Montmorillonite-supramolecular hydrogel hybrid for fluorocolorimetric sensing of polyamines.

Fluorescent sensor materials for rapidly and conveniently detecting polyamines in biological fluids are highly desirable for cancer diagnosis. We herein describe the hybridization of a supramolecular hydrogel with a layered inorganic host adsorbing a fluorescent dye which produces a fluorocolorimetric sensor for spermine and spermidine, important biomarkers for cancers, in artificial urine.

Supramolecular hydrogel exhibiting four basic logic gate functions to fine-tune substance release.

Logic-gate operations displaying macroscopic outputs are promising systems for the development of intelligent soft materials that can perform effective functions in response to various input patterns. A supramolecular hydrogel comprising the phosphate-type hydrogelator 1 exhibits macroscopic gel-sol behavior in response to four distinct input stimuli: temperature, pH, Ca(2+), and light. We characterized this performance through microscopic, spectroscopic, and rheological measurements. On the basis of its multiple-stimulus responsiveness, we constructed gel-based supramolecular logic gates from hydrogelator 1 that demonstrated AND, OR, NAND, and NOR types of stimulus-responsive gel-sol behavior in the presence of various combinations of the four stimuli. Implementation of such logic-gate functions into semiwet soft materials (e.g., supramolecular hydrogels) is an important step toward the design of controlled drug delivery and release systems. Indeed, we demonstrate herein that one of our gel-based supramolecular logic gates is capable of holding and releasing bioactive substances in response to logic triggers. Furthermore, combining our supramolecular gel-based AND logic gate with a photoresponsive supramolecular gel could temporarily modulate the release rate of the bioactive substance.

Three-dimensional encapsulation of live cells by using a hybrid matrix of nanoparticles in a supramolecular hydrogel.

From a library of glyco-lipid mimics with muconic amide as the spacer, we found that 1, a glyco-lipid that has N-acetyl glucosamine and methyl cyclohexyl groups as its hydrophilic head and hydrophobic tails, respectively, formed a stable hydrogel (0.05 wt %) through hierarchical self-assembly of the lipid molecules into supramolecular nanofibers. The formation of the supramolecular hydrogel was verified by rheological measurements, and the supramolecular nanofiber was characterized as the structural element by transmission electron microscopy and atomic force microscopy observations. Absorption and circular dichroism spectroscopic measurements revealed that the muconic amide moieties of 1 are arranged in a helical, stacked fashion in the self-assembled nanofibers. Moreover, we unexpectedly found that the homogeneous distribution of the supramolecular nanofibers of 1 was greatly facilitated by the addition of polystyrene nanobeads (100-500 nm in diameter), as evaluated by confocal laser scanning microscopic observations. It is interesting that the obtained supramolecular hybrid matrix can efficiently encapsulate and distribute live Jurkat cells in three dimensions under physiological conditions. This supramolecular hybrid matrix is intriguing as a unique biomaterial.

Organization and localization of the dnaA and dnaK gene regions on the multichromosomal genome of Burkholderia multivorans ATCC 17616.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.

Stiff, multistimuli-responsive supramolecular hydrogels as unique molds for 2D/3D microarchitectures of live cells.

Supramolecular hydrogels constructed through molecular self-assembly of small molecules have unique stimuli-responsive properties; however, they are mechanically weak in general, relative to conventional polymer gels. Very recently, we developed a zwitterionic amino acid tethered amphiphilic molecule 1, which gave rise to a remarkably stiff hydrogel comparable with polymer-based agarose gel, retaining reversible thermal-responsive properties. In this study, we describe that rational accumulation of multiple and orthogonal noncovalent interactions in the supramolecular nanofibers of 1 played crucial roles not only in the mechanical reinforcement but also in the multistimuli responsiveness. That is, the zwitterionic amino acid moiety and the C-C double bond unit of the hydrogelator 1 can function as a pH-responsive unit and a light-responsive unit, respectively. We also demonstrated that this stiff and multistimuli-responsive supramolecular hydrogel 1 is applied as a unique mold for 2D and 3D-patterning of various substances. More significantly, we succeeded in the fabrication of a collagen gel for spatial patterning, culturing, and differentiation of live cells by using hydrogel 1 molds equipped with 2D/3D microspace channels (100-200 µm in diameter).

Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia multivorans.

The fur (ferric uptake regulator) gene of Burkholderia multivorans ATCC 17616 was identified by transposon mutagenesis analysis. The fur deletion mutant of strain ATCC 17616 (i) constitutively produced siderophores, (ii) was more sensitive to reactive oxygen species (ROS) than the wild-type strain, (iii) showed lower superoxide dismutase and catalase activities than the wild-type strain, (iv) was unable to grow on M9 minimal agar plates containing several substrates that can be used as sole carbon sources by the wild-type strain, and (v) was hypersensitive to nitrite and nitric oxide under microaerobic and aerobic conditions, respectively. These results clearly indicate that the Fur protein in strain ATCC 17616 plays pleiotropic roles in iron homeostasis, removal and/or resistance to ROS and nitrosative stress, and energy metabolism. Furthermore, employment of an in vivo Fur titration assay system led to the isolation from the ATCC 17616 genome of 13 Fur-binding DNA regions, and a subsequent electrophoretic mobility-shift assay confirmed the direct binding of Fur protein to all of these DNA regions. Transcriptional analysis of the genes located just downstream of the Fur-binding sites demonstrated that Fur acts as a repressor for these genes. Nine of the 13 regions were presumed to be involved in the acquisition and utilization of iron.

Mechanical reinforcement of a supramolecular hydrogel comprising an artificial glyco-lipid through supramolecular copolymerization.

Supramolecular copolymer hydrogels were prepared by mixing 1 and the additives 2-8, and their rheological properties were evaluated. It was found that additive 3 reinforced the mechanical strength of the resultant hydrogel most efficiently, increasing the yield stress of SCH 1+3 about fourfold. The optimal mixing between the glyco-lipid hydrogelator 1 and the additive 3 sufficiently enhanced the mechanical strength of the resultant SCH, which improved the handling of the SH on the large scale. These results indicate that supramolecular copolymerization can provide the supramolecular hydrogel with desired properties and/or functions.

Photo gel-sol/sol-gel transition and its patterning of a supramolecular hydrogel as stimuli-responsive biomaterials.

In a focused library of glycolipid-based hydrogelators bearing fumaric amide as a trans-cis photoswitching module, several new photoresponsive supramolecular hydrogelators were discovered, the gel-sol/sol-gel transition of which was pseudo-reversibly induced by light. Studying the optimal hydrogel by NMR spectroscopy and various microscopy techniques showed that the trans-cis photoisomerization of the double bond of the fumaric amide unit effectively caused assembly or disassembly of the self-assembled supramolecular fibers to yield the macroscopic hydrogel or the corresponding sol, respectively. The entanglement of the supramolecular fibers produced nanomeshes, the void space of which was roughly evaluated to be 250 nm based on confocal laser scanning microscopy observations of the size-dependent Brownian motion of nanobeads embedded in the supramolecular hydrogel. It was clearly shown that such nanomeshes become a physical obstacle that captures submicro- to micrometer-sized substrates such as beads or bacteria. By exploiting the photoresponsive property of the supramolecular nanomeshes, we succeeded in off/on switching of bacterial movement and rotary motion of bead-tethered F(1)-ATPase, a biomolecular motor protein, in the supramolecular hydrogel. Furthermore, by using the photolithographic technique, gel-sol photopatterning was successfully conducted to produce sol spots within the gel matrix. The fabricated gel-sol pattern not only allowed regulation of bacterial motility in a limited area, but also off/on switching of F1-ATPase rotary motion at the single-molecule level. These results demonstrated that the photoresponsive supramolecular hydrogel and the resulting nanomeshes may provide unique biomaterials for the spatiotemporal manipulation of various biomolecules and live bacteria.

High-temperature-induced transposition of insertion elements in burkholderia multivorans ATCC 17616.

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42 degrees C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.

Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.