Discovery of long-chain polyamines embedded in the biosilica on the Bacillus cereus spore coat.

Biosilicification is the process by which organisms incorporate soluble, monomeric silicic acid, Si(OH), in the form of polymerized insoluble silica, SiO. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification has only recently begun to be studied. We previously reported that biosilicification occurs in the gram-positive, spore-forming bacterium Bacillus cereus, and that silica is intracellularly deposited on the spore coat as a protective coating against acids, although the underlying mechanism is not yet fully understood.

Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes.

Objectives: This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs).

Results: We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs.

A novel salt- and organic solvent-tolerant phosphite dehydrogenase from sp. ATCC 51142.

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium sp. ATCC 51142 (Ct-PtxD).

Gatekeeper Residue Replacement in a Phosphite Transporter Enhances Mutational Robustness of the Biocontainment Strategy.

Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO)-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO) transporters. This strategy did not allow to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.

Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly and Robust NADPH Regeneration System.

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from sp.

Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles.

Extracellular vesicles (EVs), such as exosomes, have garnered increasing interest because of their potential clinical applications that range from diagnostics to therapeutics. The development of an automated and reproducible EV purification platform would therefore aid the introduction of EV biomarkers and therapies into the clinic. Here, we demonstrate that K8- as well as K-16 peptides (containing 8 and 16 lysine residues with dissociation constants of 102 nM and 11.

Arginine-mediated dissociation of single cells and cell sheets from a polystyrene culture dish.

Here, we report a novel non-enzymatic cell dissociation method, based on our finding that adherent cells dissociate rapidly from the polystyrene culture dish when incubated in an l- or d-arginine-containing solution. We also demonstrate the successful detachment of confluent NIH/3T3 cell monolayers from the culture dish as a cell sheet by the addition of an arginine solution.

Live-cell imaging of macrophage phagocytosis of asbestos fibers under fluorescence microscopy.

Background: Frustrated phagocytosis occurs when an asbestos fiber > 10 μm in length is engulfed imperfectly by a macrophage, and it is believed to be associated with chromosomal instability. Few studies have focused on dynamic cellular imaging to assess the toxicity of hazardous inorganic materials such as asbestos. One reason for this is the relative lack of fluorescent probes available to facilitate experimental visualization of inorganic materials.

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic β cells.

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic β cells.

High photodynamic activities of water-soluble inclusion complexes of 5,15-diazaporphyrins in cyclodextrin.

Water-soluble inclusion complexes of 5,15-diazaporphyrin derivatives in the cavities of two trimethyl-β-cyclodextrins (TMe-β-CDxs) were synthesised. In the 2 : 1 complexes, two aryl groups of the diazaporphyrins protruded from the upper rims of two TMe-β-CDxs. The complexes displayed high photodynamic activity under photoirradiation at wavelengths longer than 620 nm.

Stabilisation of lipid membrane-incorporated porphyrin derivative aqueous solutions and their photodynamic activities.

Lipid membrane-incorporated porphyrin derivatives (LMIPors) having three phenyl moieties and one pyridyl or pyridinium moiety at the meso-positions were more stable than LMIPors having phenyl and/or pyridyl moieties. The former two LMIPors showed high photodynamic activity toward cancer cells under photoirradiation at wavelengths over 600 nm, which are the most suitable for photodynamic therapy. The photodynamic activities were greater than that of Photofrin, which is currently the main drug employed clinically as a photosensitiser.

Synthetic Phosphorus Metabolic Pathway for Biosafety and Contamination Management of Cyanobacterial Cultivation.

Recent progress in genetic engineering and synthetic biology have greatly expanded the production capabilities of cyanobacteria, but concerns regarding biosafety issues and the risk of contamination of cultures in outdoor culture conditions remain to be resolved. With this dual goal in mind, we applied the recently established biological containment strategy based on phosphite (HPO, Pt) dependency to the model cyanobacterium Synechococcus elongatus PCC 7942 ( Syn 7942). Pt assimilation capability was conferred on Syn 7942 by the introduction of Pt dehydrogenase (PtxD) and hypophosphite transporter (HtxBCDE) genes that allow the uptake of Pt, but not phosphate (HPO, Pi).

Improvement of Photodynamic Activity of Lipid-Membrane-Incorporated Fullerene Derivative by Combination with a Photo-Antenna Molecule.

