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Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.

Purpose Oncolytic viral therapy for neuroblastoma (NB) cells with Sindbis virus (SINV) is a promising strategy for treating high-risk NB. Here, we evaluated the possibility of using SINV structural proteins as therapeutic agents for NB since UV-inactivated SINV could induce cytopathogenic effects. Methods The cytotoxicity of UV-inactivated SINV toward human NB cell lines NB69, NGP, GOTO, NLF, SK-N-SH, SH-SY5Y, CHP134, NB-1, IMR32, and RT-BM-1 were analyzed. Apoptosis was confirmed by TUNEL assays. To determine the components of SINV responsible for the cytotoxicity of UV-inactivated SINV, expression vectors encoding the structural proteins, namely capsid, E2, and E1, were transfected in NB cells. Cytotoxicity was evaluated by MTT assays. Results UV-inactivated SINV elicited more significant cytotoxicity in NB69, NGP, and RT-BM-1 than in normal human fibroblasts. Results of the transfection experiments showed that all NB cell lines susceptible to UV-inactivated SINV were highly susceptible to the E1 protein, whereas fibroblasts transfected with vectors harboring capsid, E1, or E2 were not. Conclusions We demonstrated that the cytotoxicity of the UV-inactivated SINV is due to apoptosis induced by the E1 structural protein of SINV, which can be used selectively as a therapeutic agent for NB.

Biomineralization is an elaborate process that controls the deposition of inorganic materials in living organisms with the aid of associated proteins. Magnetotactic bacteria mineralize magnetite (Fe3O4) nanoparticles with finely tuned morphologies in their cells. Mms6, a magnetosome membrane specific (Mms) protein isolated from the surfaces of bacterial magnetite nanoparticles, plays an important role in regulating the magnetite crystal morphology. Although the binding ability of Mms6 to magnetite nanoparticles has been speculated, the interactions between Mms6 and magnetite crystals have not been elucidated thus far. Here, we show a direct adsorption ability of Mms6 on magnetite nanoparticles in vitro. An adsorption isotherm indicates that Mms6 has a high adsorption affinity (Kd = 9.52 µM) to magnetite nanoparticles. In addition, Mms6 also demonstrated adsorption on other inorganic nanoparticles such as titanium oxide, zinc oxide, and hydroxyapatite. Therefore, Mms6 can potentially be utilized for the bioconjugation of functional proteins to inorganic material surfaces to modulate inorganic nanoparticles for biomedical and medicinal applications.

DNA microarrays are useful to detect microorganisms for various purposes including clinical testing and food safety. However, conventional DNA microarrays need complicated operations such as amplification, fluorescence labeling, and washing steps. To address this issue, we previously developed the signaling probe-based DNA microarray system that can eliminate these steps, and demonstrated a direct detection of bacterial genes. Nonetheless, this system requires well-designed probe sets due to the fluorescence resonance energy transfer (FRET)-based mode of action. Up to date, the probe design was highly dependent on the trial-and-error processes. In this study, we propose a strategy to rationally design the sequences of signaling probes based on the thermodynamic analysis. This analysis aided to improve the probe performance approximately 2.8 times, without experiments, by suppressing the secondary structure formation of the probes. We successfully demonstrated the specific and amplification-free detection of 5S rRNA from total RNA extracted from Escherichia coli within 30 min.

In this study, we developed a novel DNA microarray system that does not require fluorophore-labeling, amplification, or washing of the target nucleic acid fragments. Two types of DNA probes (so-called “signaling probes”) labeled with a fluorescence dye (Cy3) and quencher molecule (BHQ2) were spotted on the DNA microarray such that fluorescent signals of Cy3 could be quenched by BHQ2 due to duplex formation between the probes. The addition of the target DNA or RNA fragments disrupted the duplex formed by the probes, resulting in the generation of fluorescence signals. We examined the assay conditions of the signaling probe-based DNA microarray, including the design of the probes, hybridization temperatures, and methods for fragmentation of target molecules. Since this approach does not require time-consuming processes, including labeling, amplification, and washing, the assay achieved specific detection of 16S rDNA and 16S rRNA extracted from Escherichia coli within 60 min, which was significantly rapid compared to conventional PCR-dependent DNA microarrays.

