An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Construction of a spheroid array culture system on a suspended permeable hydrogel membrane scaffold for improving the expression of a liver-specific drug-metabolizing enzyme of HepG2 cells.

Herein, we constructed a spheroid array culture system on a flexible hydrogel membrane suspended in the culture medium. When we applied this culture system to HepG2 cells, the results suggested that an aerobic culture environment was implemented, and the gene expression of a liver-specific drug-metabolizing enzyme was improved in comparison with that of the conventional immobilized monolayer culture.

Phototunable Cell Killing by Photochromic Diarylethene of Thiazoyl and Thienyl Derivatives.

We report a unique phototunable cell killing technique using diarylethene molecules as photo-isomerizing-molecular switches. These molecules were delivered to DNA in the cell nucleus due to closed-form generated by UV light, and then blue light triggered cell killing. A UV light irradiation switches the open form, having no DNA intercalation activity, to the closed form to induce intercalation in DNA. This isomer, thus prepared ready for the action, exerts photocytotoxicity upon the subsequent blue light irradiation. Molecular biological analysis clarifies that photocytotoxicity is due to DNA double-strand breaks. Since cell death is observed only when irradiated with light where both the open- and closed-ring isomers have absorption, the possible mechanism of cell death is assumed to be due to the repeated photocyclization and photocycloreversion reactions of the diarylethene molecules, which induce irreparable damage to DNA. This unique photo-controllable action in a cell system can provide the basis of a novel scheme of phototherapy.

Rapid and Highly Sensitive Method for Evaluating Surface Coatings against an Enveloped RNA Virus.

The COVID-19 pandemic has increased public health vigilance worldwide. The coronavirus (SARS-CoV-2) can spread via aerosols, and droplet-borne viruses remain viable on nonliving surfaces for long duration. Hence, effective antiviral coatings are highly useful in eliminating viral persistence on nonliving surfaces. Although innovative antiviral coatings have been designed, conventional procedures for antiviral assays are generally laborious, time-consuming, and have a high limit of detection. In the present study, we report a rapid and highly sensitive method for evaluating antiviral coatings by measuring the luciferase activity derived from recombinant Sendai virus (SeV). The physicochemical characteristics of SeV, which has a single-stranded RNA genome encapsulated within a lipid envelope, allow us to exploit it as an indicator of the physicochemical potential of coating materials against enveloped RNA viruses in general. We demonstrate that SeV-based assay systems allow for the rapid and quantitative evaluation of the surface coatings composed of iodine solubilized in polyvinyl acetate. Additionally, we have investigated the effect of mucins, the dominant protein component of saliva, on the antiviral activity of surface coatings. The presence of mucins in the SeV suspension considerably rescues luciferase activity at the viral-surface interface, presumably due to mucin-mediated viral protection. Our findings provide insights into a procedure capable of the rapid evaluation and optimization of surface coatings, and suggest an important role of the mucin in the valid evaluation of antiviral agents.

Photoinduced cytotoxicity of photochromic symmetric diarylethene derivatives: the relation of structure and cytotoxicity.

Photopharmacology has been attracting attention for the development of drugs with fewer side effects and lower toxicity by introducing a photoswitch structure in the drug and controlling its spatiotemporal effects by light irradiation. Ideally, to achieve precise spatiotemporal control, it is desirable to use photoresponsive molecules that act as anticancer agents based on molecular switch mechanisms at the molecular level. However, very few reports on photoinduced cytotoxicity have used photoresponsive molecules with simple structures. Here, we investigate the photoinduced cytotoxicity of twelve diarylethene derivatives having thiazole or pyridine rings in their molecules and evaluate them in terms of molecular structure and size. Our results provide insight into molecular design principles for diarylethene with a simple structure toward achieving precise control based on molecular-level switch mechanisms.

