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)4, in the form of polymerized insoluble silica, SiO2. 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. In eukaryotic biosilicifying organisms, such as diatoms and siliceous sponges, several relevant biomolecules are embedded in biogenic silica (biosilica). These biomolecules include peptides, proteins, and long-chain polyamines. In this study, we isolated organic compounds embedded in B. cereus biosilica to investigate the biomolecules involved in the prokaryotic biosilicification process and identified long-chain polyamines with a chemical structure of H2N-(CH2)4-[NH-(CH2)3]n-NH2 (n: up to 55). Our results demonstrate the common presence of long-chain polyamines in different evolutionary lineages of biosilicifying organisms, i.e., diatoms, siliceous sponges, and B. cereus, suggesting a common mechanism underlying eukaryotic and prokaryotic biosilicification.

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. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol. CONCLUSION: The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

A novel salt- and organic solvent-tolerant phosphite dehydrogenase from Cyanothece 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 Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0-10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na+, K+, and NH4 +. It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD+. Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD+-binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD+ under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L-tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent.

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 (HPO32-)-dependent biocontainment strategy, by expressing a phosphite-specific transporter HtxBCDE and phosphite dehydrogenase in bacteria devoid of their indigenous phosphate (HPO42-) transporters. This strategy did not allow Escherichia coli to generate escape mutants (EMs) in growth media containing phosphate as a phosphorus source using an assay with a detection limit of 1.9 × 10-13. In this study, we found that the coexistence of a high dose of phosphate (>0.5 mM) with phosphite in the growth medium allows the phosphite-dependent E. coli strain to generate EMs at a frequency of approximately 5.4 × 10-10. In all EMs, the mutation was a single amino acid substitution of phenylalanine to cysteine or serine at position 210 of HtxC, the transmembrane domain protein of the phosphorus compound transporter HtxBCDE. Replacement of the HtxC F210 residue with the other 17 amino acids revealed that HtxC F210 is crucial in determining substrate specificity of HtxBCDE. Based on the finding of the role of HtxC F210 as a "gatekeeper" residue for this transporter, we demonstrate that the replacement of HtxC F210 with amino acids resulting from codons that require two simultaneous point mutations to generate phosphate permissive HtxC mutants can reduce the rate of EM generation to an undetectable level. These findings also provide novel insights into the functional classification of HtxBCDE as a noncanonical ATP-binding cassette transporter in which the transmembrane domain protein participates in substrate recognition.

Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient 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 Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174-Pro178, located at the C-terminus of ß7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxDHARRA for NADP (K cat/K M)NADP was 44.1 µM-1 min-1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxDHARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

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 11.6 nM for phosphatidylserine, respectively) immobilized on magnetic beads can capture small EVs (< 0.2 µm) from culture supernatants of MCF7 human breast cancer cells. Importantly, the bound EVs could be dissociated from the beads under mild conditions (e.g. 0.5 M NaCl), and the isolated EVs had the typical shapes of EVs under SEM and TEM with a mean particle size of 99 nm. Using the peptide-immobilized beads, we adapted a pre-existing bench top instrument for magnetic separation to perform automated EV purification with higher purity and yield than that obtained using the standard ultracentrifugation method.

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. We recently developed asbestos-specific fluorescent probes based on asbestos-binding proteins, and achieved efficient fluorescent labeling of asbestos. RESULTS: Live-cell imaging with fluorescent asbestos probes was successfully utilized to dynamically analyze asbestos phagocytosis. The fluorescently labeled asbestos fibers were phagocytosed by RAW 264.7 macrophages. Internalized fibers of < 5 µm in length were visualized clearly via overlaid phase contrast and fluorescence microscopy images, but they were not clearly depicted using phase contrast images alone. Approximately 60% of the cells had phagocytosed asbestos fibers after 2 h, but over 96% of cells remained alive even 24 h after the addition of asbestos fibers. Immediate cell death was only observed when an asbestos fiber was physically pulled from a cell by an external force. Notably, at 24 h after the addition of asbestos fibers an approximately 4-fold increase in the number of binucleated cells was observed. Monitoring of individual cell divisions of cells that had phagocytosed asbestos suggested that binucleated cells were formed via the inhibition of cell separation, by asbestos fibers of > 10 µm in length that were localized in the proximity of the intercellular bridge. CONCLUSIONS: Fluorescently labeled asbestos facilitated visualization of the dynamic biological processes that occur during and after the internalization of asbestos fibers, and indicated that (i) frustrated phagocytosis itself does not lead to immediate cell death unless the asbestos fiber is physically pulled from the cell by an external force, and (ii) macrophages that have phagocytosed asbestos can divide but sometimes the resulting daughter cells fuse, leading to the formation of a binucleated cell. This fusion only seemed to occur when a comparatively long asbestos fiber (> 10 µm) was shared by two daughter cells.