Genetic analysis of single-cells is widely recognized as a powerful tool for understanding cellular heterogeneity and obtaining genetic information from rare populations. Recently, many kinds of single-cell isolation systems have been developed to facilitate single-cell genetic analysis. However, these systems mainly target non-adherent cells or cells in a cell suspension. Thus, it is still challenging to isolate single-adherent cells of interest from a culture dish using a microscope. We had previously developed a single-cell isolation technique termed "gel-based cell manipulation" (GCM). In GCM, single-cells could be visualized by photopolymerizable-hydrogel encapsulation that made it easier to isolate the single-cells. In this study, GCM-based isolation of single-adherent cancer cells from a culture dish was demonstrated. Single-adherent cells were encapsulated in a photopolymerizable hydrogel using a microscope and isolated with high efficiency. Furthermore, whole genome amplification and sequencing for the isolated single-adherent cell could be achieved. We propose that the GCM-based approach demonstrated in this study has the potential for efficient analysis of single-adherent cells at the genetic level.

Background and Objective: The relationship between periodontal sensation and Myofascial Pain (MP) is not yet fully clarified. The aim of this study was to test the null hypothesis that there is no difference in the periodontal sensation threshold between subjects with MP and subjects with no Temporomandibular Disorders (TMD). Methods: Participants have clinically assessed in accordance with the Research Diagnostic Criteria for Temporomandibular Disorders version 1.0 guidelines and assigned to the MP group (mean age 54.8 ± 14.8 years; 1 male and 11 females) or the control group (mean age: 63.9 ± 13.2 years; 1 male and 15 females). The Passive Periodontal Sensation Threshold (PPST) was evaluated using impulsive mechanical stimulation on the occlusal surface parallel to the tooth axis of the maxillary first molar, if present. The difference in the mean PPST between the MP group and the control group was evaluated using the Student t-test after checking for homoscedasticity. Results: The mean PPST value was 1050.1 ± 480.3 mN in the MP group and 712.3 ± 288.5 mN in the control group. A significant difference was observed between these mean PPST values (p = 0.045). Conclusion: There was a significant difference in PPST between the MP group and the control group. Although the etiology of the change of PPST is still unknown, the higher PPST value observed in MP patients suggests that future study on occlusal dysesthesia or occlusal sensation-related pathosis is warranted.

Omega-3 fatty acids (ω3 FAs) have attracted attention because they have various health benefits for humans. Fish oils are currently major sources of ω3 FAs, but a sustainable supply of ω3 FAs based on fish oils is problematic because of the increasing demand. In this study, the production potential of a genetically engineered marine cyanobacterium, Synechococcus sp. strain NKBG 15041c, was examined as an alternative source of ω3 FAs. A change in fatty acid composition of this cyanobacterium was successfully induced by the expression of a heterologous Δ6-desaturase, and the transformants synthesized stearidonic acid, which the wild type cannot produce. As a result of optimization of culture conditions, maximal contents of stearidonic acid and total ω3 FAs reached 12.2 ± 2.4 and 118.1 ± 3.5 mg/g, respectively. The maximal ω3 FA productivity was 4.6 ± 0.7 mg/(L⋅day). These are the highest values of the contents of stearidonic acid and ω3 FAs in genetically engineered cyanobacteria reported thus far. Therefore, genetically engineered Synechococcus sp. strain NKBG 15041c may be a promising sustainable source of ω3 fatty acids.

The contamination of foods and beverages by fungi is a severe health hazard. The rapid identification of fungi species in contaminated goods is important to avoid further contamination. To this end, we developed a fungal discrimination method based on the bioimage informatics approach of colony fingerprinting. This method involves imaging and visualizing microbial colonies (referred to as colony fingerprints) using a lens-less imaging system. Subsequently, the quantitative image features were extracted as discriminative parameters and subjected to analysis using machine learning approaches. Colony fingerprinting has been previously found to be a promising approach to discriminate bacteria. In the present proof-of-concept study, we tested whether this method is also useful for fungal discrimination. As a result, 5 fungi belonging to the Aspergillus, Penicilium, Eurotium, Alternaria, and Fusarium genera were successfully discriminated based on the extracted parameters, including the number of hyphae and their branches, and their intensity distributions on the images. The discrimination of 6 closely-related Aspergillus spp. was also demonstrated using additional parameters. The cultivation time required to generate the fungal colonies with a sufficient size for colony fingerprinting was less than 48 h, shorter than those for other discrimination methods, including MALDI-TOF-MS. In addition, colony fingerprinting did not require any cumbersome pre-treatment steps prior to discrimination. Colony fingerprinting is promising for the rapid and easy discrimination of fungi for use in the ensuring the safety of food manufacturing.