Live-cell imaging of microRNA expression with post-transcriptional feedback control.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate complex gene expression networks in eukaryotic cells. Because of their unique expression patterns, miRNAs are potential molecular markers for specific cell states. Although a system capable of imaging miRNA in living cells is needed to visually detect miRNA expression, very few fluorescence signal-on sensors that respond to expression of target miRNA (miR-ON sensors) are available. Here we report an miR-ON sensor containing a bidirectional promoter-driven Csy4 endoribonuclease and green fluorescent protein, ZsGreen1, for live-cell imaging of miRNAs with post-transcriptional feedback control. Csy4-assisted miR-ON (Csy4-miR-ON) sensors generate negligible background but respond sensitively to target miRNAs, allowing high-contrast fluorescence detection of miRNAs in various human cells. We show that Csy4-miR-ON sensors enabled imaging of various miRNAs, including miR-21, miR-302a, and miR-133, in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications.

Membrane properties of ether-type phosphatidylcholine bearing partially fluorinated C18-monoacetylenic chains and their applicability to membrane protein reconstitution matrices.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

Fabrication of Hollow Structures in Photodegradable Hydrogels Using a Multi-Photon Excitation Process for Blood Vessel Tissue Engineering.

Engineered blood vessels generally recapitulate vascular function in vitro and can be utilized in drug discovery as a novel microphysiological system. Recently, various methods to fabricate vascular models in hydrogels have been reported to study the blood vessel functions in vitro; however, in general, it is difficult to fabricate hollow structures with a designed size and structure with a tens of micrometers scale for blood vessel tissue engineering. This study reports a method to fabricate the hollow structures in photodegradable hydrogels prepared in a microfluidic device. An infrared femtosecond pulsed laser, employed to induce photodegradation via multi-photon excitation, was scanned in the hydrogel in a program-controlled manner for fabricating the designed hollow structures. The photodegradable hydrogel was prepared by a crosslinking reaction between an azide-modified gelatin solution and a dibenzocyclooctyl-terminated photocleavable tetra-arm polyethylene glycol crosslinker solution. After assessing the composition of the photodegradable hydrogel in terms of swelling and cell adhesion, the hydrogel prepared in the microfluidic device was processed by laser scanning to fabricate linear and branched hollow structures present in it. We introduced a microsphere suspension into the fabricated structure in photodegradable hydrogels, and confirmed the fabrication of perfusable hollow structures of designed patterns via the multi-photon excitation process.

Effect of the fluorination degree of partially fluorinated octyl-phosphocholine surfactants on their interfacial properties and interactions with purple membrane as a membrane protein model.

Interfacial properties and membrane protein solubilization activity of a series of partially fluorinated octyl-phosphocholine (PC) surfactants were investigated from the viewpoint of the fluorination degree of the hydrophobic chain. The critical micelle concentration (CMC), surface tension lowering activity, molecular occupied area at the CMC and free energy changes of micellization as well as adsorption to the air-water interface for each PC surfactant were estimated from surface tension measurements at 25 °C. The PCs with higher degree of fluorination exhibited low CMC and high surface activity, while the single trifluoromethyl group at the end of the chain appeared to enhance the hydrophilicity of the surfactant molecule. Under conditions where conventional short-chain surfactants, n-octyl-ß-D-glucoside, Triton X-100 and dioctanoylphosphatidylcholine significantly solubilize purple membranes (PM), none of the fluorinated-PCs solubilized PM. This suggests that fluorinated-PCs are low-invasive enough to maintain the structure of lipids/protein assemblies like PM.

Photolithographic Fabrication of Semi 3-D Microstructures Composed of Flexible Hydrogel Sheet for in Vivo-like Cell Culture System.

Insufficient reliability of current drug screening by cell-based assay has been one of the factors in the poor success rate in drug development. To improve the situation, we proposed a cell culture system using semi 3-D hydrogel microstructures as cell culture scaffolds. Because of its flexibility and permeability, the microstructure was expected to enhance the physiological function of cells. We developed a simple method of fabricating the unique hydrogel microstructures composed of hydroxypropyl cellulose through photolithography. Functionalization with poly(styrene-co-maleic anhydride) gained their cell adhesion, and it was demonstrated that several kinds of cells were incubated on the cell culture scaffolds.

