Polarization-sensitive intensity diffraction tomography.

Optical anisotropy, which is an intrinsic property of many materials, originates from the structural arrangement of molecular structures, and to date, various polarization-sensitive imaging (PSI) methods have been developed to investigate the nature of anisotropic materials. In particular, the recently developed tomographic PSI technologies enable the investigation of anisotropic materials through volumetric mappings of the anisotropy distribution of these materials. However, these reported methods mostly operate on a single scattering model, and are thus not suitable for three-dimensional (3D) PSI imaging of multiple scattering samples. Here, we present a novel reference-free 3D polarization-sensitive computational imaging technique-polarization-sensitive intensity diffraction tomography (PS-IDT)-that enables the reconstruction of 3D anisotropy distribution of both weakly and multiple scattering specimens from multiple intensity-only measurements. A 3D anisotropic object is illuminated by circularly polarized plane waves at various illumination angles to encode the isotropic and anisotropic structural information into 2D intensity information. These information are then recorded separately through two orthogonal analyzer states, and a 3D Jones matrix is iteratively reconstructed based on the vectorial multi-slice beam propagation model and gradient descent method. We demonstrate the 3D anisotropy imaging capabilities of PS-IDT by presenting 3D anisotropy maps of various samples, including potato starch granules and tardigrade.

Comprehensive Transcriptomic Analysis of Cordyceps militaris Cultivated on Germinated Soybeans.

The ascomycete fungus Cordyceps militaris infects lepidopteran larvae and pupae and forms characteristic fruiting bodies. Owing to its immune-enhancing effects, the fungus has been used as a medicine. For industrial application, this fungus can be grown on geminated soybeans as an alternative protein source. In our study, we performed a comprehensive transcriptomic analysis to identify core gene sets during C. militaris cultivation on germinated soybeans. RNA-Seq technology was applied to the fungal cultures at seven-time points (2, 4, and 7-day and 2, 3, 5, 7-week old cultures) to investigate the global transcriptomic change. We conducted a time-series analysis using a two-step regression strategy and chose 1460 significant genes and assigned them into five clusters. Characterization of each cluster based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed that transcription profiles changed after two weeks of incubation. Gene mapping of cordycepin biosynthesis and isoflavone modification pathways also confirmed that gene expression in the early stage of GSC cultivation is important for these metabolic pathways. Our transcriptomic analysis and selected genes provided a comprehensive molecular basis for the cultivation of C. militaris on germinated soybeans.

Microarrays Incorporating Gold Grid Patterns for Protein Quantification.

Protein microarrays are miniaturized two-dimensional arrays, incorporating thousands of immobilized proteins, typically printed in minute amounts on functionalized solid substrates, which can be analyzed in a high-throughput fashion. Irreproducibility of the printing techniques adopted, resulting in inconsistently and nonuniformly deposited microscopic spots, nonuniform signal intensities from the printed microspots, and significantly high background noise are some of the critical issues that affect protein analysis using traditional protein microarrays. To overcome such issues, in this study, we introduced a novel gold grid pattern-based protein microarray. The grid patterns incorporated in our microarray are equivalent to the spots used for protein analysis in conventional protein microarrays. We utilized the signal intensities from the grid patterns acting as spots for quantifying the protein concentration levels. To demonstrate the utility of our novel design concept, we quantified as low as 66.7 ng/mL of bovine serum albumin using our gold grid pattern-based protein microarray. Our grid pattern-based design concept for protein quantification overcame the signal nonuniformity issues and ensured that the dominance of any distorted signal from a single spot did not affect the overall protein quantification results as encountered in conventional protein microarrays.

DNA aptamer release from the DNA-SWNT hybrid by protein recognition.

Here we show the formation of the complex between a DNA aptamer and a single-walled carbon nanotube (SWNT) and its reaction with its target protein. The aptamer, which is specifically bound with thrombin, the target protein in this study, easily wraps and disperses the SWNT by noncovalent π-π stacking.

Cell response induced by internalized bacterial magnetic nanoparticles under an external static magnetic field.

Magnetic nanoparticles are widely used in bioapplications such as imaging and targeting tool. Their magnetic nature allows for the more efficient bioapplications by an external field gradient. However their combined effects have not yet been extensively characterized. Herein, we first demonstrate the biological effects of the communications between internalized bacterial magnetic nanoparticles (BMPs) and an external static magnetic field (SMF) on a standard human cell line. Combination of the BMPs and SMF act as the key factor leading to the alteration of cell structure and the enhanced cell growth. Also, their interaction reduced the apoptotic efficiency of human tumor cells induced by anticancer drugs. Microarray analysis suggests that these phenomena were caused by the alterations of GPCRs-mediated signal transduction originated in the interaction of internalized BMPs and the external SMF. Our findings may offer new approach for targeted cell therapy with the advantage of controlling cell viability by magnetic stimulation.

Amyloidogenesis of type III-dependent harpins from plant pathogenic bacteria.

Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His(6)-HpaG formed biologically active spherical oligomers, protofibrils, and beta-sheet-rich fibrils, whereas the null HR mutant His(6)-HpaG(L50P) did not. Biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from an alpha-helix to beta-sheet-rich fibrils is important for the biological activity of protein. The fibrillar form of His(6)-HpaG is an amyloid protein based on positive staining with Congo red to produce green birefringence under polarized light, increased protease resistance, and beta-sheet fibril structure. Other harpins, such as HrpN from Erwinia amylovora and HrpZ from Pseudomonas syringae pv. syringae, also formed curvilinear protofibrils or fibrils under plant apoplast-like conditions, suggesting that amyloidogenesis is a common feature of harpins. Missense and deletion mutagenesis of HpaG indicated that the rate of HpaG fibril formation is modulated by a motif present in the C terminus. The plant cytotoxicity of HpaG is unique among the amyloid-forming proteins that occur in several microorganisms. Structural and morphological analogies between HpaG and disease-related amyloidogenic proteins, such as Abeta protein, suggest possible common biochemical characteristics in the induction of plant and animal cell death.

Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island.

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.