Discrimination of food-contaminating microorganisms is an essential technology to secure the safety in manufacturing of foods and beverages. Conventionally, discrimination of the microorganisms has been performed by morphological observation, genetic analysis, and more recently, biochemical fingerprinting using mass spectrometry. However, several drawbacks exist in these methods, such as long assay time, cumbersome operations, and expensive equipment. To address these issues, we have proposed a novel method for discrimination of food-contaminating microorganisms, termed “colony fingerprinting”, based on bioimage informatics. In colony fingerprinting, growth of bacterial colonies were monitored using a lens-less imaging system. The characteristic images of colonies, referred to as colony fingerprints (CFPs), were obtained over time, and subsequently used to extract discriminative parameters. We demonstrated to discriminate 20 bacterial species by analyzing the extracted parameters with machine learning approaches, namely support vector machine and random forest. Colony fingerprinting is a promising method for rapid and easy discrimination of food-contaminating microorganisms.

Living organisms can produce inorganic materials with unique structure and properties. The biomineralization process is of great interest as it forms a source of inspiration for the development of methods for production of diverse inorganic materials under mild conditions. Nonetheless, regulation of biomineralization is still a challenging task. Magnetotactic bacteria produce chains of a prokaryotic organelle comprising a membrane-enveloped single-crystal magnetite with species-specific morphology. Here, we describe regulation of magnetite biomineralization through controlled expression of the mms7 gene, which plays key roles in the control of crystal growth and morphology of magnetite crystals in magnetotactic bacteria. Regulation of the expression level of Mms7 in bacterial cells enables switching of the crystal shape from dumbbell-like to spherical. The successful regulation of magnetite biomineralization opens the door to production of magnetite nanocrystals of desired size and morphology.

Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated an account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independent of magnetite crystal biomineralization when grown in a medium containing selenium oxyanion (SeO 3 2− ). Quantitative analysis shows that magnetotactic bacteria accumulate the largest amount of target molecules (Se) per cell compared with any other previously reported nonferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated than Te taken up into cells and Cd 2+ adsorbed onto the cell surface, respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. The biomagnetic recovery and the effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganisms has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report an example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the largest amount of Se compared to other foreign elements. More importantly, the Se-accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water.