Automated adherent cell elimination by a high-speed laser mediated by a light-responsive polymer.

Conventional cell handling and sorting methods require manual labor, which decreases both cell quality and quantity. To purify adherent cultured cells, cell purification technologies that are high throughput without dissociation and can be utilized in an on-demand manner are expected. Here, we developed a Laser-induced, Light-responsive-polymer-Activated, Cell Killing (LiLACK) system that enables high-speed and on-demand adherent cell sectioning and purification. This system employs a visible laser beam, which does not kill cells directly, but induces local heat production through the trans-cis-trans photo-isomerization of azobenzene moieties. Using this system in each passage for sectioning, human induced pluripotent stem cells (hiPSCs) maintained their pluripotency and self-renewal during long-term culture. Furthermore, combined with deep machine-learning analysis on fluorescent and phase contrast images, a label-free and automatic cell processing system has been developed by eliminating unwanted spontaneously differentiated cells in undifferentiated hiPSC culture conditions.

Fabrication of pocket-like hydrogel microstructures through photolithography.

Photolithographic fabrication of unique microstructures composed of flexible hydrogel sheets is proposed and demonstrated by using photo-acid-generating poly(methyl methacrylate). Crosslinking of a hydroxyl-rich polymer and lifting off of the crosslinked polymer layer from the substrate are controlled respectively in an area-selective manner upon micropatterned light irradiation, and various pocket-like microstructures are fabricated resultantly.

Photoresponsive Aqueous Dissolution of Poly( N-Isopropylacrylamide) Functionalized with o-Nitrobenzaldehyde through Phase Transition.

We report a sharp photoinduced aqueous dissolution of the copolymer through phase transition based on the photochemical reaction of o-nitrobenzaldehyde (NBA) and the principle of polymer effect. We synthesized the copolymers having poly( N-isopropylacrylamide) main chain and NBA side chain at 4, 7, and 10 mol % functionalizations and analyzed their photoresponsive characteristics. Light with 365 nm wavelength converted NBA groups at copolymer side chains to carboxylic acid efficiently at the rate of 7.3 cm2/J, and in the case of 10 mol % functionalization, the irradiation dosage no more than 56 mJ/cm2 induced sharp aqueous dissolution of the copolymer thin layer in pH 7.4 at 25 °C. As example applications, we demonstrated on-demand release of polyethylene beads and fluorescent-labeled albumins, which had been immobilized on a substrate surface via the copolymers, by the precisely controlled light irradiation using a microprojection system. Also, we examined application of the copolymers to the selective recovery of living cells from culture substrate under microscopic observation. As a result, mild light irradiation at room temperature triggered immediate detachment of the cultured adherent cells only in the irradiated areas without critical influence on their viability.

Photo- and Thermoresponsive Dehydration of Spiropyran-Functionalized Polymer Regulated by Molecular Recognition.

The photo- and thermoresponse of poly(N-isopropylacrylamide) (pNIPAAm) functionalized with spiropyran chromophore is examined with respect to the influence of molecular recognition by cyclodextrin (CD). Characterization in aqueous solutions of spiropyran-functionalized poly(N,N-dimethylacrylamide) under coexistence of α-, ß-, or γ-CD reveals that ß-CD selectively includes the ring-closing isomer of the chromophore, which is dominant under light irradiation, while no inclusion is observed for the protonated ring-opening isomer, which is dominant in the dark before irradiation. As a result, it is shown that the selective inclusion of the chromophore at a polymer side chain is switched by light irradiation. Further, drastic photoresponsive dehydration of spiropyran-functionalized pNIPAAm is inhibited only by ß-CD out of three examined CDs, demonstrating that the molecular recognition regulates the dehydration of the whole polymer triggered by the photoswitching of the chromophore introduced at only 1 mol% functionalization.

