Search

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimation method and a water-annealing treatment. The pore structure of the 3D LS-aero-sponge was constructed by the branched fibrous substrates and the films with small holes. It was assumed that the branched fibrous structure was mainly comprised of fibroin whereas the films were comprised of the fibroin interacting with the sericin fractions. Most of the WAXD peaks from the as-molded 3D LS-aerosponge could be assigned to the silk1 crystal diffraction peaks of fibroin. The silk1 crystal modification of fibroin formed in the course of the production of the 3D LS-aero-sponge could bring about a good thermal shrinkproof property. Furthermore, a gamma ray irradiation treatment applied prior to the cell proliferation tests of the 3D LS-aero-sponges was found to be effective to form cross-linking in the sericin fractions. As a result, the yield stress in the compression tests of the 3D LS-aero-sponges was increased. The 3D LS-aerosponge exhibited a good living cell proliferation properly in the culture tests using NH3T3 cells (Swiss albino mice embryonic fibroblasts) although that of the 2D LS-cast films was very poor. We suggest the good cell proliferation property of the 3D LS-aerosponge was due to the continuous branched fibrous structure comprised of fibroin.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.

Mesoscopic structures formed during an ordering process in thermodynamically unstable, isometric, binary molecular mixtures were explored by time-resolved scattering (TRS) and laser scanning confocal microscopy (LSCM). Three-dimensional (3D) bicontinuous structures, which were constructed for the first time by time-resolved LSCM, were found to have a sponge-like structure composed of two phases. The structure factor obtained by 3D Fourier transformation of the sponge was found to be identical to that obtained by TRS, confirming that the sponge truly reflects the structural entities evolving in the system. Furthermore, the sponge was shown for the first time to be theoretically predictable by using 3D computer simulations based on the time-dependent Ginzburg-Landau theory. The sponge was subjected to differential geometrical analysis: its Gaussian curvature K, mean curvature H, and their distributions were successfully determined for the first time. The result revealed that the sponge has hyperbolic interfaces with area-averaged curvatures satisfying ≅ 0 and that its interface has some deviations from a minimal surface. The sponge was found to be strikingly similar to that occurring in oil/water/surfactant systems at the hydrophile-lipophile-balance, though their characteristic length scales are diversely different (pm vs nm), implying universality of the sponge.

An overview is given of the three-dimensional microdomain structure of the star ABC composed of PS, PI, and PDMS with the arm-length (Mn) ratio of almost 1:1:1 (PS: 19.5K; PI: 20.7K; PDMS: 19.0K). In this case, strong repulsive interactions among the three different blocks yield a three-phase microdomain structure at room temperature.

A striated muscle fiber consists of thousands of myofibrils with crystalline hexagonal myofilament lattices. Because the lattices are randomly oriented, the fiber gives rise to an equatorial x-ray diffraction pattern, which is essentially a rotary-averaged "powder diffraction," carrying only information about the distance between the lattice planes. We were able to record an x-ray diffraction pattern from a single myofilament lattice, very likely originating from a single myofibril from the flight muscle of a bumblebee, by orienting the incident x-ray microbeam along the myofibrillar axis (end-on diffraction). The pattern consisted of a number of hexagonally symmetrical diffraction spots whose originating lattice planes were readily identified. This also held true for some of the weak higher order reflections. The spot-like appearance of reflections implies that the lattice order is extremely well maintained for a distance of millimeters, covering up to a thousand of approximately 2.5-microm-long sarcomeres connected in series. The results open the possibility of applying the x-ray microdiffraction technique to study many other micrometer-sized assemblies of functional biomolecules in the cell.