Interests in safety and security of foods are more growing than ever with rising global populations. Manufacturing processes for food production need to be strictly managed to exclude the possible contamination by toxic microorganisms including bacteria and fungi. Upon occurrence of microbial contamination, the species of the contaminating microorganisms should be rapidly discriminated to limit further expansion of contamination. Pre-testing of microbial contamination of food ingredients prior to manufacturing processes is also carried out to ensure that specific toxic microorganisms are not detected from the ingredients. Conventionally, sequencing of the barcode regions in the microbial genomes including the genes encoding 16S and 18S rRNA (for prokaryotes and eukaryotes, respectively) has been widely employed for microbial discrimination. More recently, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based discrimination method emerged. However, these approaches have several drawbacks such as requirements of expensive equipment, highly skilled operators, and relatively long assay time for several days. Therefore, it is not easy to employ these methods in food manufacturers, majorities of which are small-sized companies. To address this issue, we have developed a novel bioimaging-based approach for discrimination of bacteria and fungi, termed “colony fingerprinting”. In colony fingerprinting, bacterial or fungal colonies were formed on transparent agar media. The colonies were irradiated by light emitting diode (LED), and complementary metal-oxide-semiconductor (CMOS) image sensors are used to detect the light penetrating or dispersed by the colonies which generate microbial species-specific optical patterns, termed “colony fingerprints”. We extracted a number of discriminative parameters representing the features of morphology and intensity-distribution of the colony fingerprints with the aid of bioimage informatics tools. Discrimination of microbial species were carried out by machine learning-based analyses of the extracted parameters. As a proof-of-concept study, we developed a colony fingerprinting platform consisting of a CMOS image sensor (pixel size: 3.2 μm, imaging area: 6.55 × 4.92 mm2), pinhole, and blue LED. Colonies of 5 closely related Staphylococcus spp. (S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. simulans) were visualized, and 14 types of discriminative parameters such as roundness, solidity, Zernike moment, and image entropy were extracted using MATLAB software. Simple linear discrimination analysis (LDA) using the 14 parameters classified the 25 colony fingerprints of 5 Staphylococcus spp. (a total of 125 colony fingerprints) into correct species with 79.4% accuracy. Further analyses with powerful machine learning approaches including artificial neural network (ANN), support vector machine (SVM), and random forest (RF) resulted in the accuracies of 99.2%, 98.4%, and 100.0%, respectively. It was possible to obtain the colony fingerprints used for discrimination within 11 h on average. This result suggests that colony fingerprinting is promising for accurate and rapid discrimination of closely related bacterial species. We successfully demonstrated the discrimination of 20 bacterial species (5 gram-negative and 15 gram-positive bacteria) based on colony fingerprinting by employing up to 65 discrimination parameters obtained by a size-dependent manner. Therefore, accumulation of discriminative parameters could be useful to improve the discrimination accuracy. Besides bacteria, fungi are another major cause of food contamination. However, colony fingerprinting-based discrimination of fungi is challenging because fungi extend the hyphae (fungal filaments) to random directions, and branch them at arbitrary positions. These morphological features are much different from bacterial colonies. Therefore, the discriminative parameters employed for bacterial discrimination were not applicable, and a novel set of parameters need to be developed. Given the growth features of fungal colonies, we computed the number of hyphae and their branches, and intensity distribution on the images as discrimination parameters. As a result of SVM- and RF-based analyses with 7 types of parameters, 6 closely-related Aspergillus spp. were distinguished with 95% and 100%, respectively. Discrimination of fungi based on colony fingerprinting was completed within 48 h, which was shorter than time required by conventional fugal discrimination methods including MALDI-TOF-MS, Raman spectrometry and FTIR spectrometry. Given the promising features of colony fingerprinting, we newly developed a practicable platform for colony fingerprinting which equips a line imaging sensor with scanning system enabling the visualization of a number of culture plates, leading to high throughput analysis. We examined accuracy and robustness of colony fingerprinting-based bacterial discrimination by changing the colony formation conditions such as temperatures and cell densities on the culture plates. In summary, discrimination of microorganisms was successfully demonstrated with the machine learning-guided colony fingerprinting. In the future study, development of the colony fingerprint library with hundreds of microbial species will make this rapid and robust system a powerful tool to secure food safety.

Lipid tubules are of particular interest for many potential applications in nanotechnology. Among various lipid tubule fabrication techniques, the morphological regulation of membrane structure by proteins mimicking biological processes may provide the chances to form lipid tubes with highly tuned structures. Magnetotactic bacteria synthesize magnetosomes (a unique prokaryotic organelle comprising a magnetite crystal within a lipid envelope). MamY protein is previously identified as the magnetosome protein responsible for magnetosome vesicle formation and stabilization. Furthermore, MamY is shown in vitro liposome tubulation activity. In this study, the interaction of MamY and phospholipids is investigated by using a lipids-immobilized membrane strip and a peptide array. Here, the binding of MamY to the anionic phospholipid, cardiolipin, is found and enhanced liposome tubulation efficiency. The authors propose the interaction is responsible for recruiting and locating cardiolipin to elongate liposome in vitro. The authors also suggest a similar mechanism for the invagination site in magnetosomes vesicle formation, where the lipid itself contributes further to increasing the curvature. These findings are highly important to develop an effective biomimetic synthesis technique of lipid tubules and to elucidate the unique prokaryotic organelle formation in magnetotactic bacteria.

