Stoichiometrically Controlled Immobilization of Multiple Enzymes on Magnetic Nanoparticles by the Magnetosome Display System for Efficient Cellulose Hydrolysis

The immobilization of multiple cellulase complexes receiving attention for use in the efficient hydrolysis of celluloses. In this study, the magnetosome display system was employed for the preparation of systems mimicking natural multiple cellulase complexes (cellulosomes) on magnetic nanoparticles (MNPs). Initially, two fluorescent proteins, namely, green fluorescent protein and mCherry, were immobilized on MNPs. Fluorescence analysis revealed the close proximity of two different proteins on the MNPs. Enzyme-linked immunosorbent assay analysis showed that stoichiometrically equivalent amounts of the proteins were immobilized on the MNPs. Next, endoglucanase (EG) and β-glucosidase (BG) were immobilized on MNPs to give EG/BG-MNPs. The resulting MNPs were applied for the hydrolysis of celluloses, with rapid hydrolysis of carboxymethyl cellulose being observed. Furthermore, the fusion of the cellulose-binding domain to EG/BG-MNPs promoted improved hydrolysis activity against the insoluble cellulose. We could therefore conclude that the magnetosome display system can expand the possibilities of mimicking natural cellulosome organization on MNPs

High-Throughput Manipulation of Circulating Tumor Cells Using a Multiple Single-Cell Encapsulation System with a Digital Micromirror Device

Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms

Manipulation of a Single Circulating Tumor Cell Using Visualization of Hydrogel Encapsulation toward Single-Cell Whole-Genome Amplification

Genetic characterization of circulating tumor cells (CTCs) could guide the choice of therapies for individual patients and also facilitate the development of new drugs. We previously developed a CTC recovery system using a microcavity array, which demonstrated highly efficient CTC recovery based on differences in cell size and deformability. However, the CTC recovery system lacked an efficient cell manipulation tool suitable for subsequent genetic analysis. Here, we resolve this issue and present a simple and rapid manipulation method for single CTCs using a photopolymerized hydrogel, polyethylene glycol diacrylate (PEGDA), which is useful for subsequent genetic analysis. First, PEGDA was introduced into the cells entrapped on the microcavity array. Then, excitation light was projected onto the target single cells for encapsulation of each CTC by confocal laser-scanning microscopy. The encapsulated single CTCs could be visualized by the naked eye and easily handled with tweezers. The single CTCs were only partially encapsulated on the PEGDA hydrogel, which allowed for sufficient whole-genome amplification and accurate genotyping. Our proposed methodology is a valuable tool for the rapid and simple manipulation of single CTCs and is expected to become widely utilized for analyses of mammalian cells and microorganisms in addition to CTCs

Microfluidic Device with Chemical Gradient for Single-Cell Cytotoxicity Assays

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0–100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research

of Peptide-mediated microalgae harvesting method for efficient biofuel production

of Peptide-mediated microalgae harvesting method for efficient biofuel productio

Additional file 2: of Homoeolog expression bias in allopolyploid oleaginous marine diatom Fistulifera solaris

Table S1. Codon usage in genome and subgenome of F. solaris. Codon usage in (A) genome and (B) subgenome of F. solaris in per mil. (A) The table includes the codon usage of all genes predicted in F. solaris, including homoeologous genes, non-homoeologous genes, and genes predicted on scaffolds not assembled on chromosomes. (B) The table includes the codon usage of homoeologous genes and non-homoeologous genes in each subgenome. The values corresponding to Fso_h and Fso_l were written in left (bold) and right (italic) of each column, respectively. Values in parentheses represent the ratio of codon usage between Fso_h and Fso_l (Fso_h / Fso_l); Table S2. Top 10 and bottom 10 loadings of PC1 and PC2 from the PCA based on codon usage frequency; Table S3. tRNA genes discovered in each subgenome of F. solaris; Table S4. Nucleotides sequence motifs discovered in promoter region of consistently biased homoeologous genes in F. solaris; Table S5. Lists of genes which contained the motif TABASTA in promoter region. (XLSX 183 kb

Benzene toxicity in human leukocytes from Hu-NOG mice.