Insulin sensor cells for the analysis of insulin secretion responses in single living pancreatic ß 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 ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor 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-ß-cyclodextrins (TMe-ß-CDxs) were synthesised. In the 2 : 1 complexes, two aryl groups of the diazaporphyrins protruded from the upper rims of two TMe-ß-CDxs. The complexes displayed high photodynamic activity under photoirradiation at wavelengths longer than 620 nm. Although the substituents on the two aryl groups protruding from TMe-ß-CDx barely affected intracellular uptake by HeLa cells, the cellular uptake of these complexes was as high as that of a TMe-ß-CDx-tetra(4-hydroxyphenyl)porphyrin complex. Furthermore, the diazaporphyrins in the complexes with TMe-ß-CDxs were able to generate high levels of singlet oxygen because of their strong absorption of light with wavelengths greater 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. The results represent significant progress toward the optimisation of LMIPors as photosensitisers.

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 (H3PO3, 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 (H3PO4, Pi). We then identified and disrupted the two indigenous Pi transporters, pst (Synpcc7942_2441 to 2445) and pit (Synpcc7942_0184). The resultant strain failed to grow on any media containing various types of P compounds other than Pt. The strain did not yield any escape mutants for at least 28 days with a detection limit of 3.6 × 10-11 per colony forming unit, and rapidly lost viability in the absence of Pt. Moreover, growth competition of the Pt-dependent strain with wild-type cyanobacteria revealed that the Pt-dependent strain could dominate in cultures containing Pt as the sole P source. Because Pt is rarely available in aquatic environments this strategy can contribute to both biosafety and contamination management of genetically engineered cyanobacteria.

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

The weak absorbance of pristine C60 , C70 , 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 C60 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. The photodynamic activity was found to be much higher than those of dyad systems using pristine C60 and C70 .

Photodynamic Activities of Porphyrin Derivative-Cyclodextrin Complexes by Photoirradiation.

Water-soluble cyclodextrin (CyD) complexed with porphyrin derivatives with different substituents in the meso-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. These results represent a significant step toward the optimization of porphyrin derivatives as photosensitizers.

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 (H3PO2, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (H3PO3, Pt) but not phosphate (H3PO4, 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. Challenge on non-permissive growth medium revealed that no escape mutants appeared for at least 21 days with a detection limit of 1.94 × 10-13 per colony forming unit. This represents, to the best of our knowledge, the lowest escape frequency among reported strategies. Since Pt/HPt are ecologically rare and not available in amounts sufficient for the growth of the Pt/HPt-dependent bacteria, this strategy offers a reliable and practical method for biocontainment.

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

[60]Fullerene (C60) 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 C60 derivative bearing a polar group showed much higher photodynamic activity than the analogous system incorporating pristine C60.

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. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner.

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. We developed a single signal-transducing molecule with a split Gq-based DNA-nano tweezers (NT) structure that self-assembles from three single-stranded DNAs through simple mixing, and detects its target without requiring any washing steps. A model target, a partial norovirus mRNA (NV-RNA), was specifically recognized by the split Gq-based DNA-NT, causing it to undergo a structural change that restored its peroxidase activity. The peroxidase activity was measured by following the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the NV-RNA concentration. The lower detection limit was 4 nM. Our results demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT structure as a nucleic acid signal-transducing molecule in a homogenous assay format. Also the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device.