Thermoresponsive magnetic nanoparticles (MNPs) were synthesized using a magnetosome display system. An elastin-like polypeptide decamer of VPGVG (ELP10), which is hydrophobic above the transition temperature (Tt) and can form an insoluble aggregation, was immobilized on biogenic MNPs in the magnetotactic bacterium, Magnetospirillum magneticum AMB-1. It was suggested that hydrophobicity of the MNP surface increased at 60 °C compared with 20 °C by the immobilization of ELP10. Size distribution analysis indicated that the immobilization of ELP10 onto MNPs induced the increased hydrophobicity with increasing temperatures up to 60 °C, promoting aggregation of the particles by hydrophobic and magnetic interactions. These results suggest that the acceleration of magnetic collection at 60 °C was caused by particle aggregation promoted by hydrophobic interaction between ELP-MNPs. Furthermore, the immobilization of ELP on MNPs gave a quick magnetic collection at 60 °C by external magnetic field. The thermoresponsiv...

Identifying the most appropriate polymorph of active pharmaceutical ingredients is one of the important steps in drug development, since their bioactivities are largely dependent on their solid forms. However, the sample preparation for the characterization of crystal forms is time-consuming and requires large quantities of sample. Here, we introduce a microfluidic device-based method to prepare a sub-millimeter-sized single aspirin crystal from a small quantity of material. For the crystal preparation, a device equipped with a solution flow system and temperature controller was placed under the microscope. To use the device, concentration–temperature phase diagrams were generated, and regions where dominant nucleation or crystal growth with specific directions were clearly determined. By observing time-dependent changes of crystal number and size with solution temperature, a pathway to grow a single crystal of aspirin was determined and applied to prepare a sub-millimeter-sized crystal from 250 μg of asp...

Rapid and sensitive detection of microorganisms is important in cosmetics, foods, and pharmacy manufacturing from the aspect of biocontrol. The PCR test could be a potential alternative technology enabling rapid detection. However, it also meets technical difficulties that genome of microorganisms should be amplified from extremely low DNA concentration samples. To overcome these difficulties, DNA recovery and condensation process is a key step for the PCR-based detection. Our research group has developed a DNA extraction method using polyamidoamine dendron-modified magnetic nanoparticles (PAMAM-MNPs), and successfully demonstrated adsorption of high amount of DNA at the level of microgram, and efficient DNA desorption greater than 95%. In contrast, the potential of PAMAM-MNPs for recovery of tiny amount of DNA was unknown. In this study, we applied the PAPAM-MNPs to DNA recovery at less than 10 3 bacterial genome. DNA desorption behavior from the particles was characterized by adding a variety of organic solvents in the desorption buffer, and we identified that dimethyl sulfoxide (DMSO) addition much improved the DNA recovery. With the improved DNA recovery procedure using DMSO, 45.3% of 10 3 bacterial genome was recovered. Because commercialized magnetic particles for DNA extraction demonstrated only 7.5% of DNA recovery, our method showed much higher performance for small amount of DNA recovery. Furthermore, recovery of bacterial DNA at a single cell was successfully performed using the same procedure. We believe that the DNA recovery procedure presented in this study is promising for efficient DNA extraction from a single bacterial cell.

Circulating tumor cells (CTCs) are well recognized as useful biomarker for cancer diagnosis and potential target of drug discovery for metastatic cancer. Efficient and precise recovery of extremely low concentrations of CTCs from blood has been required to increase the detection sensitivity. Here, an automated system equipped with a microcavity array (MCA) was demonstrated for highly efficient and reproducible CTC recovery. The use of MCA allows selective recovery of cancer cells from whole blood on the basis of differences in size between tumor and blood cells. Intra- and inter-assays revealed that the automated system achieved high efficiency and reproducibility equal to the assay manually performed by well-trained operator. Under optimized assay workflow, the automated system allows efficient and precise cell recovery for non-small cell lung cancer cells spiked in whole blood. The automated CTC recovery system will contribute to high-throughput analysis in the further clinical studies on large cohort of cancer patients.

