Hirotaka Tajima

Mlp24 (McpX) of Vibrio cholerae Implicated in Pathogenicity Functions as a Chemoreceptor for Multiple Amino Acids

ABSTRACT The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18 l-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison with Vibrio parahaemolyticus, we speculated that at least 17 MLPs of V. cholerae may mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection. mlp24 deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity of V. cholerae.

Ligand Specificity Determined by Differentially Arranged Common Ligand-binding Residues in Bacterial Amino Acid Chemoreceptors Tsr and Tar

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.

Fabrication of Microstructures Embedding Controllable Particles Inside Dielectrophoretic Microfluidic Devices

This paper presents a method of particle manipulation by dielectrophoresis (DEP) and immobilization using photo-crosslinkable resin inside microfluidic devices. High speed particle manipulation, including patterning and concentration control by DEP was demonstrated. Immovable and movable microstructures embedding particles were fabricated on-chip. Several microelectrodes were fabricated using Indium Tin Oxides (ITO) and Cr/Au. The two kinds of DEP responses of yeast cells (W303) and other particles were experimentally confirmed. Based on negative DEP phenomenon, cell traps generated by microelectrodes were demonstrated. Position control, transportation and patterning of cells were performed with cell traps. The on-chip fabrication of arbitrary shapes of microstructures based on Poly(ethylene glycol) diacrylate (PEGDA) was reported. With cell patterning by DEP and immobilization using on-chip fabrication, microstructures embedding line patterned cells were fabricated inside microfluidic channels. A novel microfluidic device was designed to separate patterning and fabrication areas and movable microstructures embedding concentration controllable particles were fabricated inside this device.

Biodegradable porous sheet-like scaffolds for soft-tissue engineering using a combined particulate leaching of salt particles and magnetic sugar particles

Scaffolds serving as artificial extracellular matrixes (ECMs) play a pivotal role in the process of tissue regeneration by providing optimal cellular environments for penetration, ingrowth, and vascularization. Stacks of sheet-like scaffold can be engineered to become artificial ECMs, suggesting a great potential for achieving complex 3-D tissue regeneration to support cell survival and growth. In this study, we proposed and investigated a combined particulate leaching of magnetic sugar particles (MSPs) and salt particles for the development of a sheet-like scaffold. MSPs were fabricated by encapsulating NdFeB particles inside sugar spheres and were controlled using magnetic fields as a porogen to control pore size, pore structure and pore density while fabricating the scaffold. We studied the influence of the strength of the magnetic fields in controlling the coating thickness of the unmagnetized MSPs during the fabrication of the sheet-like scaffolds. The experimental relationship between magnetic flux density and the thickness of the MSP layer was illustrated. Furthermore, we investigated the infiltration capacity of different concentrations of poly(L-lactide-co-ɛ-caprolactone) (PLCL) as a scaffold material on MSP clusters. Following polymer casting and removal of the sugar template, spherical pores were generated inside the scaffolds. Cultivation of NIH/3T3 fibroblasts on the fabricated scaffold proves that the proposed method can be applied in the cell sheet fabrication.

Local nano-injection of fluorescent nano-beads inside C. elegans based on nanomanipulation

This paper presents a novel selective nano-injection method using fluorescent nanobeads based on nanomanipulation for in-vivo single cell analysis. The nanomanipulation system was constructed under hybrid microscope. The hybrid microscope consists of the optical microscope (OM) and environmental-scanning electron microscope (E-SEM) to realize biological specimen analysis by optical imaging including fluorescent imaging, and nano-scale manipulation by E-SEM imaging. Based on the bio-nano manipulation system, we propose a nanoinjection method by nanoprobe insertion and nanobead embeddedness. The nanoprobe was designed to have four tips to fix single nanobead. It is fabricated by focused ion beam (FIB) process. Its nano-scale size is considered to be important for minimal damage to a biological specimen. In this work, as biological specimen, the Caenorhabditis elegans (C. elegans) was used. It is one of the important model organisms for various diseases analysis. The nanoinjection technique is applied to transport fluorescent materials or specific biological organism into a specific cell for in-vivo experiment of C. elegans. The proposed system is considered to be important as future nano-surgery system for life innovation using model organism.