<p>(A) Human leukocytes collected from the peripheral blood and hematopoietic organs of Hu-NOG mice. Upper panel: histogram of hCD45⁺mCD45⁻ cells in Hu-NOG mice administered 0 (gray), 30 (red), or 300 mg (blue-lined) benzene/kg-b.w./day. Lower panel: numbers of hCD45⁺mCD45⁻ cells in Hu-NOG mice. Each point represents the mean ± SD of each group (n = 7 or n = 8). * <i>p</i><0.05 and ** <i>p</i><0.01 represent significant differences compared with untreated mice, as determined by <i>t</i> tests. (B) Numbers of human myeloid and lymphoid cells in the bone marrow or peripheral blood of Hu-NOG mice. Human myeloid cells were identified as hCD45⁺mCD45⁻hCD33⁺ cells (open square). Human lymphoid cells were identified as hCD45⁺mCD45⁻hCD33⁻ cells (solid square). Each point represents the mean of each group (n = 7 or n = 8). * <i>p</i><0.05 and ** <i>p</i><0.01 represent significant differences compared with untreated mice as determined by <i>t</i> tests. (C) The percentage of each T cell population in the thymus of Hu-NOG mice. The value was calculated based on the ratio of hCD45⁺mCD45⁻hCD33⁻ cells. Individual types of T cells were determined by using combinations of anti-hCD4 and hCD8 antibodies. Values represent means (n = 7 or n = 8).</p

Proteomics Analysis of Oil Body-Associated Proteins in the Oleaginous Diatom

For biodiesel production from microalgae, it is desirable to understand the entire triacylglycerol (TAG) metabolism. TAG accumulation occurs in oil bodies, and although oil body-associated proteins could play important roles in TAG metabolism, only a few microalgal species have been studied by a comprehensive analysis. Diatoms are microalgae that are promising producers of biodiesel, on which such proteomics analysis has not been conducted to date. Herein, we identified oil body-associated proteins in the oleaginous diatom <i>Fistulifera</i> sp. strain JPCC DA0580. The oil body fraction was separated by cell disruption with beads beating and subsequent ultracentrifugation. Contaminating factors could be removed by comparing proteins from the oil body and the soluble fractions. This novel strategy successfully revealed 15 proteins as oil body-associated protein candidates. Among them, two proteins, which were parts of proteins predicted to have transmembrane domains, were indeed confirmed to specifically localize to the oil bodies in this strain by observation of GFP fusion proteins. One (predicted to be a potassium channel) was also detected from the ER, suggesting that oil bodies might originate from the ER. By utilizing this novel subtraction method, we succeeded in identifying the oil body-associated proteins in the diatom for the first time

Size-Selective Microcavity Array for Rapid and Efficient Detection of Circulating Tumor Cells

Circulating tumor cells (CTCs) are tumor cells circulating in the peripheral blood of patients with metastatic cancer. Detection of CTCs has clinical significance in cancer therapy because it would enable earlier diagnosis of metastasis. In this research, a microfluidic device equipped with a size-selective microcavity array for highly efficient and rapid detection of tumor cells from whole blood was developed. The microcavity array can specifically separate tumor cells from whole blood on the basis of differences in the size and deformability between tumor and hematologic cells. Furthermore, the cells recovered on the microcavity array were continuously processed for image-based immunophenotypic analysis using a fluorescence microscope. Our device successfully detected approximately 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked with 10−100 NCI-H358 cells. In addition, breast, gastric, and colon tumor cells lines that include EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic separation, were successfully recovered on the microcavity array with high efficiency (more than 80%). On an average, approximately 98% of recovered cells were viable. Our microfluidic device has high potential as a tool for the rapid detection of CTCs and can be used to study CTCs in detail

Comparison of conserved motifs in Δ9 desaturases from diatoms.

<p>Comparison of conserved motifs in Δ9 desaturases from diatoms.</p