The weak absorbance of pristine C , C , and fullerene derivatives at wavelengths over 600 nm hampers the use of these molecules as photosensitizers (PSs) for photodynamic therapy (PDT). The coexistence of light-harvesting antenna molecules with a fullerene derivative in lipid membrane bilayers solved this issue. By controlling the location of the C derivative in the lipid membrane, the liposomal dyad system for PDT improved the photodynamic activity via an efficient photoenergy transfer from antenna molecules to the fullerene derivative.

Photodynamic Activities of Porphyrin Derivative-Cyclodextrin Complexes by Photoirradiation.

Water-soluble cyclodextrin (CyD) complexed with porphyrin derivatives with different substituents in the -positions showed different photodynamic activities toward cancer cells under illumination at wavelengths over 600 nm, the most suitable wavelengths for photodynamic therapy (PDT). In particular, aniline- and phenol-substituted derivatives had high photodynamic activity because of the efficient intracellular uptake of the complexes by tumor cells. These complexes showed greater photodynamic activity than photofrin, currently the main drug in clinical use as a photosensitizer.

A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite.

There is a growing demand to develop biocontainment strategies that prevent unintended proliferation of genetically modified organisms in the open environment. We found that the hypophosphite (HPO, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (HPO, Pt) but not phosphate (HPO, Pi), suggesting the potential for engineering a Pt/HPt-dependent bacterial strain as a biocontainment strategy. We disrupted all Pi and organic Pi transporters in an Escherichia coli strain expressing HtxABCDE and a Pt dehydrogenase, leaving Pt/HPt uptake and oxidation as the only means to obtain Pi.

Improved photodynamic activities of liposome-incorporated [60]fullerene derivatives bearing a polar group.

[60]Fullerene (C) derivatives were incorporated into liposomes using a fullerene exchange method involving the transfer of the fullerene from the cavity of two γ-cyclodextrin molecules to a liposome. A lipid-membrane-incorporated C derivative bearing a polar group showed much higher photodynamic activity than the analogous system incorporating pristine C.

Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model.

A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids.

A portable method of specific nucleic acid detection would be very useful for monitoring public health in a variety of settings for point-of-care and point-of-need testing. However, conventional methods for the detection of nucleic acids are not ideal for use in the field, as they require skilled operators and complex equipment. Here, we constructed a method for specific nucleic acid detection using a split G-quadruplex (Gq) structure that can recognize target nucleic acids without competitive reactions in a bimolecular reaction and directly produce a detectable signal based on peroxidase activity.

A BRET-based homogeneous insulin assay using interacting domains in the primary binding site of the insulin receptor.

A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulin-sensing probe proteins Nluc-αCT and L1-YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay.

A FRET-based DNA nano-tweezer technique for the imaging analysis of specific mRNA.

A DNA nano-tweezer (DNA-NT) structure-based target mRNA detection probe, which uses fluorescence resonance energy transfer (FRET) as a detection signal and works as a single molecule, has been developed. This FRET-paired fluorescent dye-modified DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from open to closed states and produces a FRET signal in response to in vitro transcripts of Hes-1 mRNA. Our results showed that the FRET-based DNA-NT detected both GLUT1 mRNA as a pre-fixed target mRNA model and Hes-1 mRNA as a model expressed inside a living cell.

Assembly of zinc finger motif-fused enzymes on a dsDNA scaffold for catalyzing consecutive reactions with a proximity effect.

The feasibility of assembling enzymes, catalyzing consecutive reactions, on to a double-stranded DNA (dsDNA) scaffold utilizing zinc finger motifs is described. The catalytic activities of two zinc finger motif-fused enzymes catalyzing a bioluminescence reaction with energy recycling, namely pyruvate phosphate dikinase and firefly luciferase, have been evaluated. Bioluminescence measurements with dsDNA scaffolds coding a different distance between the binding sites for each zinc finger motif-fused enzyme confirmed the effect of the distance, proving the proximity effect of ATP recycling presumed to be the result of efficient intermediate diffusion.

A universal DNA-based protein detection system.

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system.

Potential use of Lactobacillus cell density in feces as a non-invasive bio-indicator for evaluating environmental stress during mouse breeding.

From the viewpoint of the quality assurance of laboratory animals, it is important to guarantee that they have not accidentally been exposed to any stress during breeding. In this study, we investigated non-invasive indicators of the exposure of mice to stress. The stress of horizontal shaking and no-bedding was applied to mice and the intestinal bacterial flora in their feces was analyzed.