An In Vivo Dual-Observation Method to Monitor Tumor Mass and Tumor-Surface Blood Vessels for Developing Anti-Angiogenesis Agents against Submillimeter Tumors.

Managing metastasis at the early stage and detecting and treating submillimeter tumors at early metastasis are crucial for improving cancer prognosis. Angiogenesis is a critical target for developing drugs to detect and inhibit submillimeter tumor growth; however, drug development remains challenging because there are no suitable models for observing the submillimeter tumor mass and the surrounding blood vessels in vivo. We have established a xenograft subcutaneous submillimeter tumor mouse model with HT-29-RFP by transplanting a single spheroid grown on radiation-crosslinked gelatin hydrogel microwells. Here, we developed an in vivo dual-observation method to observe the submillimeter tumor mass and tumor-surface blood vessels using this model. RFP was detected to observe the tumor mass, and a fluorescent angiography agent FITC-dextran was administered to observe blood vessels via stereoscopic fluorescence microscopy. The anti-angiogenesis agent regorafenib was used to confirm the usefulness of this method. This method effectively detected the submillimeter tumor mass and tumor-surface blood vessels in vivo. Regorafenib treatment revealed tumor growth inhibition and angiogenesis downregulation with reduced vascular extremities, segments, and meshes. Further, we confirmed that tumor-surface blood vessel areas monitored using in vivo dual-observation correlated with intratumoral blood vessel areas observed via fluorescence microscopy with frozen sections. In conclusion, this method would be useful in developing anti-angiogenesis agents against submillimeter tumors.

A Radiation-Crosslinked Gelatin Hydrogel That Promotes Tissue Incorporation of an Expanded Polytetrafluoroethylene Vascular Graft in Rats.

A prosthetic vascular graft that induces perigraft tissue incorporation may effectively prevent serious sequelae such as seroma formation and infection. Radiation-crosslinked gelatin hydrogel (RXgel) mimics the chemical and physical properties of the in vivo extracellular matrix and may facilitate wound healing by promoting tissue organization. Fibroblasts cultured on RXgel actively migrated into the gel for up to 7 days. RXgels of three different degrees of hardness (Rx[10], soft; Rx[15], middle; Rx[20], hard) were prepared, and small disc-like samples of RXgels were implanted into rats. In vitro and in vivo results indicated that Rx[10] was too soft to coat vascular grafts. Thus, expanded polytetrafluoroethylene (ePTFE) vascular grafts coated with RXgel were developed using Rx[15] and Rx[20] gels, and ring-shaped slices of the graft were implanted into rats. Alpha-smooth muscle actin (αSMA) and type III collagen (Col-III) levels were detected by immunohistochemistry. Immunohistochemical staining for αSMA and Col-III demonstrated that RXgel-coated vascular grafts induced more granulation tissue than non-coated grafts on days 14 and 28 after implantation. RXgel-coated ePTFE vascular grafts may provide a solution for patients by reducing poor perigraft tissue incorporation.

Collagen hydrogels with controllable combined cues of elasticity and topography to regulate cellular processes.

The elasticity, topography, and chemical composition of cell culture substrates influence cell behavior. However, the cellular responses toin vivoextracellular matrix (ECM), a hydrogel of proteins (mainly collagen) and polysaccharides, remain unknown as there is no substrate that preserves the key features of native ECM. This study introduces novel collagen hydrogels that can combine elasticity, topography, and composition and reproduce the correlation between collagen concentration (C) and elastic modulus (E) in native ECM. A simple reagent-free method based on radiation-cross-linking altered ECM-derived collagen I and hydrolyzed collagen (gelatin or collagen peptide) solutions into hydrogels with tunable elastic moduli covering a broad range of soft tissues (E= 1-236 kPa) originating from the final collagen density in the hydrogels (C= 0.3%-14%) and precise microtopographies (⩾1 µm). The amino acid composition ratio was almost unchanged by this method, and the obtained collagen hydrogels maintained enzyme-mediated degradability. These collagen hydrogels enabled investigation of the responses of cell lines (fibroblasts, epithelial cells, and myoblasts) and primary cells (rat cardiomyocytes) to soft topographic cues such as thosein vivounder the positive correlation betweenCandE. These cells adhered directly to the collagen hydrogels and chose to stay atop or spontaneously migrate into them depending onE, that is, the density of the collagen network,C. We revealed that the cell morphology and actin cytoskeleton organization conformed to the topographic cues, even when they are as soft asin vivoECM. The stiffer microgrooves on collagen hydrogels aligned cells more effectively, except HeLa cells that underwent drastic changes in cell morphology. These collagen hydrogels may not only reducein vivoandin vitrocell behavioral disparity but also facilitate artificial ECM design to control cell function and fate for applications in tissue engineering and regenerative medicine.