Morphology-based optical separation of subpopulations from a heterogeneous murine breast cancer cell line.

Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated in vitro by using a spheroid assay and in vivo by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics.

Click-crosslinkable and photodegradable gelatin hydrogels for cytocompatible optical cell manipulation in natural environment.

This paper describes the generation of "click-crosslinkable" and "photodegaradable" gelatin hydrogels from the reaction between dibenzocycloctyl-terminated photoclevable tetra-arm polyethylene glycol and azide-modified gelatin. The hydrogels were formed in 30 min through the click-crosslinking reaction. The micropatterned features in the hydrogels were created by micropatterned light irradiation; the minimum resolution of micropatterning was 10-µm widths for line patterns and 20-µm diameters for circle patterns. Cells were successfully encapsulated in the hydrogels without any loss of viability across a wide concentration range of crosslinker. In contrast, an activated-ester-type photocleavable crosslinker, which we previously used to prepare photodegradable gelatin hydrogels, induced a decrease in cell viability at crosslinker concentrations greater than 1.8 mM. We also observed morphology alteration and better growth of cancer cells in the click-crosslinked photodegradable gelatin hydrogels that included matrigel than in the absence of matrigel. We also demonstrated micropatterning of the hydrogels encapsulating cells and optical cell separation. Both of the cells that remained in the non-irradiated area and the cells collected from the irradiated area maintained their viability.

Photoinduced cytotoxicity of a photochromic diarylethene via caspase cascade activation.

The photo-generated closed-ring isomer of bis(5-methyl-2-phenylthiazoyl)perfluorocyclopentene shows cytotoxicity to Madin-Darby canine kidney (MDCK) cells through a caspase cascade and induces apoptosis of cells.

Activated-ester-type photocleavable crosslinker for preparation of photodegradable hydrogels using a two-component mixing reaction.

Photodegradable hydrogels have emerged as powerful platforms for studying and directing cellular behavior in a spatiotemporally controlled manner. Photodegradable hydrogels have previously been formed by free radical polymerizations, Michael-type addition reactions, and orthogonal click reactions. Here, an ester-activated photocleavable crosslinker is presented for preparing photodegradable hydrogels by means of a one-step mixing reaction between the crosslinker and a biocompatible polymer containing amino moieties (amino-terminated tetra-arm poly(ethylene glycol) or gelatin). It is demonstrated that photodegradable hydrogels micropatterned by photolithography can be used to culture cells with high viability and proliferation rates. The resulting micropatterned cell-laden structures can potentially be used to create 3D biomaterials for various tissue-engineering applications.

Partially photodegradable hybrid hydrogels with elasticity tunable by light irradiation.

This paper reports a simple technique to synthesize elasticity tunable hybrid hydrogels using photocleavable (N-hydroxysuccinimide terminated photocleavable tetra-arm poly(ethylene glycol); NHS-PC-4armPEG) and non-photocleavable (N-hydroxysuccinimide terminated tetra-arm poly(ethylene glycol); NHS-4armPEG) activated-ester type crosslinkers. Partially photodegradable hybrid hydrogels were synthesized by reacting the crosslinker mixture with amino-terminated tetra-arm poly(ethylene glycol) (amino-4armPEG). The photocleavable crosslinks are cleaved by irradiating light while the non-photocleavable crosslinks remain intact, resulting in decreased elasticity. We demonstrate that hydrogel elasticity can be controlled by adjusting the ratio of photocleavable NHS-PC-4armPEG and non-photocleavable NHS-4armPEG, and by varying the light exposure energy. We also show how micropatterned elasticity can be obtained in the hydrogels by irradiating with micropatterned light. These techniques could provide a novel platform to tailor the elasticity of hydrogels with microscale precision for biological studies in the near future.