Magnetotactic bacteria are a group of microorganism producing nano-sized magnetic particles. The bacterial cells accumulate a large amount of iron ion from aquatic environment and synthesize single-crystal magnetic nano-particles under ambient conditions. The size, shape, and composition of the magnetic nano-particles are precisely regulated in individual bacterial cell types. Thus, the understanding of molecular mechanism should provide ideas to design and create magnetic nano-materials with environmentally friendly synthetic routes. This chapter describes the molecular mechanism of magnetic nano-particle formation that has been clarified based on comprehensive molecular analyses of magnetotactic bacteria. Identified proteins from the basic studies are shown to be available for the development of novel magnetic nano-materials. The strategy and fundamental technologies that are useful for the understanding of biomineralization mechanisms are also introduced.

Magnetotactic bacteria synthesize nano-sized magnetic particles in the cells. The bacterial magnetic particles have a core of magnetite (Fe3O4) and are surrounded by a lipid bilayer membrane containing a number of proteins, referred to as magnetosome. Since the bioengineering methodology for magnetotactic bacteria was established, expression of a wide range of functional proteins onto magnetic particles has successfully been performed in which the native proteins in the lipid membrane can serve as anchors for the protein display. The expression system, here we call “magnetosome-display system,” has enabled us to reduce the cost of production of protein-magnetic particle complexes. These advantages lead to creation of a variety of magnetic particles displaying functional proteins, including membrane proteins and disulfide-bonded proteins, which the basic approach cannot match. This review provides an overview of the developmental status of magnetic particles in the field of bioassays, summarizes magnetosome display system by magnetotactic bacteria, and discusses their usefulness and prospects in the medical and environmental fields. The novel system has shown considerable promise for improving the display efficiency of the difficult-to-express proteins and thus is expected to contribute to further development of functional magnetic particles toward biotechnological applications.

Pathogenic viral infections are an international public health concern, and viral disinfection has received increasing attention. Electrochemical treatment has been used for treatment of water contaminated by bacteria for several decades, and although in recent years several reports have investigated viral inactivation kinetics, the mode of action of viral inactivation by electrochemical treatment remains unclear. Here, we demonstrated the inactivation of feline calicivirus (FCV), a surrogate for human noroviruses, by electrochemical treatment in a developed flow-cell equipped with a screen-printed electrode. The viral infectivity titer was reduced by over 5 orders of magnitude after 15 min of treatment at 0.9V vs. Ag/AgCl. Proteomic study of electrochemically inactivated virus revealed oxidation of peptides located in the viral particles; oxidation was not observed in the non-treated sample. Furthermore, transmission electron microscopy revealed that viral particles in the treated sample had irregular structures. These results suggest that electrochemical treatment inactivates FCV via oxidation of peptides in the structural region, causing structural deformation of virus particles. This first report of viral protein damage through electrochemical treatment will contribute to broadening the understanding of viral inactivation mechanisms.

Marine microalgae are recognized as promising feedstocks for biofuels and chemicals owing to their higher growth rates than those of terrestrial crop plants. We aimed to summarize the production of biofuels and chemicals by marine microalgae and to discuss their advantages and potential from the aspect of bioprocess. The present circumstances of the microalgae industry were briefly described and large-scale industrial plants for microalgae production, where some marine microalgae are cultivated, were introduced. The advantages of marine microalgae in terms of water and land usage were also discussed. Finally, novel genome editing tools that could further exploit the potential of marine microalgae were reviewed. The present study provided comprehensive information regarding current biotechnology using marine microalgae.

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H–perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H–perfluorododecanoic acid (2H,2H,8H,8H–PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H–perfluorodecanoic acid (6H,6H–PFDeA) concentration, and decreases in 2H,2H,8H,8H–PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.

Summary Fistulifera sp. strain JPCC DA0580, a marine pennate diatom, contains extremely high levels of intracellular triglyceride and has been suggested as a promising source of feedstock for biodiesel fuels. JPCC DA0580 was isolated from a mangrove swamp located in Sumiyo Bay below the mouths of the Sumiyo and Yakugachi Rivers in Amami-Ohshima, Kagoshima, Japan. In this study, the morphology and the 18S rDNA sequence of JPCC DA0580 were compared with those of other Fistulifera strains. JPCC DA0580 possesses morphological characters of the genus Fistulifera, namely lightly silicified frustules, a distinct median costa (raphe sternum), and a wart-like central pore (fistula). Morphometric analysis revealed that JPCC DA0580 differs from other Fistulifera species by the presence of a valve with coarser striation and coarser areolation. On the basis of 18S rDNA phylogeny, JPCC DA0580 formed a well-supported clade with other members of the Fistulifera species complex, although the number of nucleotide substitutions was highest in JPCC DA0580. Our results led us to propose the taxonomic name Fistulifera solaris sp. nov. for JPCC DA0580.