Fabrication of thermoresponsive gel blocks using hysteresis towards cell assembly

In this paper, we propose a new method to assemble microstructures made of a thermoresponsive gel using hysteresis character of the thermoresponsive polymer solution. This method can be used for 3 dimensional cell assembly by embedding cells in the thermoresponsive gel structures. Gel blocks can be maintained the gel condition by the hysteresis character and the microstructures can be formed by assembling the gel blocks. The temperature distribution around a microheater was analyzed to generate the thermoresponsive gel and avoid thermal damages to cells. The generation of thermoresponsive gel was conducted using the microheater which was embedded in a probe tip and manipulated by a micromanipulator. The fabrication of a gel block was achieved using the hysteresis character and the fabricated gel block was picked and placed by the probe. Cells were embedded in the gel by controlling the position of microheater to avoid the influence of thermal convection flow. The positioning of the gel blocks can be precisely controlled by the micromanipulator. The results indicate the method we propose has a great possibility to achieve 3D cell assembly without large stress to cells during the assembly and cell culture.

Fabrication of 3D photo-resistive structure for artificial capillary blood vessel

We propose a new method to fabricate artificial capillary blood vessel using direct laser writing. IP-L and Ormocomp is used as the photo-resistive material. A hollow pipe microstructure, a 3×3 array twig microstructure and array of hollow twig microstructure is fabricated with IP-L as photo-resistive material. Ormocomp, a biocompatible photoresist is used to fabricate a similar design. These designs are purposely chosen because these structures can resemble capillary blood vessel. SU-8 is employed as the developer for both photoresist. The fabrication time and the comparison between IP-L and Ormocomp fabrication are discussed. Fabrication time is related to the fabrication model chosen during direct laser writing process. Combined model is recommended as the fabrication takes a shorter time compare to solid model and the microstructure is more likely to sustain on the substrate after development. Laser power is another important parameter during fabrication. IP-L can use up to 100% laser power while Ormocomp microstructure must be fabricated with laser power less than 100%. The fabrication of artificial capillary blood vessel has the potential application in testing new drugs for its glomerular filtration rate.

Local injection probe of functional micro-nano gel tools into Caenorhabditis elegans

Model organism is widely used in the biology and medical research fields for investigating the biological system, evaluating the drugs affectivities, and so on. The importance of model organism is 1) easy to culture, 2) well-known decoding of genes, 3) similarity with human, and so on. As model organism, we interests in Caenorhabditis elegans (C. elegans) is considered to be useful biological organism for various diseases analysis, because it is one of the smallest animal and they have many homologues genes with the human disease genes.

Fabrication of thermoresponsive gel blocks using hysteresis for cell assembly

In this paper, we propose a new method to assemble microstructures made of a thermoresponsive gel using hysteresis character of the thermoresponsive polymer solution. This method can be used for 3 dimensional cell assembly by embedding cells in the thermoresponsive gel structures. Gel blocks can be maintained the gel condition by the hysteresis and the microstructures can be formed by assembling the gel blocks. The temperature distribution around the microheater was analyzed to avoid thermal damages to cells. The generation of thermoresponsive gel is conducted using the microheater which is manipulated by a micromanipulator. The fabrication of a gel block was conducted using the hysteresis character and cells were embedded in the gel. The positioning of the gel blocks can be precisely controlled by the micromanipulator. The results indicate the method we propose has a great possibility to achieve 3D cell assembly without large stress to cells during the assembly and cell culture.

Fabrication and self-assembly of movable microstructures embedding cells with concentration control inside microfluidic devices

Currently the research about large quantities cells assembly is seriously regarded, since it can provide high efficiency methods for artificial tissue engineering. In this paper, we report a novel cell assembly method based on cell immobilization by photo-crosslinkable resin, cell manipulation by dielectrophoresis (DEP) and microfluidic self-assembly inside microfluidic devices. The on-chip fabrication of movable microstructures embedding yeast cells (W303) based on Poly (ethylene glycol) Diacrylate (PEGDA) was reported. Novel microelectrodes are fabricated by Cr/Au for manipulating cells before immobilization. A microfluidic chip for controlling cell concentration with separated patterning and fabrication areas is fabricated. Movable microstructures embedding microbeads of which the concentration is controllable are fabricated in the microfluidic channel, inside PEGDA and NaCl solution. A 2-layer microfluidic device is fabricated by Polydimethylsiloxane (PDMS) and self-assembly method of the fabricated movable microstructures via this device is preliminarily demonstrated.