Cell fiber-based 3D tissue array for drug response assay.

For the establishment of a reproducible and sensitive assay system for three-dimensional (3D) tissue-based drug screening, it is essential to develop 3D tissue arrays with uniform shapes and high cell numbers that prevent cell death in the center of the tissue. In recent years, 3D tissue arrays based on spheroids have attracted increased attention. However, they have only been used in specific tissues with hypoxic regions, such as cancer tissues, because nutrient deprivation and hypoxic regions are formed in the core as spheroids grow. Herein, we propose a method to array cell-encapsulated tube-like tissue (cell fiber (CF)) with diameters < 150 µm to prevent nutrient deprivation and hypoxia using a device that can fix the CFs, section them in uniform sizes, and transfer them to a 96-well plate. We fabricated the arrays of CF fragments from cell lines (GT1-7), cancer cells (HeLa), mouse neural stem cells (mNSCs) and differentiated mNSCs, and performed drug response assays. The array of CF fragments assessed the drug response differences among different cell types and drug responses specific to 3D tissues. The array of CF fragments may be used as a versatile drug screening system to detect drug sensitivities in various types of tissues.

Long-Term Continuous Glucose Monitoring Using a Fluorescence-Based Biocompatible Hydrogel Glucose Sensor.

Fluorescence-based hydrogel glucose sensors with boronic acid-based glucose recognition are promising regarding their potential to improve continuous glucose monitoring by facilitating long-lasting accuracy. However, these sensors typically become encapsulated after implantation, and the dynamic range decreases following long-term implantation and use. Herein, a four-arm polyethylene glycol (PEG) hydrogel is developed with an immobilized glucose-responsive fluorescence dye (GF-PEG-gel); this hydrogel is effective in mitigating foreign body reactions (FBRs) that hinder stable glucose responses in vivo. The GF-PEG-gel attached to an implantable device successfully traces blood glucose concentrations in diabetic model rats for 45 days. A pig experiment shows that the device measures the glucose concentration with an equivalent accuracy to that required for clinically available settings.

Hydrogel Glucose Sensor with In Vivo Stable Fluorescence Intensity Relying on Antioxidant Enzymes for Continuous Glucose Monitoring.

Hydrogel glucose sensors with boronic acid-based fluorescence intensity theoretically hold promise to improve in vivo continuous glucose monitoring (CGM) by facilitating long-lasting accuracy. However, these sensors generally degrade after implantation and the fluorescence intensity decreases immediately over time. Herein, we describe a hydrogel glucose sensor with in vivo stability based on boronic acid-based fluorescence intensity, integrating two antioxidant enzymes, superoxide dismutase (SOD), and catalase. These protected the arylboronic acid from being degraded by hydrogen peroxide in vitro and preserved the boronic acid-based fluorescence intensity of the hydrogel glucose sensors in rats for 28 days. These antioxidant enzymes also allowed the hydrogel glucose sensor attached to a homemade semi-implantable CGM device to trace blood glucose concentrations in rats for 5 h with the accuracy required for clinical settings. Hydrogel glucose sensors with boronic acid-based fluorescence intensity containing SOD and catalase could comprise a new strategy for in vivo CGM.

Enantiopure O-ethyl phenylphosphonothioic acid: a solvating agent for the determination of enantiomeric excesses.

An improved method, which is highly reproducible, was developed for the enantioseparation of racemic O-ethyl phenylphosphonothioic acid (1a) with brucine by introducing seeding to a supersaturated solution of the diastereomeric salt mixture. The present method gave both diastereomeric salts in high yields with a diastereomeric ratio of >99.5:0.5 upon choosing the crystallization solvent (MeOH for the (R)-1a salt and MeOH/H2 O for the (S)-1a salt). The enantiopure acid showed a good chirality recognition ability for not only strong bases, such as amines and amino alcohols, but also weakly basic alcohols and was applicable as a solvating agent to the (1) H NMR determination of the enantiomeric excess of chiral amines, amino alcohols, and alcohols, including aliphatic substrates.

Dry micromanipulation of supramolecular giant vesicles on a silicon substrate: highly stable hydrogen-bond-directed nanosheet membrane.

Guanosine derivative 1 forms hydrogen-bond-directed giant vesicles. On a silicon substrate, the vesicles retain their shape and internal water phase even after removal of external water under vacuum. Dry manipulation of the micrometer-sized vesicles was carried out via AFM-tip-induced partition and fusion of the vesicles. For larger vesicles (5-10 µm), external solutions were successfully injected through a microcapillary inserted into the vesicle in air.

One-step synthesis of differently bis-functionalized isoxazoles by cycloaddition of carbamoylnitrile oxide with ß-keto esters.