Magnetotactic bacteria synthesize magnetosomes comprised of membrane-enveloped single crystalline magnetite (Fe3 O4 ). The size and morphology of the nano-sized magnetite crystals (

An electrochemical disinfection system employing a honeycombed platinum coated titanium electrode was developed for the disinfection of seawater. Cell suspensions (2 l, 10³ cells/ml) of the fish pathogens, Vibrio alginolyticus, Edwardsiella tarda, Lactococcus garvieae and Vibrio anguillarum were circulated in a reactor equipped with 10 sets of these electrodes at a flow rate of 200 ml/min with an applied potential of 1.0 V vs. Ag/AgCl reference electrode. The circulated cells were completely disinfected after 3 h of treatment, whereas free residual chlorine generated due to seawater electrolysis was below 0.1 ppm. In addition, a diphenyl-1-pyrenylphosphine fluorescent assay revealed that lipid peroxidation in the cell membranes of disinfected bacteria was induced probably by reactive oxygen species generated during electrochemical treatment.

SiC fiber-bonded ceramics, SA-Tyrannohex®, (SA-THX) with perpendicular and parallel fiber orientations were brazed using Ag-, Ni- and Pd-base brazes, and four Si–X (X: Ti, Cr, Y, Ta) eutectics. Outcomes were variable, ranging from bonded joints through partially bonded to un-bonded joints. Prominent Ti- and Si-rich interfaces developed with Cusil-ABA, Ticusil, and Copper-ABA and Ni- and Si-rich layers with MBF-20. Stress rupture tests at 650 and 750°C on Cusil-ABA-bonded joints revealed a temperature-dependent behavior for the perpendicular joints but not for the parallel joints with failure occurring at brazed interface. Higher-use temperatures can be targeted with eutectic Si–Ti and Si–Cr alloys.

In this study, we present a method for efficient enrichment of small-sized circulating tumor cells (CTCs) such as those found in the blood of small-cell lung cancer (SCLC) patients using a microcavity array (MCA) system. To enrich CTCs from whole blood, a microfabricated nickel filter with a rectangular MCA (10(4) cavities/filter) was integrated with a miniaturized device, allowing for the isolation of tumor cells based on differences in size and deformability between tumor and blood cells. The shape and porosity of the MCA were optimized to efficiently capture small tumor cells on the microcavities under low flow resistance conditions, while allowing other blood cells to effectively pass through. Under optimized conditions, approximately 80% of SCLC (NCI-H69 and NCI-H82) cells spiked in 1 mL of whole blood were successfully recovered. In clinical samples, CTCs were detectable in 16 of 16 SCLC patients. In addition, the number of leukocytes captured on the rectangular MCA was significantly lower than that on the circular MCA (p0.001), suggesting that the use of the rectangular MCA diminishes a considerable number of carryover leukocytes. Therefore, our system has potential as a tool for the detection of CTCs in small cell-type tumors and detailed molecular analyses of CTCs.

Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

Spodoptera litura (Fabricius) males were monitored with sex-pheromone traps from spring to early summer in 1999, 2002, 2003, 2009, and 2010 in four regions in East Asia: western Japan, South Korea, China, and Taiwan. The backward trajectory analysis was conducted to investigate whether any air current arrived over the monitoring sites around the dates when distinguished increases in catches of >100 insects per day occurred. The catch data showed abrupt catch increases. Some of the catch increases coincidentally appeared among monitoring sites several hundreds of kilometers apart. These catch increases were found to coincide with the arrival of southwesterly air currents within 24–36 h from southern China and/or Taiwan. Other evidence related to the overseas migration are discussed in detail. The results of this and previous studies suggest that S. litura males immigrate into western Japan and South Korea in spring to early summer using air currents from southern China and/or Taiwan.