A simple method for production of hydrophilic, rigid, and sterilized multi-layer 3D integrated polydimethylsiloxane microfluidic chips.

Polydimethylsiloxane (PDMS) has many desirable features for microfluidics applications, particularly in diagnostics and pharmaceuticals, but its hydrophobicity and the lack of a practical method for bonding PDMS layers limit its use. Moreover, the flexibility of PDMS causes unwanted deformation during use in some applications. Here, we report a simple method for solving these problems simultaneously using an electron beam (EB) or γ-rays, which are commonly used for sterilizing medical products. Simply by applying EB or γ-ray irradiation to stacked PDMS layers, we can not only bond the interfaces between the layers by forming Si-O-Si covalent bonds but also achieve long-lasting hydrophilization and sterilization of the internal microchannels and chambers, prevent nonspecific adsorption and absorption of hydrophobic small molecules, and enhance the mechanical strength of the material by converting bulk PDMS into a Si-Ox-rich (where x is 3 or 4) structure though crosslinking. Unlike the one-at-a-time plasma process, EBs and γ-rays can penetrate through many stacked layers of PDMS sealed in their final package, enabling batch modification and bonding. The method requires no chemical crosslinkers, adhesive agents, or fillers; hence, it does not undermine the advantages of PDMS such as ease of molding in soft lithography, biocompatibility, and optical transparency. Furthermore, bonding is achieved with high-throughput yield because it occurs after re-adjustable alignment. We demonstrate that this method is applicable in the mass production of 3D integrated PDMS microfluidic chips with some glass-like properties as well as for 3D structures with complex shapes that are difficult to fabricate with plastic or glass.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis.

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼ 7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.

Fabrication of nanobeads from nanocups by controlling scission/crosslinking in organic polymer materials.

The development of several kinds of micro/nanofabrication techniques has resulted in many innovations in the micro/nanodevices that support today's science and technology. With feature miniaturization, the fabrication tools have shifted from light to ionizing radiation. Here, we propose a simple micro/nanofabrication technique for organic materials using a scanning beam (SB) of ionizing radiation. By controlling the scission/crosslinking of the material via three-dimensional energy-deposition distribution of the SB, appropriate solvents can easily peel off only the crosslinked region from the bulk material. The technique was demonstrated using a focused ion beam and a chlorinated organic polymer. The polymer underwent main-chain scission upon irradiation, but it crosslinked after high-dose irradiation. Appropriate solvents could easily peel off only the crosslinked region from the bulk material. The technique, 'nanobead from nanocup', enabled the production of desired structures such as nanowires and nanomembranes. It can be also applied to the micro/nanofabrication of functional materials.

MEP-1 and a homolog of the NURD complex component Mi-2 act together to maintain germline-soma distinctions in C. elegans.

A rapid cascade of regulatory events defines the developmental fates of embryonic cells. However, once established, these developmental fates and the underlying transcriptional programs can be remarkably stable. Here, we describe two proteins, MEP-1 and LET-418/Mi-2, required for maintenance of somatic differentiation in C. elegans. In animals lacking MEP-1 and LET-418, germline-specific genes become derepressed in somatic cells, and Polycomb group (PcG) and SET domain-related proteins promote this ectopic expression. MEP-1 and LET-418 interact in vivo with the germline-protein PIE-1. Our findings support a model in which PIE-1 inhibits MEP-1 and associated factors to maintain the pluripotency of germ cells, while at later times MEP-1 and LET-418 remodel chromatin to establish new stage- or cell-type-specific differentiation potential.