analysis system.

We recently introduced a new molecular description factor, interaction profile Factor (IPF) that is useful for evaluating molecular interactions. IPF is a data set of interaction energies calculated by the Comparative Molecular Interaction Profile Analysis system (CoMIPA). CoMIPA utilizes AutoDock 3.0 docking program, and the system has shown to be a powerful tool in clustering the interacting properties between small molecules and proteins. In this report, we describe the application of CoMIPA for protein clustering. A sample set of 15 proteins that share less than 20% homology and have no common functional motifs in primary structure were chosen. Using CoMIPA, we were able to cluster proteins that bound to the same small molecule. Other structural homology-based clustering programs such as PSI-BLAST or PFAM were unable to achieve the same classification. The results are striking because it is difficult to find any common features in the active sites of these proteins that share the same ligand. CoMIPA adds new dimensions for protein classification and has the potential to be a helpful tool in predicting and analyzing molecular interactions.

Structural Features and Transcriptional Activity of Chicken PPARs (α, ß, and γ).

While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α, ß, and γ) that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ) gene is shorter than that in humans and lacks a γ2 isoform. Moreover, in serum-free media, cPPARγ shows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARα and ß functions. Moreover, we found that bezafibrate induces the transactivation function of cPPARß, but not human PPARδ (human PPARß ortholog). This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444). These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

A water-soluble carbon nanotube network conjugated by nanoparticles with defined nanometre gaps.

Cage-shaped proteins with an affinity for carbonaceous materials were constructed and used to assemble a nanostructure in which single-walled carbon nanotubes are surrounded by cobalt oxide nanoparticles with nanometre gaps. By changing the size of proteins and materials incorporated inside the cavity, similar structures with distinctively different properties can be fabricated.

Expression and functional analysis of Nr2e3, a photoreceptor-specific nuclear receptor, suggest common mechanisms in retinal development between avians and mammals.

The photoreceptor-specific nuclear receptor (PNR; Nr2e3) is a transcription factor important for retinal development. We report here the identification and expression analysis of the avian Nr2e3. Nr2e3 mRNA is expressed in the photoreceptor layer of the neural retina during early stages of chick embryogenesis. Its temporal expression is distinct from that of a related nuclear receptor, Tlx. Chick Nr2e3 recognizes and binds to the same target DNA sequence as its vertebrate orthologs. Functional assays revealed that chick Nr2e3 acts as a transcriptional repressor. Our results suggest that Nr2e3 plays a common role in retinal development in vertebrates.

Conservation of functional domain structure in bicarbonate-regulated "soluble" adenylyl cyclases in bacteria and eukaryotes.

Soluble adenylyl cyclase (sAC) is an evolutionarily conserved bicarbonate sensor. In mammals, it is responsible for bicarbonate-induced, cAMP-dependent processes in sperm required for fertilization and postulated to be involved in other bicarbonate- and carbon dioxide-dependent functions throughout the body. Among eukaryotes, sAC-like cyclases have been detected in mammals and in the fungi Dictyostelium; these enzymes display extensive similarity extending through two cyclase catalytic domains and a long carboxy terminal extension. sAC-like cyclases are also found in a number of bacterial phyla (Cyanobacteria, Actinobacteria, and Proteobacteria), but these enzymes generally possess only a single catalytic domain and little, if any, homology with the remainder of the mammalian protein. Database mining through a number of recently sequenced genomes identified sAC orthologues in additional metazoan phyla (Arthropoda and Chordata) and additional bacterial phyla (Chloroflexi). Interestingly, the Chloroflexi sAC-like cyclases, a family of three enzymes from the thermophilic eubacterium, Chloroflexus aurantiacus, are more similar to eukaryotic sAC-like cyclases (i.e., mammalian sAC and Dictyostelium SgcA) than they are to other bacterial adenylyl cyclases (ACs) (i.e., from Cyanobacteria). The Chloroflexus sAC-like cyclases each possess two cyclase catalytic domains and extensive similarity with mammalian enzymes through their carboxy termini. We cloned one of the Chloroflexus sAC-like cyclases and confirmed it to be stimulated by bicarbonate. These data extend the family of organisms possessing bicarbonate-responsive ACs to numerous phyla within the bacterial and eukaryotic kingdoms.

Molecular evaluation using in silico protein interaction profiles.

MOTIVATION: To find a correlation between the activities and structures of molecules is one of the most important subjects for molecular evaluation study. Traditional quantitative structure-activity relationship (QSAR) methodologies represent those attempts using physicochemical descriptors. Creating a new molecular description factor based on the results of a computational docking study will add new dimensions to molecular evaluation. RESULTS: We propose a new molecular description factor analysis system called the Comparative Molecular Interaction Profile Analysis (CoMIPA) system in which the AutoDock program is used for docking evaluation of small molecule compound-protein complexes. Interaction energies are calculated, and the data sets obtained are called interaction profiles (IPFs). Using the IPF as a scoring indicator, the system could be a powerful tool to cluster the interacting properties between small molecules and bio macromolecules such as ligand-receptor bindings. Further development of the system will enable us to predict the adverse effects of a drug candidate.