A new protocol for synthesizing different functionalized isoxazoles is provided. Carbamoylnitrile oxide generated from nitroisoxazolone underwent inverse electron-demand 1,3-dipolar cycloaddition with 1,3-dicarbonyl compounds in the presence of magnesium acetate that formed magnesium enolate in situ. Although electron-deficient trifluoroacetoacetate did not undergo this cycloaddition under the same conditions, conversion to sodium enolate furnish the corresponding bis-functionalized trifluoromethylisoxazole. The DFT calculations using B3LYP 6-31G+(d,p) also supported the aforementioned reactivity.

Inverse electron-demand 1,3-dipolar cycloaddition of nitrile oxide with common nitriles leading to 3-functionalized 1,2,4-oxadiazoles.

A carbamoyl-substituted nitrile oxide was generated upon treatment of easily available 2-methyl-4-nitro-3-isoxazolin-5(2H)-one with THF (not dried); the reaction proceeded efficiently even in the absence of any special reagents and reaction conditions. The nitrile oxide caused 1,3-dipolar cycloaddition with common aliphatic nitriles or electron-rich aromatic nitriles to afford 3-functionalized 1,2,4-oxadiazoles, which are expected to serve as precursors for the preparation of a variety of functional materials by the chemical transformation of the carbamoyl group. While conventional preparative methods for 1,2,4-oxadiazoles involve the cycloaddition of an electron-rich nitrile oxide with an electron-deficient nitrile or a nitrile activated by a Lewis acid, our method employs the complementary combination of an electron-rich nitrile and an electron-deficient nitrile oxide- the inverse electron-demand 1,3-cycloaddition. The DFT calculations using B3LYP 6-31G* supported the abovementioned inverse reactivity, and also suggested the presence of an accelerating effect by the carbamoyl group as a result of hydrogen bond formation with a dipolarophilic nitrile.

Hydrogen-bond-directed giant unilamellar vesicles of guanosine derivative: preparation, properties, and fusion.

By mixing a small volume of THF containing guanosine derivative 1 and tetraethylenegrycol dodecyl ether (TEGDE) with water and subsequently removing TEGDE by gel permeation chromatography, micrometer-sized giant unilamellar vesicles (GUV) of 1 were successfully prepared. The vesicle membrane was a 2-D sheet assembly of thickness 2.5 nm, composed of a 2-D inter-guanine hydrogen-bond network. The GUV dispersion showed high stability because of a large negative zeta potential, which allowed repeated sedimentation and redispersion by centrifugation and subsequent gentle agitation. TEGDE-triggered fusion of GUVs took place within 350 ms, which proceeded by fusion of the vesicle membranes in contact. These unique static and dynamic properties of the GUV membrane assembled by the 2-D hydrogen-bond network are discussed.

Bicyclization involving pseudo-intramolecular imination with diamines.

α-Nitro-δ-keto nitriles and α-nitro-δ-keto ester were readily converted to diazabicyclo compounds having vicinal functionality upon treatment with diamines. The keto nitrile attracts the diamine nearby to an acidic hydrogen to cause the pseudo-intramolecular imination which proceeds efficiently without any catalyst at room temperature.

An anomalous hydration/dehydration sequence for the mild generation of a nitrile oxide.

A nitrile oxide containing a carbamoyl group is readily generated upon the treatment of 2-methyl-4-nitro-3-isoxazolin-5(2H)-one with water under mild reaction conditions, even in the absence of special reagents. The obtained nitrile oxide undergoes cycloaddition with dipolarophiles, alkynes and alkenes, to afford the corresponding isoxazol(in)es, which are useful intermediates in the synthesis of polyfunctionalized compounds. A plausible mechanism underlying the formation of the nitrile oxide is proposed, which involves an anomalous hydration/dehydration sequence. DFT calculations were also performed to support this mechanism.

Hydrogen-bond-directed giant vesicles of guanosine derivatives in water: formation, structure, and stability.

Hydrogen-bond-directed giant supramolecular vesicles (diameter 1.20 +/- 0.30 microm (SD)) of an alkylsilylated deoxyguanosine derivative, 2a, were prepared faciley by mixing a small volume of a 2a/THF solution with water. The formation of 2-D inter-guanine hydrogen-bond networks of 2a within the vesicles was indicated by IR spectra. The vesicle solution was stable enough for more than 30 days, in a wide range of temperatures, and between pH 4 and 10 without showing lysis, fusion, precipitation, or leakage of the encapsulated fluorescent probe. In a typical micrometer-sized vesicle, a sufficiently large internal water phase for encapsulating water-soluble substances was surrounded by a multilamellar membrane 15-20 nm in thickness, which was composed of 6-9 layers of 2-D hydrogen-bond-directed sheet assemblies. AC-mode AFM observation of the vesicle on a silicon substrate further demonstrated the high stability and deformable properties of the vesicle membrane under vacuum or mechanical stress. The formation and properties of the vesicle membrane in water were analyzed from the viewpoint of the 2-D hydrogen-bond-directed sheet assemblies, and the scope of the design principle to use nonpolar soft segments as the shielding units of the hydrogen-bond networks in water is discussed.