Adsorption of Biomineralization Protein Mms6 on Magnetite (Fe3O4) Nanoparticles.

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.

Lensless imaging-based discrimination between tumour cells and blood cells towards circulating tumour cell cultivation.

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.

Sindbis viral structural protein cytotoxicity on human neuroblastoma cells.

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.

Gel-based cell manipulation method for isolation and genotyping of single-adherent cells.

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.

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.

Biosynthesis of Thermoresponsive Magnetic Nanoparticles by Magnetosome Display System.

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 thermoresponsive properties will further expand the utility of biotechnological applications of biogenic MNPs.

Colony Fingerprint-Based Discrimination of Staphylococcus species with Machine Learning Approaches.

Detection and discrimination of bacteria are crucial in a wide range of industries, including clinical testing, and food and beverage production. Staphylococcus species cause various diseases, and are frequently detected in clinical specimens and food products. In particular, S. aureus is well known to be the most pathogenic species. Conventional phenotypic and genotypic methods for discrimination of Staphylococcus spp. are time-consuming and labor-intensive. To address this issue, in the present study, we applied a novel discrimination methodology called colony fingerprinting. Colony fingerprinting discriminates bacterial species based on the multivariate analysis of the images of microcolonies (referred to as colony fingerprints) with a size of up to 250 µm in diameter. The colony fingerprints were obtained via a lens-less imaging system. Profiling of the colony fingerprints of five Staphylococcus spp. (S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. simulans) revealed that the central regions of the colony fingerprints showed species-specific patterns. We developed 14 discriminative parameters, some of which highlight the features of the central regions, and analyzed them by several machine learning approaches. As a result, artificial neural network (ANN), support vector machine (SVM), and random forest (RF) showed high performance for discrimination of theses bacteria. Bacterial discrimination by colony fingerprinting can be performed within 11 h, on average, and therefore can cut discrimination time in half compared to conventional methods. Moreover, we also successfully demonstrated discrimination of S. aureus in a mixed culture with Pseudomonas aeruginosa. These results suggest that colony fingerprinting is useful for discrimination of Staphylococcus spp.

Evaluation of cancer cell deformability by microcavity array.

A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was achieved within 3 min. Protrusions were formed at the bottom surface of the array with a pore size of 3 µm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which can provide insight into the mechanisms underlying tumor cell malignancy.

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H-perfluorododecanol in activated sludge.

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.

Production of ω3 fatty acids in marine cyanobacterium Synechococcus sp. strain NKBG 15041c via genetic engineering.

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.

Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

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.

Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals.

UNLABELLED: The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. IMPORTANCE: Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial regulation mechanism within the linear structure of magnetosomes. This discovery provides evidence of a highly regulated protein localization mechanism for this bacterial organelle development.

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.

Biomagnetic Recovery and Bioaccumulation of Selenium Granules in Magnetotactic Bacteria.

UNLABELLED: 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 (SeO3 (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.

Co-ordinated functions of Mms proteins define the surface structure of cubo-octahedral magnetite crystals in magnetotactic bacteria.

Magnetotactic bacteria synthesize magnetosomes comprised of membrane-enveloped single crystalline magnetite (Fe3 O4 ). The size and morphology of the nano-sized magnetite crystals (< 100 nm) are highly regulated and bacterial species dependent. However, the control mechanisms of magnetite crystal morphology remain largely unknown. The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo-octahedral magnetite crystals in Magnetospirillum magneticum strain AMB-1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co-ordinated functions of Mms proteins regulate the morphology of the cubo-octahedral magnetite crystals in magnetotactic bacteria.

Oncolytic viral therapy for neuroblastoma cells with Sindbis virus AR339 strain.

PURPOSE: With current treatment regimens, high-risk neuroblastoma (NB) remains largely incurable. Oncolytic viral therapy uses replication-competent viruses, like Sindbis virus (SINV), to kill cancers. The SINV AR339 strain is blood borne and relatively non-virulent. We evaluated the feasibility of SINV AR339 for treating human NB. METHODS: The cytotoxicity and viral growth of SINV AR339 were evaluated for five human NB cell lines, SK-N-SH, IMR-32, LAN-5, GOTO, and RT-BM-1. SINV-induced apoptosis was confirmed by TUNEL assays and PARP-1 cleavage. In vivo effects of SINV on neuroblastoma cell xenografts in nude mice were assessed by intratumoral or intravenous SINV inoculation. RESULTS: In five human NB cell lines, SINV infections induced remarkable cytotoxicity. The mRNA expressions of anti-apoptotic genes, Bcl-2 and Bcl-xL, in LAN-5 and RT-BM-1, which were less sensitive to SINV infection, increased in response to SINV infection, while the other NB cell lines sensitive to SINV infection failed to respond. In nude mice, intratumoral and intravenous SINV inoculations caused significant regression of NB xenograft tumors. CONCLUSION: Our results suggested that SINV AR339 was significantly oncolytic against human NB. Thus, SINV showed promise as a novel therapy for treating NB.

Functional expression of an scFv on bacterial magnetic particles by in vitro docking.

A Gram-negative, magnetotactic bacterium, Magnetospirillum magneticum AMB-1 produces nano-sized magnetic particles (BacMPs) in the cytoplasm. Although various applications of genetically engineered BacMPs have been demonstrated, such as immunoassay, ligand-receptor interaction or cell separation, by expressing a target protein on BacMPs, it has been difficult to express disulfide-bonded proteins on BacMPs due to lack of disulfide-bond formation in the cytoplasm. Here, we propose a novel dual expression system, called in vitro docking, of a disulfide-bonded protein on BacMPs by directing an immunoglobulin Fc-fused target protein to the periplasm and its docking protein ZZ on BacMPs. By in vitro docking, an scFv-Fc fusion protein was functionally expressed on BacMPs in the dimeric or trimeric form. Our novel disulfide-bonded protein expression system on BacMPs will be useful for efficient screening of potential ligands or drugs, analyzing ligand-receptor interactions or as a magnetic carrier for affinity purification.

Line image sensor-based colony fingerprinting system for rapid pathogenic bacteria identification.

The rapid identification of pathogenic bacteria is crucial across various industries, including food or beverage manufacturing. Bacterial microcolony image-based classification has emerged as a promising approach to expedite identification, automate inspections, and reduce costs. However, conventional imaging methods have significant practical limitations, namely low throughput caused by the limited imaging range and slow imaging speed. To address these challenges, we developed an imaging system based on a line image sensor for rapid and wide-field imaging compared to existing colony imaging methods. This system can image a standard Petri dish (92 mm in diameter) completely within 22 s, successfully acquiring bacterial microcolony images. This process yielded a set of discrimination parameters termed as colony fingerprints, which were employed for machine learning. We demonstrated the performance of our system by identifying Staphylococcus aureus in food products using a machine learning model trained on a colony fingerprint dataset of 15 species from 9 genera, including foodborne pathogens. While conventional mass spectrometry-based methods require 24 h of incubation, our colony fingerprinting approach achieved 96% accuracy in just 10 h of incubation. Line image sensor offer high imaging speeds and scalability, allowing for swift and straightforward microbiological testing, eliminating the need for specialized expertise and overcoming the limitations of conventional methods. This innovation marks a transformative shift in industrial applications.

Microcavity array system for size-based enrichment of circulating tumor cells from the blood of patients with small-cell lung cancer.

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 (p < 0.001), 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.