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.

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.

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.

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.

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 .

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 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. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 µM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by high-throughput screening.

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. These results confirm the feasibility of using the FRET-based DNA-NT for imaging analysis of target mRNA.

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. Thus, fusion to zinc finger motifs offers a promising option for the assembly of bi-enzymes, catalyzing a consecutive reaction, onto a dsDNA scaffold with a proximity effect.

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.

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. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as ß-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.

Targeted delivery of a decoy oligodeoxynucleotide to a single ES cell by femtoinjection.

Femtoinjection has been proposed as a feasible approach for the targeted delivery of a decoy oligodeoxynucleotide (ODN) into a single ES cell for the study of transcription factor activity. Here, we evaluated the utility of decoy ODN delivery via femtoinjection in an ES cell model in which Venus fluorescent protein was expressed under the control of the tet-off system. Femtoinjection of a control decoy (Con-decoy) and a tetracycline response element decoy (TRE-decoy) into the cytoplasm had no apparent effect on Venus fluorescent protein expression; however, femtoinjection of the TRE-decoy into the nucleus successfully suppressed expression of the Venus fluorescent protein. We therefore conclude that it is feasible to suppress the activity of a transcription factor in a single ES cell by the delivery of a decoy ODN into the nucleus using the femtoinjection technique. FROM THE CLINICAL EDITOR: The authors of this novel basic science study successfully demonstrate a femtoinjection technique to deliver a decoy oligodeoxynucleotide into a single ES cell.

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.

A femto-injection technique for dynamic analysis of protein function in living embryonic stem cells.

The potential of a femto-injection technique for use in analyzing protein dynamics in embryonic stem (ES) cells was investigated. First, we showed that fluorescent proteins could be injected in a quantitative fashion into individual mouse ES cells. Second, we demonstrated that the technique could identify functional differences between proteins by analyzing the effect of a nuclear localization signal on the behavior of glutathione S-transferase conjugated to green fluorescent protein. The analysis showed a clear difference in the distribution of the protein when the nuclear localization signal was present. Our results confirm that the non-destructive, quantitative and time controllable aspects of the technique provide considerable advantages for the analysis of protein behavior in living ES cells. To the best of our knowledge, this is the first report of the successful introduction of proteins into living ES cells by an injection technique.

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.

Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells.

In this report, the effects of two DNA nuclear targeting sequence (DTS) candidates on the gene expression efficiency in ES cells were investigated. Reporter plasmids containing the simian virus 40 (SV40) promoter/enhancer sequence (SV40-DTS), a DTS for various types of cells but not being reported yet for ES cells, and the 81 base pairs of Sox2 regulatory region 2 (SRR2) where two transcriptional factors in ES cells, Oct3/4 and Sox2, are bound (SRR2-DTS), were introduced into cytoplasm in living cells by femtoinjection. The gene expression efficiencies of each plasmid in mouse insulinoma cell line MIN6 cells and mouse ES cells were then evaluated. Plasmids including SV40-DTS and SRR2-DTS exhibited higher gene expression efficiency comparing to plasmids without these DTSs, and thus it was concluded that both sequences work as a DTS in ES cells. In addition, it was suggested that SRR2-DTS works as an ES cell-specific DTS. To the best of our knowledge, this is the first report to confirm the function of DTSs in ES cells.

A cell-free protein-producing gel.

Proteins are important biomaterials and are generally produced in living cells. Here, we show a novel DNA hydrogel that is capable of producing functional proteins without any living cells. This protein-producing gel (termed 'the P-gel system' or 'P-gel') consists of genes as part of the gel scaffolding. This is the first time that a hydrogel has been used to produce proteins. The efficiency was about 300 times higher than current, solution-based systems. In terms of volumetric yield, the P-gel produced up to 5 mg ml(-1) of functional proteins. The mechanisms behind the high efficiency and yield include improved gene stability, higher local concentration and a faster enzyme turnover rate due to a closer proximity of genes. We have tested a total of 16 different P-gels and have successfully produced all 16 proteins including membrane and toxic proteins, demonstrating that the P-gel system can serve as a general protein production technology.